Human primordial germ cell commitment <i>in vitro</i> associates with a unique PRDM14 expression profile

Sugawa, Fumihiro,Araú,zo-Bravo, Marcos J,Yoon, Juyong,Kim, Kee-Pyo,Aramaki, Shinya,Wu, Guangming,Stehling, Martin,Psathaki, Olympia E,Hü,bner, Karin,Schö,ler, Hans R BlackWell Publishing Ltd 2015 The EMBO journal Vol.34 No.8

<P>Primordial germ cells (PGCs) develop only into sperm and oocytes <I>in vivo</I>. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of <I>in vitro</I> models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of <I>de novo</I> DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3–6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.</P>

Oct4‐induced oligodendrocyte progenitor cells enhance functional recovery in spinal cord injury model

Kim, Jeong Beom,Lee, Hyunah,Araú,zo‐,Bravo, Marcos J,Hwang, Kyujin,Nam, Donggyu,Park, Myung Rae,Zaehres, Holm,Park, Kook In,Lee, Seok‐,Jin EMBO 2015 The EMBO journal Vol.34 No.23

<P>The generation of patient-specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4-mediated direct reprogramming, using anchorage-independent growth to ensure high purity. Homogeneous iOPCs exhibit typical small-bipolar morphology, maintain their self-renewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild-type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self-renewing iOPCs and yields insights for the in-depth study of demyelination and regenerative medicine.</P>

Direct reprogramming of human neural stem cells by OCT4

Kim, Jeong Beom,Greber, Boris,Araú,zo-Bravo, Marcos J.,Meyer, Johann,Park, Kook In,Zaehres, Holm,Schö,ler, Hans R. Macmillan Publishers Limited. All rights reserved 2009 Nature Vol.461 No.7264

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells

최나영,박요셉,유재성,이혜정,Marcos J. Araúzo-Bravo,고기성,한동욱,Hans R. Schöler,고기남 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.6

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

Direct conversion of mouse fibroblasts into induced neural stem cells

Kim, Sung Min,Flaßkamp, Hannah,Hermann, Andreas,Araú,zo-Bravo, Marcos Jesú,s,Lee, Seung Chan,Lee, Sung Ho,Seo, Eun Hye,Lee, Seung Hyun,Storch, Alexander,Lee, Hoon Taek,Schö,ler, Hans R Nature Publishing Group, a division of Macmillan P 2014 Nature protocols Vol.9 No.4

Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of cells with this method. Here we describe a protocol to directly convert mouse fibroblasts into self-renewing induced neural stem cells (iNSCs) that can be expanded in vitro, thereby overcoming the limitations associated with low reprogramming efficiency. The four transcription factors required for direct conversion into iNSCs (Sox2, Klf4, Myc (also known as c-Myc) and Pou3f4 (also known as Brn4)) do not generate a pluripotent cell state, and thus the risk for tumor formation after transplantation is reduced. By following the current protocol, iNSCs are observed 4–5 weeks after transduction. Two additional months are required to establish clonal iNSC cell lines that exhibit retroviral transgene silencing and that differentiate into neurons, astrocytes and oligodendrocytes.

Reactivation of the inactive X chromosome and post-transcriptional reprogramming of <i>Xist</i> in iPSCs

Kim, Jong Soo,Choi, Hyun Woo,Araú,zo-Bravo, Marcos J.,Schö,ler, Hans R.,Do, Jeong Tae The Company of Biologists Ltd. 2015 Journal of cell science Vol.128 No.1

<P>Direct reprogramming of somatic cells to pluripotent stem cells entails the obliteration of somatic cell memory and the reestablishment of epigenetic events. Induced pluripotent stem cells (iPSCs) have been created by reprogramming somatic cells through the transduction of reprogramming factors. During cell reprogramming, female somatic cells must overcome at least one more barrier than male somatic cells in order to enter a pluripotent state, as they must reactivate an inactive X chromosome (Xi). In this study, we investigated whether the sex of somatic cells affects reprogramming efficiency, differentiation potential and the post-transcriptional processing of <I>Xist</I> RNA after reprogramming. There were no differences between male and female iPSCs with respect to reprogramming efficiency or their differentiation potential <I>in vivo</I>. However, reactivating Xi took longer than reactivating pluripotency-related genes. We also found that direct reprogramming leads to gender-appropriate post-transcriptional reprogramming – like male embryonic stem cells (ESCs), male iPSCs expressed only the long <I>Xist</I> isoform, whereas female iPSCs, like female ESCs, expressed both the long and short isoforms.</P>