Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2012; 109(7):2527-32. DOI: 10.1073/pnas.1121003109
Source: PubMed


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.

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    • "Reprogramming adult cells directly into a neural cell identity is now possible. This occurs very rapidly, within 72 hours, and can be used to generate a variety of committed cells or even NSPCs (Vierbuchen et al., 2010; Lujan et al., 2012). These virtues of reprogramming patient-specifi c cells into a desired phenotype, rapidly and without viruses, suggests that the clinical application of cell transplantation strategies in treating SCI may soon advance exponentially. "
    Jared T Wilcox · RV · MGF ·
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    ABSTRACT: Stem cell transplantation offers an attractive potential therapy for neurological and neurodegenerative disorders such as spinal cord injury (SCI). Given the demand and expectations for the success of regenerative medicines, newly developed cellular therapeutics must be carefully designed and executed, informed by strong preclinical rationale available. This chapter will address the current state of preclinical scientific research for translation into clinical use of stem cell therapy. Focus will be given to current advances in cell transplantation strategies, with specific attention paid to critical assessment of mechanism and efficacy. Specific cell types will be discussed with respect to pathophysiological processes of SCI and their proposed targets. These therapeutics targets include: 1) trophic support and reducing cell loss, 2) remyelination and neuroprotection, 3) tissue modification, and 4) regeneration through neuroplasticity. Combinatorial strategies consisting of co-administering cell types, neurotrophins and tissue modification will also be addressed. Pressing considerations and challenges of translating preclinical findings into clinical therapy exist, and ought to be critically assessed with respect to the best current animal model data. Clinical trials of cell transplantation in human participants with spinal injuries are of significant importance, and will be critically appraised. Taken as a whole—while expectations must be measured—the current status of knowledge on stem cell transplantation for SCI has evolved substantially over the past decade. While translational knowledge gaps continue to exist with regard to cervical and chronic SCI, carefully conducted early phase clinical trials with cellular therapies are warranted, but must be complemented by a robust preclinical research strategy.
    Stem Cells and Neurological Disorders, Edited by L Lescaundron, 01/2015: chapter 3: pages 61-92; Science Publishers.
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    • "To functionally characterize the iNSCs, we assessed their capacity to differentiate into neurons, astrocytes, and oligodendrocytes by growing them in previously defined conditions (Thier et al., 2012; Lujan et al., 2012). When EGF/FGF were withdrawn from the growth medium and replaced by BDNF, NT3, and ascorbic acid, the cells differentiated into neurons that stained for TUJ1 and MAP2 (Figure 1F). "
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    ABSTRACT: Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 11/2014; 3(6). DOI:10.1016/j.stemcr.2014.10.001 · 5.37 Impact Factor
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    • "iNSCs have worked probably by restoring the functions of the endogenous DA neural circuits. Since the report of iNSCs in 2012 (Sheng et al., 2012a,b), several other groups have also published studies on iNSCs, using different starter cells and reprogramming factors (Kim et al., 2014; Cheng et al., 2014a; Cheng et al., 2014b; Cheng, 2014; Maucksch et al., 2012; Mitchell et al., 2014; Zou et al., 2014; Han et al., 2012; Lujan et al., 2012). Nevertheless, none of those studies had tested the efficacy of iNSCs in a PD model. "
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    ABSTRACT: Lmx1a plays a central role in the specification of dopaminergic (DA) neurons, which potentially could be employed as a key factor for trans-differentiation to DA neurons. In our previous study, we have converted somatic cells directly into neural stem cell-like cells, namely induced neural stem cells (iNSCs), which further can be differentiated into subtypes of neurons and glia in vitro. In the present study, we continued to test whether these iNSCs have therapeutic effects when transplanted into a mouse model of Parkinson's disease (PD), especially when Lmx1a was introduced into these iNSCs under a Nestin enhancer. iNSCs that over-expressed Lmx1a (iNSC-Lmx1a) gave rise to an increased yield of dopaminergic neurons and secreted a higher level of dopamine in vitro. When transplanted into mouse models of PD, both groups of mice showed decreased ipsilateral rotations; yet mice that received iNSC-Lmx1a vs. iNSC-GFP exhibited better recovery. Although few iNSCs survived 11weeks after transplantation, the improved motor performance in iNSC-Lmx1a group did correlate with a greater tyrosine hydroxylase (TH) signal abundance in the lesioned area of striatum, suggesting that iNSCs may have worked through a non-autonomous manner to enhance the functions of remaining endogenous dopaminergic neurons in brain. Copyright © 2014. Published by Elsevier B.V.
    Stem Cell Research 10/2014; 14(1):1-9. DOI:10.1016/j.scr.2014.10.004 · 3.69 Impact Factor
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