Intercellular Cytosolic Transfer Correlates With Mesenchymal Stromal Cell (MSC) Rescue of Umbilical Cord Blood (UCB) Cell Viability During Ex Vivo UCB Expansion

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Mesenchymal Stromal Cell (MSC) co-culture enhances ex vivo expansion of hematopoietic stem and progenitor cells from thawed Umbilical Cord Blood (UCB) without prior CD34+/CD133+ cell selection. We hypothesized that the superior ex vivo expansion is due to reversal of UCB c apoptosis via intercellular cytosolic transfer from the MSC to the UCB cells. MSC endogenously expressing green fluorescent protein (GFP) or exogenously labeled with quantum dots (QDs) were co-cultured with UCB cells positive for a hematopoietic-specific CD45 marker. Cultured UCB cells without MSC layer served as control. Annexin-V, JC-1 dye & caspase inhibitors were used to investigate mitochondria-associated apoptosis pathway. Flow cytometry & confocal imaging were used to detect for the transfer of the GFP or QD in co-cultured UCB cells. Thawed UCB cells had high percentage of early apoptotic cells (defined by Annexin-V positivity) which progressed to dead cells (defined by 7AAD positivity). MSC co-culture resulted in reversal of Annexin-V positivity and reached a maximum on day 3 (viability defined by double negative staining for Annexin-V and 7AAD) (p < 0.05). A significant reduction in caspase 3/7, 8 & 9 activity as well as preservation of mitochondrial membrane potential were observed in the co-cultured UCB cells (p < 0.05). Upon co-culture with GFP-expressing or QD-labelled MSC, unlabelled UCB cells were found positive for these markers as early as 24 hours post co-culture. The number of GFP & CD45 positive UCB cells increased over the first 3 days of co-culturing, which correlated with viability increase. Direct contact between MSC & UCB was necessary for the transfer, as negligible transfer was observed when the two populations were separated by transwell inserts. Separation also showed a significant decrease in viability. The UCB cells that tightly adhered to the MSC layer exhibited optimal transfer & cell viability. DNA analysis of the viable, GFP transfer-positive UCB cells, as determined by Variable Number Tandem Repeat analysis ruled out the possibilities of MSC trans-differentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB cell viability (p < 0.0001). The above data provides evidence that intercellular transfer of cytosolic materials could be one novel mechanism preventing UCB cell death in MSC co-culture. Mesenchymal Stromal Cell (MSC) co-culture enhances ex vivo expansion of hematopoietic stem and progenitor cells from thawed Umbilical Cord Blood (UCB) without prior CD34+/CD133+ cell selection. We hypothesized that the superior ex vivo expansion is due to reversal of UCB c apoptosis via intercellular cytosolic transfer from the MSC to the UCB cells. MSC endogenously expressing green fluorescent protein (GFP) or exogenously labeled with quantum dots (QDs) were co-cultured with UCB cells positive for a hematopoietic-specific CD45 marker. Cultured UCB cells without MSC layer served as control. Annexin-V, JC-1 dye & caspase inhibitors were used to investigate mitochondria-associated apoptosis pathway. Flow cytometry & confocal imaging were used to detect for the transfer of the GFP or QD in co-cultured UCB cells. Thawed UCB cells had high percentage of early apoptotic cells (defined by Annexin-V positivity) which progressed to dead cells (defined by 7AAD positivity). MSC co-culture resulted in reversal of Annexin-V positivity and reached a maximum on day 3 (viability defined by double negative staining for Annexin-V and 7AAD) (p < 0.05). A significant reduction in caspase 3/7, 8 & 9 activity as well as preservation of mitochondrial membrane potential were observed in the co-cultured UCB cells (p < 0.05). Upon co-culture with GFP-expressing or QD-labelled MSC, unlabelled UCB cells were found positive for these markers as early as 24 hours post co-culture. The number of GFP & CD45 positive UCB cells increased over the first 3 days of co-culturing, which correlated with viability increase. Direct contact between MSC & UCB was necessary for the transfer, as negligible transfer was observed when the two populations were separated by transwell inserts. Separation also showed a significant decrease in viability. The UCB cells that tightly adhered to the MSC layer exhibited optimal transfer & cell viability. DNA analysis of the viable, GFP transfer-positive UCB cells, as determined by Variable Number Tandem Repeat analysis ruled out the possibilities of MSC trans-differentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB cell viability (p < 0.0001). The above data provides evidence that intercellular transfer of cytosolic materials could be one novel mechanism preventing UCB cell death in MSC co-culture.