The Supportive Role Of Mesenchymal Stem Cells On Normal Hematopoietic Stem Cells Is Impaired In Leukemic Bone Marrow

Mesenchymal stem cells (MSCs) have been shown to be able to form a niche that maintains hematopoietic stem cells (HSCs). However, how the phonotypical characteristics and biological functions of MSCs are affected and especially whether their supporting role on normal HSC is impaired in leukemic microenvironment, are not well defined. Here, we have examined the changes of biological characteristics of mouse MSCs and in particular their supporting function on normal HSCs in Notch1-induced acute T cell lymphocytic leukemia (T-ALL) mice (Hu et al, Blood 2009; 114:3783-3792). MSCs were sorted based on the phenotypic markers, PDGFRα⁺Sca-1⁺CD45^-TER119^- (PαS) (Houlihan et al, Nat Protoc 2012) from control or T-ALL mice and flow cytometric analysis was conducted at different time points during leukemia development. Our results showed there was no significant difference between T-ALL and control mice regarding the absolute number and percentage of PαS MSCs in total bone marrow mono-nuclear cells (BMMNCs), cell cycling status and the percentage of apoptosis of freshly sorted PαS MSCs. Moreover, PαS MSCs from both normal and T-ALL leukemic marrow had similar morphology (spindle and polymorphic shaped), and uniformly expressed known cell surface markers for cultured mouse MSCs (CD29, CD44, Sca-1). However, the number of T-ALL PαS MSCs colony forming unit-fibroblastic (CFU-F) formation was 3-fold lower than control CFU-F (p<0.001). Levels of mRNA expression of genes associated with adipogenic (adipsin, mLP, PPARγ), osteogenic (Bmp4, Sp7, Bglap) and chondrogenic (collagen II, collagen X, aggrecan) differentiation in PαS MSCs were significantly decreased in T-ALL mice compared with control mice (p<0.05). These results suggest that the proliferation potential and the differentiation potential of MSC were decreased in the leukemic environment. To investigate whether the decrease in the proliferation of T-ALL PαS MSCs was associated with a change in cellular senescence, β-galactosidase activity and quantitative RT-PCR analysis of genes associated with senescence were performed. Our finding showed a significant increase in the number of β-galactosidase–positive cells (control/T-ALL; 3.11±0.20%/3.99±0.08%, p<0.05) and in the mRNA expression level of senescence-related gene p16 in PαS MSCs T-ALL mice compared with control mice (control/T-ALL; 1.00±0.03/1.57±0.17, p<0.05). These results imply that p16 plays an important role in PαS MSCs senescence in T-ALL microenvironment. To determine whether the supporting function of T-ALL PαS MSCs on normal HSCs proliferation is impaired, the cobble-stone area forming cell (CAFC) assay, an in vitro surrogate for HSCs, was applied. Normal control or T-ALL PαS MSCs were co-cultured with the same pool of normal HSCs. After 5 weeks, co-cultures containing normal PαS MSC formed multiple large CAFCs. On the contrast, T-ALL PαS MSCs supported 7-fold less hypocellular CAFCs (p<0.05). This data indicate that T-ALL PαS MSCs have a reduced ability to support normal HSCs proliferation in vitro. To further validate the change of PαS MSCs’ supporting function on normal HSCs proliferation in leukemic microenvironment observed in vitro, in vivo co-transplantation study were performed. Our preliminary results further indicated that the supporting function of MSCs on normal hematopoiesis in the leukemic microenvironment was compromised. In conclusion, dysfunction of MSC, an important component of HSC microenvironment, may play a crucial role in the suppression of HSC during leukemia development. Improving the function of MSC may serve as a new strategy to enhance normal hematopoiesis in leukemic marrow.