CD133⁺ Purified Hematopoietic Stem Cells in Co-Culture with Mesenchymal Stromal Cells - The Cell to Cell Contact Matters.

Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became aware that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD133 purified HSC expansion and differentiation in terms of phenotype, (CXCR4) chemokine receptor expression, migration capacity and clonogenicity. After one week of co-culture with MSC adherent and non-adherent HSC were isolated and analyzed separately. As expected in co-culture experiments with MSC a significant higher expansion of TNC was observed in comparison to the expansion culture without MSC (cytokine driven), 37±6 fold vs. 19±3 fold, p<0.001. However the co-culture expansion was accompanied by a significant increase of differentiation and maturation of the progenitor cells. Therefore we were focusing on the adherent cell fraction of the co-culture system. According to FACS analysis results, adherent HSC in the co-culture display a more immature phenotype than non adherent cells (CD34⁺CD38⁻). Next colony assays and LTC-IC were performed showing that the adherent cell fraction contained a significant higher proportion of primary and secondary CFU-C, indicating that this cell fraction contains the majority of clonogenic HSC. The CXCR4 - SDF-1 axis plays an important rule in homing, mobilization and engraftment of the hematopoietic stem cells. Therefore we looked for CXCR4 expression in the two cell fractions using FACS analysis. The adherent cells expressed consistently and significantly more CXCR4 than the non adherent cells; 43±4% vs 15±2%, p<0.001. Data could be confirmed by performing western blot. Due to this data we evaluated the capacity of HSC migration toward SDF-1 gradient using transwell migration assay. The migration capacity of the adherent cells was significantly higher than that of the non adherent cell fraction correlating with the CXCR4 expression; 13±1% vs. 6±1%, p<0.001. We checked the expression of different cell adhesion molecules by FACS analysis. CD44 and CD49e (integrin α5) were significantly higher expressed in adherent cells comparing with non-adherent cells. Our data indicate that the cell-to-cell interaction mediated impact on CD133⁺ selected HSC leads to an enhanced clonogenic capacity and migratory potential. Further experiments are needed to investigate engraftment and NOD/SCID repopulation potential of the adherent and non-adherent cells after ex-vivo expansion.