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  • Master of Science, Stanford University, BIOE-MS (2014)

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All Publications

  • Transdifferentiation of human adult peripheral blood T cells into neurons. Proceedings of the National Academy of Sciences of the United States of America Tanabe, K., Ang, C. E., Chanda, S., Olmos, V. H., Haag, D., Levinson, D. F., Sudhof, T. C., Wernig, M. 2018

    Abstract

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

    View details for DOI 10.1073/pnas.1720273115

    View details for PubMedID 29866841

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

    View details for DOI 10.1038/nbt.4161

    View details for Web of Science ID 000434689200014

    View details for PubMedID 29874211

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

    Abstract

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

    View details for DOI 10.1016/j.celrep.2017.09.011

    View details for Web of Science ID 000411684400020

    View details for PubMedID 28954238

    View details for PubMedCentralID PMC5646379

  • 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

  • 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

  • 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

  • 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

  • Induced neuronal reprogramming. journal of comparative neurology Ang, C. E., Wernig, M. 2014; 522 (12): 2877-2886

    Abstract

    Cellular differentiation processes during normal embryonic development are guided by extracellular soluble factors such as morphogen gradients and cell contact signals, eventually resulting in induction of specific combinations of lineage-determining transcription factors. The young field of epigenetic reprogramming takes advantage of this knowledge and uses cell fate determination factors to convert one lineage into another such as the conversion of fibroblasts into pluripotent stem cells or neurons. These induced cell fate conversions open up new avenues for studying disease processes, generating cell material for therapeutic intervention such as drug screening and potentially also for cell-based therapies. However, there are still limitations that have to be overcome to fulfill these promises, centering on reprogramming efficiencies, cell identity, and maturation. In this review, we discuss the discovery of induced neuronal reprogramming, ways to improve the conversion process, and finally how to define properly the identity of those converted neuronal cells.

    View details for DOI 10.1002/cne.23620

    View details for PubMedID 24771471

    View details for PubMedCentralID PMC4099045

  • 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., Südhof, 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 PubMedID 25254342

    View details for PubMedCentralID PMC4176533

  • 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