Cell Autonomous Defects of Mesenchymal Stem Progenitor Cells of Patients with CMML Contribute to Impaired Hematopoietic Supportive Activity

Objective:Increasing evidence indicates a critical role of the hematopoietic microenvironment in the pathophysiology of myeloid malignancies. Mesenchymal stem/progenitor cells (MSPCs) represent an indispensible cellular component of the bone marrow microenvironment and play an important role in supporting and regulating the proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs). Abnormal biological characteristics of MSPCs involved in the initiating and development of hematopoietic disorders, such as chronic myelogenous leukemia and myelodysplastic syndromes. However, the characteristics and the role of primary MSPC defects in the pathogenesis of chronic myelomonocytic leukemia (CMML) remain poorly understood.

Methods:MSPCs were generated from bone marrow aspirates of patients with CMML and that of healthy controls. To investigate the frequency of the MSPCs in the bone marrow of CMML patients and healthy controls in vivo, a colony-forming unit fibroblast (CFU-F) assay was performed. The phenotypic evaluation of the MSPCs was performed with flow cytometric analysis after staining with a cocktail of cell surface markers, including CD34, CD45, CD29, CD44, CD73 and CD105. To determine the functional difference between the CMML- and control-MSPCs, senescence, cell proliferation, and cell cycle were assessed by EDU incorporation assays, β-galactosidase activity, and flow cytometric analysis (following BrdU/7AAD staining, respectively. To determine the multi-lineage potential of MSPCs, MSPCs were subjected osteoblast, chondrocyte and adipocyte differentiation cultures. The hematopoietic supportive activity was assessed using cobblestone-area forming cell (CAFC) assays by coculturing of CD34+ cord blood cells with CMML- or control- MSPCs. Furthermore, to evaluate the soluble factors of these MSPCs in HSPC supportive activity, the transwell assay was performed with a subsequent flow cytometric analysis further determine the multi-lineage differentiation alteration. Cytokine array was used to determine the cytokines/growth factors in the MSPC conditioned media.

Results: The expression levels of the surface markers were identical between CMML-MSPCs and control MSPCs. The frequency of CFU-F is identical between healthy control and CMML patients. However, CMML-MSPCs exhibited a poor proliferative capacity compared to controls MSPCs. CMML MSPCs exhibited an increased senescence and a S phase retardation in the cell cycle as assessed microscopically and flow cytometrically, respectively. Differentiation assay revealed that CMML MSPCs had increased chondrocyte differentiation and reduced adipocyte differentiation, while no alteration of osteoblast differentiation was noticed. In vitro co-cultures of MSPCs and CD34⁺ umbilical cord blood cells demonstrated that CMML-MSPCs had an impaired hematopoietic supportive capability as compared to control MSPCs as evidenced by reduced numbers of cobble-stone and reduced numbers of CFU-C. Furthermore, a reduced CFU-C and a reduced CFU-GEMM were observed in CAFC and transwell cultures containing CMML-MSPCs compared to that of control MSPCs. In contrast, an increased myeloid cells (CD33+/CD14-/CD15+) and an increased CFU-GM were observed in CD34⁺ cells and CMML-MSPC co-cultures as compared to that in CD34⁺cells and control-MSPC co-cultures. Cytokine array showed that the conditional media of CMML-MSPCs has lower levels of multiple cytokines (IL-6, IL-8, and GRO-β) compared to that of control MSPC conditional media.

Conclusion: Collectively, our study indicated that MSPCs of CMML patients had reduced proliferation, increased senescence and reduced cytokine production, while differentiation was not markedly altered. CMML-MSPCs display less hematopoietic supportive activity. Furthermore, CMML-MSPCs induced CD34⁺ cell differentiation favoring granulomonocytic lineage over erythroid cells. Our study suggests that the cell autonomous defects of CMML-MSPCs contribute to the pathogenesis of CMML and the interaction between hematopoietic compartment and microenvironment could serve as targets for therapeutic interventions.