radiation fibrosis resistant SMAD3 -/- mice demonstrate superior donor bone marrow stem cell transplantation capacity by competitive repopulation assay

Smad3 -/- mice are resistant to radiation fibrosis (Flanders, Am J. Path, 163:2247-2257, 2003; Epperly, In Vivo 20:573-582, 2006) attributable to inhibition of Smad3 pathway -mediated TGF-B signal transduction. We tested the hypothesis that abrogation of TGF-B signaling in Smad3 -/-(129/Sv) marrow stem cells increased engraftment capacity into the TBF-B induced marrow microenvironment of irradiated recipient mice. The number of bone marrow stem cells in situ was determined by flow cytometry. TGF-B sensitivity was determined by measuring CFU-GEMM inhibition in vitro by TGF-B treatment of freshly explanted marrow from Smad3 -/-(129/Sv) compared to C57BL/6NTac mice. Competitive repopulation assay of Smad3 -/- and C57BL/6 bone marrow stem cells were performed by mixing bone marrow from male and female mice, injecting into TBI irradiated mice and PCR performed to detect marrow male Y chromosome to demonstrate increased stem cell repopulation capacity. Smad3 -/- mice showed 95.0 stem cells per 107 bone marrow cells compared to 35.7 stem cells per 107 bone marrow cells for C57BL/6NTac mice (p = 0.0342). CFU-GEMM formed by Smad3 -/- bone marrow was not inhibited by TGF-B or altered by the TGF-B receptor antagonist SD208 while C57BL/6NTac bone marrow CFU-GEMM was inhibited by TGF-B ( 20 and 50 ng/ml) (p = 0.0084 and 0.0069, respectively). Competitive repopulation assay demonstrated that Smad3 -/- bone marrow had increased repopulation capacity by Y chromosome detected in all mice injected with as few as 0.1 X 106 male Smad3-/- bone marrow cells mixed with 0.9 X 106 female C57BL/6NTac bone marrow cells. In contrast, C57BL/6 male Y chromosome was not detected in mice injected with significantly higher ratios of control marrow (0.25 X 106 male C57BL/6 bone marrow cells mixed with 0.75 X 106 female Smad3 -/- bone marrow cells ) (p = 0.0460). Smad3 -/- mice have increased baseline bone marrow stem numbers, resistance to inhibition of committed multilineage progenitor cell proliferation by TGFB in vitro, and superior in vivo engraftment capacity by competitive repopulation compared to control C57BL/6NTac mice. The data support the hypothesis that inhibition TGFB signaling may be of therapeutic benefit to marrow transplant recipients. Supported by NIAID/NIH U19-A1068021.