Hematopoietic Stem Cells Overexpressing Smad7 Exhibit Increased Self-Renewal and Regeneration Capacity in Vivo.

The hematopoietic stem cell (HSC) resides in the bone marrow (BM) and can self-renew to generate more stem cells as well as differentiate into all hematopoietic lineages. The precise molecular mechanisms, which govern HSC fate decisions are poorly understood. The Transforming Growth Factor-β (TGF-β) superfamily of ligands, including the TGF-βs, Activins and Bone Morphogenetic Proteins (BMPs), encompasses an important group of growth factors, many of which have been shown to modulate and regulate hematopoiesis. Smad7 is known to block the phosphorylation event of receptor-activated Smads, thus creating a block in the entire signaling cascade downstream of TGF-β and related factors. To assess the effect of blocking the entire Smad signaling pathway downstream of TGF-β/Activin and BMP in HSCs in vivo, we have overexpressed the inhibitory Smad7 by a retroviral gene transfer approach. Both control and Smad7 vectors were MSCV based and expression was driven by the LTR promoter. The Smad7 vector contained the cDNA sequence for murine Smad7 and an internal ribosomal entry site (IRES) followed by GFP, whereas the control vector contained IRES and GFP only. In these experiments BM from wild type C57/B6 mice could efficiently be transduced with Smad7 or control vectors respectively. Upon transduction, cells (Ly5.2) were transplanted in a competitive fashion into lethally irradiated recipients (Ly5.1) and transduced cells were monitored by GFP fluorescence. Smad7 overexpressing cells were able to long-term reconstitute as well as give rise to both lymphoid and myeloid compartments at normal distributions. When self-renewal was assessed by secondary transplantations, Smad7 overexpressing cells showed significantly increased reconstitution ability compared to control transduced cells (blood samples at 12 weeks post transplant: 37.3 ± 5,9 for Smad7 vs. 5.06 ± 1,7 for control. Data represent % GFP positive cells ± SEM). Furthermore, Western blot analysis showed efficient expression of Smad7 protein in BM cells originating from transduced cells of transplanted mice. In addition, Smad2 and Smad1 phosphorylation was blocked upon TGF-β, Activin or BMP stimulation in BM cells, suggesting that Smad7 was functionally active in BM cells in vivo. However, when cultured under serum-free conditions in vitro, Smad7 overexpressing cells exhibited reduced proliferative capacity as compared to control transduced cells (3.5 times fewer cells by day 12 post transduction), suggesting that the in vivo phenotype was dependent on the BM microenvironment. Taken together, our data indicate that blocking of several TGF-β pathways simultaneously increases the self-renewal ability of HSCs in vivo, but not in vitro.