Expansion of umbilical cord blood mesenchymal stem cells

Abstract

7103 Background: Umbilical cord blood (UCB) is known to harbor 2 major types of stem cells, the hematopoietic stem cells (HSC) & the non-hematopoietic or mesenchymal stem cells (MSC). Under appropriate conditions, MSCs can give rise to cells of bone, fat, hepatic lineages, etc. Based on this potential, MSC hold promise for clinical applications in regenerative medicine. Methods: Stroma-free liquid culture: UCB cryopreserved mononuclear cells (MNC) were cultured in the presence of early growth factors: Flt-3 & SCF (25ng/ml), MGDF (10ng/ml) & human serum (10%). MNC derived adherent MSC were passaged at day 14 during HSC expansion & after enriching in MesenCult medium. Results: We developed a technology to generate & expand HSC & stromal/ MSC from all UCB units (5/5) at the same time using one culture system (stroma-free liquid culture). Following repeated passages, MSC count increased 357- 600-folds & CFU-Fibroblasts colonies (CFU-F) increased too (61–513 & 648–697) after 10 and 20 passages respectively. We used the CFU-F assay to demonstrate MSC activity in stromal cell formation in vitro. Phenotypically, MSC were negative for hematopoietic antigens (CD45, CD34 & CD14) & MHC class-II but >95% + for CD73, CD105, CD29, CD44 & MHC class I. To demonstrate MSC differentiation capacity in vitro, cells were incubated in various induction media to differentiate into adipocytes (fat)), osteoblasts (bone) and hepatocytes (liver) at passage 5. Following induction, positive staining with oil red O for cells of adipocyte and with alkaline phosphatase for cells of osteoblsts lineages was observed. The identity of hepatocytes was verified by the characteristic hexagonal hepatocytic shape as well as albumin, cytokeratin (CK) 18 and CK14 expression, as assessed by flow cytometry. Our data were corroborated by RT-PCR analysis. Conclusions: MSC described herein exhibit in vitro properties of multipotent stem cells. The established, stroma-free culture system facilitates expansion of MSC from all tested UCB units. Our data underline that it will be possible in the future to substitutes properly differentiated hepatocytes which might lead to efficient applications in patients suffering from various end stage liver disease. No significant financial relationships to disclose.