Expansion of Umbilical Cord Blood Hematopoietic Stem Cells for Clinical Use.

Abstract

Umbilical cord blood (UCB) is an alternative donor source for allogeneic hematopoietic stem cell transplantation. However, transplantation in adults is frequently limited by the small number of cells available in a unit. We have previously developed the technology to expand hematopoietic stem cells (HSC) and stromal/mesenchymal stem cells (SMSC) from all UCB frozen samples (13, 3). The incubation of thawed UCB mononuclear cells (MNC) in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB HSC (non-adherent). Also, long-term expansion of SMSC (adherent cells) was successful. In order to upscale the expansion to use for clinical applications, we analyzed 3 frozen UCB comparing the expansion from 24-well plates (previous) versus expansion in bags (VueLife™) after 10 and 14 days of culture. Results show substantial expansion in total cell count (TCC, 4.8, 10.9: 2.4, 3.8 fold) at d10 and d14 from wells & bags respectively. TCC increased further in the presence of SMSC (38% & 33% in CD34+ cell count cultured in wells). CD133+CD34+CD38- HSC multiplied (11, 25 fold, d10/d14 & colony forming cells (CFC) 19 fold at d14 bags respectively). Heterogeneous cell populations were detected after d 14 in bags: T and B -lymphoid (%CD3/%CD19; 65/4:50w/3), megakaryocytic (%CD61; 7:4) and myeloid (%CD33; 31:43) at d0/d14 respectively. Further, expanded cells (250,000) containing a small number of CD133+CD34+CD38- (15,000–30,000) were injected into the liver of sub-lethally irradiated newborn Balb/C Rag2-Cγ−/− mice. Our preliminary data show no engraftment from cells expanded in wells and bags after 6 weeks of transplantation from d10 cultures (human CD45 + <1%). However, positive engraftment in mouse PB was detected from cells expanded for 14 d (wells, 1.16–2.5% & bags, 1.21–3.9%) as compared to control mice (CD45; 30% PB & 70% BM) receiving selected CD34 + (300,000 CD34+ at d0). Primitive repopulating cells (PRC), and multilineage human CFC were detected after transplantation. On d14 of HSC expansion, UCB MNC derived adherent cells (SMSC) were enriched by trypsinization. SMSC were established in serum free and serum plus culture as well. The immunophenotype of harvested SMSC was CD29+, CD44+ and CD45−, CD34− and CD133− at percentages + >90%. Following repeated trypsinization, SMSC count increased 41–96 folds. CFU-Fibroblast colonies (92–173) were generated from 104 SMSC after 2 weeks in MesenCult medium. We have previously differentiated SMSC into hepatocytes. Now we also generated adipocytes in an induction medium containing, Insulin, dexamethasone & indomethacin. SMSC formed Oil-droplet vacuoles in the cytoplasm in 3 weeks. The culture conditions we defined to maintain UCB PRC should be developed clinically. SMSC described herein exhibit in vitro properties of multipotent stem cells.