Marius Wernig

All Publications

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

  Abstract

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

  View details for DOI 10.1038/nmeth.4291

  View details for PubMedID 28504679

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

  Abstract

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

  View details for DOI 10.1038/nbt.3835

  View details for Web of Science ID 000400809800019

  View details for PubMedID 28398344

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

  Abstract

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

  View details for DOI 10.1038/nature21722

  View details for Web of Science ID 000398897900040

  View details for PubMedID 28379941

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

  Abstract

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

  View details for DOI 10.1038/srep44840

  View details for Web of Science ID 000396983300001

  View details for PubMedID 28327614

  View details for PubMedCentralID PMC5361100

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2016.11.018

  View details for PubMedID 28089908

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

  Abstract

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

  View details for DOI 10.1016/j.cell.2016.12.044

  View details for PubMedID 28111074

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

  Abstract

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

  View details for DOI 10.1073/pnas.1607079113

  View details for Web of Science ID 000380346200057

  View details for PubMedID 27402759

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

  Abstract

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

  View details for DOI 10.1038/nature18323

  View details for Web of Science ID 000377856800037

  View details for PubMedID 27281220

  View details for PubMedCentralID PMC4928860

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

  Abstract

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

  View details for DOI 10.1126/science.aaf2669

  View details for Web of Science ID 000375417100028

  View details for PubMedID 26966193

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

  Abstract

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

  View details for DOI 10.1084/jem.20150951

  View details for PubMedID 27001749

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

  Abstract

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

  View details for DOI 10.1038/ncomms10862

  View details for Web of Science ID 000372218700001

  View details for PubMedID 26983594

  View details for PubMedCentralID PMC4800432

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

  Abstract

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

  View details for DOI 10.1038/mp.2015.20

  View details for PubMedID 25778475

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

  View details for DOI 10.1038/nature15749

  View details for Web of Science ID 000366053300032

  View details for PubMedID 26659182

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

  Abstract

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

  View details for DOI 10.1016/j.ceb.2015.10.001

  View details for Web of Science ID 000367112800010

  View details for PubMedID 26520682

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

  View details for DOI 10.1098/rstb.2014.0368

  View details for PubMedID 26416679

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

  View details for DOI 10.1038/nature15515

  View details for Web of Science ID 000361599900041

  View details for PubMedID 26399828

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2015.07.017

  View details for PubMedID 26279266

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

  Abstract

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

  View details for DOI 10.1172/JCI78612

  View details for PubMedID 26280581

  View details for PubMedCentralID PMC4588304

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2015.07.017

  View details for Web of Science ID 000364011000010

  View details for PubMedCentralID PMC4560990

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

  Abstract

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

  View details for DOI 10.1172/JCI78612

  View details for Web of Science ID 000362303600030

  View details for PubMedCentralID PMC4588304

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

  Abstract

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

  View details for DOI 10.1038/nature14274

  View details for Web of Science ID 000354816500056

  View details for PubMedID 25830878

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2015.01.015

  View details for PubMedID 25748935

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2014.10.019

  View details for PubMedID 25467916

  View details for PubMedCentralID PMC4389904

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2014.09.019

  View details for PubMedID 25456834

  View details for PubMedCentralID PMC4278749

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

  Abstract

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

  View details for DOI 10.1126/scitranslmed.3009540

  View details for Web of Science ID 000345595200003

  View details for PubMedCentralID PMC4428910

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

  View details for DOI 10.1002/cne.23620

  View details for Web of Science ID 000337791000014

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

  Abstract

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

  View details for DOI 10.1016/j.stemcr.2014.05.020

  View details for Web of Science ID 000340882400008

  View details for PubMedCentralID PMC4176533

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

  Abstract

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

  View details for DOI 10.1016/j.neuron.2014.02.015

  View details for PubMedID 24698271

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

  View details for DOI 10.1038/nm.3425

  View details for PubMedID 24309657

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

  Abstract

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

  View details for DOI 10.1016/j.cell.2013.09.028

  View details for Web of Science ID 000326571800016

  View details for PubMedID 24243019

  View details for PubMedCentralID PMC3871197

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

  Abstract

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

  View details for DOI 10.1073/pnas.1316240110

  View details for PubMedID 24046374

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

  Abstract

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

  View details for DOI 10.1016/j.celrep.2013.06.035

  View details for PubMedID 23891001

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

  Abstract

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

  View details for DOI 10.1016/j.jneumeth.2013.03.020

  View details for Web of Science ID 000321168200004

  View details for PubMedCentralID PMC4009399

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

  Abstract

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

  View details for DOI 10.1016/j.neuron.2013.05.029

  View details for PubMedID 23764284

  View details for PubMedCentralID PMC3751803

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

  View details for DOI 10.1038/nbt.2564

  View details for PubMedID 23584610

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

  View details for DOI 10.1038/nmeth.2325

  View details for Web of Science ID 000312810100033

  View details for PubMedID 23269377

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

  Abstract

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

  View details for DOI 10.1016/j.gde.2012.07.002

  View details for Web of Science ID 000311470900017

  View details for PubMedID 22868177

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

  Abstract

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

  View details for DOI 10.1016/j.molcel.2012.09.008

  View details for Web of Science ID 000309312900003

  View details for PubMedID 23020854

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

  Abstract

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

  View details for DOI 10.1073/pnas.1121003109

  View details for Web of Science ID 000300489200073

  View details for PubMedID 22308465

  View details for PubMedCentralID PMC3289376

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

  Abstract

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

  View details for DOI 10.1038/nprot.2011.430

  View details for Web of Science ID 000299108900011

  View details for PubMedID 22193304

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2011.11.015

  View details for Web of Science ID 000297895000012

  View details for PubMedID 22136927

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

  Abstract

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

  View details for DOI 10.1523/JNEUROSCI.4218-11.2011

  View details for Web of Science ID 000296799700005

  View details for PubMedID 22072658

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

  Abstract

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

  View details for DOI 10.1002/stem.718

  View details for Web of Science ID 000296565500009

  View details for PubMedID 21898685

  View details for PubMedCentralID PMC3285772

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2011.09.002

  View details for Web of Science ID 000296041200015

  View details for PubMedID 21962918

  View details for PubMedCentralID PMC3218088

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

  Abstract

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

  View details for DOI 10.1038/nbt.1946

  View details for Web of Science ID 000296273000016

  View details for PubMedID 21997635

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

  Abstract

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

  View details for DOI 10.1038/nature10202

  View details for Web of Science ID 000293731900039

  View details for PubMedID 21617644

  View details for PubMedCentralID PMC3159048

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

  Abstract

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

  View details for DOI 10.1038/nature10084

  View details for Web of Science ID 000291647100050

  View details for PubMedID 21602826

  View details for PubMedCentralID PMC3155806

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

  View details for DOI 10.1038/cr.2010.117

  View details for Web of Science ID 000281483800002

  View details for PubMedID 20697429

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

  Abstract

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

  View details for DOI 10.1096/fj.09-145177

  View details for Web of Science ID 000285005400015

  View details for PubMedID 20371616

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

  Abstract

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

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

  View details for Web of Science ID 000277923600008

  View details for PubMedID 20233975

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

  Abstract

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

  View details for DOI 10.1038/nature08797

  View details for Web of Science ID 000275108400027

  View details for PubMedID 20107439

  View details for PubMedCentralID PMC2829121

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

  Abstract

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

  View details for DOI 10.1096/fj.08-128546

  View details for Web of Science ID 000272193700013

  View details for PubMedID 19703934

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

  Abstract

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

  View details for DOI 10.1159/000227815

  View details for Web of Science ID 000267650600009

  View details for PubMedID 19590195

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

  Abstract

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

  View details for DOI 10.1016/j.cell.2008.07.020

  View details for Web of Science ID 000258665500024

  View details for PubMedID 18692474

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

  Abstract

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

  View details for DOI 10.1038/nature07107

  View details for Web of Science ID 000258228000045

  View details for PubMedID 18600261

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

  Abstract

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

  View details for Web of Science ID 000258325500026

  View details for PubMedID 18594521

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

  Abstract

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

  View details for DOI 10.1038/nature07056

  View details for Web of Science ID 000257308300033

  View details for PubMedID 18509334

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

  Abstract

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

  View details for DOI 10.1016/j.cell.2008.03.028

  View details for Web of Science ID 000255052000015

  View details for PubMedID 18423197

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

  Abstract

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

  View details for DOI 10.1073/pnas.0801677105

  View details for Web of Science ID 000255237200038

  View details for PubMedID 18391196

  View details for PubMedCentralID PMC2311361

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

  Abstract

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

  View details for DOI 10.1016/j.stem.2008.01.004

  View details for Web of Science ID 000253301900010

  View details for PubMedID 18371436

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

  View details for DOI 10.1016/j.stem.2007.12.001

  View details for Web of Science ID 000252606400006

  View details for PubMedID 18371415

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

  Abstract

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

  View details for DOI 10.1126/science.1152092

  View details for Web of Science ID 000251786600058

  View details for PubMedID 18063756

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

  Abstract

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

  View details for DOI 10.1038/nbt1335

  View details for Web of Science ID 000250226600030

  View details for PubMedID 17724450

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

  Abstract

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

  View details for DOI 10.1038/nature06008

  View details for Web of Science ID 000248446700035

  View details for PubMedID 17603471

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

  Abstract

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

  View details for DOI 10.1038/nature05944

  View details for Web of Science ID 000248118300050

  View details for PubMedID 17554336

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

  Abstract

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

  View details for DOI 10.1038/nature04733

  View details for Web of Science ID 000237593200048

  View details for PubMedID 16625203

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

  Abstract

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

  View details for DOI 10.1016/j.cell.2006.02.041

  View details for Web of Science ID 000237241500021

  View details for PubMedID 16630819

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

  Abstract

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

  View details for DOI 10.1073/pnas.0502507102

  View details for Web of Science ID 000231317000051

  View details for PubMedID 16085712

  View details for PubMedCentralID PMC1187970

• Functional integration of transplanted ES cell-derived neurons Symposium of the Nordrhein-Westfalischen-Akademie-der-Wissenschaften Benninger, F., Wernig, M., Schmandt, T., Rade, M., Bussow, H., Scheffler, B., Beck, H., Brustle, O. VERLAG FERDINAND SCHONINGH. 2005: 79–81

  View details for Web of Science ID 000231307500026

• Functional integration of embryonic stem cell-derived neurons in vivo JOURNAL OF NEUROSCIENCE Wernig, M., Benninger, F., Schmandt, T., Rade, M., Tucker, K. L., Bussow, H., Beck, H., Brustle, O. 2004; 24 (22): 5258-5268

  Abstract

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

  View details for DOI 10.1523/JNEUROSCI.0428-04.200

  View details for Web of Science ID 000221883100020

  View details for PubMedID 15175396

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

  Abstract

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

  View details for Web of Science ID 000184587100012

  View details for PubMedID 12904468

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

  Abstract

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

  View details for Web of Science ID 000181601300007

  View details for PubMedID 12634414

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

  Abstract

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

  View details for DOI 10.1002/jnr.10395

  View details for Web of Science ID 000177792600024

  View details for PubMedID 12205684

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

  Abstract

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

  View details for Web of Science ID 000173768100001

  View details for PubMedID 11853015

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

  Abstract

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

  View details for Web of Science ID 000172524400025

  View details for PubMedID 11731781

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

  Abstract

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

  View details for Web of Science ID 000084976500009

  View details for PubMedID 10607833