Induction of human neuronal cells by defined transcription factors. Nature

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 265 Campus Drive, Stanford, California 94305, USA.
Nature (Impact Factor: 41.46). 05/2011; 476(7359):220-3. DOI: 10.1038/nature10202
Source: PubMed


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.

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    • "Furthermore, the numerous cell divisions required for the reprogramming process and differentiation may dilute any accumulated macromolecular damage. The direct transcription factor-based conversion of fibroblasts into induced neurons (iNs) represents an alternative avenue for generating human neurons in vitro (Pang et al., 2011; Vierbuchen et al., 2010). Induction of only two transcription factors in combination with a cocktail of small molecular enhancers was shown to directly yield functional iNs from human fibroblasts with high efficiencies (Ladewig et al., 2012; Liu et al., 2013). "
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    ABSTRACT: Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.
    Cell stem cell 10/2015; DOI:10.1016/j.stem.2015.09.001 · 22.27 Impact Factor
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    • "To ensure the specificity of the amplification, titrations of total human brain RNA were used to eliminate primers without a linear amplification relation. Single-cell qRT-PCR was performed using fluidigm methods (Pang et al., 2011). For details and PCR primer sequences, see Supplemental Experimental Procedures. "
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    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. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell Stem Cell 08/2015; 17(3). DOI:10.1016/j.stem.2015.07.017 · 22.27 Impact Factor
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    • "The present work described a mechanism that converts fibroblast to neurons with high efficiency, even within 3–10 days after neuron induction. Recent studies showed that various sets of transcription factors, such as Ascl1/Brn2/Mytl1 or miR9- 124+Ascl1/Neurod2/Mytl1 could also induce fibroblast-neuron conversion (Yoo et al., 2011; Pang et al., 2011). Consistently, expression of these two sets of transcription factors in IMR90 cells induced neuron conversion in IMR90 cells (Figures 4C and S4C). "
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    ABSTRACT: Differentiated somatic cells have been reprogrammed to a pluripotent state by forced expression of a set of transcription factors (Takahashi et al., 2007), indicating that terminally differentiated cells can be induced to undergo cell fate change. Recent studies further demonstrated that direct conversion from fibroblast to neuron, a potential cell replacement therapy for neurological disorders, can be induced by a set of transcription factors without passing through a pluripotent state (Caiazzo et al., 2011, Vierbuchen et al., 2010, Pfisterer et al., 2011, Pang et al., 2011, Yoo et al., 2011 and Ambasudhan et al., 2011). However, the mechanism underlying this conversion process remains largely unclear. As a result, a variety of combinations of transcription factors have been tried but generally with low percentages and very slow time course of conversion.
    Cell Reports 12/2014; 9(6). DOI:10.1016/j.celrep.2014.11.040 · 8.36 Impact Factor
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