p38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells

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

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

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 beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-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/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.info:eu-repo/semantics/publishedVersio

The amount of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition.

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

コレステロール生合成阻害によるヒトiPS細胞の腫瘍形成リスク除去技術の確立

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。我々はコレステロール生合成阻害剤が無血清培地という条件の中で、増殖過程にある未分化な多能性幹細胞ならびに未成熟分化細胞のほぼ全てをアポトーシスにより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の生存・機能には全く影響を与えなかった点にある。本成果は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となることが期待される。Tumour formation by residual undifferentiated cells is still a major problem in human iPS cell therapy. We found that the cholesterol biosynthesis inhibitor killed almost all undifferentiated pluripotent stem cells and immature differentiated cells by apoptosis in serum-free medium. More importantly, the survival and function of mature differentiated cells that had stopped proliferating were not affected at all. These results are expected to serve as a basic technology for the "elimination of undifferentiated cells that may become tumours" and the "purification and enrichment of mature differentiated cells", which are the most important issues in transplantation medicine using iPS cells.研究分野：発生生物

The technology to eliminate the risk of tumor formation in human iPS cells by inhibiting cholesterol biosynthesis.

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。我々はコレステロール生合成阻害剤が無血清培地という条件の中で、増殖過程にある未分化な多能性幹細胞ならびに未成熟分化細胞のほぼ全てをアポトーシスにより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の生存・機能には全く影響を与えなかった点にある。本成果は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となることが期待される。Tumour formation by residual undifferentiated cells is still a major problem in human iPS cell therapy. We found that the cholesterol biosynthesis inhibitor killed almost all undifferentiated pluripotent stem cells and immature differentiated cells by apoptosis in serum-free medium. More importantly, the survival and function of mature differentiated cells that had stopped proliferating were not affected at all. These results are expected to serve as a basic technology for the "elimination of undifferentiated cells that may become tumours" and the "purification and enrichment of mature differentiated cells", which are the most important issues in transplantation medicine using iPS cells.研究分野：発生生物