Modulation of Interaction of Human Osteoprogenitor Cells with Hematopoietic Stem and Progenitor Cells

Osteoprogenitor cells (OPCs) are marrow microenvironmental cells known to modulate hematopoietic stem and progenitor cells (HSPCs). Specifically, OPCs regulate HSPCs in response to Parathyroid hormone (PTH) treatment in murine models. However, the role of OPCs in human HSPC regulation and whether human OPCs can be manipulated is poorly understood. Niche stimulation is an appealing strategy to aid in the treatment of hematopoietic dysfunction. Myelodysplastic syndromes (MDS) are clonal disorders with ineffective hematopoiesis resulting in cytopenias and risk of transformation to acute leukemia (AML). In mouse models, disruption of the osteolineage cells can contribute to initiation of ineffective hematopoiesis with phenotypic features of MDS. Our long term goal is to utilize microenvironmental stimulation as a therapeutic tool to improve hematopoietic disorders. We hypothesized that human cells isolated from the marrow fraction containing spicules harbor HSPC supportive cells, which can be manipulated to improve HSPC support. Moreover we hypothesized that OPC number and function is impaired by dysplasia-initiated microenvironmental disruption as a potential mechanism for reduced support of HSPCs and ineffective hematopoiesis. Our objective was to isolate human bone marrow spicule associated cells (SACs) and define their ability to support HSPCs, determine the impact of PTH treatment of SAC/HSPCs interactions and characterize dysplasia-induced osteolineage changes in human MDS and AML bone marrow. To achieve this objective, we used normal as well as MDS/AML patient-derived OPCs using a mouse-human co-culture system.

Human bone marrow SACs isolated by collagenase digestion were either used for co-culture, analyzed with flow cytometry or cultured in mineralization media in limited dilutions.

To assess the potential impact of PTH on human OPC interaction with HSPCs, we developed a 7 day co-culture of human bone marrow SACs treated with either vehicle or PTH, with mouse Lineage- Sca1+ c-Kit+ (LSK) hematopoietic progenitor cells. At the end of the co-culture, all cells present were used for competitive transplantation. Transplant experiments demonstrated that PTH treatment of the human bone marrow SACs leads to improved function of the co-cultured LSK cells as demonstrated by significantly improved engraftment of the LSK cells after transplant into irradiated C57/bl6 recipient mice when sampled at pre-specified time points over a 20-week period (N=12, 2-way ANOVA; p < 0.05).

Flow cytometry analysis showed that mature (Lin- CD31- CD146+ CD105-) and immature osteolineage (Lin- CD31- CD146+ CD105+) cells were present in SACs and more abundant compared to within BMMCs (1% vs 0.1% and 0.24% vs 0.12% for the same patient). Notably, the putative HSC-supportive MSC pool was increased in SACs vs BMMCs (0.052% vs 0.019%). The presence of OPCs was functionally confirmed using colony forming unit osteoblasts (CFU-OBs). CFU-OB frequency was calculated using L-Calc ^TM (StemCell technologies). Among normal donors the frequency of CFU-OBs was low in marrow donors >50 years old compared to <50 years old donors (2.240e-005 ± 3.300e-006 N=2 vs. 0.0001146 ± 4.163e-005 N=9). We identified non-statistically significant decrease in the frequency of CFU-OBs in bone marrow SACs from MDS patients compared to normal donors (1.090e-005 ± 1.400e-006 N=2 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.179); and similar decrease in frequency of osteoprogenitor cells in the bone marrow aspirates from AML patients compared to normal donors (2.303e-005 ± 9.371e-006 N=3 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.251). These data support our hypothesis that OPCs in patients with MDS and AML are negatively impacted compared to normal bone marrow.

These data demonstrate that human SACs contain HSPC-supportive cells which can be stimulated to improve HSPC function. Human SACs comprise MSCs and osteolineage cells including osteoprogenitor cells. Aging decreases OPC pools in SACs. Our data in our small sample also suggest that dysplastic bone marrow microenvironment may negatively impact OPCs, which may in turn decrease OPC support of HPSCs. PTH treatment in our in-vitro model shows the potential to improve the interaction between the OPCs and HSPCs, resulting in amelioration of HSC function. Together these data suggest a strategy where targeting the MDS microenvironment may add to the currently available treatment modalities.