HIF1α-shRNA MSC–derived extramedullary bones show reduced leukemic cell engraftment. (A) After puromycin selection, the NS-shRNA MSCs (i) and HIF1α-shRNA (ii) MSCs exhibited similar morphology. Both of the MSCs differentiate along multiple mesenchymal lineages with induction of special media, including adipocytes (first row), chondrocytes (second row), and osteoblasts (last row; iii). The degrees of differentiation were determined by quantitative RT-PCR analysis for the expression of lineage markers, ABL1 was used as reference housekeeping gene (iv). No significant differences were found between NS-shRNA MSCs and HIF1α-shRNA MSCs. (B) NS-shRNA MSCs and HIF1α-shRNA MSCs were exposed to hypoxic conditions (1% oxygen) for different times. Western blot analysis showed significant down regulation of the HIF1α protein levels (i). SDF-1α transcription levels were diminished (∼ 30%; P < .01) in HIF1α-shRNA MSCs compared with control NS-shRNA MSCs (ii). Consistent with quantitative RT-PCR, HIF1α-shRNA MSCs secreted lower levels of SDF-1α than NS-shRNA MSCs (P < .05; n = 4; iii). (C) HIF1α accumulated in TRE HIF1α MSCs with the doxycycline administration in both hypoxia (1% pO₂) and normoxia (21% pO₂; i). This correlated with the significantly up-regulation of SDF-1α transcription and secretion (ii-iii). (D) Extramedullary bones with similar morphology and osteoblastic activity developed from NS-shRNA MSCs and HIF1α-shRNA MSCs. (E) Representative H&E (i-ii) and anti-GFP staining (iii-iv) shows different cell densities in the cavities of 2 types of extramedullary bones. (F) Slides stained with anti-GFP antibody were analyzed in a CRi system. Five images per slide were quantified and averaged at 3 different focal depths within the tissue section. The results showed a significant reduction of GFP⁺ leukemic cell density in HIF1α-shRNA MSC-derived extramedullary bones compared with the control MSC-derived bones (P = .0006). (1) NS-shRNA MSCs in panels A and B and extramedullary bones derived from NS-shRNA MSC-ECFC-Matrigel in panels D through F. (2) HIF1α-shRNA MSCs in panels A and B and extramedullary bones derived from HIF1α-shRNA MSC-ECFC-Matrigel in panels D through F.