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  • Academic
    yihanng@stanford.edu
    University - Academic staff Department: Department of Molecular and Cellular Physiology Position: Basic Life Research Scientist

Additional Info

  • Mail Code: 5453

All Publications

  • Transcription Factor-Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells. Methods in molecular biology (Clifton, N.J.) Ng, Y. H., Janas, J. A. 2023; 2683: 39-51

    Abstract

    The ability to differentiate pluripotent stem cells and to generate specific cell types is a long-standing goal of regenerative medicine. This can be accomplished by recreating the developmental trajectories using sequential activation of the corresponding signaling pathways, or more recently-by direct programming of cell identities using lineage-specific transcription factors. Notably, to be functional in cell replacement therapies, generation of complex cell types, such as specialized neuronal sub-types of the brain, requires precise induction of molecular profiles and regional specification of the cells. However, the induction of the correct cellular identity and marker gene expression can be hampered by technical challenges, one of which is the robust co-expression of multiple transcription factors that is often required for correct cell identity specification. Here, we describe in detail a method for co-expression of seven transcription factors required for efficient induction of dopaminergic neurons with midbrain characteristics from human embryonic and induced pluripotent stem cells.

    View details for DOI 10.1007/978-1-0716-3287-1_4

    View details for PubMedID 37300765

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • A Synaptic Circuit Required for Acquisition but Not Recall of Social Transmission of Food Preference. Neuron Wang, C. Y., Liu, Z. n., Ng, Y. H., Südhof, T. C. 2020

    Abstract

    During social transmission of food preference (STFP), the combination of an olfactory sensory input with a social cue induces long-term memory of a food odor. How a social cue produces long-term learning of an olfactory input, however, remains unknown. Here we show that the neurons of the anterior olfactory nucleus (AON), which form abundant synaptic projections onto granule cells in the olfactory bulb (OB), express the synaptogenic molecule C1ql3. Deletion of C1ql3 in the dorsolateral AON impaired synaptic AON→OB connections and abolished acquisition, but not recall, of STFP memory without significantly affecting basal olfaction. Moreover, deletion in granule cells of the OB of Bai3, a postsynaptic GPCR that binds C1ql3, similarly suppressed synaptic transmission at AON→OB projections and abolished acquisition, but not recall, of STFP memory. Thus, synaptic AON→OB connections are selectively required for STFP memory acquisition and are formed by an essential interaction of presynaptic C1ql3 with postsynaptic Bai3.

    View details for DOI 10.1016/j.neuron.2020.04.004

    View details for PubMedID 32369733

  • 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

    Abstract

    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

  • A central amygdala to zona incerta projection is required for acquisition and remote recall of conditioned fear memory. Nature neuroscience Zhou, M., Liu, Z., Melin, M. D., Ng, Y. H., Xu, W., Sudhof, T. C. 2018

    Abstract

    The formation and retrieval of conditioned fear memories critically depend on the amygdala. Here we identify an inhibitory projection from somatostatin-positive neurons in the central amygdala to parvalbumin-positive neurons in the zona incerta that is required for both recent and remote fear memories. Thus, the amygdala inhibitory input to parvalbumin-positive neurons in the zona incerta, a nucleus not previously implicated in fear memory, is an essential component of the fear memory circuitry.

    View details for PubMedID 30349111

  • 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

    Abstract

    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

    Abstract

    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-?

    Abstract

    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

  • 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-?

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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