Mall M, Kareta MS, Chanda S, Ahlenius H, Perotti N, Zhou B, Grieder SD, Ge X, Drake S, Euong Ang C, Walker BM, Vierbuchen T, Fuentes DR, Brennecke P, Nitta KR, Jolma A, Steinmetz LM, Taipale J, Südhof TC, Wernig M

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.

Chao MP, Gentles AJ, Chatterjee S, Lan F, Reinisch A, Corces MR, Xavy S, Shen J, Haag D, Chanda S, Sinha R, Morganti RM, Nishimura T, Ameen M, Wu H, Wernig M, Wu JC, Majeti R

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.

Rivetti di Val Cervo P, Romanov RA, Spigolon G, Masini D, Martín-Montañez E, Toledo EM, La Manno G, Feyder M, Pifl C, Ng YH, Sánchez SP, Linnarsson S, Wernig M, Harkany T, Fisone G, Arenas E

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.

Yang N, Chanda S, Marro S, Ng YH, Janas JA, Haag D, Ang CE, Tang Y, Flores Q, Mall M, Wapinski O, Li M, Ahlenius H, Rubenstein JL, Chang HY, Buylla AA, Südhof TC, Wernig M

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.

Osorio MJ, Rowitch DH, Tesar P, Wernig M, Windrem MS, Goldman SA

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

Huang YA, Zhou B, Wernig M, Südhof TC

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.

Chanda S, Aoto J, Lee SJ, Wernig M, Südhof TC

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.

Carlson AL, Bennett NK, Francis NL, Halikere A, Clarke S, Moore JC, Hart RP, Paradiso K, Wernig M, Kohn J, Pang ZP, Moghe PV

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.

Ahlenius H, Chanda S, Webb AE, Yousif I, Karmazin J, Prusiner SB, Brunet A, Südhof TC, Wernig M

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.

Treutlein B, Lee QY, Camp JG, Mall M, Koh W, Shariati SA, Sim S, Neff NF, Skotheim JM, Wernig M, Quake SR

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.

Patzke C, Acuna C, Giam LR, Wernig M, Südhof TC

Hundreds of L1CAM gene 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 conditional L1CAM loss-of-function mutation and produced precisely matching control and L1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion of L1CAM dramatically 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 the L1CAM mutation 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 of L1CAM mutations 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.

De Los Angeles A, Ferrari F, Fujiwara Y, Mathieu R, Lee S, Lee S, Tu HC, Ross S, Chou S, Nguyen M, Wu Z, Theunissen TW, Powell BE, Imsoonthornruksa S, Chen J, Borkent M, Krupalnik V, Lujan E, Wernig M, Hanna JH, Hochedlinger K, Pei D, Jaenisch R, Deng H, Orkin SH, Park PJ, Daley GQ

De Los Angeles A, Ferrari F, Xi R, Fujiwara Y, Benvenisty N, Deng H, Hochedlinger K, Jaenisch R, Lee S, Leitch HG, Lensch MW, Lujan E, Pei D, Rossant J, Wernig M, Park PJ, Daley GQ

Yi F, Danko T, Botelho SC, Patzke C, Pak C, Wernig M, Südhof TC

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 hyperpolarization-activated cation (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) that form Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest that SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.

Cheloufi S, Elling U, Hopfgartner B, Jung YL, Murn J, Ninova M, Hubmann M, Badeaux AI, Euong Ang C, Tenen D, Wesche DJ, Abazova N, Hogue M, Tasdemir N, Brumbaugh J, Rathert P, Jude J, Ferrari F, Blanco A, Fellner M, Wenzel D, Zinner M, Vidal SE, Bell O, Stadtfeld M, Chang HY, Almouzni G, Lowe SW, Rinn J, Wernig M, Aravin A, Shi Y, Park PJ, Penninger JM, Zuber J, Hochedlinger K

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

Kareta MS, Gorges LL, Hafeez S, Benayoun BA, Marro S, Zmoos AF, Cecchini MJ, Spacek D, Batista LF, O'Brien M, Ng YH, Ang CE, Vaka D, Artandi SE, Dick FA, Brunet A, Sage J, Wernig M

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.

Pak C, Danko T, Zhang Y, Aoto J, Anderson G, Maxeiner S, Yi F, Wernig M, Südhof TC

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.

Chen G, Wernig M, Berninger B, Nakafuku M, Parmar M, Zhang CL

Cell reprogramming technologies have enabled the generation of various specific cell types including neurons from readily accessible patient cells, such as skin fibroblasts, providing an intriguing novel cell source for autologous cell transplantation. However, cell transplantation faces several difficult hurdles such as cell production and purification, long-term survival, and functional integration after transplantation. Recently, in vivo reprogramming, which makes use of endogenous cells for regeneration purpose, emerged as a new approach to circumvent cell transplantation. There has been evidence for in vivo reprogramming in the mouse pancreas, heart, and brain and spinal cord with various degrees of success. This mini review summarizes the latest developments presented in the first symposium on in vivo reprogramming glial cells into functional neurons in the brain and spinal cord, held at the 2014 annual meeting of the Society for Neuroscience in Washington, DC.

De Los Angeles A, Ferrari F, Xi R, Fujiwara Y, Benvenisty N, Deng H, Hochedlinger K, Jaenisch R, Lee S, Leitch HG, Lensch MW, Lujan E, Pei D, Rossant J, Wernig M, Park PJ, Daley GQ

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

Lujan E, Zunder ER, Ng YH, Goronzy IN, Nolan GP, Wernig M

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.

De Los Angeles A, Ferrari F, Fujiwara Y, Mathieu R, Lee S, Lee S, Tu HC, Ross S, Chou S, Nguyen M, Wu Z, Theunissen TW, Powell BE, Imsoonthornruksa S, Chen J, Borkent M, Krupalnik V, Lujan E, Wernig M, Hanna JH, Hochedlinger K, Pei D, Jaenisch R, Deng H, Orkin SH, Park PJ, Daley GQ

Tanabe K, Haag D, Wernig M

The predominant view of embryonic development and cell differentiation has been that rigid and even irreversible epigenetic marks are laid down along the path of cell specialization ensuring the proper silencing of unrelated lineage programmes. This model made the prediction that specialized cell types are stable and cannot be redirected into other lineages. Accordingly, early attempts to change the identity of somatic cells had little success and was limited to conversions between closely related cell types. Nuclear transplantation experiments demonstrated, however, that specialized cells even from adult mammals can be reprogrammed into a totipotent state. The discovery that a small combination of transcription factors can reprogramme cells to pluripotency without the need of oocytes further supported the view that these epigenetic barriers can be overcome much easier than assumed, but the extent of this flexibility was still unclear. When we showed that a differentiated mesodermal cell can be directly converted to a differentiated ectodermal cell without a pluripotent intermediate, it was suggested that in principle any cell type could be converted into any other cell type. Indeed, the work of several groups in recent years has provided many more examples of direct somatic lineage conversions. Today, the question is not anymore whether a specific cell type can be generated by direct reprogramming but how it can be induced.

Kareta MS, Sage J, Wernig M

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.

Patzke C, Han Y, Covy J, Yi F, Maxeiner S, Wernig M, Südhof TC

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.

Zunder ER, Lujan E, Goltsev Y, Wernig M, Nolan GP

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.