Hedgehog Signaling and Hematopoiesis Regulation: Preliminary Study in Myelosuppressed Adult Non Human Primate model.

Abstract 4603

The precise role of hedgehog signaling (Shh) in adult hematopoiesis is still debated. One one hand its implication in differentiation and proliferation of hematopoietic stem cells has been reported by different teams. Thus Bhatia et al showed using an immunocompromized mouse model that Shh allows in vitro amplification of CD34⁺/38^- pool. Shh may be especially invoved in megakaryocytopoiesis. In addition transgenic mice (ptc1/-) exhibiting an increased Shh activity has been shown to recover more rapidly than controls following myelosuppression and prolonged Shh stimulation appeared to deplete the stem cell pool due to cell cycle acceleration. On the other hand, recent studies also suggest that hedgehog signaling could be dispensable for adult hematopoieisis (Hoffmann-Zhang, Blood 2008 abstr 1391. The goal of this study was to set up an adult non human primate model to clarify some of these points. We chose a gene therapy strategy based on short term expression of Shh in bone marrow of highly irradiated monkeys (efficient stimulation and reduction of side effects). We first demonstrated the feasibility of transducing multipotent fibroblastic stem cells as vector cells using a piRES-2-Shh-eGFP plasmid and Nucleofector technology (Amaxa). As bone marrow nonhuman primate mesenchymal stem cells are known to be frequently contaminated by retroviruses, we used adipocyte derived stem cells as vector cells. In this model, Shh protein was detected during 5 days in the transduced cells and was secreted during 3 days in accordance with our short term secretion model. Then Shh-ASC were injected to rhesus monkeys the day after a global and frontal irradiation at the dose of 8Gy ⁶⁰Co gamma. At this dose, monkeys exhibited a severe and prolonged neutropenia and thrombocytopenia allowing us to evaluate the therapeutic benefit of the grafts. In a first grafted monkey (9kg), 2×10⁶ Shh-ASC (about 43% GFP positive cells) were injected in the femur. Blood cell counts did not significantly differ from irradiated control animals (n=4). Further experiments will contribute to determine whether Hedgehog signaling is involved in adult primate recovery following myelosuppression.