Biological importance of OCT transcription factors in reprogramming and development

Kim Kee-Pyo,Han Dong Wook,Kim Johnny,Schöler Hans R. 생화학분자생물학회 2021 Experimental and molecular medicine Vol.53 No.-

Ectopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences.

Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells without Microenvironment of Feeder Cells

이승원,Guangming Wu,최나영,이혜정,방진석,이유경,이민성,고기성,Hans R. Schöler,고기남 한국분자세포생물학회 2018 Molecules and cells Vol.41 No.7

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into plu-ripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows long-term SSC prolifera-tion without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self reprogrammed into 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.

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

Han, Dong Wook,Im, Young Bin,Do, Jeong Tae,Gupta, Mukesh Kumar,Uhm, Sang Jun,Kim, Jin-Hoi,Schö,ler, Hans R.,Lee, Hoon Taek Wiley Subscription Services, Inc., A Wiley Company 2008 Molecular reproduction and development Vol.75 No.5

<P>This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer. Mol. Reprod. Dev. 75: 777–784, 2008. © 2008 Wiley-Liss, Inc.</P>

Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors

Hermann, Andreas,Kim, Jeong Beom,Srimasorn, Sumitra,Zaehres, Holm,Reinhardt, Peter,Schö,ler, Hans R.,Storch, Alexander Hindawi Publishing Corporation 2016 Stem cells international Vol.2016 No.-

<P>Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC<SUB>1F-NSC</SUB>) or two (OCT4, KLF4; hiPSC<SUB>2F-NSC</SUB>) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC<SUB>3F-FIB</SUB>) or four reprogramming factors (hiPSC<SUB>4F-FIB</SUB>). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC<SUB>1F-NSC</SUB> and hiPSC<SUB>2F-NSC</SUB> was as efficient as iPSC<SUB>3F-FIB</SUB> or iPSC<SUB>4F-FIB</SUB>. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.</P>

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>

Two-Step Generation of Oligodendrocyte Progenitor Cells From Mouse Fibroblasts for Spinal Cord Injury

Lee, Yukyeong,Kim, C-Yoon,Lee, Hye Jeong,Kim, Jae Gon,Han, Dong Wook,Ko, Kisung,Walter, James,Chung, Hyung-Min,Schö,ler, Hans R.,Bae, Young Min,Ko, Kinarm Frontiers Media S.A. 2018 Frontiers in cellular neuroscience Vol.12 No.-

<P>Oligodendrocyte progenitor cells (OPCs) are attracting attention as the ideal cell therapy for spinal cord injury (SCI). Recently, advanced reprogramming and differentiation techniques have made it possible to generate therapeutic cells for treating SCI. In the present study, we used directly-induced neural stem cells (DNSCs) from fibroblasts to establish OPCs (DN-OPCs) capable of proliferation and confirmed their OPC-specific characteristics. Also, we evaluated the effect of transplanted DN-OPCs on SCI in rats. The DN-OPCs exhibited an OPC-specific phenotype and electrophysiological function and could be differentiated into oligodendrocytes. In the SCI model, transplanted DN-OPCs improved behavior recovery, and showed engraftment into the host spinal cord with expression of myelin basic protein. These results suggest that DN-OPCs could be a new source of potentially useful cells for treating SCI.</P>

Metastable Reprogramming State of Single Transcription Factor-Derived Induced Hepatocyte-Like Cells

Hwang, Seon In,Kwak, Tae Hwan,Kang, Ji Hyun,Kim, Jonghun,Lee, Hyunseong,Kim, Kee-Pyo,Ko, Kinarm,Schö,ler, Hans R.,Han, Dong Wook Hindawi 2019 Stem cells international Vol.2019 No.-

<P>We previously described the generation of induced hepatocyte-like cells (iHeps) using the hepatic transcription factor <I>Hnf1a</I> together with small molecules. These iHeps represent a hepatic state that is more mature compared with iHeps generated with multiple hepatic factors. However, the underlying mechanism of hepatic conversion involving transgene dependence of the established iHeps is largely unknown. Here, we describe the generation of transgene-independent iHeps by inducing the ectopic expression of <I>Hnf1a</I> using both an episomal vector and a doxycycline-inducible lentivirus. In contrast to iHeps with sustained expression of <I>Hnf1a</I>, transgene-independent <I>Hnf1a</I> iHeps lose their typical morphology and <I>in vitro</I> functionality with rapid downregulation of hepatic markers upon withdrawal of small molecules. Taken together, our data indicates that the reprogramming state of single factor <I>Hnf1a-</I>derived iHeps is metastable and that the hepatic identity of these cells could be maintained only by the continuous supply of either small molecules or the master hepatic factor <I>Hnf1a</I>. Our findings emphasize the importance of a factor screening strategy for inducing specific cellular identities with a stable reprogramming state in order to eventually translate direct conversion technology to the clinic.</P>

Conversion of genomic imprinting by reprogramming and redifferentiation

Kim, Min Jung,Choi, Hyun Woo,Jang, Hyo Jin,Chung, Hyung Min,Arauzo-Bravo, Marcos J.,Schö,ler, Hans R.,Do, Jeong Tae The Company of Biologists Limited 2013 Journal of cell science Vol.126 No.11

<P>Induced pluripotent stem cells (iPSCs), generated from somatic cells by overexpression of transcription factors Oct4, Sox2, Klf4 and c-Myc have the same characteristics as pluripotent embryonic stem cells (ESCs). iPSCs reprogrammed from differentiated cells undergo epigenetic modification during reprogramming, and ultimately acquire a similar epigenetic state to that of ESCs. In this study, these epigenetic changes were observed in reprogramming of uniparental parthenogenetic somatic cells. The parthenogenetic pattern of imprinted genes changes during the generation of parthenogenetic maternal iPSCs (miPSCs), a process referred to as pluripotent reprogramming. We determined whether altered imprinted genes are maintained or revert to the parthenogenetic state when the reprogrammed cells are redifferentiated into specialized cell types. To address this question, we redifferentiated miPSCs into neural stem cells (miPS-NSCs) and compared them with biparental female NSCs (fNSCs) and parthenogenetic NSCs (pNSCs). We found that pluripotent reprogramming of parthenogenetic somatic cells could reset parthenogenetic DNA methylation patterns in imprinted genes, and that alterations in DNA methylation were maintained even after miPSCs were redifferentiated into miPS-NSCs. Notably, maternally methylated imprinted genes (<I>Peg1</I>, <I>Peg3</I>, <I>Igf2r</I>, <I>Snrpn</I> and <I>Ndn</I>), whose differentially methylated regions were fully methylated in pNSCs, were demethylated and their expression levels were found to be close to the levels in normal biparental fNSCs after reprogramming and redifferentiation. Our findings suggest that pluripotent reprogramming of parthenogenetic somatic cells followed by redifferentiation leads to changes in DNA methylation of imprinted genes and the reestablishment of gene expression levels to those of normal biparental cells.</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.