Connective Tissue Growth Factor (CTGF) Regulates Mesenchymal Stromal Cell De-Differentiation Into Adipocyte Progenitors and Facilitates Leukemic Cell Homing to Extra-Medullary Bone Marrow

Abstract 2391

Connective tissue growth factor (CTGF) regulates extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Recent reports suggest that recombinant CTGF transforms mesenchymal stromal cells (MSCs) into fibroblast like cells and inhibits their differentiation potential. We have recently shown that stable knockdown of CTGF expression in bone marrow derived MSCs rendered them quiescent and increased their adipocyte differentiation potential (Battula et al., ASH 2010 abstract 3845, Blood, Vol. 116). Based on these findings, we hypothesized that inhibition of CTGF expression modifies stem cell properties of MSCs and may affect leukemic cell homing. To test this hypothesis, bone marrow derived MSCs were transduced with lentivirus expressing either CTGFshRNA or control vector. Down-regulation of CTGF mRNA by ∼75% was observed in CTGF-KD-MSCs compared to empty vector control-MSCs. To examine the stem cell potential of CTGF-KD-MSCs, embryonic stem cell markers including oct-4, nanog, sox2 were analyzed. Real-time RT-PCR analysis revealed 2–3 fold up regulation of these genes in CTGF-KD-MSCs compared to controls indicating increased stemness. To test if leukemic cells home differentially to CTGF-KD-MSCs, we utilized our recently developed extra-medullary human bone marrow model by implanting a carrier impregnated with BM-MSC mixed with endothelial progenitor cells (EPCs) subcutaneously into NOG mice. These implants generate extra-medullary bone marrow (EXM-BM) in 6–8 weeks and resemble the normal bone marrow microenvironment. In-vivo imaging confirmed that both cell types formed EXM-BM with positive Osteosense binding which reflects the presence of hydroxyapatite. H&E staining of bone sections revealed that CTGF-KD-MSCs derived EXM-BM displayed more spongy bone compared to control-MSCs. To examine the extent of adipocytic differentiation in CTGF-KD-MSCs derived bone pellets, expression of adipocyte-specific markers PPAR-γ and C/EBPα was analyzed. Cells residing in the spongy EXM-BM region expressed high levels of nuclear PPAR-γ and C/EBPα, confirming differentiation into mature adipocytes. These markers were negative in the stromal/endothelial compartment of CTGF-KD-MSCs derived EXM-BM or control EXM-BM. When Nalm-6 (B-ALL cell line) cells expressing firefly luciferase and YFP were transplanted into these mice, leukemic cells migrated 10 fold more avidly towards CTGF-KD-MSCs derived EXM-BM compared to control EXM-BM in the same mouse (n=5). Immunohisto-chemical analysis demonstrated preferential homing of leukemic cells inside spongy regions of the EXM-BM derived from CTGF-KD-MSCs and control-MSCs. To investigate the mechanism behind the leukemic cell homing into CTGF-KD-MSCs derived EXM-BM, expression of stromal cell-derived factor-1α (SDF1α), a major factor involved in leukemic cell homing to bone marrow, was analyzed in both cell types by qRT-PCR. CTGF-KD-MSCs expressed 3-fold higher levels of SDF-1α compared to control cells indicating that SDF1α secretion by CTGF-KD-MSCs derived bone marrow might enhance the leukemia cell homing. In summary, these findings suggest that CTGF plays a crucial role in the regulation of stemness and differentiation of MSCs and affects the homing of leukemic cells to the bone marrow microenvironment. Targeting adipocyte progenitors may reduce the lodging of leukemic cells in the bone marrow niche and enhance chemosensitivity of leukemic cells within the BM microenvironment.