Meiotic sex chromosome inactivation (MSCI) is an essential event in meiotic progression in mammalian spermatogenesis. We found that La Ribonucleoprotein 7 (LARP7) is involved in MSCI. LARP7 plays a role in fetal germ cells to promote their proliferation, but is once abolished in postnatal gonocytes and re-expressed in spermatocytes at the onset of meiosis. In spermatocytes, LARP7 localizes to the XY body, a compartmentalized chromatin domain on sex chromosomes. In germline-specific Larp7-deficient mice, spermatogenesis is arrested in spermatocytes, and transcription of the genes on sex chromosomes remained active, which suggests failure of meiotic sex chromosome inactivation (MSCI). Furthermore, the XY body in spermatocytes lacking Larp7 shows accumulation of H4K12ac and elimination of H3K9me2, suggesting defective chromatin silencing by abnormal epigenetic controls. These results indicate a new functional role for LARP7 in MSCI.

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Mouse embryonic stem cells (mESCs) have been widely used as a model system to study the basic biology of pluripotency and to develop cell-based therapies. Traditionally, mESCs have been cultured in a medium supplemented with fetal bovine serum (FBS). However, serum with its inconsistent chemical composition has been problematic for reproducibility and for studying the role of specific components. While some serum-free media have been reported, these media contain commercial additives whose detailed components have not been disclosed. Recently, we developed a serum-free medium, DA-X medium, which can maintain a wide variety of adherent cancer lines. In this study, we modified the DA-X medium and established a novel serum-free condition for both naïve mESCs in which all components are chemically defined and disclosed (DA-X-modified medium for robust growth of pluripotent stem cells: DARP medium). The DARP medium fully supports the normal transcriptome and differentiation potential in teratoma and the establishment of mESCs from blastocysts that retain the developmental potential in all three germ layers, including germ cells in chimeric embryos. Utility of chemically defined DA-X medium for primed mouse epiblast stem cells (mEpiSCs) revealed that an optimal amount of cholesterol is required for the robust growth of naïve-state mESCs, but is dispensable for the maintenance of primed-state mEpiSCs. Thus, this study provides reliable and reproducible culture methods to investigate the role of specific components regulating self-renewal and pluripotency in a wide range of pluripotent states.

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-β, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

To produce muscle fibers for cultured meat on a large scale, it is important to expand myoblasts in a serum-reduced or serum-free medium to avoid cost, ethical, and environmental issues. Myoblasts such as C2C12 cells differentiate quickly into myotubes and lose their ability to proliferate when the serum-rich medium is replaced with a serum-reduced medium. This study demonstrates that Methyl-β-cyclodextrin (MβCD), a starch-derived agent that depletes cholesterol, can inhibit further differentiation of myoblasts at the MyoD-positive stage by reducing plasma membrane cholesterol on C2C12 cells and primary cultured chick muscle cells. Furthermore, MβCD efficiently blocks cholesterol-dependent apoptotic cell death of myoblasts, which is one of the mechanisms by which it inhibits the differentiation of C2C12 myoblast cells, as dead cells of myoblast are necessary for the fusion of adjacent myoblasts during the differentiation process into myotubes. Importantly, MβCD maintains the proliferative capacity of myoblasts only under differentiation conditions with a serum-reduced medium, suggesting that its mitogenic effect is due to its inhibitory effect on myoblast differentiation into myotube. In conclusion, this study provides significant insights into ensuring the proliferative capacity of myoblasts in a future serum-free condition for cultured meat production.

Pluripotent stem cells (PSCs) are in vitro adaptations of in vivo pluripotency continuum and can be broadly classified into naive state characteristic of pre-implantation epiblast and primed state resembling peri-gastrulation epiblasts. Naive and primed PSCs differ in their cellular and molecular characteristics, e.g., molecular mechanisms for maintaining undifferentiated state. Naive-to-primed PSC transition provides a tractable in vitro model to study pluripotency development in vivo. We previously developed a protocol that enabled high-efficient (100%) and homogenous derivation of ground state of primed epiblast stem cells (rsEpiSCs) by culturing the isolated post-implantation mouse epiblast under the culture condition containing FGF2 and a Wnt signaling inhibitor (IWR1) (F/R1 condition). Based on F/ R1 condition, in this study, we developed three naive-to-primed conversion methods for generating rsEpiSCs from naive ground state of mouse ESCs (2i/LIF condition). We found that stepwise methods, but not directly, were effective for bona fide rsEpiSCs conversion from mouse ESCs. In sum, we established a robust and efficient ground states of naive-to-primed PSC conversion strategy that will facilitate the study of genetic, epigenetic and metabolic processes involved in pluripotency progression in vivo. (c) 2021 Elsevier Inc. All rights reserved.

Cell competition involves a conserved fitness-sensing process during which fitter cells eliminate neighbouring less-fit but viable cells1. Cell competition has been proposed as a surveillance mechanism to ensure normal development and tissue homeostasis, and has also been suggested to act as a barrier to interspecies chimerism2. However, cell competition has not been studied in an interspecies context during early development owing to the lack of an in vitro model. Here we developed an interspecies pluripotent stem cell (PSC) co-culture strategy and uncovered a previously unknown mode of cell competition between species. Interspecies competition between PSCs occurred in primed but not naive pluripotent cells, and between evolutionarily distant species. By comparative transcriptome analysis, we found that genes related to the NF-κB signalling pathway, among others, were upregulated in less-fit ‘loser’ human cells. Genetic inactivation of a core component (P65, also known as RELA) and an upstream regulator (MYD88) of the NF-κB complex in human cells could overcome the competition between human and mouse PSCs, thereby improving the survival and chimerism of human cells in early mouse embryos. These insights into cell competition pave the way for the study of evolutionarily conserved mechanisms that underlie competitive cell interactions during early mammalian development. Suppression of interspecies PSC competition may facilitate the generation of human tissues in animals.

Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. To date, however, no known PSCs have demonstrated dual competency for chimera formation and direct responsiveness to primordial germ cell (PGC) specification, a unique functional feature of formative pluripotency. Here, by modulating fibroblast growth factor (FGF), transforming growth factor β (TGF-β), and WNT pathways, we derived PSCs from mice, horses, and humans (designated as XPSCs) that are permissive for direct PGC-like cell induction in vitro and are capable of contributing to intra- or inter-species chimeras in vivo. XPSCs represent a pluripotency state between naive and primed pluripotency and harbor molecular, cellular, and phenotypic features characteristic of formative pluripotency. XPSCs open new avenues for studying mammalian pluripotency and dissecting the molecular mechanisms governing PGC specification. Our method may be broadly applicable for the derivation of analogous stem cells from other mammalian species. Yu, Wu, and colleagues report the derivation of intermediate PSCs from mice, horses, and humans (designated as XPSCs) that are permissive for direct PGC-like cell induction in vitro and capable of contributing to intra- or inter-species chimeras in vivo. XPSCs harbor molecular, cellular, and phenotypic features characteristic of formative pluripotency.

Teratomas are tumors consisting of components of the three germ layers that differentiate from pluripotent stem cells derived from germ cells. In the normal mouse testis, teratomas rarely form, but a deficiency in Dead-end1 (Dnd1) in mice with a 129/Sv genetic background greatly enhances teratoma formation. Thus, DND1 is crucial for suppression of teratoma development from germ cells. In the Dnd1 mutant testis, nascent teratoma cells emerge at E15.5. To understand the nature of early teratoma cells, we established cell lines in the presence of serum and leukemia inhibitory factor (LIF) from teratoma-forming cells in neonatal Dnd1 mutant testis. These cells, which we designated cultured Dnd1 mutant germ cells (CDGCs), were morphologically similar to embryonic stem cells (ESCs) and could be maintained in the naïve pluripotent condition. In addition, the cells expressed pluripotency genes including Oct4, Nanog, and Sox2; differentiated into cells of the three germ layers in culture; and contributed to chimeric mice. The expression levels of pluripotency genes and global transcriptomes in CDGCs as well as these cells’ adaption to culture conditions for primed pluripotency suggested that their pluripotent status is intermediate between naïve and primed pluripotency. In addition, the teratoma-forming cells in the neonatal testis from which CDGCs were derived also showed gene expression profiles intermediate between naïve and primed pluripotency. The results suggested that germ cells in embryonic testes of Dnd1 mutants acquire the intermediate pluripotent status during the course of conversion into teratoma cells.

Pluripotency refers to the potential of cells to generate all cell types of the embryo proper. Pluripotency spans a spectrum of cellular states. At one polar extreme is naïve pluripotency, which is identified based on the potential to form germline chimeras. At the other polar extreme is primed pluripotency, in which pluripotent cells are primed to differentiate. Mouse naïve PS cells can be derived from preimplantation embryos. Primed epiblast stem (EpiS) cells are typically isolated from epiblasts of early postimplantation mouse embryos. In this chapter, we describe protocols for highly efficient derivation and propagation of murine naïve and primed PS cell lines in serum-free conditions from preimplantation and postimplantation embryos. We describe generation of mouse naïve PS cells using LIF and inhibitors of MEK and GSK3 kinases and of mouse primed PS cells using FGF2 and IWR1 compound which induces the stabilization of Axin proteins.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naive hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PaperClip

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells - pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs. © 2013 The Authors.

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic. © 2013 Macmillan Publishers Limited. All rights reserved.

Embryonic stem cells and primordial germ cells (PGCs) express many pluripotency-associated genes, but embryonic stem cells do not normally undergo conversion into primordial germ cells. Thus, we predicted that there is a mechanism that represses primordial germ cell-related gene expression in embryonic stem cells. Here we identify genes involved in this putative mechanism, by using an embryonic stem cell line with a Vasa reporter in an RNA interference screen of transcription factor genes expressed in embryonic stem cells. We identify five genes that result in the expression of Vasa when silenced. Of these, Max is the most striking. Transcriptome analysis reveals that Max knockdown in embryonic stem cells results in selective, global derepression of germ cell-specific genes. Max interacts with histone H3K9 methyltransferases and associates with the germ cell-specific genes in embryonic stem cells. In addition, Max knockdown results in a decrease in histone H3K9 dimethylation at their promoter regions. We propose that Max is part of protein complex that acts as a repressor of germ cell-related genes in embryonic stem cells. © 2013 Macmillan Publishers Limited. All rights reserved.

In mouse embryos, some primordial germ cells (PGCs) are eliminated by apoptosis, but the molecular pathways that lead to PGC survival versus apoptosis have not been fully characterized. Here, we found that REST (repressor element 1-silencing transcription factor), a transcription factor that binds a conserved regulatory element, NRSE/RE1, played a role in PGC survival. REST expression was higher in PGCs than in surrounding somatic cells. Moreover, in mouse embryos with a PGC-specific conditional REST mutation, the PGC population experienced more apoptosis and was significantly smaller than that in control embryos; these findings indicated that REST functioned in a cell-autonomous fashion that was critical for PGC survival. Several anti-apoptotic genes were among the previously identified REST-target gene candidates; moreover, some of these genes were downregulated in the REST-deficient PGCs. Mek5, which encodes a component in the a MAP kinase cascade, was one of these downregulated REST-target gene candidates, and a Mek5 mutation, like the REST mutation, caused an increase in PGC apoptosis; these finding suggested that REST promoted PGC survival via regulation of the Mek5 expression. Importantly, there were a normal number of PGCs in the REST mutants at birth, and both the male and female REST-mutant adults were fertile; these final observations revealed that the PGC population was very robust and could recover from a genetically induced reduction in cell number. © 2012 Elsevier Inc.

In mammalian embryos, PGCs (primordial germ cells) are specified from a pluripotent epiblast cell population after implantation. In this study, we demonstrated an essential role for the germline-specific transcription factor Oct3/4 in PGC specification. We generated chimeric embryos with ZHBTc4 ES cells lacking both alleles of the Oct3/4 gene (pou5f1). Pluripotency was maintained by an Oct3/4 transgene, and its expression was suppressed by doxycycline (Dox). Transcription of the Oct3/4 transgene in the ES-derived cells unexpectedly suffered constitutive suppression in chimeric embryos without Dox, and the ES-derived cells contributed to PGC precursor-like cells, but failed to form PGCs. We then attempted to rescue Oct3/4 expression in the ES-derived cells in the chimeric embryos by introducing an additional Oct3/4 transgene. The ES cell-derived cells indeed recovered Oct3/4 transcription in these chimeric embryos, and were successfully specified to PGCs. We further confirmed the requirement of Oct3/4 by using another derivative of ZHBTc4 ES cells in which a Dex (dexamethasone)-dependent Oct3/4 transgene was introduced. In the presence of Dox, Oct3/4 protein was absent in the nuclei of the ES-derived cells, which failed to form PGCs. In contrast, the ES-derived cells could be specified to PGCs after activation of Oct3/4 function in the presence of Dex. © 2008 Elsevier Inc. All rights reserved.

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the β-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of β-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/β-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo. Copyright © 2005 by Elsevier Inc.

The mode and timing of germ-cell specification has been studied in diverse organisms, however, the molecular mechanism regulating germ-cell-fate determination remains to be elucidated. In some model organisms, maternal germ-cell determinants playa key role. In mouse embryos, some germ-line-specific gene products exist as maternal molecules and play critical roles in a pluripotential cell population at preimplantation stages. From those cells, primordial germ cells (PGCs) are specified by extracellular signaling mediated by tissue, as well as cell-cell interaction during gastrulation. Thus, establishment of germ-cell lineage in mammalian embryos appears to be regulated by a multistep process, including formation and maintenance of a pluripotential cell population, as well as specification of PGCs. PGCs can be generated from pluripotential embryonic stem (ES) cells in a simple monolayer culture in which tissue interaction does not occur. This raises the possibility that ES cells, as well as, possibly, pluripotential cells in preimplantation embryos, are more closely related to the PGC precursors than pluripotential cells after implantation. © 2005 Wiley Periodicals, Inc.

As an in vitro assay system for the identification of human imprinted genes, a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from normal human fibroblasts, has been previously generated by microcell-mediated chromosome transfer (MMCT). To supplement this assay system, we constructed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo selection marker. To validate the A9 hybrids, we screened them with chromosome-specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching paternal and maternal chromosome pairs of A9 hybrids were identified for chromosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc finger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hydatidiform mole-associated and imprinted transcript), and the maternal-specific methylation of a CpG island within an imprinted domain on human chromosome 6q24, were maintained in A9 hybrids. For an example, we profiled the expression of expressed sequence tags (ESTs) and the methylation of CpG islands in the 300-kb imprinted domain around 6q24, which may be associated with cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.

In order to examine their roles in carcinogenesis or in progression of colorectal carcinoma, we investigated telomerase activity and microsatellite instability in 67 nonfamilial colorectal cancers and in 18 adenomas. The incidence of detectable telomerase activity increased from 22% of normal colorectal mucosas adjacent to carcinoma, and 33% of adenomas, to 75% of carcinomas. On the other hand, the incidence of detectable microsatellite instability in carcinomas (30%) was almost the same as in adenomas (22%). No significant correlation was detected in the incidence of telomerase activity and microsatellite instability in carcinomas or in adenomas. Moreover, the incidence of telomerase activity and microsatellite instability did not increase during the progression of carcinomas. These results indicate that telomerase activity and microsatellite instability are independent events in colorectal carcinogenesis, and that telomerase activity and microsatellite instability are not correlated with the progression of colorectal carcinoma. However, in 13 multiple cancers, the incidence of telomerase activity (92%) and the incidence of microsatellite instability (54%) was higher than that of telomerase activity (70%) and that of microsatellite instability (24%) in 54 sporadic cancers. Moreover, the incidence of telomerase activity and that of microsatellite instability in adenomas with carcinomas (45% and 36% respectively) was higher than that of telomerase activity and microsatellite instability in adenomas without carcinomas (14% and 0% respectively). These results indicate that telomerase activity and microsatellite instability may play an important role in multicentric carcinogenesis in colorectal carcinoma.

本研究は、体細胞核移植（SCNT）胚の発生能を改善するヒストン脱アセチル化酵素阻害剤(HDACi)がSCNT後のドナー細胞核からのヒストンH2A.Zの除去作用をもつことに着目し、その責任因子を探索することを目的とする。 ［課題Ⅰ］受精卵におけるヒストンH2A.Zの除去過程で相互作用する因子の探索と機能解析 MII期卵内に存在し、受精後ヒストンH2A.Zの除去過程で相互作用する因子を共免疫沈降（Co-IP）で捕捉し質量分析により抽出することを目的として、GFP-H2A.Z、FLAG-H2A.Zおよび3xFLAG-H2A.Z mRNAを調製するための発現ベクターを構築した。 ［課題Ⅱ］ 受精卵におけるHDAC阻害剤と相互作用する因子の探索と機能解析 HDAC阻害剤（SAHA）を固定化した磁気ビーズを用いたchemical pull-downにより、卵細胞質中の相互作用因子をMII期卵・活性化卵で比較し選定する。初年度である令和3年度は、卵子のような微量サンプルから本法により既知の相互作用因子の検出が可能かを検討した。卵子100個分のタンパク質からSAHA固定化ビーズを用いて精製したタンパク質の中に、SAHAがターゲットとしているHDACが検出された。そこで、本法を用いて卵子中に存在するSAHAのターゲット因子の探索を質量解析により行った。その結果、線維芽細胞からは864因子、MⅡ期卵子からは139因子、活性化6時間後の卵子(6hpa)からは101因子が検出された。質量解析のデータから、MⅡ期卵子と6hpa卵子で共通して検出され、かつ線維芽細胞では検出されない因子として16因子が抽出された。その中からE2酵素を介したタンパク質のユビキチン化に関与する分子等、いくつか興味深い候補分子が抽出された。

ヒトiPS細胞を用いた細胞治療を行う際、「残存する未分化細胞による腫瘍形成」が今なお大きな課題として残されている。最近我々はコレステロール生合成阻 害剤が、増殖過程にある未分化な多能性幹細胞のみならず、未成熟分化細胞のほぼ全てを死滅させることを見出した。より重要なことは増殖を止めた成熟分化細 胞(心筋細胞)の生存・機能には全く影響を与えなかった点にある。本研究提案はコレステロール生合成系の代謝に着目し、iPS細胞を用いた移植医療を行う上 でもっとも大きな課題である「腫瘍化の恐れのある未分化細胞を除去」ならびに「成熟分化細胞」を精製/純化するための基盤技術の確立を目指すものである。 2021年度はこれまでの心筋細胞の分化誘導時に加え、心筋以外のどのような組織・細胞で適用されるかを重点的に検証した。今回ターゲットとした組織は、移植を伴うiPS治療の臨床研究に既に承認が得られた組織・細胞(ドーパミン産生ニューロン・網膜・心筋・角膜など)の中から、ドーパミン作動性ニューロンである。ヒトiPS細胞からドーパミン作動性ニューロンを分化誘導する際、一定期間に限ってコレステロール生合成阻害剤を添加することにより、ある特定の細胞集団に細胞死が誘導されることが分かった。詳細に解析したところ、細胞分裂マーカーを有する細胞が特異的に除去されていることが判明した。コレステロール生合成阻害剤による細胞死誘導のメカニズムを探る上でも、増殖性細胞を特異的に細胞死に至らしめることを突き止めた成果はインパクトがあるものと考えられる。

絶滅危惧動物であるトゲネズミの幹細胞の樹立を目的として、異種間顕微授精を用いた雄性2倍体胚由来ES細胞の樹立について検討した。ラットおよびモリアカネズミ精子をマウス除核卵子に顕微注入したが胚発生は停止した。そこで、マウス卵子の異種精子の受容能についてハイブリッド胚による解析を行った。その結果、モリアカネズミ精子では雄性前核の形成が遅延すること、先体酵素の除去処理により異種雄性前核の形成遅延が改善されることが明らかとなった。アマミトゲネズミ－マウス異種間体細胞核移植胚の作製とES細胞の樹立を試みた。しかし、胚盤胞への発生率は極めて低く、発生した胚盤胞からもntES細胞の樹立には至らなかった。

今回我々は, エピジェネティック修飾分子として機能する種々の低分子化合物を培養下に添加することで, マウス始原生殖細胞から従来のEG細胞とは大きく異なる性質を持つ多能性幹細胞を作製することに成功した。今回の我々の結果は, 始原生殖細胞が従来の「再プログラム化」のみならず, 「多様な多能性スペクトル」を研究する上でも高い潜在性を持つことを示しており, 今後その有用なモデルになることが期待される。

vcPGCsは新規性の高い多能性幹細胞であると考えられるので、RNAシークエンスを行い遺伝子プロファイルの比較解析が必須であると考える。しかしここで重要となるのが、比較対象であるEG細胞やvcPGC細胞株の元となる始原生殖細胞の発生ステージの違いも含めた比較である。 分化運命決定後のマウス始原生殖細胞では、DNA脱メチル化やヒストン修飾など、様々なエピジェネティックな変化がゲノムワイドで認められる。それらは、周りの体細胞では認められない変化のため、始原生殖細胞が将来全能性を獲得するための再プログラム化に重要なプロセスであると考えられてはいるが、現在までにその変化の一つ一つが、始原生殖細胞のどのような性質の変化に相関したものなのか、その生物学的な意義も含めほとんど分かっていない。そこで、PGCの運命決定直後の7.5日胚から生殖巣へと移動した12.5日胚の始原生殖細胞まで、あらゆるステージのPGCとその派生細胞株（EG細胞、vcPGC細胞）を揃えるために、発生ステージごと（7.5日胚から12.5日胚まで1日刻み）にvcPGCsとEG細胞の樹立を試みた。その結果、全ての発生ステージにおけるPGCより、EG細胞とvcPGC細胞株の樹立に成功した。今後は採取した胚内のPGCsとの比較も含め、各細胞株の発生ステージによる発現遺伝子の変化をプロファイリングすることで、vcPGCsやEG細胞に特異的に発現する遺伝子等の特徴が発生ステージを追って詳細に見えてくると考える。

本研究では生殖細胞の形成と分化の制御機構に関して次の3点を明らかにした。(1)転写制御因子のRESTとLarp7が、胎仔に存在する未分化な生殖細胞である始原生殖細胞(PGC)の生存と増殖に重要であることを明らかにした。(2)PGCでの特異的な遺伝子発現にDNA脱メチル化が重要であることがわかった。(3)転写制御因子のMaxの機能阻害により多能性幹細胞であるES細胞が生殖細胞特異的遺伝子を発現するようになることを明らかにした。

分化決定期に始原生殖細胞で発現を開始する分子をスクリーニングした結果、Rest遺伝子が見出された。Rest遺伝子欠損胚を解析を行った結果、Rest欠損胚では発生初期(移動期)における始原生殖細胞の数が、正常胚と比較して、有意に減少していることを見出した。分化決定後の始原生殖細胞の増殖には影響が認められず、移動期において、アポトーシスによる細胞死が引き起こされていることが原因であることが分かった。

前年度、始原生殖細胞特異的に発現する遺伝子のスクリーニングの結果、発現パターンの特異性、また構造上の特製から、2つの候補遺伝子に絞った。2年計画の最終年度である今年度は、先で示した2つの候補遺伝子について、機能解析を進めた。一つの候補遺伝子は、Zn fingerモチーフを有しており、転写抑制に働く事が示されている既知の遺伝子であるが、現在までに生殖細胞での機能は全く報告されていない。当該研究領域において、生殖細胞内での体細胞特異的な遺伝子発現の抑制が注目されており、その観点からも注目される候補遺伝子である。また最近、この遺伝子がES細胞内における未分化性の維持にも必須な機能を持つ事が報告され、生殖細胞の「全能性獲得」にも機能しうる可能性が期待出来る。もう一つの候補遺伝子は、RNA bindingモチーフを有しているが、現在までにマウス始原生殖細胞において、同構造を持った分子の機能的な関与は示されておらず、始原生殖細胞分化における、新しい機能分子として注目している。 両遺伝子ともに、始原生殖細胞以外での発現も認められるため、機能解析の手段としては、Cre-loxPシステムを使った、分化決定期の始原生殖細胞特異的な遺伝子欠損胚の作製によって、その機能を解析する。現在までに、当該遺伝子に対するES細胞の相同組み替え体を作製する為に、ターゲティングベクターの作製を完了、複数の相同組み替え体を同定した。現在、キメラマウスを作製する前の品質確認作業を進めている。