Evaluating the Mesenchymal Stromal Cell (MSCs) Contribution in Pre-Leukemic Myelodysplastic Syndromes in Different Patient Risk Groups

Introduction: Our current understanding of hematological cancers and methods for their treatment are largely derived from investigations of the hematopoietic system. However, studies in murine animal models now indicate a significant contribution from non-hematopoietic bone marrow mesenchymal stem/stromal cells (BM-MSCs) in the initiation and propagation of hematopoietic defects that resemble early leukemia (myelodysplastic syndromes). To study this possibility in human leukemia, we have established a repository of MDS patient derived MSCs and early studies have identified a group of these MSCs that can cause aberrant hematopoietic differentiation of human BM CD34⁺ in in vitro and in vivo systems. Future work will be focused on vigorous MSC characterization to elucidate mechanisms responsible the stroma contribution towards cancer formation.

Methods: BM MDS samples (n=20) were obtained from the Department of Hematology repository at Singapore General Hospital. Osteogeneic and adipogeneic differentiation as well as proliferation capacity, immunophenotyping and the ability of MDS-MSCs to support hematopoiesis in co-cultures were tested. Gene expression and methylation studies were performed on MDS-MSCs to investigate the degree of correlation with their ability to support hematopoiesis.

Results: Of the 20 MDS-MSC samples collected, 11 were classified as lower-risk MDS patients (< 5% marrow blasts) and 9 were higher-risk MDS patients (> 5% marrow blasts). Compared to healthy MSCs, cells from all MDS patients present non-fibroblast morphology and the early colonies also appear more disorganized. All MDS-MSCs express uniform levels of expression of known MSC markers such as CD73, CD90, CD105, CD166 and CD140B and are negative for CD45 and CD34. Expression of CD44 (n= 20; range 26% - 90%) and CD106 (n=20; range: 10% - 26.7%) was varied across MDS samples. MDS-MSCs have significantly reduced proliferative capacities (p=0.021) as well as reduced osteogeneic differentiation potentials (p<0.0001) compared to healthy MSCs. The total numbers of HSPCs or CD34+ cell fraction was reduced in MDS co-cultures compared to healthy co-cultures (3.13 x10⁶vs4.31x10⁶; n=8; p<0.05) and significantly reduced compared to expansion without MSCs (3.5x10⁵; n=3; p=0.0003 and p=0.0021). Co-culture of higher-risk MDS-MSCs showed a significant reduction in CFU-GM (16.6 ± 1.3; n=7; p<0.05) and CFU-GEMM (6.4 ± 0.4; n=7; p<0.05) lineages in comparison to healthy co-cultures (CFU-GM: 39.0 ± 1.0 and CFU-GEMM: 12.0 ± 1.0) respectively. However, these effects were not as evident in lower-risk MDS-MSC co-cultures. Following transplantation of co-cultured healthy HSPCs into NSG mice, we observed reduced HSPC engraftment in the higher-risk MDS experimental group compared to healthy controls at 8 weeks. qPCR analysis of these MDS-MSCs showed a consistent attenuation of gene expression associated with hematopoietic support (CXCL12, SCF, TPO, etc).

Conclusion: We show that MSCs derived from all MDS patients have phenotypic abnormalities but not all may have the potential support cancer development. In the identified higher-risk MDS-MSC group that impairs healthy hematopoiesis, we observed a more consistent attenuation of gene expression associated with hematopoiesis. We are further investigating the methylome and exosome-mediated transfer of RNA (from MSCs to HSPCs) as possible mechanisms for our observations.