Kinarm Ko

Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors

Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1–11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.

OCT4-INDUCED PLURIPOTENCY IN ADULT NEURAL STEM CELLS

The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

Conserved and Divergent Roles of FGF Signaling in Mouse Epiblast Stem Cells and Human Embryonic Stem Cells

Mouse epiblast stem cells (EpiSCs) are cultured with FGF2 and Activin A, like human embryonic stem cells (hESCs), but the action of the associated pathways in EpiSCs has not been well characterized. Here, we show that activation of the Activin pathway promotes self-renewal of EpiSCs via direct activation of Nanog, whereas inhibition of this pathway induces neuroectodermal differentiation, like in hESCs. In contrast, the different roles of FGF signaling appear to be only partially conserved in the mouse. Our data suggest that FGF2 fails to cooperate with SMAD2/3 signaling in actively promoting EpiSC self-renewal through Nanog, in contrast to its role in hESCs. Rather, FGF appears to stabilize the epiblast state by dual inhibition of differentiation to neuroectoderm and of media-induced reversion to a mouse embryonic stem cell-like state. Our data extend the current model of cell fate decisions concerning EpiSCs by clarifying the distinct roles played by FGF signaling.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Proteome Quantitation of Mouse Embryonic Stem Cells to a Depth of 5,111 Proteins

Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as OCT4, NANOG, SOX2, and UTF1 along with the embryonic form of RAS (ERAS). We also quantified the proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and membrane/chromatin fractions. We compared two different preparation approaches, cell fractionation followed by one-dimensional gel separation and in-solution digestion of total cell lysate combined with isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatics analysis of the ES cell proteome revealed a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compared the proteome with a recently published map of chromatin states of promoters in ES cells and found excellent correlation between protein expression and the presence of active and repressive chromatin marks.

Induction of Pluripotency in Adult Unipotent Germline Stem Cells

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

Direct reprogramming of fibroblasts into epiblast stem cells

Epiblast stem cells (EpiSCs) derived from epiblast tissue of post-implantation embryos are pluripotent and can give rise to all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4, Sox2, Klf4 and c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs) that are very similar to embryonic stem cells (ESCs) in a number of characteristics. However, generation of EpiSCs by the direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here, we show that these transcription factors can be used to directly generate induced EpiSCs (iEpiSCs) under EpiSC culture conditions. iEpiSCs resemble EpiSCs in morphology, gene expression pattern, epigenetic status and chimaera-forming capability. This study demonstrates that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We therefore hypothesize that it will eventually be possible to shape the identity of a directly reprogrammed cell simply by modulating culture conditions.

CD49f Enhances Multipotency and Maintains Stemness Through the Direct Regulation of OCT4 and SOX2

CD49f (integrin subunit α6) regulates signaling pathways in a variety of cellular activities. However, the role of CD49f in regulating the differentiation and pluripotency of stem cells has not been fully investigated. Therefore, in this study, human mesenchymal stem cells (hMSCs) were induced to form spheres under nonadherent culture conditions, and we found that the CD49f‐positive population was enriched in MSC spheres compared with MSCs in a monolayer. The expression of CD49f regulated the ability of hMSCs to form spheres and was associated with an activation of the phosphatidylinositol 3‐kinase (PI3K)/AKT signaling pathway. Furthermore, the forced expression of CD49f modulated the proliferation and differentiation potentials of hMSCs through prolonged activation of PI3K/AKT and suppressed the level of p53. We showed that the pluripotency factors OCT4 and SOX2 were recruited to the putative promoter region of CD49f, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. Indeed, CD49f expression was upregulated in human embryonic stem cells (hESCs) compared with hMSCs. The elevated level of CD49f expression was significantly decreased upon embryoid body formation in hESCs. In hESCs, the knockdown of CD49f downregulated PI3K/AKT signaling and upregulated the level of p53, inducing differentiation into three germ layers. Taken together, our data suggest that the cell‐surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency. STEM CELLS 2012;30:876–887

Human adult germline stem cells in question

Arising from: S. Conrad et al. 456, 344–349 (2008)10.1038/nature07404; Conrad et al. replyConrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues’ haGSCs is therefore called into question.

Conversion of adult mouse unipotent germline stem cells into pluripotent stem cells

Germline stem cells (GSCs), often called spermatogonial stem cells, are unipotent stem cells that can give rise only to gametes. Under defined culture conditions, unipotent GSCs can be converted into pluripotent stem cells, termed as germline-derived pluripotent stem (gPS) cells. gPS cells can be differentiated into cells forming all three germ layers and germ cells. In this study, we describe a simple and robust protocol for the derivation of GSCs from adult mouse testis and the rapid and reproducible conversion of GSCs into gPS cells. Under our defined culture conditions, GSCs can be converted into gPS cells in ∼1 month. The initial number of plated GSCs and the culture time are two conditions that are critical for the successful conversion of GSCs into gPS cells. gPS cells are similar to embryonic stem cells, as judged by molecular and cellular properties and its development potential. Thus, generation of gPS cells holds potential for tissue regenerative medicine.

Neural Induction Intermediates Exhibit Distinct Roles of Fgf Signaling

Formation of the neural plate is an intricate process in early mammalian embryonic development mediated by cells of the inner cell mass and involving a series of steps, including development of the epiblast. Here, we report on the creation of an embryonic stem (ES) cell‐based system to isolate and identify neural induction intermediates with characteristics of epiblast cells and neural plate. We demonstrate that neural commitment requires prior differentiation of ES cells into epiblast cells that are indistinguishable from those derived from natural embryos. We also demonstrate that epiblast cells can be isolated and cultured as epiblast stem cell lines. Fgf signaling is shown to be required for the differentiation of ES cells into these epiblast cells. Fgf2, widely used for maintenance of both human ES cells and epiblast stem cells, inhibits formation of early neural cells by epiblast intermediates in a dose‐dependent manner and is sufficient to promote transient self‐renewal of epiblast stem cells. In contrast, Fgf8, the endogenous embryonic neural inducer, fails to promote epiblast self‐renewal, but rather promotes more homogenous neural induction with transient self‐renewal of early neural cells. Removal of Fgf signaling entirely from epiblast cells promotes rapid neural induction and subsequent neurogenesis. We conclude that Fgf signaling plays different roles during the differentiation of ES cells, with an initial requirement in epiblast formation and a subsequent role in self‐renewal. Fgf2 and Fgf8 thus stimulate self‐renewal in different cell types. STEM CELLS 2010;28:1772–1781