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: 9.81). 02/2012; 109(7):2527-32. DOI: 10.1073/pnas.1121003109
Source: PubMed

ABSTRACT 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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Available from: Henrik Ahlenius, Aug 21, 2015
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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 When EGF/FGF were withdrawn from the growth medium and replaced by BDNF, NT3, and ascorbic acid, the cells differentiated into neurons that stained for TUJ1 and MAP2 (Figure 1F).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 · 5.64 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 · 3.91 Impact Factor
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    • "Therefore, neural stem cells have enormous potential for regenerative therapies directed toward neurodegenerative diseases. Recent studies, including those conducted in our labs, have reported the direct conversion of mouse and human somatic cells into functional, expandable iNSCs that show all the major properties of primary NSCs (Corti et al., 2012; Han et al., 2012; Kim et al., 2011; Lujan et al., 2012; Ring et al., 2012; Sheng et al., 2012; Thier et al., 2012). However, in vivo long-term survival rates, multilineage differentiation, and the functional integration of iNSCs have not been analyzed in detail among these cells. "
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    ABSTRACT: Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications.
    Stem Cell Reports 09/2014; 3(3):423-31. DOI:10.1016/j.stemcr.2014.06.017 · 5.64 Impact Factor
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