Impaired Reconstitution Potential of TGFβ-Hypersensitive Human Hematopoietic Stem Cells

Abstract 394

Hematopoietic stem cells (HSCs) are primitive, tissue-specific cells that can self-renew and differentiate along all lineages of the blood system. These properties make the HSCs critical for tissue regeneration and clinical applications in cell therapy. Cord blood (CB) is an accessible source for HSCs. However, the yield of HSCs from one cord is too low in order to successfully transplant adult patients. The expansion of HSCs in vitro has met with limited success due to incomplete knowledge regarding the mechanisms regulating self-renewal. Members of the transforming growth factor-β (TGF-β) superfamily have been shown to regulate HSCs through the downstream Smad signaling pathway. TGF-β1 potently inhibits HSC growth in vitro, and overexpression of the inhibitory Smad7 has been demonstrated to increase in vivo self-renewal of murine HSC, indicating that the Smad pathway negatively regulates self-renewal (Blank et al. Blood, 2006). However, disruption of the entire Smad pathway in HSCs through conditional deletion of the common Smad4 resulted in reduced repopulative capacity (Karlsson et al. JEM, 2007). These findings demonstrate the complexity of Smad signaling and highlight the importance to investigate it further. Therefore, we asked whether enforced expression of Smad4 could reveal a role for TGF-β in human HSCs regulation in vivo or affect self-renewal and regenerative ability of HSCs in vitro.

To investigate the effect of Smad4 overexpression in hematopoiesis, full-length cDNA of human Smad4 was cloned in to a lentiviral vector carrying a GFP reporter gene, referred to as Smad4 vector. As control, a lentiviral vector carrying GFP only, referred to as control vector, was generated. Human CB HSCs overexpressing Smad4 displayed increased sensitivity to TGF-β in colony assays (TGF-β treated-/untreated growth: 0.22 ±0.04 vs. 0.32 ±0.04 for Smad4 vector and control vector, respectively P=.0197). Importantly, the addition of a TGF-β inhibitor targeting ALK4, 5 and 7 receptors (SB431542) rescued the colony forming capacity (TGF-β treated-/untreated growth: 0.6 ±0.046 vs. 0.72 ±0.078 for Smad4 vector and control vector, respectively) demonstrating the functional overactivity of the TGF-β pathway in Smad4 overexpressing cells. Since TGF-β is a well-known growth inhibitor of hematopoietic progenitors (Batard et al. JCS, 2000; Cashman et al. Blood, 1990; Sitnicka et al. Blood, 1996) we further analyzed cell cycle status of transduced cells. Cells with enforced expression of Smad4 and increased TGF-β sensitivity were to a larger extent in the quiescent state of the cell cycle (G₀) compared to control cells when cultured for six days (16.54 ±5.70% vs. 7.84 ±0.51% for Smad4 vector and control vector, respectively P=.0286) but could be released from G₀ when treated with the inhibitor SB431542. Moreover, as TGF-β also is known to induce apoptosis (Jacobsen et al. Blood, 1995) we further investigated if enforced expression of Smad4 would affect apoptosis in cultured CB cells. After six days of culture Smad4 overexpressing cells had significantly higher AnnexinV expression compared to control cells (25.74 ±3.81% vs. 15.45 ±4.44% for Smad4 vector and control vector, respectively P=.0281), an effect that also was decreased when adding the inhibitor SB431542 to the culture (20.38 ±5.96% vs. 16.25 ±6.35% for Smad4 vector and control vector, respectively). Furthermore, we transplanted transduced CB cells into NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ mice. Interestingly, despite having similar transduction efficiency as the empty vector control (30 ±16% vs. 29 ±13% for Smad4 vector and control vector, respectively) CD34⁺ CB HSCs transduced with the Smad4 vector had impaired engraftment as measured by FACS analysis of peripheral blood (PB) (Smad4 vector 1.03 ±1.3% GFP vs. control vector 2.94 ±1.97% P=.0035) and bone marrow 6 months post transplantation (Smad4 vector 1.5 ±0.88% GFP vs. control vector 5.60 ±1.54% P=.0029). Expression of lineage surface markers (CD13, CD15 and CD19) in PB 3 month post transplantation was unaltered.

In summary, our results demonstrate that increased Smad4 expression sensitizes human CB HSCs to TGF-β. This leads to growth arrest and apoptosis in vitro and reduced HSC reconstitution capacity in vivo with no effect on lineage distribution. Together, these findings demonstrate an important role for TGF-β signaling in the regulation of human HSCs in vivo.