Direct reprogramming of mouse fibroblasts to neural progenitors

Abstract

The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineagespecific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factorbased reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.A lthough successful transdifferentiation from one cell type to another by overexpressing lineage-specific genes in vivo (1, 2) and in vitro (3, 4) has been reported, until recently these methods were only effective for fate switching within the major lineages, i.e., ectoderm, mesoderm, and endoderm. However, the generation of iN cells (5) using neural-specific transcription factors has established that interlineage transdifferentiation is also possible in vitro. These transdifferentiation schemes entail overexpression of different sets of lineage-specific transcription factors. A more recent example reported single-factor transdifferentiation of fibroblasts into blood precursors using long-term ectopic expression of OCT4 (6); through extensive binding to the regulatory regions of key hematopoietic genes, OCT4 also appears to be participating in regulating hematopoietic programs acting as a lineage-specific transcription factor in this context. An important aspect of this study is the ability to generate a mitotically active progenitor population that can be further differentiated into a variety of blood cells-a critical feat that has yet to be accomplished in transdifferentiation to neural and endoderm lineages.In an effort to devise a more general transdifferentiation strategy that might give rise to a broad array of unrelated cell typesincluding lineage-specific precursors-we attempted to direct conventional four iPSC-factor-based reprogramming (7, 8) toward alternative outcomes. Specifically, studies indicating that iPSCs are generated in a sequential and stochastic manner (9-11) led us to hypothesize that we might be able to manipulate cells at an early and epigenetically highly unstable state induced by the reprogramming factors. Different conditions could potentially give rise to a multitude ...

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

“…3). Kim et al (2011a) investigated whether iNS cells could be generated from somatic cells by reprogramming cells with the same four reprogramming factors used to generate iPS cells followed by culture in neural stem (NS) cells medium. They successfully reprogrammed fibroblasts to iNS cells over an abbreviated induction period, and these iNS cells expressed several NS cell-specific markers, including promyelocytic leukemia zinc finger (Plzf), a rosette NSC marker (Elkabetz et al, 2008), and Pax6, an early neural transcription factor (Walther and Gruss, 1991).…”

“…Furthermore, somatic cells other than fibroblasts have been directly converted into other cell types, and even direct endoderm-to-ectoderm switching has been achieved . It has been also demonstrated that fibroblasts can be reprogrammed into neural stem cells (Kim et al, 2011a;Han et al, 2012;Thier et al, 2012). In this review, we will discuss these recent studies in iN cells and iNS cells conversion from terminally differentiated cells by different combinations of transcription factors (Fig.…”

The evolving biology of small molecules: controlling cell fate and identity

“…On the other hand, direct reprogramming to various progenitor cells-and perhaps terminally differentiated cells as well-is a feasible outcome, especially if (i) culture conditions allow and/or promote their formation and (ii) establishment of pluripotency is prevented by using a small-molecule inhibitor of JAK/STAT signalling. adapt the process to the rapid and efficient generation of NPCs from fibroblasts [96]. We believe that we have thus established a new transdifferentiation paradigm, and small molecules have once again played a prominent role in the process (figure 2).…”