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

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.induced neural precursor cells D uring development, the creation of distinct cell types depends upon tightly regulated spatiotemporal expression of lineage-specific transcription factors. A key question is whether cells retain their competence to respond to such transcription factors even after differentiation and after their cell-type-specific phenotype has been stabilized by epigenetic mechanisms (1). A number of classic and recent studies have provided powerful evidence that the differentiated state of at least some somatic cells is more flexible than assumed. For instance, transfer of somatic nuclei into oocytes has been shown to impose an early embryonic program on somatic cells (2, 3). Similarly, aberrant cell-type-specific genes could be induced following cell fusion (4), and misexpression of defined transcription factors has been shown to induce conversion of cells in closely related cell types (5). For instance, the basic helix-loop-helix (bHLH) transcription factor MyoD has been shown to induce muscle-specific properties in fibroblasts but not in hepatocytes (6, 7); expression of Cebpα in B cells induces features of macrophages (8); loss of Pax5 in B cells induces dedifferentiation to a common lymphoid progenitor (9); and the bHLH transcription factor Ngn3 or NeuroD1, in combination with Pdx1 and MafA, efficientl...

Induced neural stem cells: a new tool for studying neural development and neurological disorders

“…Later, the same strategy was successfully applied to mouse tail tip fibroblasts and generated iNSCs with neurogenesis potency both in vitro and in vivo, which further confirmed the feasibility of this method [6]. Meanwhile, Wernig and colleagues also reported a similar screening of 11 factors using a Sox2-EGFP reporter MEF system, and found that as few as two factors, FoxG1 and Sox2, were able to generate selfrenewing, bipotent iNSCs [3]. When Brn2 was added to the combination, tripotent iNSCs, which gave rise to functional neurons, astrocytes as well as oligodendrocytes, were obtained [3].…”

“…However, the innate limitations of iNs may restrict their applications in the clinic. Very recent publications describe the direct conversion of mouse somatic cells into induced neural stem cells (iNSCs) [1][2][3][4][5][6]. Together with other reports [7,8], they constitute the basis for a rising concept that priming and retaining reprogrammed cells into an expandable neural progenitor stage may render them to be more amenable and with great potential for both disease modeling and possible therapeutic treatment for neurological diseases.…”

Specification of functional neurons and glia from human pluripotent stem cells

“…Transdifferentiation from other type of somatic cells into functional neurons has allowed the generation of mature neurons in a relatively shorter period, which seems to be an ideal source of cells for transplantation, nevertheless, the efficiency of transdifferentiation is low, and the iNs are not expandable for large quantity. Alternative procedures that convert other type of somatic cells into expandable neural stem cells are also under extensive exploration (Kim et al, 2011;Han et al, 2012;Lujan et al, 2012;Ring et al, 2012;Sheng et al, 2012;Thier et al, 2012). In contrast to the iNs, the induced neural stem cells (iNSCs) are capable to proliferate, thus allow yield of larger quantity of cells through selectively amplification of the induced neural stem cells.…”

Direct lineage conversion: induced neuronal cells and induced neural stem cells

“…Lujan et al recently reported the generation of self-renewing induced neural precursor (iNP) cells from mouse embryonic fibroblasts using five factors (Rfx4, ID4, FoxG1, Lhx2, and Sox2) (Lujan et al, 2012). The transfection of these factors into mouse embryonic fibroblasts (MEFs) was sufficient to induce Sox2-EGFP+ colonies, as assessed 24 d after infection.…”