Dr. Wernig is a Professor in the Departments of Pathology and Chemical and Systems Biology and Co-Director of the Institute for Stem Cell Biology and Regenerative Medicine at Stanford University. He graduated with an M.D. Ph.D. from the Technical University of Munich where he trained in developmental genetics in the lab of Rudi Balling. After completing his residency in Neuropathology and General Pathology at the University of Bonn, he then became a postdoctoral fellow in the lab of Dr. Rudolf Jaenisch at the Whitehead Institute for Biomedical Research/ MIT in Cambridge, MA.

He received an NIH Pathway to Independence Award, the Cozzarelli Prize for Outstanding Scientific Excellence from the National Academy of Sciences U.S.A., the Outstanding Investigator Award from the International Society for Stem Cell Research, the New York Stem Cell Foundation Robertson Stem Cell Prize, and more recently was awarded the Ogawa-Yamanaka Stem Cell Prize presented by the Gladstone Institutes and has been named a HHMI Faculty Scholar.

Dr. Wernig’s lab is interested in pluripotent stem cell biology and the molecular determinants of neural cell fate decisions. His laboratory was the first to generate functional neuronal cells reprogrammed directly from skin fibroblasts, which he termed induced neuronal (iN) cells. The lab is now working on identifying the molecular mechanisms underlying induced lineage fate changes, the phenotypic consequences of disease-causing mutations in human neurons and other neural lineages as well as the development of novel therapeutic gene targeting and cell transplantation-based strategies for a variety of monogenetic diseases.

Administrative Appointments

  • Faculty Senate, Department of Pathology (2017 - Present)
  • Assistant Professor, Institute for Stem Cell Biology and Regenerative Medicine (2008 - 2014)

Honors & Awards

  • Ogawa-Yamanaka Stem Cell Prize, The Gladstone Institutes (2018)
  • HHMI Faculty Scholar Award, Howard Hughes Medical Institute (2016)
  • New York Stem Cell Foundation Robertson Stem Cell Prize, New York Stem Cell Foundation (2014)
  • The Outstanding Young Investigator Award, International Society for Stem Cell Research (2013)
  • Ascina Award, Republic of Austria (2010)
  • Cozzarelli Prize for Outstanding Scientific Excellence, National Academy of Sciences USA (2009)
  • New Scholar in Aging, Ellison Medical Foundation (2010)
  • Robertson Investigator Award, New York Stem Cell Foundation (2010)
  • Donald E. and Delia B. Baxter Faculty Scholarship, Stanford University (2009)
  • Margaret and Herman Sokol Award, Biomedical Research (2007)
  • Longterm fellowship Human Frontiers Science Program Organisation, HFSP (2004-2006)

Boards, Advisory Committees, Professional Organizations

  • Member, Society for Neuroscience (2003 - Present)
  • Member, International Society for Stem Cell Research (2004 - Present)
  • Editorial Board Member, Cell Stem Cell (2012 - Present)
  • Editorial Board Member, Stem Cell Reports (2013 - Present)
  • Member, Program Committee, Society for Neuroscience (2016 - Present)
  • Chair, Program Committee, International Society for Stem Cell Research (2017 - Present)

Professional Education

  • M.D., Technical University of Munich, Medicine (2000)

Current Research and Scholarly Interests

Our laboratory is generally interested in the molecular mechanisms that determine specific cell fates.

Recently, we have identified a pool of transcription factors that are sufficient to convert skin fibroblasts directly into functional neuronal cells that we termed induced neuronal (iN) cells. This was a surprising finding and indicated that direct lineage reprogramming may be applicable to many somatic cell types and many different directions. Indeed, following our work others have identified transcription factors that could induce cardiomyocytes, blood progenitors, and hepatocytes from fibroblasts.

We are now focussing on two major aspects of iN and iPS cell reprogramming:
(i) we are fascinated by the puzzle how a hand full of transcription factors can so efficiently reprogram the entire epigenome of a cell so that it changes identity. To that end we are applying genome-wide expression analysis, chromatin immunoprecipitation, protein biochemistry, proteomics and functional screens.
(ii) it is equally exciting to now use reprogramming methods as tools to study or treat certain diseases. iPS cells have the great advantage that they can easily be genetically manipulated rendering them ideal for treating monogenetic disorders when combined with cell transplantation-based therapies. In particular we are working on Dystrophic Epidermolysis Bullosa in collaboration with Stanford's Dermatology Department. An exciting application of iN cell technology will be to try modeling neurological diseases in vitro. We perform both mouse and human experiments hoping to identify quantifiable phenotypes correlated with genotype and in a second step evaluate whether this assay could be used to discover novel drugs improve the disease progression.

Clinical Trials

  • Characteristics of Patients With Recessive Dystrophic Epidermolysis Bullosa Recruiting

    Recessive dystrophic epidermolysis bullosa (RDEB) is a disease caused by genetic mutations in the gene for type VII collagen. Patients with RDEB develop large, severely painful blisters and open wounds from minor trauma to their skin. We are screening subjects with RDEB to evaluate characteristics of the subjects and their cells in order to develop new strategies of therapy and determine whether subjects could be candidates for treatment studies.

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  • Study to Create Potential Cell-Based Therapies to Treat Human Disease and Disability Not Recruiting

    This is a research study in which your cells will be used for somatic cell nuclear transfer (SCNT), and/or genetic reprogramming research which may result in the production of stem cell lines. This study does not provide treatment.

    Stanford is currently not accepting patients for this trial.

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  • Study to Create Potential Cell-Based Therapies to Treat Human Disease and Disability Not Recruiting

    This is a research study in which your cells will be used for somatic cell nuclear transfer (SCNT), and/or genetic reprogramming research which may result in the production of stem cell lines. This study does not provide treatment.

    Stanford is currently not accepting patients for this trial. For more information, please contact Tasha Kalista, (650) 725 - 9977.

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2023-24 Courses

Stanford Advisees

All Publications

  • A scalable and cGMP-compatible autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa. Nature communications Neumayer, G., Torkelson, J. L., Li, S., McCarthy, K., Zhen, H. H., Vangipuram, M., Mader, M. M., Gebeyehu, G., Jaouni, T. M., Jacków-Malinowska, J., Rami, A., Hansen, C., Guo, Z., Gaddam, S., Tate, K. M., Pappalardo, A., Li, L., Chow, G. M., Roy, K. R., Nguyen, T. M., Tanabe, K., McGrath, P. S., Cramer, A., Bruckner, A., Bilousova, G., Roop, D., Tang, J. Y., Christiano, A., Steinmetz, L. M., Wernig, M., Oro, A. E. 2024; 15 (1): 5834


    We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.

    View details for DOI 10.1038/s41467-024-49400-z

    View details for PubMedID 38992003

    View details for PubMedCentralID PMC11239819

  • Author Correction: Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates. Nature Mall, M., Kareta, M. S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S. D., Ge, X., Drake, S., Ang, C. E., Walker, B. M., Vierbuchen, T., Fuentes, D. R., Brennecke, P., Nitta, K. R., Jolma, A., Steinmetz, L. M., Taipale, J., Südhof, T. C., Wernig, M. 2024

    View details for DOI 10.1038/s41586-024-07594-8

    View details for PubMedID 38834754

  • Human tNeurons reveal aging-linked proteostasis deficits driving Alzheimer's phenotypes. Research square Chou, C. C., Vest, R., Prado, M. A., Wilson-Grady, J., Paulo, J. A., Shibuya, Y., Moran-Losada, P., Lee, T. T., Luo, J., Gygi, S. P., Kelly, J. W., Finley, D., Wernig, M., Wyss-Coray, T., Frydman, J. 2024


    Aging is a prominent risk factor for Alzheimer's disease (AD), but the cellular mechanisms underlying neuronal phenotypes remain elusive. Both accumulation of amyloid plaques and neurofibrillary tangles in the brain1 and age-linked organelle deficits2-7 are proposed as causes of AD phenotypes but the relationship between these events is unclear. Here, we address this question using a transdifferentiated neuron (tNeuron) model directly from human dermal fibroblasts. Patient-derived tNeurons retain aging hallmarks and exhibit AD-linked deficits. Quantitative tNeuron proteomic analyses identify aging and AD-linked deficits in proteostasis and organelle homeostasis, particularly affecting endosome-lysosomal components. The proteostasis and lysosomal homeostasis deficits in aged tNeurons are exacerbated in sporadic and familial AD tNeurons, promoting constitutive lysosomal damage and defects in ESCRT-mediated repair. We find deficits in neuronal lysosomal homeostasis lead to inflammatory cytokine secretion, cell death and spontaneous development of Aß and phospho-Tau deposits. These proteotoxic inclusions co-localize with lysosomes and damage markers and resemble inclusions in brain tissue from AD patients and APP-transgenic mice. Supporting the centrality of lysosomal deficits driving AD phenotypes, lysosome-function enhancing compounds reduce AD-associated cytokine secretion and Aβ deposits. We conclude that proteostasis and organelle deficits are upstream initiating factors leading to neuronal aging and AD phenotypes.

    View details for DOI 10.21203/

    View details for PubMedID 38853828

    View details for PubMedCentralID PMC11160905

  • Myeloid cell replacement is neuroprotective in chronic experimental autoimmune encephalomyelitis. Nature neuroscience Mader, M. M., Napole, A., Wu, D., Atkins, M., Scavetti, A., Shibuya, Y., Foltz, A., Hahn, O., Yoo, Y., Danziger, R., Tan, C., Wyss-Coray, T., Steinman, L., Wernig, M. 2024


    Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination of the central nervous system (CNS). Autologous hematopoietic cell transplantation (HCT) shows promising benefits for relapsing-remitting MS in open-label clinical studies, but the cellular mechanisms underlying its therapeutic effects remain unclear. Using single-nucleus RNA sequencing, we identify a reactive myeloid cell state in chronic experimental autoimmune encephalitis (EAE) associated with neuroprotection and immune suppression. HCT in EAE mice results in an increase of the neuroprotective myeloid state, improvement of neurological deficits, reduced number of demyelinated lesions, decreased number of effector Tcells and amelioration of reactive astrogliosis. Enhancing myeloid cell incorporation after a modified HCT further improved these neuroprotective effects. These data suggest that myeloid cell manipulation or replacement may be an effective therapeutic strategy for chronic inflammatory conditions of the CNS.

    View details for DOI 10.1038/s41593-024-01609-3

    View details for PubMedID 38514857

  • Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1. Proceedings of the National Academy of Sciences of the United States of America Ang, C. E., Olmos, V. H., Vodehnal, K., Zhou, B., Lee, Q. Y., Sinha, R., Narayanaswamy, A., Mall, M., Chesnov, K., Dominicus, C. S., Sudhof, T., Wernig, M. 2024; 121 (11): e2308401121


    Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

    View details for DOI 10.1073/pnas.2308401121

    View details for PubMedID 38446849

  • Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming. Science advances Song, Y., Soto, J., Wong, S. Y., Wu, Y., Hoffman, T., Akhtar, N., Norris, S., Chu, J., Park, H., Kelkhoff, D. O., Ang, C. E., Wernig, M., Kasko, A., Downing, T. L., Poo, M. M., Li, S. 2024; 10 (7): eadk0639


    We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

    View details for DOI 10.1126/sciadv.adk0639

    View details for PubMedID 38354231

    View details for PubMedCentralID PMC10866547

  • Stress response silencing by an E3 ligase mutated in neurodegeneration. Nature Haakonsen, D. L., Heider, M., Ingersoll, A. J., Vodehnal, K., Witus, S. R., Uenaka, T., Wernig, M., Rapé, M. 2024


    Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

    View details for DOI 10.1038/s41586-023-06985-7

    View details for PubMedID 38297121

    View details for PubMedCentralID 8997189

  • RAPIDASH: A tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues and stimulated macrophages. bioRxiv : the preprint server for biology Susanto, T. T., Hung, V., Levine, A. G., Kerr, C. H., Yoo, Y., Chen, Y., Oses-Prieto, J. A., Fromm, L., Fujii, K., Wernig, M., Burlingame, A. L., Ruggero, D., Barna, M. 2023


    Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translational control. However, a lack of technologies to enrich RAPs across many sample types has prevented systematic analysis of RAP number, dynamics, and functions. Here, we have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development that is linked to the translation of genes with long coding sequences. Finally, we characterized ribosome composition remodeling during immune activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs ranging from those with neuroregulatory functions to those activated by immune stimuli, thereby providing critical insights into how ribosomes are remodeled.

    View details for DOI 10.1101/2023.12.07.570613

    View details for PubMedID 38106052

    View details for PubMedCentralID PMC10723405

  • Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases. Nature genetics Guo, M. G., Reynolds, D. L., Ang, C. E., Liu, Y., Zhao, Y., Donohue, L. K., Siprashvili, Z., Yang, X., Yoo, Y., Mondal, S., Hong, A., Kain, J., Meservey, L., Fabo, T., Elfaki, I., Kellman, L. N., Abell, N. S., Pershad, Y., Bayat, V., Etminani, P., Holodniy, M., Geschwind, D. H., Montgomery, S. B., Duncan, L. E., Urban, A. E., Altman, R. B., Wernig, M., Khavari, P. A. 2023


    Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

    View details for DOI 10.1038/s41588-023-01533-5

    View details for PubMedID 37857935

    View details for PubMedCentralID 4112379

  • Insights and applications of direct neuronal reprogramming. Current opinion in genetics & development Schaukowitch, K., Janas, J. A., Wernig, M. 2023; 83: 102128


    Direct neuronal reprogramming converts somatic cells of a defined lineage into induced neuronal cells without going through a pluripotent intermediate. This approach not only provides access to the otherwise largely inaccessible cells of the brain for neuronal disease modeling, but also holds great promise for ultimately enabling neuronal cell replacement without the use of transplantation. To improve efficiency and specificity of direct neuronal reprogramming, much of the current efforts aim to understand the mechanisms that safeguard cell identities and how the reprogramming cells overcome the barriers resisting fate changes. Here, we review recent discoveries into the mechanisms by which the donor cell program is silenced, and new cell identities are established. We also discuss advancements that have been made toward fine-tuning the output of these reprogramming systems to generate specific types of neuronal cells. Finally, we highlight the benefit of using direct neuronal reprogramming to study age-related disorders and the potential of in vivo direct reprogramming in regenerative medicine.

    View details for DOI 10.1016/j.gde.2023.102128

    View details for PubMedID 37862835

  • Ascl1 and Ngn2 convert mouse embryonic stem cells to neurons via functionally distinct paths. Nature communications Vainorius, G., Novatchkova, M., Michlits, G., Baar, J. C., Raupach, C., Lee, J., Yelagandula, R., Wernig, M., Elling, U. 2023; 14 (1): 5341


    Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, and it remains unknown whether cells are converted via distinct mechanisms. Here we show that Ascl1 and Ngn2 induce mutually exclusive side populations by binding and activating distinct lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we find that Ascl1 relies more on factors regulating pluripotency and the cell cycle, such as Tcf7l1. In the absence of Tcf7l1, Ascl1 still represses core pluripotency genes but fails to exit the cell cycle. However, overexpression of Cdkn1c induces cell cycle exit and restores the generation of neurons. These findings highlight that cell type conversion can occur through two distinct mechanistic paths, even when induced by closely related transcription factors.

    View details for DOI 10.1038/s41467-023-40803-y

    View details for PubMedID 37660160

    View details for PubMedCentralID PMC10475046

  • Transdifferentiation: A Novel Tool for Disease Modeling and Translational Applications in Alzheimer's Disease Chou, C., Vest, R., Prado, M. A., Wilson-Grady, J., Paulo, J. A., Shibuya, Y., Moran-Losada, P., Lee, T., Luo, J., Gygi, S. P., Kelly, J. W., Finley, D. P., Wernig, M., Wyss-Coray, T., Frydman, J. WILEY. 2023: S205-S206
  • Efficient generation of functional neurons from mouse embryonic stem cells via neurogenin-2 expression. Nature protocols Liu, Y., Wang, J., Südhof, T. C., Wernig, M. 2023


    The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.

    View details for DOI 10.1038/s41596-023-00863-2

    View details for PubMedID 37596357

    View details for PubMedCentralID 3032267

  • A cell therapy approach to restore microglial Trem2 function in a mouse model of Alzheimer's disease. Cell stem cell Yoo, Y., Neumayer, G., Shibuya, Y., Marc-Daniel Mader, M., Wernig, M. 2023; 30 (8): 1043-1053.e6


    Alzheimer's disease (AD) remains one of the grand challenges facing human society. Much controversy exists around the complex and multifaceted pathogenesis of this prevalent disease. Given strong human genetic evidence, there is little doubt, however, that microglia play an important role in preventing degeneration of neurons. For example, loss of function of the microglial gene Trem2 renders microglia dysfunctional and causes an early-onset neurodegenerative syndrome, and Trem2 variants are among the strongest genetic risk factors for AD. Thus, restoring microglial function represents a rational therapeutic approach. Here, we show that systemic hematopoietic cell transplantation followed by enhancement of microglia replacement restores microglial function in a Trem2 mutant mouse model of AD.

    View details for DOI 10.1016/j.stem.2023.07.006

    View details for PubMedID 37541210

  • Augmentation of a neuroprotective myeloid state by hematopoietic cell transplantation. bioRxiv : the preprint server for biology Marc-Daniel Mader, M., Napole, A., Wu, D., Shibuya, Y., Scavetti, A., Foltz, A., Atkins, M., Hahn, O., Yoo, Y., Danziger, R., Tan, C., Wyss-Coray, T., Steinman, L., Wernig, M. 2023


    Multiple sclerosis (MS) is an autoimmune disease associated with inflammatory demyelination in the central nervous system (CNS). Autologous hematopoietic cell transplantation (HCT) is under investigation as a promising therapy for treatment-refractory MS. Here we identify a reactive myeloid state in chronic experimental autoimmune encephalitis (EAE) mice and MS patients that is surprisingly associated with neuroprotection and immune suppression. HCT in EAE mice leads to an enhancement of this myeloid state, as well as clinical improvement, reduction of demyelinated lesions, suppression of cytotoxic T cells, and amelioration of reactive astrogliosis reflected in reduced expression of EAE-associated gene signatures in oligodendrocytes and astrocytes. Further enhancement of myeloid cell incorporation into the CNS following a modified HCT protocol results in an even more consistent therapeutic effect corroborated by additional amplification of HCT-induced transcriptional changes, underlining myeloid-derived beneficial effects in the chronic phase of EAE. Replacement or manipulation of CNS myeloid cells thus represents an intriguing therapeutic direction for inflammatory demyelinating disease.

    View details for DOI 10.1101/2023.03.10.532123

    View details for PubMedID 36945385

    View details for PubMedCentralID PMC10028976

  • A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa. bioRxiv : the preprint server for biology Neumayer, G., Torkelson, J. L., Li, S., McCarthy, K., Zhen, H. H., Vangipuram, M., Jackow, J., Rami, A., Hansen, C., Guo, Z., Gaddam, S., Pappalardo, A., Li, L., Cramer, A., Roy, K. R., Nguyen, T. M., Tanabe, K., McGrath, P. S., Bruckner, A., Bilousova, G., Roop, D., Bailey, I., Tang, J. Y., Christiano, A., Steinmetz, L. M., Wernig, M., Oro, A. E. 2023


    Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing.iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice.DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

    View details for DOI 10.1101/2023.02.28.529447

    View details for PubMedID 36909618

    View details for PubMedCentralID PMC10002612

  • The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1. Scientific reports Haddad Derafshi, B., Danko, T., Chanda, S., Batista, P. J., Litzenburger, U., Lee, Q. Y., Ng, Y. H., Sebin, A., Chang, H. Y., Sudhof, T. C., Wernig, M. 2022; 12 (1): 22425


    The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

    View details for DOI 10.1038/s41598-022-23614-x

    View details for PubMedID 36575212

  • Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. Mabe, N. W., Huang, M., Schaefer, D. A., Dalton, G. N., Digiovanni, G., Alexe, G., Geraghty, A. C., Khalid, D., Mader, M. M., Sheffer, M., Linde, M. H., Ly, N., Rotiroti, M., Smith, B. H., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. AMER ASSOC CANCER RESEARCH. 2022
  • Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. Mabe, N. W., Huang, M., Schaefer, D. A., Dalton, G. N., Digiovanni, G., Alexe, G., Geraghty, A. C., Khalid, D., Mader, M. M., Sheffer, M., Linde, M. H., Ly, N., Rotiroti, M., Smith, B. H., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. AMER ASSOC CANCER RESEARCH. 2022: 3
  • Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation. Molecular cell Janas, J. A., Zhang, L., Luu, J. H., Demeter, J., Meng, L., Marro, S. G., Mall, M., Mooney, N. A., Schaukowitch, K., Ng, Y. H., Yang, N., Huang, Y., Neumayer, G., Gozani, O., Elias, J. E., Jackson, P. K., Wernig, M. 2022


    Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

    View details for DOI 10.1016/j.molcel.2022.11.002

    View details for PubMedID 36417913

  • Directly induced human retinal ganglion cells mimic fetal RGCs and are neuroprotective after transplantation invivo. Stem cell reports Luo, Z., Chang, K., Wu, S., Sun, C., Xia, X., Nahmou, M., Bian, M., Wen, R. R., Zhu, Y., Shah, S., Tanasa, B., Wernig, M., Goldberg, J. L. 2022


    Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated gamma-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation invitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.

    View details for DOI 10.1016/j.stemcr.2022.10.011

    View details for PubMedID 36368332

  • Synaptogenic effect of APP-Swedish mutation in familial Alzheimer's disease. Science translational medicine Zhou, B., Lu, J. G., Siddu, A., Wernig, M., Sudhof, T. C. 2022; 14 (667): eabn9380


    Mutations in beta-amyloid (Abeta) precursor protein (APP) cause familial Alzheimer's disease (AD) probably by enhancing Abeta peptides production from APP. An antibody targeting Abeta (aducanumab) was approved as an AD treatment; however, some Abeta antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the APP-Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the APP deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Abeta production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of APP-Swedish-mutant neurons. Modest elevations of Abeta, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked APP-Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Abeta. These data are consistent with the relative inefficacy of BACE1 and anti-Abeta treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Abeta but rather by a long-term effect of elevated Abeta concentrations.

    View details for DOI 10.1126/scitranslmed.abn9380

    View details for PubMedID 36260691

  • Generation of functional human oligodendrocytes from dermal fibroblasts by direct lineage conversion. Development (Cambridge, England) Tanabe, K., Nobuta, H., Yang, N., Ang, C. E., Huie, P., Jordan, S., Oldham, M. C., Rowitch, D. H., Wernig, M. 2022; 149 (20)


    Oligodendrocytes, the myelinating cells of the central nervous system, possess great potential for disease modeling and cell transplantation-based therapies for leukodystrophies. However, caveats to oligodendrocyte differentiation protocols ( Ehrlich et al., 2017; Wang et al., 2013; Douvaras and Fossati, 2015) from human embryonic stem and induced pluripotent stem cells (iPSCs), which include slow and inefficient differentiation, and tumorigenic potential of contaminating undifferentiated pluripotent cells, are major bottlenecks towards their translational utility. Here, we report the rapid generation of human oligodendrocytes by direct lineage conversion of human dermal fibroblasts (HDFs). We show that the combination of the four transcription factors OLIG2, SOX10, ASCL1 and NKX2.2 is sufficient to convert HDFs to induced oligodendrocyte precursor cells (iOPCs). iOPCs resemble human primary and iPSC-derived OPCs based on morphology and transcriptomic analysis. Importantly, iOPCs can differentiate into mature myelinating oligodendrocytes in vitro and in vivo. Finally, iOPCs derived from patients with Pelizaeus Merzbacher disease, a hypomyelinating leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene, showed increased cell death compared with iOPCs from healthy donors. Thus, human iOPCs generated by direct lineage conversion represent an attractive new source for human cell-based disease models and potentially myelinating cell grafts.

    View details for DOI 10.1242/dev.199723

    View details for PubMedID 35748297

  • Endocytosis in the axon initial segment maintains neuronal polarity. Nature Eichel, K., Uenaka, T., Belapurkar, V., Lu, R., Cheng, S., Pak, J. S., Taylor, C. A., Sudhof, T. C., Malenka, R., Wernig, M., Ozkan, E., Perrais, D., Shen, K. 2022


    Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly1. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments2,3. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism thatis likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.

    View details for DOI 10.1038/s41586-022-05074-5

    View details for PubMedID 35978188

  • Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nature cancer Mabe, N. W., Huang, M., Dalton, G. N., Alexe, G., Schaefer, D. A., Geraghty, A. C., Robichaud, A. L., Conway, A. S., Khalid, D., Mader, M. M., Belk, J. A., Ross, K. N., Sheffer, M., Linde, M. H., Ly, N., Yao, W., Rotiroti, M. C., Smith, B. A., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C. S., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. 2022


    Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

    View details for DOI 10.1038/s43018-022-00405-x

    View details for PubMedID 35817829

  • Myt1l haploinsufficiency leads to obesity and multifaceted behavioral alterations in mice. Molecular autism Wohr, M., Fong, W. M., Janas, J. A., Mall, M., Thome, C., Vangipuram, M., Meng, L., Sudhof, T. C., Wernig, M. 2022; 13 (1): 19


    BACKGROUND: The zinc finger domain containing transcription factor Myt1l is tightly associated with neuronal identity and is the only transcription factor known that is both neuron-specific and expressed in all neuronal subtypes. We identified Myt1l as a powerful reprogramming factor that, in combination with the proneural bHLH factor Ascl1, could induce neuronal fate in fibroblasts. Molecularly, we found it to repress many non-neuronal gene programs, explaining its supportive role to induce and safeguard neuronal identity in combination with proneural bHLH transcriptional activators. Moreover, human genetics studies found MYT1L mutations to cause intellectual disability and autism spectrum disorder often coupled with obesity.METHODS: Here, we generated and characterized Myt1l-deficient mice. A comprehensive, longitudinal behavioral phenotyping approach was applied.RESULTS: Myt1l was necessary for survival beyond 24h but not for overall histological brain organization. Myt1l heterozygous mice became increasingly overweight and exhibited multifaceted behavioral alterations. In mouse pups, Myt1l haploinsufficiency caused mild alterations in early socio-affective communication through ultrasonic vocalizations. In adulthood, Myt1l heterozygous mice displayed hyperactivity due to impaired habituation learning. Motor performance was reduced in Myt1l heterozygous mice despite intact motor learning, possibly due to muscular hypotonia. While anxiety-related behavior was reduced, acoustic startle reactivity was enhanced, in line with higher sensitivity to loud sound. Finally, Myt1l haploinsufficiency had a negative impact on contextual fear memory retrieval, while cued fear memory retrieval appeared to be intact.LIMITATIONS: In future studies, additional phenotypes might be identified and a detailed characterization of direct reciprocal social interaction behavior might help to reveal effects of Myt1l haploinsufficiency on social behavior in juvenile and adult mice.CONCLUSIONS: Behavioral alterations in Myt1l haploinsufficient mice recapitulate several clinical phenotypes observed in humans carrying heterozygous MYT1L mutations and thus serve as an informative model of the human MYT1L syndrome.

    View details for DOI 10.1186/s13229-022-00497-3

    View details for PubMedID 35538503

  • Is hypoimmunogenic stem cell therapy safe in times of pandemics? Stem cell reports Matheus, F., Raveh, T., Oro, A. E., Wernig, M., Drukker, M. 2022


    The manipulation of human leukocyte antigens (HLAs) and immune modulatory factors in "universal" human pluripotent stem cells (PSCs) holds promise for immunological tolerance without HLA matching. This paradigm raises concerns should "universal" grafts become virally infected. Furthermore, immunological manipulation might functionally impair certain progeny, such as hematopoietic stem cells. We discuss the risks and benefits of hypoimmunogenic PSCs, and the need to further advance HLA matching and autologous strategies.

    View details for DOI 10.1016/j.stemcr.2022.02.014

    View details for PubMedID 35334219

  • Treatment of a genetic brain disease by CNS-wide microglia replacement. Science translational medicine Shibuya, Y., Kumar, K. K., Mader, M. M., Yoo, Y., Ayala, L. A., Zhou, M., Mohr, M. A., Neumayer, G., Kumar, I., Yamamoto, R., Marcoux, P., Liou, B., Bennett, F. C., Nakauchi, H., Sun, Y., Chen, X., Heppner, F. L., Wyss-Coray, T., Südhof, T. C., Wernig, M. 2022; 14 (636): eabl9945


    Hematopoietic cell transplantation after myeloablative conditioning has been used to treat various genetic metabolic syndromes but is largely ineffective in diseases affecting the brain presumably due to poor and variable myeloid cell incorporation into the central nervous system. Here, we developed and characterized a near-complete and homogeneous replacement of microglia with bone marrow cells in mice without the need for genetic manipulation of donor or host. The high chimerism resulted from a competitive advantage of scarce donor cells during microglia repopulation rather than enhanced recruitment from the periphery. Hematopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained full microglia replacement potency equivalent to whole bone marrow. To explore its therapeutic potential, we applied microglia replacement to a mouse model for Prosaposin deficiency, which is characterized by a progressive neurodegeneration phenotype. We found a reduction of cerebellar neurodegeneration and gliosis in treated brains, improvement of motor and balance impairment, and life span extension even with treatment started in young adulthood. This proof-of-concept study suggests that efficient microglia replacement may have therapeutic efficacy for a variety of neurological diseases.

    View details for DOI 10.1126/scitranslmed.abl9945

    View details for PubMedID 35294256

  • Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation. The Journal of neuroscience : the official journal of the Society for Neuroscience Lam, D. D., Williams, R. H., Lujan, E., Tanabe, K., Huber, G., Saw, N. L., Merl-Pham, J., Salminen, A. V., Lohse, D., Spendiff, S., Plastini, M. J., Zech, M., Lochmuller, H., Geerlof, A., Hauck, S. M., Shamloo, M., Wernig, M., Winkelmann, J. 1800


    Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen alpha3 (VI) (COL6A3) C-terminal domain (CTD). These Col6a3 CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3 CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3 CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENTDystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor CB1R. Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.

    View details for DOI 10.1523/JNEUROSCI.0962-21.2021

    View details for PubMedID 34965974

  • Somatic Lineage Reprogramming. Cold Spring Harbor perspectives in biology Shelby, H., Shelby, T., Wernig, M. 2021


    Embryonic development and cell specification have been viewed as an epigenetically rigid process. Through accumulation of irreversible epigenetic marks, the differentiation process has been considered unidirectional, and once completed cell specification would be permanent and stable. However, somatic cell nuclear transfer that involved the implantation of a somatic nucleus into a previously enucleated oocyte accomplished in amphibians in the 1950s and in mammals in the late 1990s-resulting in the birth of "Dolly the sheep"-clearly showed that "terminal" differentiation is reversible. In parallel, work on lineage-determining factors like MyoD revealed surprising potential to modulate lineage identity in somatic cells. This work culminated in the discovery that a set of four defined factors can reprogram fibroblasts into induced pluripotent stem (iPS) cells, which were shown to be molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells, thus essentially showing that defined factors can induce authentic reprogramming without the need of oocytes. This concept was further extended when it was shown that fibroblasts can be directly converted into neurons, showing induced lineage conversion is possible even between cells representing two different germ layers. These findings suggest that "everything is possible" (i.e., once key lineage reprogramming factors are identified, cells should be able to convert into any desired lineage).

    View details for DOI 10.1101/cshperspect.a040808

    View details for PubMedID 34872969

  • RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development. Cell Wang, J., Miao, Y., Wicklein, R., Sun, Z., Wang, J., Jude, K. M., Fernandes, R. A., Merrill, S. A., Wernig, M., Garcia, K. C., Sudhof, T. C. 2021


    RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65A crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

    View details for DOI 10.1016/j.cell.2021.10.016

    View details for PubMedID 34758294

  • Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics. Stem cell reports Ng, Y. H., Chanda, S., Janas, J. A., Yang, N., Kokubu, Y., Sudhof, T. C., Wernig, M. 2021


    The differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We foundthat the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction.

    View details for DOI 10.1016/j.stemcr.2021.05.017

    View details for PubMedID 34171286

  • Rapid protocol for induced retinal ganglion cell differentiation from human stem cells Luo, Z., Chang, K., Tanasa, B., Wernig, M., Goldberg, J. L. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2021
  • Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons. Proceedings of the National Academy of Sciences of the United States of America Pak, C., Danko, T., Mirabella, V. R., Wang, J., Liu, Y., Vangipuram, M., Grieder, S., Zhang, X., Ward, T., Huang, Y. A., Jin, K., Dexheimer, P., Bardes, E., Mitelpunkt, A., Ma, J., McLachlan, M., Moore, J. C., Qu, P., Purmann, C., Dage, J. L., Swanson, B. J., Urban, A. E., Aronow, B. J., Pang, Z. P., Levinson, D. F., Wernig, M., Sudhof, T. C. 2021; 118 (22)


    Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

    View details for DOI 10.1073/pnas.2025598118

    View details for PubMedID 34035170

  • Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis. Cell reports Raj, N., McEachin, Z. T., Harousseau, W., Zhou, Y., Zhang, F., Merritt-Garza, M. E., Taliaferro, J. M., Kalinowska, M., Marro, S. G., Hales, C. M., Berry-Kravis, E., Wolf-Ochoa, M. W., Martinez-Cerdeno, V., Wernig, M., Chen, L., Klann, E., Warren, S. T., Jin, P., Wen, Z., Bassell, G. J. 2021; 35 (2): 108991


    Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, invitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.

    View details for DOI 10.1016/j.celrep.2021.108991

    View details for PubMedID 33852833

  • Optogenetic manipulation of cellular communication using engineered myosin motors. Nature cell biology Zhang, Z., Denans, N., Liu, Y., Zhulyn, O., Rosenblatt, H. D., Wernig, M., Barna, M. 2021


    Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.

    View details for DOI 10.1038/s41556-020-00625-2

    View details for PubMedID 33526902

  • H3.3-K27M drives neural stem cell-specific gliomagenesis in a human iPSC-derived model. Cancer cell Haag, D., Mack, N., Benites Goncalves da Silva, P., Statz, B., Clark, J., Tanabe, K., Sharma, T., Jager, N., Jones, D. T., Kawauchi, D., Wernig, M., Pfister, S. M. 2021


    Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.

    View details for DOI 10.1016/j.ccell.2021.01.005

    View details for PubMedID 33545065

  • Comparison of Acute Effects of Neurotoxic Compounds on Network Activity in Human and Rodent Neural Cultures. Toxicological sciences : an official journal of the Society of Toxicology Saavedra, L. n., Wallace, K. n., Freudenrich, T. F., Mall, M. n., Mundy, W. R., Davila, J. n., Shafer, T. J., Wernig, M. n., Haag, D. n. 2021; 180 (2): 295–312


    Assessment of neuroactive effects of chemicals in cell-based assays remains challenging as complex functional tissue is required for biologically relevant readouts. Recent in vitro models using rodent primary neural cultures grown on multielectrode arrays allow quantitative measurements of neural network activity suitable for neurotoxicity screening. However, robust systems for testing effects on network function in human neural models are still lacking. The increasing number of differentiation protocols for generating neurons from human-induced pluripotent stem cells (hiPSCs) holds great potential to overcome the unavailability of human primary tissue and expedite cell-based assays. Yet, the variability in neuronal activity, prolonged ontogeny and rather immature stage of most neuronal cells derived by standard differentiation techniques greatly limit their utility for screening neurotoxic effects on human neural networks. Here, we used excitatory and inhibitory neurons, separately generated by direct reprogramming from hiPSCs, together with primary human astrocytes to establish highly functional cultures with defined cell ratios. Such neuron/glia cocultures exhibited pronounced neuronal activity and robust formation of synchronized network activity on multielectrode arrays, albeit with noticeable delay compared with primary rat cortical cultures. We further investigated acute changes of network activity in human neuron/glia cocultures and rat primary cortical cultures in response to compounds with known adverse neuroactive effects, including gamma amino butyric acid receptor antagonists and multiple pesticides. Importantly, we observed largely corresponding concentration-dependent effects on multiple neural network activity metrics using both neural culture types. These results demonstrate the utility of directly converted neuronal cells from hiPSCs for functional neurotoxicity screening of environmental chemicals.

    View details for DOI 10.1093/toxsci/kfab008

    View details for PubMedID 33537736

  • Pro-neuronal activity of Myod1 due to promiscuous binding to neuronal genes. Nature cell biology Lee, Q. Y., Mall, M., Chanda, S., Zhou, B., Sharma, K. S., Schaukowitch, K., Adrian-Segarra, J. M., Grieder, S. D., Kareta, M. S., Wapinski, O. L., Ang, C. E., Li, R., Sudhof, T. C., Chang, H. Y., Wernig, M. 2020


    The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.

    View details for DOI 10.1038/s41556-020-0490-3

    View details for PubMedID 32231311

  • Cdk1 Controls Global Epigenetic Landscape in Embryonic Stem Cells. Molecular cell Michowski, W. n., Chick, J. M., Chu, C. n., Kolodziejczyk, A. n., Wang, Y. n., Suski, J. M., Abraham, B. n., Anders, L. n., Day, D. n., Dunkl, L. M., Li Cheong Man, M. n., Zhang, T. n., Laphanuwat, P. n., Bacon, N. A., Liu, L. n., Fassl, A. n., Sharma, S. n., Otto, T. n., Jecrois, E. n., Han, R. n., Sweeney, K. E., Marro, S. n., Wernig, M. n., Geng, Y. n., Moses, A. n., Li, C. n., Gygi, S. P., Young, R. A., Sicinski, P. n. 2020


    The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

    View details for DOI 10.1016/j.molcel.2020.03.010

    View details for PubMedID 32240602

  • APPROACHES TO TRANSCRIPTOME ANALYSIS OF HUMAN INDUCED NEURONS IN CO-CULTURE WITH MURINE GLIA TO MODEL FUNCTIONAL SYNAPSES Purmann, C., Zhang, X., Pak, C., Huang, Y., Pattni, R., Grieder, S., Wernig, M., Levinson, D., Aronow, B., Sudhof, T., Urban, A. ELSEVIER. 2019: S172–S173
  • In vitro modeling of the bipolar disorder and schizophrenia using patient-derived induced pluripotent stem cells with copy number variations of PCDH15 and RELN. eNeuro Ishii, T., Ishikawa, M., Fujimori, K., Maeda, T., Kushima, I., Arioka, Y., Mori, D., Nakatake, Y., Yamagata, B., Nio, S., Kato, T. A., Yang, N., Wernig, M., Kanba, S., Mimura, M., Ozaki, N., Okano, H. 2019


    Bipolar disorder (BP) and schizophrenia (SCZ) are major psychiatric disorders, but the molecular mechanisms underlying the complicated pathologies of these disorders remain unclear. It is difficult to establish adequate in vitro models for pathological analysis because of the heterogeneity of these disorders. In the present study, to recapitulate the pathologies of these disorders in vitro, we established in vitro models by differentiating mature neurons from human induced pluripotent stem cells (hiPSCs) derived from BP and SCZ patients with contributive copy number variations (CNVs): two BP patients with PCDH15 deletion and one SCZ patient with RELN deletion. Glutamatergic neurons and GABAergic neurons were induced from hiPSCs under optimized conditions. Both types of induced neurons from both hiPSCs exhibited similar phenotypes of MAP2-positive dendrite shortening and decreasing synapse numbers. Additionally, we analyzed isogenic PCDH15- or RELN-deleted cells. The dendrite and synapse phenotypes of isogenic neurons were partially similar to those of patient-derived neurons. These results suggest that the observed phenotypes are general phenotypes of psychiatric disorders, and our in vitro models using hiPSC-based technology may be suitable for analysis of the pathologies of psychiatric disorders.Significance Statement Useful in vitro models of psychiatric disorders such as schizophrenia and bipolar disorder are urgently required for pathological analysis and drug discovery. In this study, mature excitatory and inhibitory neurons were induced from patient-derived induced pluripotent stem cells. The patients-derived induced neurons exhibited abnormalities in dendrite and synapse formation in vitro, which are similar to the previously reported findings observed in the postmortem brains. Our in vitro model may reflect general phenotypes of psychiatric disorders and can be used to further examine therapeutic targets.

    View details for DOI 10.1523/ENEURO.0403-18.2019

    View details for PubMedID 31540999

  • Differential Signaling Mediated by ApoE2, ApoE3, and ApoE4 in Human Neurons Parallels Alzheimer's Disease Risk. The Journal of neuroscience : the official journal of the Society for Neuroscience Huang, Y. A., Zhou, B., Nabet, A. M., Wernig, M., Sudhof, T. C. 2019


    In blood, apolipoprotein E (ApoE) is a component of circulating lipoproteins and mediates the clearance of these lipoproteins from blood by binding to ApoE receptors. Humans express three genetic ApoE variants, ApoE2, ApoE3, and ApoE4, that exhibit distinct ApoE receptor-binding properties and differentially affect Alzheimer's disease (AD), such that ApoE2 protects against, and ApoE4 predisposes to AD. In brain, ApoE-containing lipoproteins are secreted by activated astrocytes and microglia, but their functions and role in AD pathogenesis are largely unknown. Ample evidence suggests that ApoE4 induces microglial dysregulation and impedes Abeta clearance in AD, but the direct neuronal effects of ApoE variants are poorly studied. Extending previous studies, we here demonstrate that the three ApoE variants differentially activate multiple neuronal signaling pathways and regulate synaptogenesis. Specifically, using human neurons (male embryonic stem cell-derived) cultured in the absence of glia to exclude indirect glial mechanisms, we show that ApoE broadly stimulates signal transduction cascades. Among others, such stimulation enhances APP synthesis and synapse formation with an ApoE4>ApoE3>ApoE2 potency rank order, paralleling the relative risk for AD conferred by these ApoE variants. Unlike the previously described induction of APP transcription, however, ApoE-induced synaptogenesis involves CREB activation rather than cFos activation. We thus propose that in brain, ApoE acts as a glia-secreted signal that activates neuronal signaling pathways. The parallel potency rank order of ApoE4>ApoE3>ApoE2 in AD risk and neuronal signaling suggests that ApoE4 may in an apparent paradox promote AD pathogenesis by causing a chronic increase in signaling, possibly via enhancing APP expression.SIGNIFICANCE STATEMENTHumans express three genetic variants of apolipoprotein E (ApoE), ApoE2, ApoE3, and ApoE4. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), whereas ApoE2 protects against AD. Significant evidence suggests that ApoE4 impairs microglial function and impedes astrocytic Abeta clearance in brain, but the direct neuronal effects of ApoE are poorly understood, and the differences between ApoE variants in these effects are unclear. Here, we report that ApoE acts on neurons as a glia-secreted signaling molecule that, among others, enhances synapse formation. In activating neuronal signaling, the three ApoE variants exhibit a differential potency of ApoE4>ApoE3>ApoE2, which mirrors their relative effects on AD risk, suggesting that differential signaling by ApoE variants may contribute to AD pathogenesis.

    View details for DOI 10.1523/JNEUROSCI.2994-18.2019

    View details for PubMedID 31331998

  • Stem cell-derived retinal ganglion cell differentiation and its transplantation Chang, K., Wu, S., Li, L., Sun, C., Xia, X., Knasel, C., Nahmou, M., Wernig, M., Goldberg, J. L. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
  • Neuroligin-4 Regulates Excitatory Synaptic Transmission in Human Neurons. Neuron Marro, S. G., Chanda, S., Yang, N., Janas, J. A., Valperga, G., Trotter, J., Zhou, B., Merrill, S., Yousif, I., Shelby, H., Vogel, H., Kalani, M. Y., Sudhof, T. C., Wernig, M. 2019


    The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human embryonic stem cell-derived neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change-of-function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting that human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved.

    View details for DOI 10.1016/j.neuron.2019.05.043

    View details for PubMedID 31257103

  • Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9. Molecular cell Shariati, S. A., Dominguez, A., Xie, S., Wernig, M., Qi, L. S., Skotheim, J. M. 2019; 74 (3): 622


    The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

    View details for PubMedID 31051141

  • Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9 MOLECULAR CELL Shariati, S., Dominguez, A., Xie, S., Wernig, M., Qi, L. S., Skotheim, J. M. 2019; 74 (3): 622-+
  • Global DNA methylation remodeling during direct reprogramming of fibroblasts to neurons. eLife Luo, C., Lee, Q. Y., Wapinski, O., Castanon, R., Nery, J. R., Mall, M., Kareta, M. S., Cullen, S. M., Goodell, M. A., Chang, H. Y., Wernig, M., Ecker, J. R. 2019; 8


    Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

    View details for PubMedID 30644360

  • TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment. Cell stem cell Li, L., Wang, Y., Torkelson, J. L., Shankar, G., Pattison, J. M., Zhen, H. H., Fang, F., Duren, Z., Xin, J., Gaddam, S., Melo, S. P., Piekos, S. N., Li, J., Liaw, E. J., Chen, L., Li, R., Wernig, M., Wong, W. H., Chang, H. Y., Oro, A. E. 2019


    Tissue development results from lineage-specific transcription factors (TFs) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we define epigenomic landscape during epidermal differentiation of human pluripotent stem cells (PSCs) and create inference networks that integrate gene expression, chromatin accessibility, and TF binding to define regulatory mechanisms during keratinocyte specification. We found two critical chromatin networks during surface ectoderm initiation and keratinocyte maturation, which are driven by TFAP2C and p63, respectively. Consistently, TFAP2C, but not p63, is sufficient to initiate surface ectoderm differentiation, and TFAP2C-initiated progenitor cells are capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively autoregulate its own expression and close a subset of the TFAP2C-initiated surface ectoderm program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

    View details for PubMedID 30686763

  • The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders. eLife Ang, C. E., Ma, Q., Wapinski, O. L., Fan, S., Flynn, R. A., Lee, Q. Y., Coe, B., Onoguchi, M., Olmos, V. H., Do, B. T., Dukes-Rimsky, L., Xu, J., Tanabe, K., Wang, L., Elling, U., Penninger, J. M., Zhao, Y., Qu, K., Eichler, E. E., Srivastava, A., Wernig, M., Chang, H. Y. 2019; 8


    Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

    View details for PubMedID 30628890

  • Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing. Nature Mahmoudi, S. n., Mancini, E. n., Xu, L. n., Moore, A. n., Jahanbani, F. n., Hebestreit, K. n., Srinivasan, R. n., Li, X. n., Devarajan, K. n., Prélot, L. n., Ang, C. E., Shibuya, Y. n., Benayoun, B. A., Chang, A. L., Wernig, M. n., Wysocka, J. n., Longaker, M. T., Snyder, M. P., Brunet, A. n. 2019; 574 (7779): 553–58


    Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.

    View details for DOI 10.1038/s41586-019-1658-5

    View details for PubMedID 31645721

  • Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation. Cell stem cell Nobuta, H. n., Yang, N. n., Ng, Y. H., Marro, S. G., Sabeur, K. n., Chavali, M. n., Stockley, J. H., Killilea, D. W., Walter, P. B., Zhao, C. n., Huie, P. n., Goldman, S. A., Kriegstein, A. R., Franklin, R. J., Rowitch, D. H., Wernig, M. n. 2019; 25 (4): 531–41.e6


    Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

    View details for DOI 10.1016/j.stem.2019.09.003

    View details for PubMedID 31585094

  • Modeling Alzheimer's disease with human iPS cells: advancements, lessons, and applications. Neurobiology of disease Essayan-Perez, S. n., Zhou, B. n., Nabet, A. M., Wernig, M. n., Huang, Y. A. 2019: 104503


    One in three people will develop Alzheimer's disease (AD) or another dementia and, despite intense research efforts, treatment options remain inadequate. Understanding the mechanisms of AD pathogenesis remains our principal hurdle to developing effective therapeutics to tackle this looming medical crisis. In light of recent discoveries from whole-genome sequencing and technical advances in humanized models, studying disease risk genes with induced human neural cells presents unprecedented advantages. Here, we first review the current knowledge of the proposed mechanisms underlying AD and focus on modern genetic insights to inform future studies. To highlight the utility of human pluripotent stem cell-based innovations, we then present an update on efforts in recapitulating the pathophysiology by induced neuronal, non-neuronal and a collection of brain cell types, departing from the neuron-centric convention. Lastly, we examine the translational potentials of such approaches, and provide our perspectives on the promise they offer to deepen our understanding of AD pathogenesis and to accelerate the development of intervention strategies for patients and risk carriers.

    View details for DOI 10.1016/j.nbd.2019.104503

    View details for PubMedID 31202913

  • Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity. Cell stem cell Chanda, S. n., Ang, C. E., Lee, Q. Y., Ghebrial, M. n., Haag, D. n., Shibuya, Y. n., Wernig, M. n., Südhof, T. C. 2019


    Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.

    View details for DOI 10.1016/j.stem.2019.04.021

    View details for PubMedID 31155484

  • Direct targeting of the mouse optic nerve for therapeutic delivery. Journal of neuroscience methods Mesentier-Louro, L. A., Dodd, R., Domizi, P., Nobuta, H., Wernig, M., Wernig, G., Liao, Y. J. 2018


    BACKGROUND: Animal models of optic nerve injury are often used to study central nervous system (CNS) degeneration and regeneration, and targeting the optic nerve is a powerful approach for axon-protective or remyelination therapy. However, the experimental delivery of drugs or cells to the optic nerve is rarely performed because injections into this structure are difficult in small animals, especially in mice.NEW METHOD: We investigated and developed methods to deliver drugs or cells to the mouse optic nerve through 3 different routes: a) intraorbital, b) through the optic foramen and c) transcranial.RESULTS: The methods targeted different parts of the mouse optic nerve: intraorbital proximal (intraorbital), intracranial middle (optic-foramen) or intracranial distal (transcranial) portion.COMPARISON WITH EXISTING METHODS: Most existing methods target the optic nerve indirectly. For instance, intravitreally delivered cells often cannot cross the inner limiting membrane to reach retinal neurons and optic nerve axons. Systemic delivery, eye drops and intraventricular injections do not always successfully target the optic nerve. Intraorbital and transcranial injections into the optic nerve or chiasm have been performed but these methods have not been well described. We approached the optic nerve with more selective and precise targeting than existing methods.CONCLUSIONS: We successfully targeted the murine optic nerve intraorbitally, through the optic foramen, and transcranially. Of all methods, the injection through the optic foramen is likely the most innovative and fastest. These methods offer additional approaches for therapeutic intervention to be used by those studying white matter damage and axonal regeneration in the CNS.

    View details for PubMedID 30389488

  • CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming. Cell stem cell Liu, Y., Yu, C., Daley, T. P., Wang, F., Cao, W. S., Bhate, S., Lin, X., Still, C. 2., Liu, H., Zhao, D., Wang, H., Xie, X. S., Ding, S., Wong, W. H., Wernig, M., Qi, L. S. 2018


    Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.

    View details for PubMedID 30318302

  • The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling. Science translational medicine Zhang, Z., Marro, S. G., Zhang, Y., Arendt, K. L., Patzke, C., Zhou, B., Fair, T., Yang, N., Sudhof, T. C., Wernig, M., Chen, L. 2018; 10 (452)


    Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.

    View details for PubMedID 30068571

  • Stem cell therapy for treatment of ischemic optic neuropathy Mesentier-Louro, L., Yang, N., Shariati, A., Domizi, P., Dodd, R., Wernig, G., Wernig, M., Liao, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
  • Transdifferentiation of human adult peripheral blood T cells into neurons. Proceedings of the National Academy of Sciences of the United States of America Tanabe, K., Ang, C. E., Chanda, S., Olmos, V. H., Haag, D., Levinson, D. F., Sudhof, T. C., Wernig, M. 2018


    Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.

    View details for PubMedID 29866841

  • Profiling DNA-transcription factor interactions NATURE BIOTECHNOLOGY Ang, C., Wernig, M. 2018; 36 (6): 501–2

    View details for PubMedID 29874211

  • Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons CELL REPORTS Wapinski, O. L., Lee, Q., Chen, A. C., Li, R., Corces, M., Ang, C., Treutlein, B., Xiang, C., Baubet, V., Suchy, F., Sankar, V., Sim, S., Quake, S. R., Dahmane, N., Wernig, M., Chang, H. Y. 2017; 20 (13): 3236–47


    How transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hr after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells is accomplished with relatively little chromatin reconfiguration. By integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming and identified Zfp238, Sox8, and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to stepwise cell fate transitions in development.

    View details for PubMedID 28954238

  • The novel tool of cell reprogramming for applications in molecular medicine. Journal of molecular medicine (Berlin, Germany) Mall, M., Wernig, M. 2017


    Recent discoveries in the field of stem cell biology have enabled scientists to "reprogram" cells from one type to another. For example, it is now possible to place adult skin or blood cells in a dish and convert them into neurons, liver, or heart cells. It is also possible to literally "rejuvenate" adult cells by reprogramming them into embryonic-like stem cells, which in turn can be differentiated into every tissue and cell type of the human body. Our ability to reprogram cell types has four main implications for medicine: (1) scientists can now take skin or blood cells from patients and convert them to other cells to study disease processes. This disease modeling approach has the advantage over animal models because it is directly based on human patient cells. (2) Reprogramming could also be used as a "clinical trial in a dish" to evaluate the general efficacy and safety of newly developed drugs on human patient cells before they would be tested in animal models or people. (3) In addition, many drugs have deleterious side effects like heart arrhythmias in only a small and unpredictable subpopulation of patients. Reprogramming could facilitate precision medicine by testing the safety of already approved drugs first on reprogrammed patient cells in a personalized manner prior to administration. For example, drugs known to sometimes cause arrhythmias could be first tested on reprogrammed heart cells from individual patients. (4) Finally, reprogramming allows the generation of new tissues that could be grafted therapeutically to regenerate lost or damaged cells.

    View details for DOI 10.1007/s00109-017-1550-4

    View details for PubMedID 28597071

  • Generation of pure GABAergic neurons by transcription factor programming. Nature methods Yang, N., Chanda, S., Marro, S., Ng, Y., Janas, J. A., Haag, D., Ang, C. E., Tang, Y., Flores, Q., Mall, M., Wapinski, O., Li, M., Ahlenius, H., Rubenstein, J. L., Chang, H. Y., Buylla, A. A., Südhof, T. C., Wernig, M. 2017; 14 (6): 621-628


    Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

    View details for DOI 10.1038/nmeth.4291

    View details for PubMedID 28504679

  • μNeurocircuitry: Establishing in vitro models of neurocircuits with human neurons. Technology Fantuzzo, J. A., De Filippis, L., McGowan, H., Yang, N., Ng, Y. H., Halikere, A., Liu, J. J., Hart, R. P., Wernig, M., Zahn, J. D., Pang, Z. P. 2017; 5 (2): 87-97


    Neurocircuits in the human brain govern complex behavior and involve connections from many different neuronal subtypes from different brain regions. Recent advances in stem cell biology have enabled the derivation of patient-specific human neuronal cells of various subtypes for the study of neuronal function and disease pathology. Nevertheless, one persistent challenge using these human-derived neurons is the ability to reconstruct models of human brain circuitry. To overcome this obstacle, we have developed a compartmentalized microfluidic device, which allows for spatial separation of cell bodies of different human-derived neuronal subtypes (excitatory, inhibitory and dopaminergic) but is permissive to the spreading of projecting processes. Induced neurons (iNs) cultured in the device expressed pan-neuronal markers and subtype specific markers. Morphologically, we demonstrate defined synaptic contacts between selected neuronal subtypes by synapsin staining. Functionally, we show that excitatory neuronal stimulation evoked excitatory postsynaptic current responses in the neurons cultured in a separate chamber.

    View details for DOI 10.1142/S2339547817500054

    View details for PubMedID 28781993

    View details for PubMedCentralID PMC5541685

  • Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model NATURE BIOTECHNOLOGY Cervo, P. R., Romanov, R. A., Spigolon, G., Masini, D., Martin-Montanez, E., Toledo, E. M., La Manno, G., Feyder, M., Pifl, C., Ng, Y., Sanchez, S. P., Linnarsson, S., Wernig, M., Harkany, T., Fisone, G., Arenas, E. 2017; 35 (5): 444-?


    Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for Parkinson's disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinson's disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinson's disease by delivery of genes rather than cells.

    View details for DOI 10.1038/nbt.3835

    View details for Web of Science ID 000400809800019

    View details for PubMedID 28398344

  • Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates NATURE Mall, M., Kareta, M. S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S. D., Ge, X., Drake, S., Ang, C. E., Walker, B. M., Vierbuchen, T., Fuentes, D. R., Brennecke, P., Nitta, K. R., Jolma, A., Steinmetz, L. M., Taipale, J., Sudhof, T. C., Wernig, M. 2017; 544 (7649): 245-?


    Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar 'many-but-one' lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.

    View details for DOI 10.1038/nature21722

    View details for PubMedID 28379941

  • Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons SCIENTIFIC REPORTS Chuang, W., Sharma, A., Shukla, P., Li, G., Mall, M., Rajarajan, K., Abilez, O. J., Hamaguchi, R., Wu, J. C., Wernig, M., Wu, S. M. 2017; 7


    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

    View details for DOI 10.1038/srep44840

    View details for Web of Science ID 000396983300001

    View details for PubMedID 28327614

    View details for PubMedCentralID PMC5361100

  • Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease. Cell stem cell Chao, M. P., Gentles, A. J., Chatterjee, S., Lan, F., Reinisch, A., Corces, M. R., Xavy, S., Shen, J., Haag, D., Chanda, S., Sinha, R., Morganti, R. M., Nishimura, T., Ameen, M., Wu, H., Wernig, M., Wu, J. C., Majeti, R. 2017; 20 (3): 329-344 e7


    Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

    View details for DOI 10.1016/j.stem.2016.11.018

    View details for PubMedID 28089908

  • Concise Review: Stem Cell-Based Treatment of Pelizaeus-Merzbacher Disease STEM CELLS Osorio, M., Rowitch, D. H., Tesar, P., Wernig, M., Windrem, M. S., Goldman, S. A. 2017; 35 (2): 311–15


    Pelizaeus-Merzbacher disease (PMD) is an X-linked disorder caused by mutation in the proteolipid protein-1 (PLP1) gene, which encodes the proteolipid protein of myelinating oligodendroglia. PMD exhibits phenotypic variability that reflects its considerable genotypic heterogeneity, but all forms of the disease result in central hypomyelination, associated in most cases with early neurological dysfunction, progressive deterioration, and ultimately death. PMD may present as a connatal, classic and transitional forms, or as the less severe spastic paraplegia type 2 and PLP-null phenotypes. These disorders are most often associated with duplications of the PLP1 gene, but can also be caused by coding and noncoding point mutations as well as full or partial deletion of the gene. A number of genetically-distinct but phenotypically-similar disorders of hypomyelination exist which, like PMD, lack any effective therapy. Yet as relatively pure CNS hypomyelinating disorders, with limited involvement of the PNS and relatively little attendant neuronal pathology, PMD and similar hypomyelinating disorders are attractive therapeutic targets for neural stem cell and glial progenitor cell transplantation, efforts at which are now underway in a number of research centers. Stem Cells 2017;35:311-315.

    View details for PubMedID 27882623

    View details for PubMedCentralID PMC5314962

  • ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aß Secretion. Cell Huang, Y. A., Zhou, B., Wernig, M., Südhof, T. C. 2017; 168 (3): 427-441 e21


    Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis.

    View details for DOI 10.1016/j.cell.2016.12.044

    View details for PubMedID 28111074

  • Unique versus Redundant Functions of Neuroligin Genes in Shaping Excitatory and Inhibitory Synapse Properties. The Journal of neuroscience : the official journal of the Society for Neuroscience Chanda, S. n., Hale, W. D., Zhang, B. n., Wernig, M. n., Südhof, T. C. 2017; 37 (29): 6816–36


    Neuroligins are evolutionarily conserved postsynaptic cell adhesion molecules that interact with presynaptic neurexins. Neurons express multiple neuroligin isoforms that are targeted to specific synapses, but their synaptic functions and mechanistic redundancy are not completely understood. Overexpression or RNAi-mediated knockdown of neuroligins, respectively, causes a dramatic increase or decrease in synapse density, whereas genetic deletions of neuroligins impair synapse function with only minor effects on synapse numbers, raising fundamental questions about the overall physiological role of neuroligins. Here, we have systematically analyzed the effects of conditional genetic deletions of all major neuroligin isoforms (i.e., NL1, NL2, and NL3), either individually or in combinations, in cultured mouse hippocampal and cortical neurons. We found that conditional genetic deletions of neuroligins caused no change or only a small change in synapses numbers, but strongly impaired synapse function. This impairment was isoform specific, suggesting that neuroligins are not functionally redundant. Sparse neuroligin deletions produced phenotypes comparable to those of global deletions, indicating that neuroligins function in a cell-autonomous manner. Mechanistically, neuroligin deletions decreased the synaptic levels of neurotransmitter receptors and had no effect on presynaptic release probabilities. Overexpression of neuroligin-1 in control or neuroligin-deficient neurons increased synaptic transmission and synapse density but not spine numbers, suggesting that these effects reflect a gain-of-function mechanism; whereas overexpression of neuroligin-3, which, like neuroligin-1 is also targeted to excitatory synapses, had no comparable effect. Our data demonstrate that neuroligins are required for the physiological organization of neurotransmitter receptors in postsynaptic specializations and suggest that they do not play a major role in synapse formation.SIGNIFICANCE STATEMENT Human neuroligin genes have been associated with autism, but the cellular functions of different neuroligins and their molecular mechanisms remain incompletely understood. Here, we performed comparative analyses in cultured mouse neurons of all major neuroligin isoforms, either individually or in combinations, using conditional knockouts. We found that neuroligin deletions did not affect synapse numbers but differentially impaired excitatory or inhibitory synaptic functions in an isoform-specific manner. These impairments were due, at least in part, to a decrease in synaptic distribution of neurotransmitter receptors upon deletion of neuroligins. Conversely, the overexpression of neuroligin-1 increased synapse numbers but not spine numbers. Our results suggest that various neuroligin isoforms perform unique postsynaptic functions in organizing synapses but are not essential for synapse formation or maintenance.

    View details for PubMedID 28607166

  • FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ahlenius, H., Chanda, S., Webb, A. E., Yousif, I., Karmazin, J., Prusiner, S. B., Brunet, A., Suedhof, T. C., Wernig, M. 2016; 113 (30): 8514-8519


    We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.

    View details for DOI 10.1073/pnas.1607079113

    View details for PubMedID 27402759

  • Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq NATURE Treutlein, B., Lee, Q. Y., Camp, J. G., Mall, M., Koh, W., Shariati, S. A., Sim, S., Neff, N. F., Skotheim, J. M., Wernig, M., Quake, S. R. 2016; 534 (7607): 391-?


    Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

    View details for DOI 10.1038/nature18323

    View details for Web of Science ID 000377856800037

    View details for PubMedID 27281220

    View details for PubMedCentralID PMC4928860

  • Autism-associated SHANK3 haploinsufficiency causes I-h channelopathy in human neurons SCIENCE Yi, F., Danko, T., Botelho, S. C., Patzke, C., Pak, C., Wernig, M., Sudhof, T. C. 2016; 352 (6286): 672-?


    Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired Ih channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) forming Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.

    View details for DOI 10.1126/science.aaf2669

    View details for PubMedID 26966193

  • Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation. journal of experimental medicine Patzke, C., Acuna, C., Giam, L. R., Wernig, M., Südhof, T. C. 2016; 213 (4): 499-515


    Hundreds ofL1CAMgene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditionalL1CAMloss-of-function mutation and produced precisely matching control andL1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion ofL1CAMdramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20-50% and ∼20-30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that theL1CAMmutation selectively decreased activity-dependent Na(+)-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations ofL1CAMmutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.

    View details for DOI 10.1084/jem.20150951

    View details for PubMedID 27001749

    View details for PubMedCentralID PMC4821644

  • Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds NATURE COMMUNICATIONS Carlson, A. L., Bennett, N. K., Francis, N. L., Halikere, A., Clarke, S., Moore, J. C., Hart, R. P., Paradiso, K., Wernig, M., Kohn, J., Pang, Z. P., Moghe, P. V. 2016; 7


    Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼ 3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼ 38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance.

    View details for DOI 10.1038/ncomms10862

    View details for Web of Science ID 000372218700001

    View details for PubMedID 26983594

    View details for PubMedCentralID PMC4800432

  • Pathogenic mechanism of an autism-associated neuroligin mutation involves altered AMPA-receptor trafficking. Molecular psychiatry Chanda, S., Aoto, J., Lee, S., Wernig, M., Südhof, T. C. 2016; 21 (2): 169-177


    Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Although the general synaptic role of neuroligins is undisputed, their specific functions at a synapse remain unclear, even controversial. Moreover, many neuroligin gene mutations were associated with autism, but the pathophysiological relevance of these mutations is often unknown, and their mechanisms of action uninvestigated. Here, we examine the synaptic effects of an autism-associated neuroligin-4 substitution (called R704C), which mutates a cytoplasmic arginine residue that is conserved in all neuroligins. We show that the R704C mutation, when introduced into neuroligin-3, enhances the interaction between neuroligin-3 and AMPA receptors, increases AMPA-receptor internalization and decreases postsynaptic AMPA-receptor levels. When introduced into neuroligin-4, conversely, the R704C mutation unexpectedly elevated AMPA-receptor-mediated synaptic responses. These results suggest a general functional link between neuroligins and AMPA receptors, indicate that both neuroligin-3 and -4 act at excitatory synapses but perform surprisingly distinct functions, and demonstrate that the R704C mutation significantly impairs the normal function of neuroligin-4, thereby validating its pathogenicity.Molecular Psychiatry advance online publication, 17 March 2015; doi:10.1038/mp.2015.20.

    View details for DOI 10.1038/mp.2015.20

    View details for PubMedID 25778475

  • The histone chaperone CAF-1 safeguards somatic cell identity NATURE Cheloufi, S., Elling, U., Hopfgartner, B., Jung, Y. L., Murn, J., Ninova, M., Hubmann, M., Badeaux, A. I., Ang, C. E., Tenen, D., Wesche, D. J., Abazova, N., Hogue, M., Tasdemir, N., Brumbaugh, J., Rathert, P., Jude, J., Ferrari, F., Blanco, A., Fellner, M., Wenzel, D., Zinner, M., Vidal, S. E., Bell, O., Stadtfeld, M., Chang, H. Y., Almouzni, G., Lowe, S. W., Rinn, J., Wernig, M., Aravin, A., Shi, Y., Park, P. J., Penninger, J. M., Zuber, J., Hochedlinger, K. 2015; 528 (7581): 218-?


    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

    View details for DOI 10.1038/nature15749

    View details for PubMedID 26659182

  • Crosstalk between stem cell and cell cycle machineries CURRENT OPINION IN CELL BIOLOGY Kareta, M. S., Sage, J., Wernig, M. 2015; 37: 68-74


    Pluripotent stem cells, defined by an unlimited self-renewal capacity and an undifferentiated state, are best typified by embryonic stem cells. These cells have a unique cell cycle compared to somatic cells as defined by a rapid progression through the cell cycle and a minimal time spent in G1. Recent reports indicate that pluripotency and cell cycle regulation are mechanistically linked. In this review, we discuss the reciprocal co-regulation of these processes, how this co-regulation may prevent differentiation, and how cellular reprogramming can re-establish the unique cell cycle regulation in induced pluripotent stem cells.

    View details for DOI 10.1016/

    View details for Web of Science ID 000367112800010

    View details for PubMedID 26520682

  • Direct somatic lineage conversion. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Tanabe, K., Haag, D., Wernig, M. 2015; 370 (1680)

    View details for DOI 10.1098/rstb.2014.0368

    View details for PubMedID 26416679

  • Hallmarks of pluripotency NATURE De Los Angeles, A., Ferrari, F., Xi, R., Fujiwara, Y., Benvenisty, N., Deng, H., Hochedlinger, K., Jaenisch, R., Lee, S., Leitch, H. G., Lensch, M. W., Lujan, E., Pei, D., Rossant, J., Wernig, M., Park, P. J., Daley, G. Q. 2015; 525 (7570): 469-478


    Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.

    View details for DOI 10.1038/nature15515

    View details for Web of Science ID 000361599900041

    View details for PubMedID 26399828

  • Failure to replicate the STAP cell phenomenon NATURE De Los Angeles, A., Ferrari, F., Fujiwara, Y., Mathieu, R., Lee, S., Lee, S., Tu, H., Ross, S., Chou, S., Minh Nguyen, Wu, Z., Theunissen, T. W., Powell, B. E., Imsoonthornruksa, S., Chen, J., Borkent, M., Krupalnik, V., Lujan, E., Wernig, M., Hanna, J. H., Hochedlinger, K., Pei, D., Jaenisch, R., Deng, H., Orkin, S. H., Park, P. J., Daley, G. Q. 2015; 525 (7570): E6-+

    View details for PubMedID 26399835

  • Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1. Cell stem cell Pak, C., Danko, T., Zhang, Y., Aoto, J., Anderson, G., Maxeiner, S., Yi, F., Wernig, M., Südhof, T. C. 2015; 17 (3): 316-328


    Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

    View details for DOI 10.1016/j.stem.2015.07.017

    View details for PubMedID 26279266

    View details for PubMedCentralID PMC4560990

  • Analysis of conditional heterozygous STXBP1 mutations in human neurons JOURNAL OF CLINICAL INVESTIGATION Patzke, C., Han, Y., Covy, J., Yi, F., Maxeiner, S., Wernig, M., Suedhof, T. C. 2015; 125 (9): 3560-3571


    Heterozygous mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes Munc18-1, a core component of the presynaptic membrane-fusion machinery, cause infantile early epileptic encephalopathy (Ohtahara syndrome), but it is unclear how a partial loss of Munc18-1 produces this severe clinical presentation. Here, we generated human ES cells designed to conditionally express heterozygous and homozygous STXBP1 loss-of-function mutations and studied isogenic WT and STXBP1-mutant human neurons derived from these conditionally mutant ES cells. We demonstrated that heterozygous STXBP1 mutations lower the levels of Munc18-1 protein and its binding partner, the t-SNARE-protein Syntaxin-1, by approximately 30% and decrease spontaneous and evoked neurotransmitter release by nearly 50%. Thus, our results confirm that using engineered human embryonic stem (ES) cells is a viable approach to studying disease-associated mutations in human neurons on a controlled genetic background, demonstrate that partial STXBP1 loss of function robustly impairs neurotransmitter release in human neurons, and suggest that heterozygous STXBP1 mutations cause early epileptic encephalopathy specifically through a presynaptic impairment.

    View details for DOI 10.1172/JCI78612

    View details for Web of Science ID 000362303600030

    View details for PubMedCentralID PMC4588304

  • Analysis of conditional heterozygous STXBP1 mutations in human neurons. journal of clinical investigation Patzke, C., Han, Y., Covy, J., Yi, F., Maxeiner, S., Wernig, M., Südhof, T. C. 2015; 125 (9): 3560-3571


    Heterozygous mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes Munc18-1, a core component of the presynaptic membrane-fusion machinery, cause infantile early epileptic encephalopathy (Ohtahara syndrome), but it is unclear how a partial loss of Munc18-1 produces this severe clinical presentation. Here, we generated human ES cells designed to conditionally express heterozygous and homozygous STXBP1 loss-of-function mutations and studied isogenic WT and STXBP1-mutant human neurons derived from these conditionally mutant ES cells. We demonstrated that heterozygous STXBP1 mutations lower the levels of Munc18-1 protein and its binding partner, the t-SNARE-protein Syntaxin-1, by approximately 30% and decrease spontaneous and evoked neurotransmitter release by nearly 50%. Thus, our results confirm that using engineered human embryonic stem (ES) cells is a viable approach to studying disease-associated mutations in human neurons on a controlled genetic background, demonstrate that partial STXBP1 loss of function robustly impairs neurotransmitter release in human neurons, and suggest that heterozygous STXBP1 mutations cause early epileptic encephalopathy specifically through a presynaptic impairment.

    View details for DOI 10.1172/JCI78612

    View details for PubMedID 26280581

    View details for PubMedCentralID PMC4588304

  • Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1 CELL STEM CELL Pak, C., Danko, T., Zhang, Y., Aoto, J., Anderson, G., Maxeiner, S., Yi, F., Wernig, M., Suedhof, T. C. 2015; 17 (3): 316-328


    Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

    View details for DOI 10.1016/j.stem.2015.07.017

    View details for Web of Science ID 000364011000010

    View details for PubMedCentralID PMC4560990

  • Early reprogramming regulators identified by prospective isolation and mass cytometry NATURE Lujan, E., Zunder, E. R., Ng, Y. H., Goronzy, I. N., Nolan, G. P., Wernig, M. 2015; 521 (7552): 352-?


    In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.

    View details for DOI 10.1038/nature14274

    View details for Web of Science ID 000354816500056

    View details for PubMedID 25830878

    View details for PubMedCentralID PMC4441548

  • A Continuous Molecular Roadmap to iPSC Reprogramming through Progression Analysis of Single-Cell Mass Cytometry. Cell stem cell Zunder, E. R., Lujan, E., Goltsev, Y., Wernig, M., Nolan, G. P. 2015; 16 (3): 323-337


    To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4(high)Klf4(high) cells transitioned to a CD73(high)CD104(high)CD54(low) partially reprogrammed state. Ki67(low) cells from this intermediate population reverted to a MEF-like phenotype, but Ki67(high) cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like Nanog(high)Sox2(high)CD54(high) population and a mesendoderm-like Nanog(low)Sox2(low)Lin28(high)CD24(high)PDGFR-α(high) population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development.

    View details for DOI 10.1016/j.stem.2015.01.015

    View details for PubMedID 25748935

  • Inhibition of pluripotency networks by the rb tumor suppressor restricts reprogramming and tumorigenesis. Cell stem cell Kareta, M. S., Gorges, L. L., Hafeez, S., Benayoun, B. A., Marro, S., Zmoos, A., Cecchini, M. J., Spacek, D., Batista, L. F., O'Brien, M., Ng, Y., Ang, C. E., Vaka, D., Artandi, S. E., Dick, F. A., Brunet, A., Sage, J., Wernig, M. 2015; 16 (1): 39-50


    Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.

    View details for DOI 10.1016/j.stem.2014.10.019

    View details for PubMedID 25467916

    View details for PubMedCentralID PMC4389904

  • m(6)A RNA Modification Controls Cell Fate Transition in Mammalian Embryonic Stem Cells. Cell stem cell Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., Carter, A. C., Flynn, R. A., Zhou, C., Lim, K., Dedon, P., Wernig, M., Mullen, A. C., Xing, Y., Giallourakis, C. C., Chang, H. Y. 2014; 15 (6): 707-719


    N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.

    View details for DOI 10.1016/j.stem.2014.09.019

    View details for PubMedID 25456834

  • Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa SCIENCE TRANSLATIONAL MEDICINE Sebastiano, V., Zhen, H. H., Derafshi, B. H., Bashkirova, E., Melo, S. P., Wang, P., Leung, T. L., Siprashvili, Z., Tichy, A., Li, J., Ameen, M., Hawkins, J., Lee, S., Li, L., Schwertschkow, A., Bauer, G., Lisowski, L., Kay, M. A., Kim, S. K., Lane, A. T., Wernig, M., Oro, A. E. 2014; 6 (264)


    Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

    View details for DOI 10.1126/scitranslmed.3009540

    View details for Web of Science ID 000345595200003

    View details for PubMedCentralID PMC4428910

  • Induced Neuronal Reprogramming JOURNAL OF COMPARATIVE NEUROLOGY Ang, C. E., Wernig, M. 2014; 522 (12): 2877-2886

    View details for DOI 10.1002/cne.23620

    View details for Web of Science ID 000337791000014

  • Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1 STEM CELL REPORTS Chanda, S., Ang, C. E., Davila, J., Pak, C., Mall, M., Lee, Q. Y., Ahlenius, H., Jung, S. W., Suedhof, T. C., Wernig, M. 2014; 3 (2): 282-296


    Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

    View details for DOI 10.1016/j.stemcr.2014.05.020

    View details for Web of Science ID 000340882400008

    View details for PubMedCentralID PMC4176533

  • Calcineurin Signaling Regulates Neural Induction through Antagonizing the BMP Pathway. Neuron Cho, A., Tang, Y., Davila, J., Deng, S., Chen, L., Miller, E., Wernig, M., Graef, I. A. 2014; 82 (1): 109-124


    Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing embryo. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. We find that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins. Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and the development of anterior structures.

    View details for DOI 10.1016/j.neuron.2014.02.015

    View details for PubMedID 24698271

  • Harnessing the stem cell potential: a case for neural stem cell therapy. Nature medicine Yang, N., Wernig, M. 2013; 19 (12): 1580-1581

    View details for DOI 10.1038/nm.3425

    View details for PubMedID 24309657

  • Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons CELL Wapinski, O. L., Vierbuchen, T., Qu, K., Lee, Q. Y., Chanda, S., Fuentes, D. R., Giresi, P. G., Ng, Y. H., Marro, S., Neff, N. F., Drechsel, D., Martynoga, B., Castro, D. S., Webb, A. E., Suedhof, T. C., Brunet, A., Guillemot, F., Chang, H. Y., Wernig, M. 2013; 155 (3): 621-635


    Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

    View details for DOI 10.1016/j.cell.2013.09.028

    View details for PubMedID 24243019

  • Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation. Proceedings of the National Academy of Sciences of the United States of America Chanda, S., Marro, S., Wernig, M., Südhof, T. C. 2013; 110 (41): 16622-16627


    Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling.

    View details for DOI 10.1073/pnas.1316240110

    View details for PubMedID 24046374

    View details for PubMedCentralID PMC3799342

  • FOXO3 Shares Common Targets with ASCL1 Genome-wide and Inhibits ASCL1-Dependent Neurogenesis. Cell reports Webb, A. E., Pollina, E. A., Vierbuchen, T., Urbán, N., Ucar, D., Leeman, D. S., Martynoga, B., Sewak, M., Rando, T. A., Guillemot, F., Wernig, M., Brunet, A. 2013; 4 (3): 477-491


    FOXO transcription factors are central regulators of longevity from worms to humans. FOXO3, the FOXO isoform associated with exceptional human longevity, preserves adult neural stem cell pools. Here, we identify FOXO3 direct targets genome-wide in primary cultures of adult neural progenitor cells (NPCs). Interestingly, FOXO3-bound sites are enriched for motifs for bHLH transcription factors, and FOXO3 shares common targets with the proneuronal bHLH transcription factor ASCL1/MASH1 in NPCs. Analysis of the chromatin landscape reveals that FOXO3 and ASCL1 are particularly enriched at the enhancers of genes involved in neurogenic pathways. Intriguingly, FOXO3 inhibits ASCL1-dependent neurogenesis in NPCs and direct neuronal conversion in fibroblasts. FOXO3 also restrains neurogenesis in vivo. Our study identifies a genome-wide interaction between the prolongevity transcription factor FOXO3 and the cell-fate determinant ASCL1 and raises the possibility that FOXO3's ability to restrain ASCL1-dependent neurogenesis may help preserve the neural stem cell pool.

    View details for DOI 10.1016/j.celrep.2013.06.035

    View details for PubMedID 23891001

  • Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts JOURNAL OF NEUROSCIENCE METHODS Davila, J., Chanda, S., Ang, C. E., Suedhof, T. C., Wernig, M. 2013; 216 (2): 104-109


    We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O₂). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O₂ tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O₂ conditions compared to 50% under high O₂ growth conditions, confirming the beneficial aspect of reprogramming under low O₂. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3β and Smad-mediated pathways. Our results showed that lowering the O₂ tension at the initiation of reprogramming is a simple and efficient strategy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

    View details for DOI 10.1016/j.jneumeth.2013.03.020

    View details for Web of Science ID 000321168200004

    View details for PubMedCentralID PMC4009399

  • Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron Zhang, Y., Pak, C., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W., Yang, N., Danko, T., Chen, L., Wernig, M., Südhof, T. C. 2013; 78 (5): 785-798


    Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.

    View details for DOI 10.1016/j.neuron.2013.05.029

    View details for PubMedID 23764284

  • Generation of oligodendroglial cells by direct lineage conversion. Nature biotechnology Yang, N., Zuchero, J. B., Ahlenius, H., Marro, S., Ng, Y. H., Vierbuchen, T., Hawkins, J. S., Geissler, R., Barres, B. A., Wernig, M. 2013; 31 (5): 434-439


    Transplantation of oligodendrocyte precursor cells (OPCs) is a promising potential therapeutic strategy for diseases affecting myelin. However, the derivation of engraftable OPCs from human pluripotent stem cells has proven difficult and primary OPCs are not readily available. Here we report the generation of induced OPCs (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to reprogram mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures resembling primary OPCs. More importantly, iOPCs gave rise to mature oligodendrocytes that could ensheath multiple host axons when co-cultured with primary dorsal root ganglion cells and formed myelin after transplantation into shiverer mice. We propose direct lineage reprogramming as a viable alternative approach for the generation of OPCs for use in disease modeling and regenerative medicine.

    View details for DOI 10.1038/nbt.2564

    View details for PubMedID 23584610

  • An indirect approach to generating specific human cell types NATURE METHODS Lujan, E., Wernig, M. 2013; 10 (1): 44-46

    View details for DOI 10.1038/nmeth.2325

    View details for Web of Science ID 000312810100033

    View details for PubMedID 23269377

  • The many roads to Rome: induction of neural precursor cells from fibroblasts CURRENT OPINION IN GENETICS & DEVELOPMENT Lujan, E., Wernig, M. 2012; 22 (5): 517-522


    The experimental induction of specific cell fates in related or unrelated lineages has fascinated developmental biologists for decades. The evaluation of altered cell fates in response to ectopic expression during embryonic development has been a standard assay for interrogating gene function. However, until recently examples of cell lineage conversions were limited to closely related and primitive cell types. The induction of pluripotency in fibroblasts prominently highlighted that combinations of transcription factors can be extremely powerful and are much more effective than single genes. On the basis of this conclusion we previously identified transcription factor combinations that directly induce functional neuronal cells from mesodermal and endodermal cells. This work has evoked numerous additional studies demonstrating direct lineage conversion into neural and other lineages. Here, we review the generation of neural progenitor cells from fibroblasts, which is the newest addition to the arena of induced cell types. Surprisingly, two fundamentally different approaches have been taken to induce this cell type, one direct approach and another that involves the intermediate generation of a partially reprogrammed pluripotent state.

    View details for DOI 10.1016/j.gde.2012.07.002

    View details for Web of Science ID 000311470900017

    View details for PubMedID 22868177

  • Molecular Roadblocks for Cellular Reprogramming MOLECULAR CELL Vierbuchen, T., Wernig, M. 2012; 47 (6): 827-838


    During development, diverse cellular identities are established and maintained in the embryo. Although remarkably robust in vivo, cellular identities can be manipulated using experimental techniques. Lineage reprogramming is an emerging field at the intersection of developmental and stem cell biology in which a somatic cell is stably reprogrammed into a distinct cell type by forced expression of lineage-determining factors. Lineage reprogramming enables the direct conversion of readily available cells from patients (such as skin fibroblasts) into disease-relevant cell types (such as neurons and cardiomyocytes) or into induced pluripotent stem cells. Although remarkable progress has been made in developing novel reprogramming methods, the efficiency and fidelity of reprogramming need to be improved in order increase the experimental and translational utility of reprogrammed cells. Studying the mechanisms that prevent successful reprogramming should allow for improvements in reprogramming methods, which could have significant implications for regenerative medicine and the study of human disease. Furthermore, lineage reprogramming has the potential to become a powerful system for dissecting the mechanisms that underlie cell fate establishment and terminal differentiation processes. In this review, we will discuss how transcription factors interface with the genome and induce changes in cellular identity in the context of development and reprogramming.

    View details for DOI 10.1016/j.molcel.2012.09.008

    View details for Web of Science ID 000309312900003

    View details for PubMedID 23020854

  • Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lujan, E., Chanda, S., Ahlenius, H., Suedhof, T. C., Wernig, M. 2012; 109 (7): 2527-2532


    We recently showed that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as "induced neuronal" (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. We hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, we infected mouse embryonic fibroblasts derived from Sox2-EGFP mice with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP(+) colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, we found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When we added the Pou and Homeobox domain-containing transcription factor Brn2 to Sox2 and FoxG1, we were able to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. Our data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord.

    View details for DOI 10.1073/pnas.1121003109

    View details for PubMedID 22308465

  • Comprehensive qPCR profiling of gene expression in single neuronal cells NATURE PROTOCOLS Citri, A., Pang, Z. P., Suedhof, T. C., Wernig, M., Malenka, R. C. 2012; 7 (1): 118-127


    A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe uses Fluidigm Biomark dynamic arrays for high-throughput expression profiling from single neuronal cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment, which can be completed within 2-3 d. The protocol enables simple and cost-effective profiling of several hundred transcripts from a single cell, and it could have numerous utilities.

    View details for DOI 10.1038/nprot.2011.430

    View details for PubMedID 22193304

  • Induced Neuronal Cells: How to Make and Define a Neuron CELL STEM CELL Yang, N., Ng, Y. H., Pang, Z. P., Suedhof, T. C., Wernig, M. 2011; 9 (6): 517-525


    Cellular plasticity is a major focus of investigation in developmental biology. The recent discovery that induced neuronal (iN) cells can be generated from mouse and human fibroblasts by expression of defined transcription factors suggested that cell fate plasticity is much wider than previously anticipated. In this review, we summarize the most recent developments in this nascent field and suggest criteria to help define and categorize iN cells that take into account the complexity of neuronal identity.

    View details for DOI 10.1016/j.stem.2011.11.015

    View details for PubMedID 22136927

  • Cellular Reprogramming: Recent Advances in Modeling Neurological Diseases JOURNAL OF NEUROSCIENCE Ming, G., Bruestle, O., Muotri, A., Studer, L., Wernig, M., Christian, K. M. 2011; 31 (45): 16070-16075


    The remarkable advances in cellular reprogramming have made it possible to generate a renewable source of human neurons from fibroblasts obtained from skin samples of neonates and adults. As a result, we can now investigate the etiology of neurological diseases at the cellular level using neuronal populations derived from patients, which harbor the same genetic mutations thought to be relevant to the risk for pathology. Therapeutic implications include the ability to establish new humanized disease models for understanding mechanisms, conduct high-throughput screening for novel biogenic compounds to reverse or prevent the disease phenotype, identify and engineer genetic rescue of causal mutations, and develop patient-specific cellular replacement strategies. Although this field offers enormous potential for understanding and treating neurological disease, there are still many issues that must be addressed before we can fully exploit this technology. Here we summarize several recent studies presented at a symposium at the 2011 annual meeting of the Society for Neuroscience, which highlight innovative approaches to cellular reprogramming and how this revolutionary technique is being refined to model neurodevelopmental and neurodegenerative diseases, such as autism spectrum disorders, schizophrenia, familial dysautonomia, and Alzheimer's disease.

    View details for DOI 10.1523/JNEUROSCI.4218-11.2011

    View details for Web of Science ID 000296799700005

    View details for PubMedID 22072658

  • In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases STEM CELLS Sebastiano, V., Maeder, M. L., Angstman, J. F., Haddad, B., Khayter, C., Yeo, D. T., Goodwin, M. J., Hawkins, J. S., Ramirez, C. L., Batista, L. F., Artandi, S. E., Wernig, M., Joung, J. K. 2011; 29 (11): 1717-1726


    The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.

    View details for DOI 10.1002/stem.718

    View details for Web of Science ID 000296565500009

    View details for PubMedID 21898685

    View details for PubMedCentralID PMC3285772

  • Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons CELL STEM CELL Marro, S., Pang, Z. P., Yang, N., Tsai, M., Qu, K., Chang, H. Y., Suedhof, T. C., Wernig, M. 2011; 9 (4): 374-382


    Several recent studies have showed that mouse and human fibroblasts can be directly reprogrammed into induced neuronal (iN) cells, bypassing a pluripotent intermediate state. However, fibroblasts represent heterogeneous mesenchymal progenitor cells that potentially contain neural crest lineages, and the cell of origin remained undefined. This raises the fundamental question of whether lineage reprogramming is possible between cell types derived from different germ layers. Here, we demonstrate that terminally differentiated hepatocytes can be directly converted into functional iN cells. Importantly, single-cell and genome-wide expression analyses showed that fibroblast- and hepatocyte-derived iN cells not only induced a neuronal transcriptional program, but also silenced their donor transcriptome. The remaining donor signature decreased over time and could not support functional hepatocyte properties. Thus, the reprogramming factors lead to a binary lineage switch decision rather than an induction of hybrid phenotypes, but iN cells retain a small but detectable epigenetic memory of their donor cells.

    View details for DOI 10.1016/j.stem.2011.09.002

    View details for PubMedID 21962918

  • Direct lineage conversions: unnatural but useful? NATURE BIOTECHNOLOGY Vierbuchen, T., Wernig, M. 2011; 29 (10): 892-907


    Classic experiments such as somatic cell nuclear transfer into oocytes and cell fusion demonstrated that differentiated cells are not irreversibly committed to their fate. More recent work has built on these conclusions and discovered defined factors that directly induce one specific cell type from another, which may be as distantly related as cells from different germ layers. This suggests the possibility that any specific cell type may be directly converted into any other if the appropriate reprogramming factors are known. Direct lineage conversion could provide important new sources of human cells for modeling disease processes or for cellular-replacement therapies. For future applications, it will be critical to carefully determine the fidelity of reprogramming and to develop methods for robustly and efficiently generating human cell types of interest.

    View details for DOI 10.1038/nbt.1946

    View details for Web of Science ID 000296273000016

    View details for PubMedID 21997635

    View details for PubMedCentralID PMC3222779

  • Induction of human neuronal cells by defined transcription factors NATURE Pang, Z. P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D. R., Yang, T. Q., Citri, A., Sebastiano, V., Marro, S., Suedhof, T. C., Wernig, M. 2011; 476 (7359): 220-U122


    Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

    View details for DOI 10.1038/nature10202

    View details for PubMedID 21617644

  • Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells NATURE Batista, L. F., Pech, M., Zhong, F. L., Nguyen, H. N., Xie, K. T., Zaug, A. J., Crary, S. M., Choi, J., Sebastiano, V., Cherry, A., Giri, N., Wernig, M., Alter, B. P., Cech, T. R., Savage, S. A., Pera, R. A., Artandi, S. E. 2011; 474 (7351): 399-?


    The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.

    View details for DOI 10.1038/nature10084

    View details for Web of Science ID 000291647100050

    View details for PubMedID 21602826

    View details for PubMedCentralID PMC3155806

  • An imprinted signature helps isolate ESC-equivalent iPSCs CELL RESEARCH Lujan, E., Wernig, M. 2010; 20 (9): 974-976

    View details for DOI 10.1038/cr.2010.117

    View details for Web of Science ID 000281483800002

    View details for PubMedID 20697429

  • Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells FASEB JOURNAL Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Saric, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. 2010; 24 (8): 2739-2751


    Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.

    View details for DOI 10.1096/fj.09-145177

    View details for Web of Science ID 000285005400015

    View details for PubMedID 20371616

  • Generation of iPSCs from cultured human malignant cells BLOOD Carette, J. E., Pruszak, J., Varadarajan, M., Blomen, V. A., Gokhale, S., Camargo, F. D., Wernig, M., Jaenisch, R., Brummelkamp, T. R. 2010; 115 (20): 4039-4042


    Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients.

    View details for DOI 10.1182/blood-2009-07-231845

    View details for Web of Science ID 000277923600008

    View details for PubMedID 20233975

  • Direct conversion of fibroblasts to functional neurons by defined factors NATURE Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Suedhof, T. C., Wernig, M. 2010; 463 (7284): 1035-U50


    Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

    View details for DOI 10.1038/nature08797

    View details for PubMedID 20107439

  • Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro FASEB JOURNAL Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., Saric, T., Wernig, M., Jaenisch, R., Hescheler, J. 2009; 23 (12): 4168-4180


    Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c-Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS-cell-based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, alphaMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac-specific voltage-gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels; CMs generated from iPS cells have a ventricular phenotype; and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC-derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.

    View details for DOI 10.1096/fj.08-128546

    View details for Web of Science ID 000272193700013

    View details for PubMedID 19703934

  • Cardiac Myocytes Derived from Murine Reprogrammed Fibroblasts: Intact Hormonal Regulation, Cardiac Ion Channel Expression and Development of Contractility CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Pfannkuche, K., Liang, H., Hannes, T., Xi, J., Fatima, A., Nguemo, F., Matzkies, M., Wernig, M., Jaenisch, R., Pillekamp, F., Halbach, M., Schunkert, H., Saric, T., Hescheler, J., Reppel, M. 2009; 24 (1-2): 73-86


    Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells.Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation.Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.

    View details for DOI 10.1159/000227815

    View details for Web of Science ID 000267650600009

    View details for PubMedID 19590195

  • Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells CELL Marson, A., Levine, S. S., Cole, M. F., Frampton, G. M., Brambrink, T., Johnstone, S., Guenther, M. G., Johnston, W. K., Wernig, M., Newman, J., Calabrese, J. M., Dennis, L. M., Volkert, T. L., Gupta, S., Love, J., Hannett, N., Sharp, P. A., Bartel, D. P., Jaenisch, R., Young, R. A. 2008; 134 (3): 521-533


    MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

    View details for DOI 10.1016/j.cell.2008.07.020

    View details for Web of Science ID 000258665500024

    View details for PubMedID 18692474

  • Genome-scale DNA methylation maps of pluripotent and differentiated cells NATURE Meissner, A., Mikkelsen, T. S., Gu, H., Wernig, M., Hanna, J., Sivachenko, A., Zhang, X., Bernstein, B. E., Nusbaum, C., Jaffe, D. B., Gnirke, A., Jaenisch, R., Lander, E. S. 2008; 454 (7205): 766-U91


    DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

    View details for DOI 10.1038/nature07107

    View details for Web of Science ID 000258228000045

    View details for PubMedID 18600261

  • A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types NATURE BIOTECHNOLOGY Wernig, M., Lengner, C. J., Hanna, J., Lodato, M. A., Steine, E., Foreman, R., Staerk, J., Markoulaki, S., Jaenisch, R. 2008; 26 (8): 916-924


    The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

    View details for Web of Science ID 000258325500026

    View details for PubMedID 18594521

  • Dissecting direct reprogramming through integrative genomic analysis NATURE Mikkelsen, T. S., Hanna, J., Zhang, X., Ku, M., Wernig, M., Schorderet, P., Bernstein, B. E., Jaenisch, R., Lander, E. S., Meissner, A. 2008; 454 (7200): 49-U1


    Somatic cells can be reprogrammed to a pluripotent state through the ectopic expression of defined transcription factors. Understanding the mechanism and kinetics of this transformation may shed light on the nature of developmental potency and suggest strategies with improved efficiency or safety. Here we report an integrative genomic analysis of reprogramming of mouse fibroblasts and B lymphocytes. Lineage-committed cells show a complex response to the ectopic expression involving induction of genes downstream of individual reprogramming factors. Fully reprogrammed cells show gene expression and epigenetic states that are highly similar to embryonic stem cells. In contrast, stable partially reprogrammed cell lines show reactivation of a distinctive subset of stem-cell-related genes, incomplete repression of lineage-specifying transcription factors, and DNA hypermethylation at pluripotency-related loci. These observations suggest that some cells may become trapped in partially reprogrammed states owing to incomplete repression of transcription factors, and that DNA de-methylation is an inefficient step in the transition to pluripotency. We demonstrate that RNA inhibition of transcription factors can facilitate reprogramming, and that treatment with DNA methyltransferase inhibitors can improve the overall efficiency of the reprogramming process.

    View details for DOI 10.1038/nature07056

    View details for Web of Science ID 000257308300033

    View details for PubMedID 18509334

  • Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency CELL Hanna, J., Markoulaki, S., Schorderet, P., Carey, B. W., Beard, C., Wernig, M., Creyghton, M. P., Steine, E. J., Cassady, J. P., Foreman, R., Lengner, C. J., Dausman, J. A., Jaenisch, R. 2008; 133 (2): 250-264


    Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.

    View details for DOI 10.1016/j.cell.2008.03.028

    View details for Web of Science ID 000255052000015

    View details for PubMedID 18423197

  • Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, M., Zhao, J., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O., Jaenisch, R. 2008; 105 (15): 5856-5861


    The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

    View details for DOI 10.1073/pnas.0801677105

    View details for Web of Science ID 000255237200038

    View details for PubMedID 18391196

    View details for PubMedCentralID PMC2311361

  • Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells CELL STEM CELL Brambrink, T., Foreman, R., Welstead, G. G., Lengner, C. J., Wernig, M., Suh, H., Jaenisch, R. 2008; 2 (2): 151-159


    Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.

    View details for DOI 10.1016/j.stem.2008.01.004

    View details for Web of Science ID 000253301900010

    View details for PubMedID 18371436

  • c-Myc is dispensable for direct reprogramming of mouse fibroblasts CELL STEM CELL Wernig, M., Meissner, A., Cassady, J. P., Jaenisch, R. 2008; 2 (1): 10-12

    View details for DOI 10.1016/j.stem.2007.12.001

    View details for Web of Science ID 000252606400006

    View details for PubMedID 18371415

  • Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin SCIENCE Hanna, J., Wernig, M., Markoulaki, S., Sun, C., Meissner, A., Cassady, J. P., Beard, C., Brambrink, T., Wu, L., Townes, T. M., Jaenisch, R. 2007; 318 (5858): 1920-1923


    It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.

    View details for DOI 10.1126/science.1152092

    View details for Web of Science ID 000251786600058

    View details for PubMedID 18063756

  • Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells NATURE BIOTECHNOLOGY Meissner, A., Wernig, M., Jaenisch, R. 2007; 25 (10): 1177-1181


    In vitro reprogramming of somatic cells into a pluripotent embryonic stem cell-like state has been achieved through retroviral transduction of murine fibroblasts with Oct4, Sox2, c-myc and Klf4. In these experiments, the rare 'induced pluripotent stem' (iPS) cells were isolated by stringent selection for activation of a neomycin-resistance gene inserted into the endogenous Oct4 (also known as Pou5f1) or Nanog loci. Direct isolation of pluripotent cells from cultured somatic cells is of potential therapeutic interest, but translation to human systems would be hindered by the requirement for transgenic donors in the present iPS isolation protocol. Here we demonstrate that reprogrammed pluripotent cells can be isolated from genetically unmodified somatic donor cells solely based upon morphological criteria.

    View details for DOI 10.1038/nbt1335

    View details for Web of Science ID 000250226600030

    View details for PubMedID 17724450

  • Genome-wide maps of chromatin state in pluripotent and lineage-committed cells NATURE Mikkelsen, T. S., Ku, M., Jaffe, D. B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T., Koche, R. P., Lee, W., Mendenhall, E., O'Donovan, A., Presser, A., Russ, C., Xie, X., Meissner, A., Wernig, M., Jaenisch, R., Nusbaum, C., Lander, E. S., Bernstein, B. E. 2007; 448 (7153): 553-U2


    We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

    View details for DOI 10.1038/nature06008

    View details for Web of Science ID 000248446700035

    View details for PubMedID 17603471

  • In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state NATURE Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E., Jaenisch, R. 2007; 448 (7151): 318-U2


    Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.

    View details for DOI 10.1038/nature05944

    View details for Web of Science ID 000248118300050

    View details for PubMedID 17554336

  • Polycomb complexes repress developmental regulators in murine embryonic stem cells NATURE Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K., Bell, G. W., Otte, A. P., Vidal, M., Gifford, D. K., Young, R. A., Jaenisch, R. 2006; 441 (7091): 349-353


    The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.

    View details for DOI 10.1038/nature04733

    View details for Web of Science ID 000237593200048

    View details for PubMedID 16625203

  • A bivalent chromatin structure marks key developmental genes in embryonic stem cells CELL Bernstein, B. E., Mikkelsen, T. S., Xie, X. H., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., Jaenisch, R., Wagschal, A., Feil, R., Schreiber, S. L., Lander, E. S. 2006; 125 (2): 315-326


    The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.

    View details for DOI 10.1016/j.cell.2006.02.041

    View details for Web of Science ID 000237241500021

    View details for PubMedID 16630819

  • The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, G., Janzen, V., SCHAFER, R., Zweyer, M., Knauf, U., Hoegemeier, O., Mundegar, R. R., Garbe, S., Stier, S., Franz, T., Wernig, M., Wernig, A. 2005; 102 (33): 11852-11857


    Bone-marrow-derived cells can contribute nuclei to skeletal muscle fibers. However, serial sectioning of muscle in mdx mice implanted with GFP-labeled bone marrow reveals that only 20% of the donor nuclei chronically incorporated in muscle fibers show dystrophin (or GFP) expression, which is still higher than the expected frequency of "revertant" fibers, but there is no overall increase above controls over time. Obviously, the vast majority of incorporated nuclei either never or only temporarily turn on myogenic genes; also, incorporated nuclei eventually loose the activation of the beta-actin::GFP transgene. Consequently, we attempted to enhance the expression of dystrophin. In vivo application of the chromatin-modifying agents 5-azadeoxycytidine and phenylbutyrate as well as local damage by cardiotoxin injections caused a small increase in dystrophin-positive fibers without abolishing the appearance of "silent" nuclei. The results thus confirm that endogenous repair processes and epigenetic modifications on a small-scale lead to dystrophin expression from donor nuclei. Both effects, however, remain below functionally significant levels.

    View details for DOI 10.1073/pnas.0502507102

    View details for Web of Science ID 000231317000051

    View details for PubMedID 16085712

    View details for PubMedCentralID PMC1187970

  • Functional integration of transplanted ES cell-derived neurons Symposium of the Nordrhein-Westfalischen-Akademie-der-Wissenschaften Benninger, F., Wernig, M., Schmandt, T., Rade, M., Bussow, H., Scheffler, B., Beck, H., Brustle, O. VERLAG FERDINAND SCHONINGH. 2005: 79–81
  • Functional integration of embryonic stem cell-derived neurons in vivo JOURNAL OF NEUROSCIENCE Wernig, M., Benninger, F., Schmandt, T., Rade, M., Tucker, K. L., Bussow, H., Beck, H., Brustle, O. 2004; 24 (22): 5258-5268


    Pluripotency and the potential for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the functional integration of transplanted ES cell-derived neurons on the single-cell level. To address this issue, ES cell-derived neural precursors exhibiting neuron-specific enhanced green fluorescent protein (EGFP) expression were introduced into the developing brain. Donor cells implanted into the cerebral ventricles of embryonic rats migrated as single cells into a variety of brain regions, where they acquired complex morphologies and adopted excitatory and inhibitory neurotransmitter phenotypes. Synaptic integration was suggested by the expression of PSD-95 (postsynaptic density-95) on donor cell dendrites, which in turn were approached by multiple synaptophysin-positive host axon terminals. Ultrastructural and electrophysiological data confirmed the formation of synapses between host and donor cells. Ten to 21 d after birth, all EGFP-positive donor cells examined displayed active membrane properties and received glutamatergic and GABAergic synaptic input from host neurons. These data demonstrate that, at the single-cell level, grafted ES cell-derived neurons undergo morphological and functional integration into the host brain circuitry. Antibodies to the region-specific transcription factors Bf1, Dlx, En1, and Pax6 were used to explore whether functional donor cell integration depends on the acquisition of a regional phenotype. Our data show that incorporated neurons frequently exhibit a lacking or ectopic expression of these transcription factors. Thus, the lack of an appropriate regional "code" does not preclude morphological and synaptic integration of ES cell-derived neurons.

    View details for DOI 10.1523/JNEUROSCI.0428-04.200

    View details for Web of Science ID 000221883100020

    View details for PubMedID 15175396

  • Functional integration of embryonic stem cell-derived neurons in hippocampal slice cultures JOURNAL OF NEUROSCIENCE Benninger, F., Beck, H., Wernig, M., Tucker, K. L., Brustle, O., Scheffler, B. 2003; 23 (18): 7075-7083


    The generation of neurons and glia from pluripotent embryonic stem (ES) cells represents a promising strategy for the study of CNS development and repair. ES cell-derived neural precursors have been shown to develop into morphologically mature neurons and glia when grafted into brain and spinal cord. However, there is a surprising shortage of data concerning the functional integration of ES cell-derived neurons (ESNs) into the host CNS tissue. Here, we use ES cells engineered to express enhanced green fluorescent protein (EGFP) only in neuronal progeny to study the functional properties of ESNs during integration into long-term hippocampal slice cultures. After incorporation into the dentate gyrus, EGFP+ donor neurons display a gradual maturation of their intrinsic discharge behavior and a concomitant increase in the density of voltage-gated Na+ and K+ channels. Integrated ESNs express AMPA and GABA(A) receptor subunits. Most importantly, neurons derived from ES cells receive functional glutamatergic and GABAergic synapses from host neurons. Specifically, we demonstrate that host perforant path axons form synapses onto integrated ESNs. These synapses between host and ES cell-derived neurons display pronounced paired-pulse facilitation indicative of intact presynaptic short-term plasticity. Thus, ES cell-derived neural precursors generate functionally active neurons capable of integrating into the brain circuitry.

    View details for Web of Science ID 000184587100012

    View details for PubMedID 12904468

  • Migration and differentiation of myogenic precursors following transplantation into the developing rat brain STEM CELLS Steffel, J., Wernig, M., Knauf, U., Kumar, S., WIESTLER, O. D., Wernig, A., Brustle, O. 2003; 21 (2): 181-189


    There is increasing evidence that muscle-derived precursor cells can, under appropriate conditions, give rise to other than myogenic cell types. Transplantation into the embryonic ventricular zone provides a unique opportunity to study the migration and differentiation of non-neural somatic progenitor cells in response to instructive cues within the developing neuroepithelium. Here, we demonstrate that myogenic cell lines grafted into the ventricles of rat embryos showed widespread migration into several host brain compartments. In contrast to incorporation patterns observed after transplantation of neural cells, grafted myoblasts incorporated virtually exclusively along endogenous blood vessels. Preferential incorporation sites included cortex, olfactory bulb, hippocampus, striatum, thalamus, hypothalamus, and tectum. While the engrafted myoblasts showed no evidence of neural differentiation, a fraction exhibited pronounced coexpression of endothelial marker antigens. These findings support the concept of a close developmental relationship between the myogenic and the endothelial lineages. Used as a delivery system, transfected myoblasts may be exploited for widespread gene transfer to the perivascular compartment of the perinatal central nervous system.

    View details for Web of Science ID 000181601300007

    View details for PubMedID 12634414

  • Tau EGFP embryonic stem cells: An efficient tool for neuronal lineage selection and transplantation JOURNAL OF NEUROSCIENCE RESEARCH Wernig, M., Tucker, K. L., Gornik, V., Schneiders, A., Buschwald, R., WIESTLER, O. D., Barde, Y. A., Brustle, O. 2002; 69 (6): 918-924


    Pluripotency and the capacity for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for cell-replacement strategies. A key prerequisite for a therapeutic application of ES cells is the generation of defined somatic cell populations. Here we demonstrate that a targeted insertion of the EGFP gene into the tau locus permits efficient fluorescence-activated cell sorting (FACS)-based lineage selection of ES cell-derived neurons. After in vitro differentiation of heterozygous tau EGFP ES cells into multipotent neural precursors, EGFP is selectively induced in postmitotic neurons of various neurotransmitter phenotypes. By using FACS, ES cell-derived neurons can be enriched to purities of more than 90%. Because neuron-specific EGFP fluorescence is also observed upon transplantation of ES cell-derived neural precursors, the tau EGFP mutant represents a useful tool for the in vivo analysis of grafted ES cell-derived neurons.

    View details for DOI 10.1002/jnr.10395

    View details for Web of Science ID 000177792600024

    View details for PubMedID 12205684

  • Fifty ways to make a neuron: Shifts in stem cell hierarchy and their implications for neuropathology and CNS repair JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY Wernig, M., Brustle, O. 2002; 61 (2): 101-110


    During embryogenesis, the developmental potential of individual cells is continuously restricted. While embryonic stem (ES) cells derived from the inner cell mass of the blastocyst can give rise to all tissues and cell types, their progeny segregates into a multitude of tissue-specific stem and progenitor cells. Following organogenesis, a pool of resident "adult" stem cells is maintained in many tissues. In this hierarchical concept, transition through defined intermediate stages of decreasing potentiality is regarded as prerequisite for the generation of a somatic cell type. Several recent findings have challenged this view. First, adult stem cells have been shown to adopt properties of pluripotent cells and contribute cells to a variety of tissues. Second, a direct transition from a pluripotent ES cell to a defined somatic phenotype has been postulated for the neural lineage. Finally, nuclear transplantation has revealed that the transcriptional machinery associated with a distinct somatic cell fate can be reprogrammed to totipotency. The possibility to bypass developmental hierarchies in stem cell differentiation opens new avenues for the study of nervous system development, disease, and repair.

    View details for Web of Science ID 000173768100001

    View details for PubMedID 11853015

  • In vitro differentiation of transplantable neural precursors from human embryonic stem cells NATURE BIOTECHNOLOGY Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., Thomson, J. A. 2001; 19 (12): 1129-1133


    The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube-like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell-derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.

    View details for Web of Science ID 000172524400025

    View details for PubMedID 11731781

  • A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression HUMAN MOLECULAR GENETICS Faiella, A., Wernig, M., Consalez, G. G., Hostick, U., Hofmann, C., Hustert, E., Boncinelli, E., Balling, R., Nadeau, J. H. 2000; 9 (2): 227-236


    Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

    View details for Web of Science ID 000084976500009

    View details for PubMedID 10607833