Short-Term Ex Vivo Expansion of CD133+ Cells by Co-Culture with Mesenchymal Stem Cells: Role of SDF-1/CXCL12

CD133 (prominin-1) is the first in a class of novel pentaspan membrane proteins identified in humans and mice, and studies have since confirmed the utility of CD133 as a marker of stem cells with hematopoietic and non-hematopoietic lineage potential. A number of human transplantation studies have documented hematopoietic reconstitution from CD133+ stem cells from mismatched donors, with a suggested advantage over standard grafts in avoidance of graft versus host disease. We have developed a novel hematopoietic culture system (Long-Term Stem Cell Culture or LTSCC) to investigate the potential of human mesenchymal stem cells (MSC) to form stroma that can support short- and long-term hematopoiesis derived from cord blood (CB)-derived CD133+ cells. In addition, we analyzed the effect of stromal derived factor-1 (SDF-1/CXCL12) on survival and short-and long-term colony-forming capacity of CD133+ hematopoiesis. LTSCC induced stroma-like changes in the MSC feeder layer, with adipocyte formation, thought to be needed for formation of stem cell niches, and supported long-term (>9 weeks) survival of CB-CD133+ cells. Cobblestone areas of active CD133-derived hematopoiesis were seen in LTSCC for up to 9 weeks of culture. SDF-1/CXCL12 acted as a survival factor for CB-CD133+ cells and induced a significant ex vivo cell expansion at weeks 3 and 4 of LTSCC (maximal 500-fold increase), while maintaining the capacity for CFU-Mix and BFU-E colony formation up to 7 weeks. Long-term hematopoiesis was assessed by enumeration of long-term culture initiating cells (LTC-IC). When SDF-1/CXCL12 was added to LTSCC, we found a significant increase in LTC-IC: 0.3% (+SDF-1/CXCL12) vs. 0.05% (-SDF-1/CXCL12). Finally, homing capacity, as defined by SDF-1/CXCL12-induced adhesion and migration of CB-CD133+ cells, was maintained and even increased during the first 3 weeks of LTSCC. In summary, MSC can be maintained in LTSCC medium, and this simplified feeder layer is able to provide niches for cobblestone area forming cells derived from CB-CD133+ cells. SDF-1/CXCL12 is critical to support the survival and expansion of CD133+ cells, either directly or indirectly by paracrinesignaled retention of CD133+ cells in contact with specialized MSC niches. We suggest that expansion of CD133+ cells from cord blood may be useful in clinical transplantation limited by insufficient numbers of stem cells.