Expansion of Cord Blood Stem Cells and Enhancing Their Mobilization and Homing Potential Using Mesenchymal Stromal Cells

Umbilical cord blood (CB) contains hematopoietic stem cells (HSC). CB can be collected easily, cryopreserved and be readily available from CB banks when needed. Transplantation with CB is associated with less graft-versus-host disease (GvHD) when compared to peripheral blood (PB) and bone marrow (BM) HSCT. Attempts have been made to overcome the issue of low and insufficient number of CB stem cells.

Mesenchymal stromal cells (MSCs) has been proven as immune modulators in GvHD and other immunological diseases. MSCs may enhance HSC engraftment to improve hematopoietic recovery. There is compelling evidence that MSCs produce a wide range of cytokines that are capable of maintaining HSC in a quiescent state and other cytokines which induces proliferation and self-renewal of HSC.

Recently, it has been shown that expansion of HSC in unfractionated CB is markedly enhanced by co-culture with MSCs, with the time to neutrophil engraftment and platelet recovery markedly shortened. CB stem cells expanded with allogeneic MSCs then transplanted into patients led to multiple times expansion for both total nucleated cells and CD34⁺ cells in vivo when compared to unmanipulated CB stem cells. Therefore, expansion of CB stem cells with MSCs has great potential. The exact mechanism of how CB cells expand at the molecular level remains unknown. This study's aim was to expand CB in an ex vivo coculture system using MSCs and selected growth factors (GFs) and to investigate expansion at the molecular level.

In this study, CB cells were expanded in a coculture system for two weeks using BM-derived MSCs with and without GFs (SCF, TPO, Flt3-L, G-CSF and IL6). A 69.9 fold expansion in CB total nucleated cells and 53.2 fold expansion in CD34⁺ cells was achieved after 12 days using MSCs as feeders in 50 ng/ml Flt3-L, SCF, TPO, G-CSF, and IL6.

The viability of CB cells was significantly higher when cocultured with MSCs regardless of GFs addition. CB expanded on MSCs expressed higher percentages of CD45⁺CD34⁺CXCR4⁺ and CD45⁺CD34⁺EpHB4⁺ populations. Interestingly, the HSC maintenance marker EpHB4 expressed by MSCs at low levels decreased significantly in the coculture system.

To investigate MSCs effect on CB expression levels of other genes that are important for expansion, stemness and pluripotency hematopoietic lineage commitment, RUNX1, SOX17, HOXC8, Myc, TP53, SOX9, FOXO1, FOXO4, GATA1 genes were examined. The study demonstrated that HOXC8, SDF-1, SOX17 and SOX9 expression was suppressed in CB cells, while the expression of other genes such as CXCR4, EpHB4, FOXO1, Myc, and HPRT1 increased.

The undetectable level of HOXC8 expression, which regulates self-renewal and differentiation of stem cells, may have caused less CB stem cell differentiation, favoring an increase in CD34⁺ cells. Equally, SOX17 and SOX9 expression, which also decreased to undetectable levels in CB cells post coculture with MSCs, plays a significant role in CB expansion. SOX17 is known as a primer of hemogenic potential, regulating hematopoietic development from hESCs/iPSCs, whereas SOX7 plays an important role in HSC differentiation. Therefore, we speculate that MSCs-CB coculture dampens the differentiation of adjacent CB cells, associated with changes of signaling in BM-like niches.

Simultaneously, MSCs may be sending signalling messages to CB cells in the ex vivo niche to maintain quiescence or self-renewal capacity through other signalling pathways. FOXO family has the ability to regulate stem cells and program them to remain quiescent through cell-cycle repression via the oxidative stress-activated P66shc-Akt-FOXO pathway. In this study, FOXO1 was upregulated in CB cells cultured with MSCs supporting our hypothesis and other earlier findings (decrease in the expression of EpHB4 on MSCs and a decline in the expression of SDF1 in CB cells), suggesting that MSCs have the ability to induce stem cell quiescence and maintain stemness.

In conclusion BM-derived MSCs support CB viability, expansion, and increased stemness potential, by modifying key hematopoietic, progenitor, differentiation and stemness signaling pathways. The study identified HOXC8, SDF-1, SOX17, SOX9, CXCR4, EpHB4, FOXO1, Myc, and HPRT1 as potential factors involved in CB expansion signaling pathways. Using MSCs as a feeder layer resulted in higher CB viability and proliferation rates which may increase the potential use of CB in hematopoietic stem cell transplantation.