Inhibition of Gap Junctional Intercellular Communication Upregulates Pluripotency Gene Expression in Endogenous Pluripotent Muse Cells.

Gap junctions (GJ) are suggested to support stem cell differentiation. The Muse cells that are applied in clinical trials are non-tumorigenic pluripotent-like endogenous stem cells, can be collected as stage-specific embryonic antigen 3 (SSEA-3+) positive cells from multiple tissues, and show triploblastic differentiation and self-renewability at a single cell level. They were reported to up-regulate pluripotency gene expression in suspension. We examined how GJ inhibition affected pluripotency gene expression in adherent cultured-Muse cells. Muse cells, mainly expressing gap junction alpha-1 protein (GJA1), reduced GJ intercellular communication from ~85% to 5-8% after 24 h incubation with 120 µM 18α-glycyrrhetinic acid, 400 nM 12-O-tetradecanoylphorbol-13-acetate, and 90 µM dichlorodiphenyltrichloroethane, as confirmed by a dye-transfer assay. Following inhibition, NANOG, OCT3/4, and SOX2 were up-regulated 2-4.5 times more; other pluripotency-related genes, such as KLF4, CBX7, and SPRY2 were elevated; lineage-specific differentiation-related genes were down-regulated in quantitative-PCR and RNA-sequencing. Connexin43-siRNA introduction also confirmed the up-regulation of NANOG, OCT3/4, and SOX2. YAP, a co-transcriptional factor in the Hippo signaling pathway that regulates pluripotency gene expression, co-localized with GJA1 (also known as Cx43) in the cell membrane and was translocated to the nucleus after GJ inhibition. Adherent culture is usually more suitable for the stable expansion of cells than is a suspension culture. GJ inhibition is suggested to be a simple method to up-regulate pluripotency in an adherent culture that involves a Cx43-YAP axis in pluripotent stem cells, such as Muse cells.

Beneficial Effects of Systemically Administered Human Muse Cells in Adriamycin Nephropathy.

Multilineage-differentiating stress-enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be collected from various organs. Intravenously administered Muse cells have been shown to spontaneously migrate to damaged tissue and replenish lost cells, but the effect in FSGS is unknown. We systemically administered human bone marrow-derived Muse cells without concurrent administration of immunosuppressants to severe combined immune-deficient (SCID) and BALB/c mouse models with adriamycin-induced FSGS (FSGS-SCID and FSGS-BALB/c, respectively). In FSGS-SCID mice, human Muse cells preferentially integrated into the damaged glomeruli and spontaneously differentiated into cells expressing markers of podocytes (podocin; 31%), mesangial cells (megsin; 13%), and endothelial cells (CD31; 41%) without fusing to the host cells; attenuated glomerular sclerosis and interstitial fibrosis; and induced the recovery of creatinine clearance at 7 weeks. Human Muse cells induced similar effects in FSGS-BALB/c mice at 5 weeks, despite xenotransplant without concurrent immunosuppressant administration, and led to improvement in urine protein, creatinine clearance, and plasma creatinine levels more impressive than that in the FSGS-SCID mice at 5 weeks. However, functional recovery in FSGS-BALB/c mice was impaired at 7 weeks due to immunorejection, suggesting the importance of Muse cell survival as glomerular cells in the FSGS kidney for tissue repair and functional recovery. In conclusion, Muse cells are unique reparative stem cells that preferentially home to damaged glomeruli and spontaneously differentiate into glomerular cells after systemic administration. Introduction of genes to induce differentiation is not required before Muse cell administration; thus, Muse cells may be a feasible therapeutic strategy in FSGS.