Anti-CD9 monoclonal antibody, clone 6H7

Cytotherapy with mesenchymal stem cells (MSCs) has been studied in many species, and often requires in vitro cell expansion to obtain therapeutic doses of stem cells. Because the characteristics of MSCs, such as self-renewal and multi-lineage differentiation, can be altered by long-term culture, it is important to maintain stemness during cultivation. This study assessed the changes in the characteristics of feline adipose tissue-derived (fAT)-MSCs during in vitro passaging. Stem cells isolated from the adipose tissue of donor cats were cultured for seven sub-passages. Proliferation capacity was analyzed by calculating the cell doubling time and by colorimetric assay. Expression of stem cell-specific markers was evaluated by quantitative reverse transcription (qRT)-PCR and immunophenotyping. Expression of adipogenic and osteogenic differentiation markers was also measured by qRT-PCR. Histochemical staining and measurement of beta-galactosidase activity were conducted to detect cellular senescence. The cell proliferation rate decreased significantly at passage 5 (P5). Gene expression levels of pluripotency markers (Sox2, Nanog and Klf4) and stem cell surface markers (CD9, CD44, CD90 and CD105) decreased during continuous culture; in most assays, statistically significant changes were observed at P5. The ability of cells to undergo adipogenic or osteogenic differentiation was inversely proportional to the number of passages. The proportion of senescent cells increased with the number of passages. These results suggest that repeated passages alter the proliferation and multipotency of fAT-MSCs. In clinical trials, early-passage cells should be used to achieve the maximum therapeutic effect.

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testisspecific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.