Sergiu P. Pasca

All Publications

• Antisense oligonucleotide therapeutic approach for Timothy syndrome. Nature Chen, X., Birey, F., Li, M. Y., Revah, O., Levy, R., Thete, M. V., Reis, N., Kaganovsky, K., Onesto, M., Sakai, N., Hudacova, Z., Hao, J., Meng, X., Nishino, S., Huguenard, J., Pașca, S. P. 2024; 628 (8009): 818-825

  Abstract

  Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

  View details for DOI 10.1038/s41586-024-07310-6

  View details for PubMedID 38658687

  View details for PubMedCentralID 1149428

• Kirigami electronics for long-term electrophysiological recording of human neural organoids and assembloids. Nature biotechnology Yang, X., Forro, C., Li, T. L., Miura, Y., Zaluska, T. J., Tsai, C., Kanton, S., McQueen, J. P., Chen, X., Mollo, V., Santoro, F., Pașca, S. P., Cui, B. 2024

  Abstract

  Realizing the full potential of organoids and assembloids to model neural development and disease will require improved methods for long-term, minimally invasive recording of electrical activity. Current technologies, such as patch clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not allow chronic recording of organoids in suspension, which is necessary to preserve architecture. Inspired by kirigami art, we developed flexible electronics that transition from a two-dimensional to a three-dimensional basket-like configuration with either spiral or honeycomb patterns to accommodate the long-term culture of organoids in suspension. Here we show that this platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids for up to 120days while preserving their morphology, cytoarchitecture and cell composition. We demonstrate integration of KiriE with optogenetic and pharmacological manipulation and modeling phenotypes related to a genetic disease. Moreover, KiriE can capture corticostriatal connectivity in assembloids following optogenetic stimulation. Thus, KiriE will enable investigation of disease and activity patterns underlying nervous system assembly.

  View details for DOI 10.1038/s41587-023-02081-3

  View details for PubMedID 38253880

• Constructing human neural circuits in living systems by transplantation. Cell Pașca, S. P. 2024; 187 (1): 8-13

  Abstract

  Our understanding of how the brain assembles its circuits and how this goes awry in disease remains incomplete. There has been great progress in generating human neurons from stem cells invitro and, more recently, in constructing circuits with human cells invivo by transplantation. Here, I highlight approaches, promises, and challenges of growing human neurons in living animals to study human development and disease.

  View details for DOI 10.1016/j.cell.2023.12.008

  View details for PubMedID 38181744

• Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment. Nature Meng, X., Yao, D., Imaizumi, K., Chen, X., Kelley, K. W., Reis, N., Thete, M. V., Arjun McKinney, A., Kulkarni, S., Panagiotakos, G., Bassik, M. C., Pașca, S. P. 2023

  Abstract

  The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.

  View details for DOI 10.1038/s41586-023-06564-w

  View details for PubMedID 37758944

  View details for PubMedCentralID 4349583

• Maturation and circuit integration of transplanted human cortical organoids. Nature Revah, O., Gore, F., Kelley, K. W., Andersen, J., Sakai, N., Chen, X., Li, M. Y., Birey, F., Yang, X., Saw, N. L., Baker, S. W., Amin, N. D., Kulkarni, S., Mudipalli, R., Cui, B., Nishino, S., Grant, G. A., Knowles, J. K., Shamloo, M., Huguenard, J. R., Deisseroth, K., Pașca, S. P. 2022; 610 (7931): 319-326

  Abstract

  Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.

  View details for DOI 10.1038/s41586-022-05277-w

  View details for PubMedID 36224417

• A nomenclature consensus for nervous system organoids and assembloids. Nature Pașca, S. P., Arlotta, P., Bateup, H. S., Camp, J. G., Cappello, S., Gage, F. H., Knoblich, J. A., Kriegstein, A. R., Lancaster, M. A., Ming, G. L., Muotri, A. R., Park, I. H., Reiner, O., Song, H., Studer, L., Temple, S., Testa, G., Treutlein, B., Vaccarino, F. M. 2022; 609 (7929): 907-910

  Abstract

  Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.

  View details for DOI 10.1038/s41586-022-05219-6

  View details for PubMedID 36171373

• Human brain organogenesis: Toward a cellular understanding of development and disease. Cell Kelley, K. W., Pașca, S. P. 2021

  Abstract

  The construction of the human nervous system is a distinctly complex although highly regulated process. Human tissue inaccessibility has impeded a molecular understanding of the developmental specializations from which our unique cognitive capacities arise. A confluence of recent technological advances in genomics and stem cell-based tissue modeling is laying the foundation for a new understanding of human neural development and dysfunction in neuropsychiatric disease. Here, we review recent progress on uncovering the cellular and molecular principles of human brain organogenesis in vivo as well as using organoids and assembloids in vitro to model features of human evolution and disease.

  View details for DOI 10.1016/j.cell.2021.10.003

  View details for PubMedID 34774127

• Chromatin accessibility dynamics in a model of human forebrain development. Science (New York, N.Y.) Trevino, A. E., Sinnott-Armstrong, N. n., Andersen, J. n., Yoon, S. J., Huber, N. n., Pritchard, J. K., Chang, H. Y., Greenleaf, W. J., Pașca, S. P. 2020; 367 (6476)

  Abstract

  Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.

  View details for DOI 10.1126/science.aay1645

  View details for PubMedID 31974223

• Generation of human striatal organoids and cortico-striatal assembloids from human pluripotent stem cells. Nature biotechnology Miura, Y. n., Li, M. Y., Birey, F. n., Ikeda, K. n., Revah, O. n., Thete, M. V., Park, J. Y., Puno, A. n., Lee, S. H., Porteus, M. H., Pașca, S. P. 2020; 38 (12): 1421–30

  Abstract

  Cortico-striatal projections are critical components of forebrain circuitry that regulate motivated behaviors. To enable the study of the human cortico-striatal pathway and how its dysfunction leads to neuropsychiatric disease, we developed a method to convert human pluripotent stem cells into region-specific brain organoids that resemble the developing human striatum and include electrically active medium spiny neurons. We then assembled these organoids with cerebral cortical organoids in three-dimensional cultures to form cortico-striatal assembloids. Using viral tracing and functional assays in intact or sliced assembloids, we show that cortical neurons send axonal projections into striatal organoids and form synaptic connections. Medium spiny neurons mature electrophysiologically following assembly and display calcium activity after optogenetic stimulation of cortical neurons. Moreover, we derive cortico-striatal assembloids from patients with a neurodevelopmental disorder caused by a deletion on chromosome 22q13.3 and capture disease-associated defects in calcium activity, showing that this approach will allow investigation of the development and functional assembly of cortico-striatal connectivity using patient-derived cells.

  View details for DOI 10.1038/s41587-020-00763-w

  View details for PubMedID 33273741

• Generation of Functional Human 3D Cortico-Motor Assembloids. Cell Andersen, J. n., Revah, O. n., Miura, Y. n., Thom, N. n., Amin, N. D., Kelley, K. W., Singh, M. n., Chen, X. n., Thete, M. V., Walczak, E. M., Vogel, H. n., Fan, H. C., Paşca, S. P. 2020

  Abstract

  Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.

  View details for DOI 10.1016/j.cell.2020.11.017

  View details for PubMedID 33333020

• Neuronal defects in a human cellular model of 22q11.2 deletion syndrome. Nature medicine Khan, T. A., Revah, O. n., Gordon, A. n., Yoon, S. J., Krawisz, A. K., Goold, C. n., Sun, Y. n., Kim, C. H., Tian, Y. n., Li, M. Y., Schaepe, J. M., Ikeda, K. n., Amin, N. D., Sakai, N. n., Yazawa, M. n., Kushan, L. n., Nishino, S. n., Porteus, M. H., Rapoport, J. L., Bernstein, J. A., O'Hara, R. n., Bearden, C. E., Hallmayer, J. F., Huguenard, J. R., Geschwind, D. H., Dolmetsch, R. E., Paşca, S. P. 2020

  Abstract

  22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

  View details for DOI 10.1038/s41591-020-1043-9

  View details for PubMedID 32989314

• Assembling human brain organoids. Science (New York, N.Y.) Pasca, S. P. 2019; 363 (6423): 126–27

  View details for PubMedID 30630918

• Human 3D cellular model of hypoxic brain injury of prematurity. Nature medicine Pașca, A. M., Park, J. Y., Shin, H. W., Qi, Q. n., Revah, O. n., Krasnoff, R. n., O'Hara, R. n., Willsey, A. J., Palmer, T. D., Pașca, S. P. 2019

  Abstract

  Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.

  View details for PubMedID 31061540

• Reliability of human cortical organoid generation. Nature methods Yoon, S. J., Elahi, L. S., Pașca, A. M., Marton, R. M., Gordon, A. n., Revah, O. n., Miura, Y. n., Walczak, E. M., Holdgate, G. M., Fan, H. C., Huguenard, J. R., Geschwind, D. H., Pașca, S. P. 2019; 16 (1): 75–78

  Abstract

  The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

  View details for PubMedID 30573846

• Generation and assembly of human brain region-specific three-dimensional cultures. Nature protocols Sloan, S. A., Andersen, J., Pașca, A. M., Birey, F., Pașca, S. P. 2018

  Abstract

  The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain-specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region-specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.

  View details for PubMedID 30202107

• The rise of three-dimensional human brain cultures. Nature Pașca, S. P. 2018; 553 (7689): 437-445

  Abstract

  Pluripotent stem cells show a remarkable ability to self-organize and differentiate in vitro in three-dimensional aggregates, known as organoids or organ spheroids, and to recapitulate aspects of human brain development and function. Region-specific 3D brain cultures can be derived from any individual and assembled to model complex cell-cell interactions and to generate circuits in human brain assembloids. Here I discuss how this approach can be used to understand unique features of the human brain and to gain insights into neuropsychiatric disorders. In addition, I consider the challenges faced by researchers in further improving and developing methods to probe and manipulate patient-derived 3D brain cultures.

  View details for DOI 10.1038/nature25032

  View details for PubMedID 29364288

• Building Models of Brain Disorders with Three-Dimensional Organoids. Neuron Amin, N. D., Paşca, S. P. 2018; 100 (2): 389–405

  Abstract

  Disorders of the nervous system are challenging to study and treat due to the relative inaccessibility of functional human brain tissue for research. Stem cell-derived 3D human brain organoids have the potential to recapitulate features of the human brain with greater complexity than 2D models and are increasingly being applied to model diseases affecting the central nervous system. Here, we review the use of human brain organoids to investigate neurological and psychiatric (neuropsychiatric) disorders and how this technology may ultimately advance our biological understanding of these conditions.

  View details for PubMedID 30359604

• Assembly of functionally integrated human forebrain spheroids NATURE Birey, F., Andersen, J., Makinson, C. D., Islam, S., Wei, W., Huber, N., Fan, H. C., Metzler, K. R., Panagiotakos, G., Thom, N., O'Rourke, N. A., Steinmetz, L. M., Bernstein, J. A., Hallmayer, J., Huguenard, J. R., Pasca, S. P. 2017; 545 (7652): 54-?

  Abstract

  The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

  View details for DOI 10.1038/nature22330

  View details for PubMedID 28445465

• Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells. Neuron Sloan, S. A., Darmanis, S. n., Huber, N. n., Khan, T. A., Birey, F. n., Caneda, C. n., Reimer, R. n., Quake, S. R., Barres, B. A., Paşca, S. P. 2017; 95 (4): 779–90.e6

  Abstract

  There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20 months in vitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

  View details for PubMedID 28817799

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods Pasca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J., O'Rourke, N. A., Nguyen, K. D., Smith, S. J., Huguenard, J. R., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015; 12 (7): 671-678

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedID 26005811

• Assembloid models of cell-cell interaction to study tissue and disease biology. Cell stem cell Onesto, M. M., Kim, J. I., Pasca, S. P. 2024

  Abstract

  Neurodevelopment involves the migration, projection, and integration of various cell types across different regions of the nervous system. Assembloids are self-organizing systems formed by the integration of multiple organoids or cell types. Here, we outline the generation and application of assembloids. We illustrate how assembloids recapitulate critical neurodevelopmental steps, like migration, axon projection, and circuit formation, and how they are starting to provide biological insights into neuropsychiatric disorders. Additionally, we review how assembloids can be used to study properties emerging from cell-cell interactions within non-neural tissues. Overall, assembloid platforms represent a powerful tool for discovering human biology and developing therapeutics.

  View details for DOI 10.1016/j.stem.2024.09.017

  View details for PubMedID 39454582

• Human assembloids reveal the consequences of CACNA1G gene variants in the thalamocortical pathway. Neuron Kim, J. I., Miura, Y., Li, M. Y., Revah, O., Selvaraj, S., Birey, F., Meng, X., Thete, M. V., Pavlov, S. D., Andersen, J., Pașca, A. M., Porteus, M. H., Huguenard, J. R., Pașca, S. P. 2024

  Abstract

  Abnormalities in thalamocortical crosstalk can lead to neuropsychiatric disorders. Variants in CACNA1G, which encodes the α1G subunit of the thalamus-enriched T-type calcium channel, are associated with absence seizures, intellectual disability, and schizophrenia, but the cellular and circuit consequences of these genetic variants in humans remain unknown. Here, we developed a human assembloid model of the thalamocortical pathway to dissect the contribution of genetic variants in T-type calcium channels. We discovered that the M1531V CACNA1G variant associated with seizures led to changes in T-type currents in thalamic neurons, as well as correlated hyperactivity of thalamic and cortical neurons in assembloids. By contrast, CACNA1G loss, which has been associated with risk of schizophrenia, resulted in abnormal thalamocortical connectivity that was related to both increased spontaneous thalamic activity and aberrant axonal projections. These results illustrate the utility of multi-cellular systems for interrogating human genetic disease risk variants at both cellular and circuit level.

  View details for DOI 10.1016/j.neuron.2024.09.020

  View details for PubMedID 39419023

• Host circuit engagement of human cortical organoids transplanted in rodents. Nature protocols Kelley, K. W., Revah, O., Gore, F., Kaganovsky, K., Chen, X., Deisseroth, K., Pașca, S. P. 2024

  Abstract

  Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell-derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30-60 days, and the transplantation procedure itself requires ~0.5-1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron-host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages.

  View details for DOI 10.1038/s41596-024-01029-4

  View details for PubMedID 39075308

  View details for PubMedCentralID 5667942

• Roadmap for developing biologically inspired therapeutics for genetic brain disorders. Trends in molecular medicine Imaizumi, K., Paşca, S. P. 2023

  View details for DOI 10.1016/j.molmed.2023.07.005

  View details for PubMedID 37586931

• Spatially controlled construction of assembloids using bioprinting. Nature communications Roth, J. G., Brunel, L. G., Huang, M. S., Liu, Y., Cai, B., Sinha, S., Yang, F., Pașca, S. P., Shin, S., Heilshorn, S. C. 2023; 14 (1): 4346

  Abstract

  The biofabrication of three-dimensional (3D) tissues that recapitulate organ-specific architecture and function would benefit from temporal and spatial control of cell-cell interactions. Bioprinting, while potentially capable of achieving such control, is poorly suited to organoids with conserved cytoarchitectures that are susceptible to plastic deformation. Here, we develop a platform, termed Spatially Patterned Organoid Transfer (SPOT), consisting of an iron-oxide nanoparticle laden hydrogel and magnetized 3D printer to enable the controlled lifting, transport, and deposition of organoids. We identify cellulose nanofibers as both an ideal biomaterial for encasing organoids with magnetic nanoparticles and a shear-thinning, self-healing support hydrogel for maintaining the spatial positioning of organoids to facilitate the generation of assembloids. We leverage SPOT to create precisely arranged assembloids composed of human pluripotent stem cell-derived neural organoids and patient-derived glioma organoids. In doing so, we demonstrate the potential for the SPOT platform to construct assembloids which recapitulate key developmental processes and disease etiologies.

  View details for DOI 10.1038/s41467-023-40006-5

  View details for PubMedID 37468483

  View details for PubMedCentralID PMC10356773

• Magnetized 3D Bioprinting To Construct Multi-Organoid Assembloids Cai, B., Roth, J. G., Brunel, L. G., Huang, M. S., Liu, Y., Pasca, S., Shin, S., Heilshorn, S. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001120356800251

• A Cross-Sectional Study of the Neuropsychiatric Phenotype of CACNA1C-Related Disorder Levy, R., Timothy, K., Underwood, J., Hall, J., Bernstein, J., Pasca, S. LIPPINCOTT WILLIAMS & WILKINS. 2023

  View details for DOI 10.1212/WNL.0000000000202194

  View details for Web of Science ID 001053672101052

• Single-cell transcriptomic landscape of the developing human spinal cord. Nature neuroscience Andersen, J., Thom, N., Shadrach, J. L., Chen, X., Onesto, M. M., Amin, N. D., Yoon, S. J., Li, L., Greenleaf, W. J., Müller, F., Pașca, A. M., Kaltschmidt, J. A., Pașca, S. P. 2023

  Abstract

  Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease.

  View details for DOI 10.1038/s41593-023-01311-w

  View details for PubMedID 37095394

  View details for PubMedCentralID 8353162

• Selection of rAAV vectors that cross the human blood-brain barrier and target the central nervous system using a transwell model MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Song, R., Pekrun, K., Khan, T. A., Zhang, F., Pasca, S. P., Kay, M. A. 2022; 27: 73-88

  Abstract

  A limitation for recombinant adeno-associated virus (rAAV)-mediated gene transfer into the central nervous system (CNS) is the low penetration of vectors across the human blood-brain barrier (BBB). High doses of intravenously delivered vector are required to reach the CNS, which has resulted in varying adverse effects. Moreover, selective transduction of various cell types might be important depending on the disorder being treated. To enhance BBB penetration and improve CNS cell selectivity, we screened an AAV capsid-shuffled library using an in vitro transwell BBB system with separate layers of human endothelial cells, primary astrocytes and/or human induced pluripotent stem cell-derived cortical neurons. After multiple passages through the transwell, we identified chimeric AAV capsids with enhanced penetration and improved transduction of astrocytes and/or neurons compared with wild-type capsids. We identified the amino acids (aa) from regions 451-470 of AAV2 associated with the capsids selected for neurons, and a combination of aa from regions 413-496 of AAV-rh10 and 538-598 of AAV3B/LK03 associated with capsids selected for astrocytes. A small interfering RNA screen identified several genes that affect transcytosis of AAV across the BBB. Our work supports the use of a human transwell system for selecting enhanced AAV capsids targeting the CNS and may allow for unraveling the underlying molecular mechanisms of BBB penetration.

  View details for DOI 10.1016/j.omtm.2022.09.002

  View details for Web of Science ID 000860483300005

  View details for PubMedID 36186955

  View details for PubMedCentralID PMC9494039

• What Have Organoids and Assembloids Taught Us About the Pathophysiology of Neuropsychiatric Disorders? Biological psychiatry Levy, R. J., Paşca, S. P. 2022

  Abstract

  Neuropsychiatric research has been impeded by limited access to human brain tissue, especially from early stages of neurodevelopment when the pathophysiology of many childhood-onset disorders is initiated. Neural organoids are 3-dimensional, self-organizing, multicellular structures generated from pluripotent stem cells that recapitulate some of the cell diversity, cytoarchitecture, and functional features of domains of the developing nervous system. Assembloids are 3-dimensional, self-organizing cultures created by the combination of two or more distinctly patterned organoids or an organoid plus additional cell or tissue type(s) that are used to model cell migration and connectivity. Here we review recent advances in neuropsychiatric disorder research using organoid and assembloid models to study the role of disease-relevant genes and mutations, as well as the impact of environmental risk factors on neural development. We also highlight some of the advantages and limitations of these model systems in bringing insights into the pathophysiology of neuropsychiatric disorders.

  View details for DOI 10.1016/j.biopsych.2022.11.017

  View details for PubMedID 36739210

• A Cross-Sectional Study of the Neuropsychiatric Phenotype of CACNA1C-Related Disorder. Pediatric neurology Levy, R. J., Timothy, K. W., Underwood, J. F., Hall, J., Bernstein, J. A., Pașca, S. P. 2022; 138: 101-106

  Abstract

  BACKGROUND: CACNA1C encodes the voltage-gated L-type calcium channel CaV1.2. A specific gain-of-function pathogenic variant in CACNA1C causes Timothy syndrome type 1 (TS1) with cardiac long QT syndrome, syndactyly, and neuropsychiatric symptoms. Our previous work found that the TS1 mutation alters neuronal activity-dependent signaling and interneuron migration. Recent case series highlighted a broader spectrum of CACNA1C-related disorder (CRD) that includes isolated cardiac disease, isolated neurologic deficits, and TS, but it is unknown how the clinical presentation of other CRD variants relates to neural defects. We surveyed individuals with CRD to define the neuropsychiatric and developmental phenotype in an effort to guide future research into the role of calcium channels in neural development.METHODS: Caregivers of and individuals with CRD completed an online survey of pre- and perinatal events, cardiac events, developmental milestones, neuropsychiatric symptoms, and neuropsychiatric diagnoses. Multiple Mann-Whitney tests were used for comparison of categorical values and Fisher exact test for comparison of categorical variables between participants with and without cardiac arrhythmia.RESULTS: Twenty-four participants with germline CACNA1C variants including TS1 completed the survey. The most common neuropsychiatric symptoms and/or diagnoses were developmental delay in 92%, incoordination in 71%, hypotonia in 67%, autism spectrum disorder in 50% (autistic features in 92%), seizures in 37.5%, and attention-deficit/hyperactivity disorder in 21% of participants. There were no significant differences in symptoms between participants with and without arrhythmia.CONCLUSIONS: In our CRD cohort, there was an increased prevalence of multiple neuropsychiatric symptoms compared with the general population. These findings indicate the key role of CaV1.2 in brain development and the clinical importance of screening and therapeutically addressing neuropsychiatric symptoms in all individuals with CRD. Future directions include deep phenotyping of neuropsychiatric symptoms and efforts to relate these symptoms to cellular defects.

  View details for DOI 10.1016/j.pediatrneurol.2022.10.013

  View details for PubMedID 36436328

• Stretchable mesh microelectronics for the biointegration and stimulation of human neural organoids. Biomaterials Li, T. L., Liu, Y., Forro, C., Yang, X., Beker, L., Bao, Z., Cui, B., Pașca, S. P. 2022; 290: 121825

  Abstract

  Advances in tridimensional (3D) culture approaches have led to the generation of organoids that recapitulate cellular and physiological features of domains of the human nervous system. Although microelectrodes have been developed for long-term electrophysiological interfaces with neural tissue, studies of long-term interfaces between microelectrodes and free-floating organoids remain limited. In this study, we report a stretchable, soft mesh electrode system that establishes an intimate in vitro electrical interface with human neurons in 3D organoids. Our mesh is constructed with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) based electrically conductive hydrogel electrode arrays and elastomeric poly(styrene-ethylene-butylene-styrene) (SEBS) as the substrate and encapsulation materials. This mesh electrode can maintain a stable electrochemical impedance in buffer solution under 50% compressive and 50% tensile strain. We have successfully cultured pluripotent stem cell-derived human cortical organoids (hCO) on this polymeric mesh for more than 3 months and demonstrated that organoids readily integrate with the mesh. Using simultaneous stimulation and calcium imaging, we show that electrical stimulation through the mesh can elicit intensity-dependent calcium signals comparable to stimulation from a bipolar stereotrode. This platform may serve as a tool for monitoring and modulating the electrical activity of in vitro models of neuropsychiatric diseases.

  View details for DOI 10.1016/j.biomaterials.2022.121825

  View details for PubMedID 36326509

• How collaboration between bioethicists and neuroscientists can advance research. Nature neuroscience Hyun, I., Scharf-Deering, J. C., Sullivan, S., Aach, J. D., Arlotta, P., Baum, M. L., Church, G. M., Goldenberg, A., Greely, H. T., Khoshakhlagh, P., Kohman, R. E., Lopes, M., Lowenthal, C., Lu, A., Ng, A. H., Pasca, S. P., Paulsen, B., Pigoni, M., Scott, C. T., Silbersweig, D. A., Skylar-Scott, M. A., Truog, R. D., Lunshof, J. E. 2022

  View details for DOI 10.1038/s41593-022-01187-2

  View details for PubMedID 36258039

• Human assembloids. Development (Cambridge, England) Kanton, S., Pasca, S. P. 2022; 149 (20)

  Abstract

  Deconstructing and then reconstructing developmental processes ex vivo is crucial to understanding how organs assemble and how physiology can be disrupted in disease. Human 3D stem cell-derived systems, such as organoids, have facilitated this pursuit; however, they often do not capture inter-tissue or inter-lineage cellular interactions that give rise to emergent tissue properties during development. Assembloids are self-organizing 3D cellular systems that result from the integration of multiple organoids or the combination of organoids with missing cell types or primary tissue explants. Here, we outline the concept and types of assembloids and present their applications for studying the nervous system and other tissues. We describe tools that are used to probe and manipulate assembloids and delineate current challenges and the potential for this new approach to interrogate development and disease.

  View details for DOI 10.1242/dev.201120

  View details for PubMedID 36317797

• Modulating miR-218 in Human Motor Neurons Using Assembloids Amin, N., Kulkarni, S., Pasca, S. WILEY. 2022: S168

  View details for Web of Science ID 000867884200294

• Mouse embryo models built from stem cells take shape in a dish. Nature Amin, N. D., Pașca, S. P. 2022; 610 (7930): 39-40

  View details for DOI 10.1038/d41586-022-03075-y

  View details for PubMedID 36192499

• Imaging neuronal migration and network activity in human forebrain assembloids. STAR protocols Birey, F., Pașca, S. P. 2022; 3 (3): 101478

  Abstract

  Assembloids generated from human pluripotent stem cells are self-organizing, multicellular in vitro models that recapitulate aspects of cell-cell interactions and circuit assembly during neural development. Here, we present protocols to functionally monitor, in forebrain assembloids, the migration of GABAergic interneurons from the ventral to the dorsal forebrain and the activity in early cortical networks. Specifically, we describe high-resolution imaging and analysis of neuronal migration as well as calcium imaging of network activity in forebrain assembloids. For complete details on the use and execution of this protocol, please refer to Birey et al. (2022).

  View details for DOI 10.1016/j.xpro.2022.101478

  View details for PubMedID 35769932

  View details for PubMedCentralID PMC9234084

• A tissue-like neurotransmitter sensor for the brain and gut. Nature Li, J., Liu, Y., Yuan, L., Zhang, B., Bishop, E. S., Wang, K., Tang, J., Zheng, Y., Xu, W., Niu, S., Beker, L., Li, T. L., Chen, G., Diyaolu, M., Thomas, A., Mottini, V., Tok, J. B., Dunn, J. C., Cui, B., Pașca, S. P., Cui, Y., Habtezion, A., Chen, X., Bao, Z. 2022; 606 (7912): 94-101

  Abstract

  Neurotransmitters play essential roles in regulating neural circuit dynamics both in the central nervous system as well as at the peripheral, including the gastrointestinal tract1-3. Their real-time monitoring will offer critical information for understanding neural function and diagnosing disease1-3. However, bioelectronic tools to monitor the dynamics of neurotransmitters in vivo, especially in the enteric nervous systems, are underdeveloped. This is mainly owing to the limited availability of biosensing tools that are capable of examining soft, complex and actively moving organs. Here we introduce a tissue-mimicking, stretchable, neurochemical biological interface termed NeuroString, which is prepared by laser patterning of a metal-complexed polyimide into an interconnected graphene/nanoparticle network embedded in an elastomer. NeuroString sensors allow chronic in vivo real-time, multichannel and multiplexed monoamine sensing in the brain of behaving mouse, as well as measuring serotonin dynamics in the gut without undesired stimulations and perturbing peristaltic movements. The described elastic and conformable biosensing interface has broad potential for studying the impact of neurotransmitters on gut microbes, brain-gut communication and may ultimately be extended to biomolecular sensing in other soft organs across the body.

  View details for DOI 10.1038/s41586-022-04615-2

  View details for PubMedID 35650358

• Mesh electrode arrays for integration with electrogenic organoids Forro, C., Li, T., Yang, X., Tsai, C., Cui, B., Pasca, S. CELL PRESS. 2022: 16

  View details for Web of Science ID 000759523000078

• Engineering brain assembloids to interrogate human neural circuits. Nature protocols Miura, Y., Li, M. Y., Revah, O., Yoon, S. J., Narazaki, G., Pașca, S. P. 2022

  Abstract

  The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.

  View details for DOI 10.1038/s41596-021-00632-z

  View details for PubMedID 34992269

• A CROSS-SECTIONAL STUDY OF THE NEUROPSYCHIATRIC PHENOTYPE OF CACNA1C-RELATED DISORDER Levy, R. J., Timothy, K., Bernstein, J., Pasca, S. BMJ PUBLISHING GROUP. 2022: 287-288

  View details for DOI 10.1136/jim-2022-WRMC.355

  View details for Web of Science ID 000737295900376

• Nanotechnology Enables Novel Modalities for Neuromodulation. Advanced materials (Deerfield Beach, Fla.) Yang, X., McGlynn, E., Das, R., Pasca, S. P., Cui, B., Heidari, H. 2021: e2103208

  Abstract

  Neuromodulation is of great importance both as a fundamental neuroscience research tool for analyzing and understanding the brain function, and as a therapeutic avenue for treating brain disorders. Here, an overview of conceptual and technical progress in developing neuromodulation strategies is provided, and it is suggested that recent advances in nanotechnology are enabling novel neuromodulation modalities with less invasiveness, improved biointerfaces, deeper penetration, and higher spatiotemporal precision. The use of nanotechnology and the employment of versatile nanomaterials and nanoscale devices with tailored physical properties have led to considerable research progress. To conclude, an outlook discussing current challenges and future directions for next-generation neuromodulation modalities is presented.

  View details for DOI 10.1002/adma.202103208

  View details for PubMedID 34668249

• Chromatin dynamics in human brain development and disease. Trends in cell biology Valencia, A. M., Pașca, S. P. 2021

  Abstract

  Chromatin-related genes are frequently mutated in neurodevelopmental disorders; yet, the mechanisms by which these perturbations disrupt brain assembly and function are not understood. Here, we describe how recent advances in transcriptional and chromatin profiling in combination with cellular models are beginning to inform our understanding of neurodevelopment and chromatinopathies.

  View details for DOI 10.1016/j.tcb.2021.09.001

  View details for PubMedID 34610892

• Advancing models of neural development with biomaterials. Nature reviews. Neuroscience Roth, J. G., Huang, M. S., Li, T. L., Feig, V. R., Jiang, Y., Cui, B., Greely, H. T., Bao, Z., Pasca, S. P., Heilshorn, S. C. 2021

  Abstract

  Human pluripotent stem cells have emerged as a promising in vitro model system for studying the brain. Two-dimensional and three-dimensional cell culture paradigms have provided valuable insights into the pathogenesis of neuropsychiatric disorders, but they remain limited in their capacity to model certain features of human neural development. Specifically, current models do not efficiently incorporate extracellular matrix-derived biochemical and biophysical cues, facilitate multicellular spatio-temporal patterning, or achieve advanced functional maturation. Engineered biomaterials have the capacity to create increasingly biomimetic neural microenvironments, yet further refinement is needed before these approaches are widely implemented. This Review therefore highlights how continued progression and increased integration of engineered biomaterials may be well poised to address intractable challenges in recapitulating human neural development.

  View details for DOI 10.1038/s41583-021-00496-y

  View details for PubMedID 34376834

• Scrutinizing disease states and regulation in human microglia. Nature genetics Kelley, K. W., Pașca, S. P. 2021

  View details for DOI 10.1038/s41588-021-00826-x

  View details for PubMedID 34083790

• Breaking Thru the Human Blood Brain Barrier: Discovering AAV Vectors Targeting the Central Nervous System Using a Transwell Model Song, R., Pekrun, K., Khan, T. A., Zhang, F., Pasca, S., Kay, M. A. CELL PRESS. 2021: 26-27

  View details for Web of Science ID 000645188700054

• Long-term maturation of human cortical organoids matches key early postnatal transitions. Nature neuroscience Gordon, A. n., Yoon, S. J., Tran, S. S., Makinson, C. D., Park, J. Y., Andersen, J. n., Valencia, A. M., Horvath, S. n., Xiao, X. n., Huguenard, J. R., Pașca, S. P., Geschwind, D. H. 2021

  Abstract

  Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.

  View details for DOI 10.1038/s41593-021-00802-y

  View details for PubMedID 33619405

• Mapping human brain organoids on a spatial atlas. Cell stem cell Miura, Y., Pașca, S. P. 2021; 28 (6): 983-984

  Abstract

  Brain organoids are tridimensional, self-organizing cultures derived from pluripotent stem cells that recapitulate aspects of human neurodevelopment and can be applied toward investigating neural disease and evolution. In this issue of Cell Stem Cell, Fleck et al. (2021) describe a computational platform for mapping cell identity in organoids.

  View details for DOI 10.1016/j.stem.2021.05.004

  View details for PubMedID 34087156

• The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C. Science translational medicine Pluvinage, J. V., Sun, J., Claes, C., Flynn, R. A., Haney, M. S., Iram, T., Meng, X., Lindemann, R., Riley, N. M., Danhash, E., Chadarevian, J. P., Tapp, E., Gate, D., Kondapavulur, S., Cobos, I., Chetty, S., Pașca, A. M., Pașca, S. P., Berry-Kravis, E., Bertozzi, C. R., Blurton-Jones, M., Wyss-Coray, T. 2021; 13 (622): eabg2919

  Abstract

  [Figure: see text].

  View details for DOI 10.1126/scitranslmed.abg2919

  View details for PubMedID 34851695

• Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution. Cell Trevino, A. E., Müller, F., Andersen, J., Sundaram, L., Kathiria, A., Shcherbina, A., Farh, K., Chang, H. Y., Pașca, A. M., Kundaje, A., Pașca, S. P., Greenleaf, W. J. 2021

  Abstract

  Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.

  View details for DOI 10.1016/j.cell.2021.07.039

  View details for PubMedID 34390642

• A matter of space and time: Emerging roles of disease-associated proteins in neural development. Neuron Panagiotakos, G., Pasca, S. P. 2021

  Abstract

  Recent genetic studies of neurodevelopmental disorders point to synaptic proteins and ion channels as key contributors to disease pathogenesis. Although many of these proteins, such as the L-type calcium channel Cav1.2 or the postsynaptic scaffolding protein SHANK3, have well-studied functions in mature neurons, new evidence indicates that they may subserve novel, distinct roles in immature cells as the nervous system is assembled in prenatal development. Emerging tools and technologies, including single-cell sequencing and human cellular models of disease, are illuminating differential isoform utilization, spatiotemporal expression, and subcellular localization of ion channels and synaptic proteins in the developing brain compared with the adult, providing new insights into the regulation of developmental processes. We propose that it is essential to consider the temporally distinct and cell-specific roles of these proteins during development and maturity in our framework for understanding neuropsychiatric disorders.

  View details for DOI 10.1016/j.neuron.2021.10.035

  View details for PubMedID 34847355

• Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome. Cell stem cell Birey, F., Li, M. Y., Gordon, A., Thete, M. V., Valencia, A. M., Revah, O., Paşca, A. M., Geschwind, D. H., Paşca, S. P. 2021

  Abstract

  Defects in interneuron migration can disrupt the assembly of cortical circuits and lead to neuropsychiatric disease. Using forebrain assembloids derived by integration of cortical and ventral forebrain organoids, we have previously discovered a cortical interneuron migration defect in Timothy syndrome (TS), a severe neurodevelopmental disease caused by a mutation in the L-type calcium channel (LTCC) Cav1.2. Here, we find that acute pharmacological modulation of Cav1.2 can regulate the saltation length, but not the frequency, of interneuron migration in TS. Interestingly, the defect in saltation length is related to aberrant actomyosin and myosin light chain (MLC) phosphorylation, while the defect in saltation frequency is driven by enhanced γ-aminobutyric acid (GABA) sensitivity and can be restored by GABA-A receptor antagonism. Finally, we describe hypersynchronous hCS network activity in TS that is exacerbated by interneuron migration. Taken together, these studies reveal a complex role of LTCC function in human cortical interneuron migration and strategies to restore deficits in the context of disease.

  View details for DOI 10.1016/j.stem.2021.11.011

  View details for PubMedID 34990580

• Primate cell fusion disentangles gene regulatory divergence in neurodevelopment. Nature Agoglia, R. M., Sun, D. n., Birey, F. n., Yoon, S. J., Miura, Y. n., Sabatini, K. n., Pașca, S. P., Fraser, H. B. 2021

  Abstract

  Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.

  View details for DOI 10.1038/s41586-021-03343-3

  View details for PubMedID 33731928

• Research and training in autism spectrum disorder to catalyze the next genomic and neuroscience revolutions. Molecular psychiatry Pasca, S. P., Veenstra-VanderWeele, J., McPartland, J. C. 2020

  View details for DOI 10.1038/s41380-020-0830-5

  View details for PubMedID 32601454

• Selection of Adeno-Associated Virus Vectors Targeting the Central Nervous System Usingan In Vitro Model of Human Blood-Brain Barrier Song, R., Pekrun, K., Khan, T. A., Zhang, F., Pasca, S., Kay, M. A. CELL PRESS. 2020: 75

  View details for Web of Science ID 000530089300144

• Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals. Science (New York, N.Y.) Liu, J. n., Kim, Y. S., Richardson, C. E., Tom, A. n., Ramakrishnan, C. n., Birey, F. n., Katsumata, T. n., Chen, S. n., Wang, C. n., Wang, X. n., Joubert, L. M., Jiang, Y. n., Wang, H. n., Fenno, L. E., Tok, J. B., Pașca, S. P., Shen, K. n., Bao, Z. n., Deisseroth, K. n. 2020; 367 (6484): 1372–76

  Abstract

  The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

  View details for DOI 10.1126/science.aay4866

  View details for PubMedID 32193327

• Aberrant calcium channel splicing drives defects in cortical differentiation in Timothy Syndrome. eLife Panagiotakos, G., Haveles, C., Arjun, A., Petrova, R., Rana, A., Portmann, T., Pasca, S. P., Palmer, T. D., Dolmetsch, R. E. 2019; 8

  Abstract

  The syndromic autism spectrum disorder (ASD) Timothy Syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally-regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.

  View details for DOI 10.7554/eLife.51037

  View details for PubMedID 31868578

• Engineering a Genetically Encoded Magnetic Protein Crystal. Nano letters Li, T. L., Wang, Z., You, H., Ong, Q., Varanasi, V. J., Dong, M., Lu, B., Pasca, S. P., Cui, B. 2019

  Abstract

  Magnetogenetics is a new field that leverages genetically encoded proteins and protein assemblies that are sensitive to magnetic fields to study and manipulate cell behavior. Theoretical studies show that many proposed magnetogenetic proteins do not contain enough iron to generate substantial magnetic forces. Here, we have engineered a genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. Each of these crystals contains more than 10 million ferritin subunits and is capable of mineralizing substantial amounts of iron. When isolated from cells and loaded with iron in vitro, these crystals generate magnetic forces that are 9 orders of magnitude larger than the forces fromthe single ferritin cages used in previous studies. These protein crystals are attracted to an applied magnetic field and move toward magnets even when internalized into cells. While additional studies are needed to realize the full potential of magnetogenetics, these results demonstrate the feasibility of engineering protein assemblies for magnetic sensing.

  View details for DOI 10.1021/acs.nanolett.9b02266

  View details for PubMedID 31552740

• Engineered materials for organoid systems. Nature reviews. Materials Kratochvil, M. J., Seymour, A. J., Li, T. L., Paşca, S. P., Kuo, C. J., Heilshorn, S. C. 2019; 4 (9): 606-622

  Abstract

  Organoids are 3D cell culture systems that mimic some of the structural and functional characteristics of an organ. Organoid cultures provide the opportunity to study organ-level biology in models that mimic human physiology more closely than 2D cell culture systems or non-primate animal models. Many organoid cultures rely on decellularized extracellular matrices as scaffolds, which are often poorly chemically defined and allow only limited tunability and reproducibility. By contrast, the biochemical and biophysical properties of engineered matrices can be tuned and optimized to support the development and maturation of organoid cultures. In this Review, we highlight how key cell-matrix interactions guiding stem-cell decisions can inform the design of biomaterials for the reproducible generation and control of organoid cultures. We survey natural, synthetic and protein-engineered hydrogels for their applicability to different organoid systems and discuss biochemical and mechanical material properties relevant for organoid formation. Finally, dynamic and cell-responsive material systems are investigated for their future use in organoid research.

  View details for DOI 10.1038/s41578-019-0129-9

  View details for PubMedID 33552558

  View details for PubMedCentralID PMC7864216

• Cell diversity in the human cerebral cortex: from the embryo to brain organoids. Current opinion in neurobiology Arlotta, P. n., Paşca, S. P. 2019; 56: 194–98

  Abstract

  The development and wiring of the central nervous system is a remarkable biological process that starts with the generation of and interaction between a large diversity of cell types. Our understanding of the developmental logic that drives cellular diversification in the mammalian brain comes, to a large extent, from studies in rodents. However, identifying the unique cellular processes underlying primate corticogenesis has been slow, due to the challenges associated with directly observing and manipulating brain tissue from these species. Recent technological advances in two areas hold promise to accelerate discovery of the mechanisms that govern human brain development, evolution, and pathophysiology of disease. Molecular profiling of large numbers of single cells can now capture cell identity and cell states within a complex tissue. Furthermore, modeling aspects of human organogenesis in vitro, even for tissues as complex as the brain, has been advanced by the use of three-dimensional organoid systems. Here, we describe how these approaches have been applied to date and how they promise to uncover the principles of cell diversification in the developing human brain.

  View details for PubMedID 31051421

• Organoid and Assembloid Technologies for Investigating Cellular Crosstalk in Human Brain Development and Disease. Trends in cell biology Marton, R. M., Pașca, S. P. 2019

  Abstract

  The biology of the human brain, and in particular the dynamic interactions between the numerous cell types and regions of the central nervous system, has been difficult to study due to limited access to functional brain tissue. Technologies to derive brain organoids and assembloids from human pluripotent stem cells are increasingly utilized to model, in progressively complex preparations, the crosstalk between cell types in development and disease. Here, we review the use of these human cellular models to study cell-cell interactions among progenitors, neurons, astrocytes, oligodendrocytes, cancer cells, and non-central nervous system cell types, as well as efforts to study connectivity between brain regions following controlled assembly of organoids. Ultimately, the promise of these patient-derived preparations is to uncover previously inaccessible features of brain function that emerge from complex cell-cell interactions and to improve our mechanistic understanding of neuropsychiatric disorders.

  View details for DOI 10.1016/j.tcb.2019.11.004

  View details for PubMedID 31879153

• Reliability of human cortical organoid generation NATURE METHODS Yoon, S., Elahi, L. S., Pasca, A. M., Marton, R. M., Gordon, A., Revah, O., Miura, Y., Walczak, E. M., Holdgate, G. M., Fan, H., Huguenard, J. R., Geschwind, D. H., Pasca, S. P. 2019; 16 (1): 75-+

  View details for DOI 10.1038/s41592-018-0255-0

  View details for Web of Science ID 000454162400033

• A framework for the investigation of rare genetic disorders in neuropsychiatry. Nature medicine Sanders, S. J., Sahin, M. n., Hostyk, J. n., Thurm, A. n., Jacquemont, S. n., Avillach, P. n., Douard, E. n., Martin, C. L., Modi, M. E., Moreno-De-Luca, A. n., Raznahan, A. n., Anticevic, A. n., Dolmetsch, R. n., Feng, G. n., Geschwind, D. H., Glahn, D. C., Goldstein, D. B., Ledbetter, D. H., Mulle, J. G., Pasca, S. P., Samaco, R. n., Sebat, J. n., Pariser, A. n., Lehner, T. n., Gur, R. E., Bearden, C. E. 2019

  Abstract

  De novo and inherited rare genetic disorders (RGDs) are a major cause of human morbidity, frequently involving neuropsychiatric symptoms. Recent advances in genomic technologies and data sharing have revolutionized the identification and diagnosis of RGDs, presenting an opportunity to elucidate the mechanisms underlying neuropsychiatric disorders by investigating the pathophysiology of high-penetrance genetic risk factors. Here we seek out the best path forward for achieving these goals. We think future research will require consistent approaches across multiple RGDs and developmental stages, involving both the characterization of shared neuropsychiatric dimensions in humans and the identification of neurobiological commonalities in model systems. A coordinated and concerted effort across patients, families, researchers, clinicians and institutions, including rapid and broad sharing of data, is now needed to translate these discoveries into urgently needed therapies.

  View details for DOI 10.1038/s41591-019-0581-5

  View details for PubMedID 31548702

• The hidden biology of the human brain. Nature medicine Pașca, S. P. 2019

  View details for DOI 10.1038/s41591-019-0621-1

  View details for PubMedID 31700168

• Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures. Nature neuroscience Marton, R. M., Miura, Y. n., Sloan, S. A., Li, Q. n., Revah, O. n., Levy, R. J., Huguenard, J. R., Pașca, S. P. 2019

  Abstract

  Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.

  View details for PubMedID 30692691

• Polarizing brain organoids. Nature biotechnology Miura, Y. n., Pașca, S. P. 2019

  View details for PubMedID 30936565

• Loss of Adaptive Myelination Contributes to Methotrexate Chemotherapy-Related Cognitive Impairment. Neuron Geraghty, A. C., Gibson, E. M., Ghanem, R. A., Greene, J. J., Ocampo, A. n., Goldstein, A. K., Ni, L. n., Yang, T. n., Marton, R. M., Paşca, S. P., Greenberg, M. E., Longo, F. M., Monje, M. n. 2019

  Abstract

  Activity-dependent myelination is thought to contribute to adaptive neurological function. However, the mechanisms by which activity regulates myelination and the extent to which myelin plasticity contributes to non-motor cognitive functions remain incompletely understood. Using a mouse model of chemotherapy-related cognitive impairment (CRCI), we recently demonstrated that methotrexate (MTX) chemotherapy induces complex glial dysfunction for which microglial activation is central. Here, we demonstrate that remote MTX exposure blocks activity-regulated myelination. MTX decreases cortical Bdnf expression, which is restored by microglial depletion. Bdnf-TrkB signaling is a required component of activity-dependent myelination. Oligodendrocyte precursor cell (OPC)-specific TrkB deletion in chemotherapy-naive mice results in impaired cognitive behavioral performance. A small-molecule TrkB agonist rescues both myelination and cognitive impairment after MTX chemotherapy. This rescue after MTX depends on intact TrkB expression in OPCs. Taken together, these findings demonstrate a molecular mechanism required for adaptive myelination that is aberrant in CRCI due to microglial activation.

  View details for DOI 10.1016/j.neuron.2019.04.032

  View details for PubMedID 31122677

• Inhibitory Interneurons in Hemimegalencephaly: A Survey of 9 Cases Lummus, S., Andersen, J., Pasca, S., Kleinschmidt-DeMasters, B., Vogel, H. OXFORD UNIV PRESS INC. 2018: 501

  View details for Web of Science ID 000434064400092

• A human cellular model of amyotrophic lateral sclerosis NATURE MEDICINE Marton, R. M., Pasca, S. P. 2018; 24 (3): 256–57

  View details for PubMedID 29509753

• The ethics of experimenting with human brain tissue. Nature Farahany, N. A., Greely, H. T., Hyman, S. n., Koch, C. n., Grady, C. n., Pașca, S. P., Sestan, N. n., Arlotta, P. n., Bernat, J. L., Ting, J. n., Lunshof, J. E., Iyer, E. P., Hyun, I. n., Capestany, B. H., Church, G. M., Huang, H. n., Song, H. n. 2018; 556 (7702): 429–32

  View details for DOI 10.1038/d41586-018-04813-x

  View details for PubMedID 29691509

• Absent forebrain replaced by embryonic stem cells. Nature Andersen, J. n., Pașca, S. P. 2018; 563 (7729): 44–45

  View details for DOI 10.1038/d41586-018-06933-w

  View details for PubMedID 30375499

• Building three-dimensional human brain organoids. Nature neuroscience 2018

  View details for PubMedID 29593345

• The Zika threat to the periphery. Nature neuroscience Khan, T. A., Paşca, S. P. 2017; 20 (9): 1191-1192

  View details for DOI 10.1038/nn.4633

  View details for PubMedID 28849792

• Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring. Proceedings of the National Academy of Sciences of the United States of America Cao, Y., Hjort, M., Chen, H., Birey, F., Leal-Ortiz, S. A., Han, C. M., Santiago, J. G., Pasca, S. P., Wu, J. C., Melosh, N. A. 2017

  Abstract

  Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

  View details for DOI 10.1073/pnas.1615375114

  View details for PubMedID 28223521

• MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis. Cell reports Madelaine, R. n., Sloan, S. A., Huber, N. n., Notwell, J. H., Leung, L. C., Skariah, G. n., Halluin, C. n., Paşca, S. P., Bejerano, G. n., Krasnow, M. A., Barres, B. A., Mourrain, P. n. 2017; 20 (7): 1533–42

  Abstract

  In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.

  View details for PubMedID 28813666

• Neural Differentiation in the Third Dimension: Generating a Human Midbrain. Cell stem cell Marton, R. M., Pasca, S. P. 2016; 19 (2): 145-146

  Abstract

  In recent years, technological improvements in three-dimensional (3D) culture systems have enabled the generation of organoids or spheroids representing a variety of tissues, including the brain. In this issue of Cell Stem Cell, Jo et al. (2016) describe a 3D culture model of the human midbrain containing dopaminergic neurons and neuromelanin.

  View details for DOI 10.1016/j.stem.2016.07.017

  View details for PubMedID 27494668

• Personalized Human Cortical Spheroids. American journal of psychiatry Pasca, S. P. 2016; 173 (4): 332-333

  View details for DOI 10.1176/appi.ajp.2016.16020133

  View details for PubMedID 27035533

• Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature biotechnology Deverman, B. E., Pravdo, P. L., Simpson, B. P., Kumar, S. R., Chan, K. Y., Banerjee, A., Wu, W., Yang, B., Huber, N., Pasca, S. P., Gradinaru, V. 2016; 34 (2): 204-209

  Abstract

  Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.

  View details for DOI 10.1038/nbt.3440

  View details for PubMedID 26829320

• A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients. eLife Sun, Y., Pasca, S. P., Portmann, T., Goold, C., Worringer, K. A., Guan, W., Chan, K. C., Gai, H., Vogt, D., Chen, Y. J., Mao, R., Chan, K., Rubenstein, J. L., Madison, D. V., Hallmayer, J., Froehlich-Santino, W. M., Bernstein, J. A., Dolmetsch, R. E. 2016; 5

  Abstract

  Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.

  View details for DOI 10.7554/eLife.13073

  View details for PubMedID 27458797

• Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature Biotechnology Deverman, B. E., Pravdo, P. L., Simpson, B. P., Kumar, S. R., Chan, K. Y., Banerjee, A., Wu, W., Yang, B., Huber, N., Pasca, S., Gradinaru, V. 2016; 34 (2): 204-9

  View details for DOI 10.1038/nbt.3440

• Generating human neurons in vitro and using them to understand neuropsychiatric disease. Annual review of neuroscience Pasca, S. P., Panagiotakos, G., Dolmetsch, R. E. 2014; 37: 479-501

  Abstract

  Recent advances in cell reprogramming enable investigators to generate pluripotent stem cells from somatic cells. These induced pluripotent cells can subsequently be differentiated into any cell type, making it possible for the first time to obtain functional human neurons in the lab from control subjects and patients with psychiatric disorders. In this review, we survey the progress made in generating various neuronal subtypes in vitro, with special emphasis on the characterization of these neurons and the identification of unique features of human brain development in a dish. We also discuss efforts to uncover neuronal phenotypes from patients with psychiatric disease and prospects for the use of this platform for drug development.

  View details for DOI 10.1146/annurev-neuro-062012-170328

  View details for PubMedID 25002278

• Alteration in basal and depolarization induced transcriptional network in iPSC derived neurons from Timothy syndrome. Genome medicine Tian, Y., Voineagu, I., Pasca, S. P., Won, H., Chandran, V., Horvath, S., Dolmetsch, R. E., Geschwind, D. H. 2014; 6 (10): 75-?

  Abstract

  Common genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2.To identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calcium-dependent co-regulation.We identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca(2+) signaling in TS patients leads to the observed molecular dysregulation.We applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca(2+) signaling dysregulation on transcription.

  View details for DOI 10.1186/s13073-014-0075-5

  View details for PubMedID 25360157

  View details for PubMedCentralID PMC4213483

• Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons NATURE NEUROSCIENCE Krey, J. F., Pasca, S. P., Shcheglovitov, A., Yazawa, M., Schwemberger, R., Rasmusson, R., Dolmetsch, R. E. 2013; 16 (2): 201-209

  Abstract

  L-type voltage gated calcium channels have an important role in neuronal development by promoting dendritic growth and arborization. A point mutation in the gene encoding Ca(V)1.2 causes Timothy syndrome, a neurodevelopmental disorder associated with autism spectrum disorders (ASDs). We report that channels with the Timothy syndrome alteration cause activity-dependent dendrite retraction in rat and mouse neurons and in induced pluripotent stem cell (iPSC)-derived neurons from individuals with Timothy syndrome. Dendrite retraction was independent of calcium permeation through the mutant channel, was associated with ectopic activation of RhoA and was inhibited by overexpression of the channel-associated GTPase Gem. These results suggest that Ca(V)1.2 can activate RhoA signaling independently of Ca(2+) and provide insights into the cellular basis of Timothy syndrome and other ASDs.

  View details for DOI 10.1038/nn.3307

  View details for Web of Science ID 000314260200017

  View details for PubMedCentralID PMC3568452

• A promoter in the coding region of the calcium channel gene CACNA1C generates the transcription factor CCAT. PloS one Gomez-Ospina, N., Panagiotakos, G., Portmann, T., Pasca, S. P., Rabah, D., Budzillo, A., Kinet, J. P., Dolmetsch, R. E. 2013; 8 (4)

  Abstract

  The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3' end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.

  View details for DOI 10.1371/journal.pone.0060526

  View details for PubMedID 23613729

  View details for PubMedCentralID PMC3628902

• Motor abnormalities as a putative endophenotype for Autism Spectrum Disorders. Frontiers in integrative neuroscience Esposito, G., Pasca, S. P. 2013; 7: 43-?

  Abstract

  Autism Spectrum Disorders (ASDs) represent a complex group of behaviorally defined conditions with core deficits in social communication and the presence of repetitive and restrictive behaviors. To date, neuropathological studies have failed to identify pathognomonic cellular features for ASDs and there remains a fundamental disconnection between the complex clinical aspects of ASDs and the underlying neurobiology. Although not listed among the core diagnostic domains of impairment in ASDs, motor abnormalities have been consistently reported across the spectrum. In this perspective article, we summarize the evidence that supports the use of motor abnormalities as a putative endophenotype for ASDs. We argue that because these motor abnormalities do not directly depend on social or linguistic development, they may serve as an early disease indicator. Furthermore, we propose that stratifying patients based on motor development could be useful not only as an outcome predictor and in identifying more specific treatments for different ASDs categories, but also in exposing neurobiological mechanisms.

  View details for DOI 10.3389/fnint.2013.00043

  View details for PubMedID 23781177

• Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome NATURE MEDICINE Pasca, S. P., Portmann, T., Voineagu, I., Yazawa, M., Shcheglovitov, A., Pasca, A. M., Cord, B., Palmer, T. D., Chikahisa, S., Nishino, S., Bernstein, J. A., Hallmayer, J., Geschwind, D. H., Dolmetsch, R. E. 2011; 17 (12): 1657-U176

  Abstract

  Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Ca(v)1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca(2+)) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker. These findings provide strong evidence that Ca(v)1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.

  View details for DOI 10.1038/nm.2576

  View details for PubMedID 22120178

• Surround modulation of neuronal responses in V1 is as stable over time as responses to direct stimulation of receptive fields CORTEX Pasca, S. P., Singer, W., Nikolic, D. 2010; 46 (9): 1199-1203

  Abstract

  In the primary visual cortex (V1) the modulation of neuronal responses by surround stimuli displays considerable variability. At present, it is not known whether this variability across neurons is due to temporal instability or to neuron-specific differences. We explored this question in the cat visual cortex by making multi-channel recordings while repeatedly presenting surround gratings of collinear and orthogonal orientation to the centre stimulus for a period of 96 h. Our results indicate that surround modulation is temporally stable to about the same degree as the responses evoked by the centre stimuli. The results support the notion that the mechanisms of surround modulation exhibit a high degree of stability and play an important role in the modulation of cortical responses.

  View details for DOI 10.1016/j.cortex.2010.05.003

  View details for Web of Science ID 000282561600014

  View details for PubMedID 20557882

• Paraoxonase 1 activities and polymorphisms in autism spectrum disorders JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Pasca, S. P., Dronca, E., Nemes, B., Kaucsar, T., Endreffy, E., Iftene, F., Benga, I., Cornean, R., Dronca, M. 2010; 14 (3): 600-607

  Abstract

  Autism spectrum disorders (ASD) comprise a complex and heterogeneous group of conditions of unknown aetiology, characterized by significant disturbances in social, communicative and behavioural functioning. Recent studies suggested a possible implication of the high-density lipoprotein associated esterase/lactonase paraoxonase 1 (PON1) in ASD. In the present study, we aimed at investigating the PON1 status in a group of 50 children with ASD as compared to healthy age and sex matched control participants. We evaluated PON1 bioavailability (i.e. arylesterase activity) and catalytic activity (i.e. paraoxonase activity) in plasma using spectrophotometric methods and the two common polymorphisms in the PON1 coding region (Q192R, L55M) by employing Light Cycler real-time PCR. We found that both PON1 arylesterase and PON1 paraoxonase activities were decreased in autistic patients (respectively, P < 0.001, P < 0.05), but no association with less active variants of the PON1 gene was found. The PON1 phenotype, inferred from the two-dimensional enzyme analysis, had a similar distribution in the ASD group and the control group. In conclusion, both the bioavailability and the catalytic activity of PON1 are impaired in ASD, despite no association with the Q192R and L55M polymorphisms in the PON1 gene and a normal distribution of the PON1 phenotype.

  View details for DOI 10.1111/j.1582-4934.2008.00414.x

  View details for Web of Science ID 000276950100011

  View details for PubMedID 18624774

• One carbon metabolism disturbances and the C677T MTHFR gene polymorphism in children with autism spectrum disorders JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Pasca, S. P., Dronca, E., Kaucsar, T., Craciun, E. C., Endreffy, E., Ferencz, B. K., Iftene, F., Benga, I., Cornean, R., Banerjee, R., Dronca, M. 2009; 13 (10): 4229-4238

  Abstract

  Autism spectrum disorders (ASDs), which include the prototypic autistic disorder (AD), Asperger's syndrome (AS) and pervasive developmental disorders not otherwise specified (PDD-NOS), are complex neurodevelopmental conditions of unknown aetiology. The current study investigated the metabolites in the methionine cycle, the transsulphuration pathway, folate, vitamin B(12) and the C677T polymorphism of the MTHFR gene in three groups of children diagnosed with AD (n= 15), AS (n= 5) and PDD-NOS (n= 19) and their age- and sex-matched controls (n= 25). No metabolic disturbances were seen in the AS patients, while in the AD and PDD-NOS groups, lower plasma levels of methionine (P= 0.01 and P= 0.03, respectively) and alpha-aminobutyrate were observed (P= 0.01 and P= 0.001, respectively). Only in the AD group, plasma cysteine (P= 0.02) and total blood glutathione (P= 0.02) were found to be reduced. Although there was a trend towards lower levels of serine, glycine, N, N-dimethylglycine in AD patients, the plasma levels of these metabolites as well as the levels of homocysteine and cystathionine were not statistically different in any of the ASDs groups. The serum levels of vitamin B(12) and folate were in the normal range. The results of the MTHFR gene analysis showed a normal distribution of the C677T polymorphism in children with ASDs, but the frequency of the 677T allele was slightly more prevalent in AD patients. Our study indicates a possible role for the alterations in one carbon metabolism in the pathophysiology of ASDs and provides, for the first time, preliminary evidence for metabolic and genetic differences between clinical subtypes of ASDs.

  View details for DOI 10.1111/j.1582-4934.2008.00463.x

  View details for Web of Science ID 000274181900015

  View details for PubMedID 19267885

• Vomiting is not an adaption for glaucoma (and Darwinian medicine is difficult) MEDICAL HYPOTHESES Pasca, S. P., Nesse, R. M. 2008; 71 (3): 472-473

  View details for DOI 10.1016/j.mehy.2008.04.009

  View details for Web of Science ID 000258641800040

  View details for PubMedID 18513879

• Serum paraoxonase 1 activities and homocysteinemia in hemodialysis patients CLINICAL CHEMISTRY AND LABORATORY MEDICINE Dronca, M., Pasca, S. P., Nemes, B., Vlase, L., Vladutiu, D. 2008; 46 (6): 880-881

  View details for DOI 10.1515/CCLM.2008.164

  View details for Web of Science ID 000257542800023

  View details for PubMedID 18601616

• Behavioral effects of corpus callosum transection and environmental enrichment in adult rats BEHAVIOURAL BRAIN RESEARCH Miu, A. C., Heilman, R. M., Pasca, S. P., Stefan, C. A., Spanu, F., Vasiu, R., Olteanu, A. I., Miclea, M. 2006; 172 (1): 135-144

  Abstract

  A common assumption about the corpus callosum transection (CCX) is that it only affects behaviors heavily relying on interhemispheric communication. However, cerebral laterality is ubiquitous across motor and perceptual, cognitive and emotional domains, and the corpus callosum is important for its establishment. Several recent studies showed that the partial denervation of the sensorimotor isocortex through CCX derepressed neural growth processes that were sensitive to motor demand (experience-dependent neural plasticity). We investigated whether the facilitatory effects of CCX on cortical neural plasticity, shaped by differential housing, extended beyond the motor domain. Adult rats were housed in enriched (EE), standard (SE) or impoverished environments (IE) for 10 weeks, that is, 2 weeks before they underwent CCX or sham surgery, and, then, 8 weeks throughout the experiments. After they recovered from surgery, the behavioral performance of rats was tested using open-field, spontaneous alternation in the T-maze, paw preference, Morris water maze, and tone fear conditioning. The results indicated that the effects of CCX and housing on open-field behavior were independent, with CCX increasing the time spent in the center of the field at the beginning of the observation (i.e., emotionality), and EE and IE increasing rearing (emotionality) and reducing teeth-chattering (habituation), respectively. CCX reduced the frequency of spontaneous alternation, denoting spatial working memory deficits, while housing did not influence this performance. Neither CCX, nor housing significantly affected paw preference lateralization, although CCX was associated with a leftward bias in paw preference. In the Morris water maze, housing had effects on spatial acquisition, while CCX reduced activity, without interfering with spatial memory. CCX did not influence tone fear conditioning, but context fear conditioning seemed to benefit from EE. We conclude that CCX in adult rats has subtle, but specific behavioral effects pertaining to emotionality, spatial working memory, and, possibly, aversively motivated exploration, and these effects are either independent or only peripherally interact with the effects of housing.

  View details for DOI 10.1016/j.bbr.2006.05.007

  View details for Web of Science ID 000239683800016

  View details for PubMedID 16764947

• High levels of homocysteine and low serum paraoxonase 1 arylesterase activity in children with autism LIFE SCIENCES Pasca, S. P., Nemes, B., Vlase, L., Gagyi, C. E., Dronca, E., Miu, A. C., Dronca, M. 2006; 78 (19): 2244-2248

  Abstract

  Autism is a behaviorally defined disorder of unknown etiology that is thought to be influenced by genetic and environmental factors. High levels of homocysteine and oxidative stress are generally associated with neuropsychiatric disorders. The purpose of this study was to compare the level of homocysteine and other biomarkers in children with autism to corresponding values in age-matched healthy children. We measured total homocysteine (tHcy), vitamin B(12), paraoxonase and arylesterase activities of human paraoxonase 1 (PON1) in plasma and glutathione peroxidase (GPx) activity in erythrocytes from 21 children: 12 with autism (age: 8.29 +/- 2.76 years) and 9 controls (age: 8.33 +/- 1.82 years). We found statistically significant differences in tHcy levels and in arylesterase activity of PON1 in children with autism compared to the control group: 9.83 +/- 2.75 vs. 7.51 +/- 0.93 micromol/L (P < or =0.01) and 72.57 +/- 11.73 vs. 81.83 +/- 7.39 kU/L (P < or =0.005). In the autistic group there was a strong negative correlation between tHcy and GPx activity and the vitamin B(12) level was low or suboptimal. In conclusion, our study shows that in children with autism there are higher levels of tHcy, which is negatively correlated with GPx activity, low PON1 arylesterase activity and suboptimal levels of vitamin B(12).

  View details for DOI 10.1016/j.lfs.2005.09.040

  View details for Web of Science ID 000236718800012

  View details for PubMedID 16297937