Effective ex vivo expansion of hematopoietic stem cells using osteoblast-differentiated mesenchymal stem cells is CXCL12 dependent

Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine‐differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast‐differentiated B10 (Ost‐B10) as a feeder layer and examined ex vivo expansion of CD34+CD38− HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost‐B10 cells exhibited similar effects on total HSC expansion; however, Ost‐B10 demonstrated a higher potency in CD34+CD38− cell‐specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony‐forming cell assay and long‐term culture initiating cell assay revealed that Ost‐B10 displayed multipotent differentiation and enabled long‐term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost‐B10 cells compared with the B10 cells. CD34+CD38− cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost‐B10, whereas expansion of CD34+CD38− cells was decreased in Ost‐B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast‐differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway.