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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    • "Neuronal transdifferentiation was first demonstrated by Vierbuchen et al. (2010), who determined that forced expression of the neuronal transcription factors Brn2, Ascl1, and Myt1l (BAM) could rapidly and efficiently convert fibroblasts into neuron-like cells (termed induced neuronal cells). Subsequent studies expanded on this work, demonstrating the generation of induced neuronal cells using variations of BAM (Kim, 2011), additional transcription factors (Pang et al., 2011), small-molecule inhibitors (Ladewig et al., 2012), and microRNAs (Yoo et al., 2011). Induced neuronal cells were also shown to be functional electrophysiologically (Ambasudhan et al., 2011; Vierbuchen et al., 2010), with examples of functional integration following transplantation into murine models (Kim et al., 2011). "
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    ABSTRACT: The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with βIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.
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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.
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    • "Furthermore, when miR-9/9*-124 were combined with DLX1 and DLX2, previously shown to be important for terminal differentiation of MSNs (Anderson et al., 1997), we detected a large percentage of GABAergic neurons (72.3% of MAP2-positive cells) (Figure 1A). It is interesting to note that MYT1L, which has previously been used by our group and others to enhance direct neuronal reprogramming with other factors (Pang et al., 2011; Yoo et al., 2011), significantly increased the number of MAP2- positive cells when tested alone with miR-9/9*-124 (Figures 1A and S3). Importantly, single transcription factors tested without miR-9/9*-124 did not induce MAP2-positive cells (data not shown). "
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    ABSTRACT: The ability to generate human neurons of specific subtypes of clinical importance offers experimental platforms that may be instrumental for disease modeling. We recently published a study demonstrating the use of neuronal microRNAs (miRNAs) and transcription factors to directly convert human fibroblasts to a highly enriched population of striatal medium spiny neurons (MSNs), a neuronal subpopulation that has a crucial role in motor control and harbors selective susceptibility to cell death in Huntington's disease. Here we describe a stepwise protocol for the generation of MSNs by direct neuronal conversion of human fibroblasts in 30 d. We provide descriptions of cellular behaviors during reprogramming and crucial steps involved in gene delivery, cell adhesion and culturing conditions that promote cell survival. Our protocol offers a unique approach to combine microRNAs and transcription factors to guide the neuronal conversion of human fibroblasts toward a specific neuronal subtype.
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