Stem cell challenges and opportunities.

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s)", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.

Trehalose Suppresses Lysosomal Anomalies in Supporting Cells of Oocytes and Maintains Female Fertility.

Supporting cells of oocytes, i.e., cumulus cells, control oocyte quality, which determines fertilization success. Therefore, the transformation of mature and immature cumulus cells (MCCs and ICCs, respectively) into dysmature cumulus cells (DCCs) with dead characteristics deteriorates oocyte quality. However, the molecular basis for this transformation remains unclear. Here, we explored the link between autophagic decline and cumulus transformation using cumulus cells from patients with infertility, female mice, and human granulosa cell-derived KGN cell lines. When human cumulus cells were labeled with LysoTracker probes, fluorescence corresponding to lysosomes was enhanced in DCCs compared to that in MCCs and ICCs. Similarly, treatment with the autophagy inhibitor chloroquine elevated LysoTracker fluorescence in both mouse cumulus cells and KGN cells, subsequently suppressing ovulation in female mice. Electron microscopy analysis revealed the proliferation of abnormal lysosomes in chloroquine-treated KGN cells. Conversely, the addition of an autophagy inducer, trehalose, suppressed chloroquine-driven problematic lysosomal anomalies and ameliorated ovulation problems. Our results suggest that autophagy maintains the healthy state of the supporting cells of human oocytes by suppressing the formation of lysosomes. Thus, our results provide insights into the therapeutic effects of trehalose on female fertility.

Advanced paternal age alone does not adversely affect pregnancy or live-birth rates or sperm parameters following intrauterine insemination.

PURPOSE: This study aimed to evaluate the effect of advanced paternal age on pregnancy outcomes and sperm parameters following intrauterine insemination (IUI). We used IUI data rather than assisted reproductive technology data, which might mask the effects of sperm impairments. METHODS: We retrospectively analyzed 1576 IUI cycles in women under 40 years old between April 2012 and May 2016 at the National Center for Child Health and Development in Japan. The main outcomes were clinical pregnancy and live birth. RESULTS: The mean male age was significantly lower in cycles that resulted in pregnancy compared with those without pregnancy (38.0 vs 39.1 years; P < 0.001), with a similar trend for live-birth cycles. However, there was no relationship between advanced paternal age and pregnancy outcomes after adjusting for confounding factors and correlations within patients using generalized estimating equations, and the age of the female partner was the only factor affecting pregnancy rate. Furthermore, advanced paternal age had no effect on sperm parameters. CONCLUSIONS: Advanced paternal age alone does not adversely affect pregnancy or live-birth rates or sperm parameters following IUI.

Autophagy-disrupted LC3 abundance leads to death of supporting cells of human oocytes.

Autophagic recycling of cell parts is generally termed as the opposite of cell death. Here, we explored the relation between cell death and autophagy by examining granulosa cell layers that control oocyte quality, which is important for the success of fertilization. Granulosa cell layers were collected from infertile women and morphologically divided into four types, viz., mature (MCCs), immature (ICCs), and dysmature cumulus cells (DCCs), and mural granulosa cells (MGCs). Microtubule-associated protein light chain 3 (LC3), which is involved in autophagosome formation, was expressed excessively in DCCs and MGCs, and their chromosomal DNA was highly fragmented. However, autophagy initiation was limited to MGCs, as indicated by the expression of membrane-bound LC3-II and autophagy-related protein 7 (ATG7), an enzyme that converts LC3-I to LC3-II. Although pro-LC3 was accumulated, autophagy was disabled in DCCs, resulting in cell death. Our results suggest the possibility that autophagy-independent accumulation of pro-LC3 proteins leads to the death of human granulosa cells surrounding the oocytes and presumably reduces oocyte quality and female fertility.