Mitochondrial abundance and genome are crucial for cellular function, with disruptions often associated with disease. However, methods to modulate these parameters for direct functional dissection remain limited. Here, we eliminate mitochondria from pluripotent stem cells (PSCs) by enforced mitophagy and show that PSCs survived for several days in culture without mitochondria. We then leverage enforced mitophagy to generate interspecies PSC fusions that harbor either human or non-human hominid (NHH) mitochondrial DNA (mtDNA). Comparative analyses indicate that human and NHH mtDNA are largely interchangeable in supporting pluripotency in these PSC fusions. However, species divergence between nuclear and mtDNA leads to subtle species-specific transcriptional and metabolic variations. By developing a transgenic enforced mitophagy approach, we further show that reducing mitochondrial abundance leads to delayed development in pre-implantation mouse embryos. Our study opens avenues for investigating the roles of mitochondria in development, disease, and interspecies biology.

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.

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we developed a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated plate (called as "DA-X condition") showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.

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.

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。

For the purpose of establishing ES cells derived from androgenic diploid embryos produced by interspecific sperm injection, either rat or wood mouse (Apodemus sylvaticus) sperm were microinjected into mouse enucleated oocytes, but embryonic development was arrested. Therefore, in order to verify the acceptance of heterologous sperm in mouse oocytes, hybrid embryos were analyzed. As a result, the formation of the male pronucleus was delayed in the wood mouse sperm, and the delay in the formation of the heterologous male pronucleus was improved by the removal of the acrosomal enzyme or the destabilization of the sperm cell membrane. Due to COVID-19, original research plan was changed to establish ES cells from the embryos of Ryukyu spiny rat using interspecific somatic cell nuclear transfer. However, its developmental ability to the blastocysts was extremely low, and ntES cells could not be established.

In this study, we examined the effect of Vitamin-C on freshly isolated PGCs. Vitamin-C, also known as L-ascorbic acid, has recently gained considerable attention largely due to its role in a Tet-dependent manner. This promoted us to test Vitamin-C’s direct effect on freshly isolated in vivo PGCs. To our surprise, while there was no visible colony observed in control culture, Vitamin-C supplementation alone led to the emergence of alkaline phosphatase positive colonies and subsequently the derivation of stable cell line from freshly isolated PGCs. We designated this newly derived cell line as VcPGCs. To our surprise, these cells can be propagated indefinitely in vitro and demonstrated pluripotency using teratoma formation assay. More interestingly, the pluripotent cell lines generated with Vitamine-C share distinct features compared to conventional EGCs and thus may represent a distinct pluripotent state arising from the germline.

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。

In this research project, we have found the following three points. (1) Transcription regulators REST and Larp7 positively control survival and proliferation of primordial germ cells (PGCs), respectively, in mouse embryos. (2) The expression of PGC-specific genes is controlled by DNA demethylation of their regulatory regions. (3) Transcription regulator Max represses the expression of germ cell-specific genes in ES cells.

We had screened the genes were first detected their expression in just specified primordial germ cells, that resulted in identification of Rest/NRSF gene. So we generated knock-out mice for Rest gene. The results showed that the number of primordial germ cells at migrating stage in Rest mutant embryos significantly decreased, compared with that in wild embryos, it caused by apoptotic cell death, but not defect of proliferation rate of primordial germ cells.

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。