Abstract

There are multiple applications for expansion of Hematopoietic Stem Cells (HSC) and progenitors in transplantation. Gene therapy for most hematopoietic diseases requires selective expansion of genetically corrected HSC to achieve therapeutic effects. Obtaining sufficient number of HSC (CD34+) is a limitation for use of cord blood (CB) in transplantation. Delayed immune recovery following HSC transplantation is associated with increased morbidity and mortality. Murine lymphoid recovery can be hastened by co-transplantation of Common Lymphoid Progenitors (CLP) with HSC, however, the rarity of human CLP in harvested products preclude their clinical use. We hypothesized that human HSC and CLP can be selectively and reversibly expanded by expression of a fusion protein comprised of the intracellular signaling domain of Thrombopoietin receptor (mpl), linked to a specific binding domain (F36V) for the chemical inducer of dimerization AP20187 (CID) (ARIAD Pharmaceuticals). Upon binding of CID to F36V, mpl signaling occurs. A lentiviral vector expressing the fusion protein (F36V-mpl) and a marker gene (GFP) was constructed and efficiently transduced and expressed in human CB HSC (CD34+CD38-CD7-) and CLP (CD34+CD38-CD7+). CID-induced mpl signaling in transduced human CLP maintained robust generation of total, B and NK cells for > 60 days, in cytokine free lymphoid cultures (N=4). Under these conditions transduced HSC cultures (N=4) maintained robust generation of total, B, NK cells for >120 day. Transduced HSC continued to generate clonogenic myelo-erythroid progenitors for > 120 days in ELTC-IC assay (N=4). These cytokine free in vitro assays indicate that CID-induced mpl signaling in human HSC and CLP induced prolonged survival and proliferation of transduced progenitors. Most of the cells generated in the presence of CID expressed GFP (mean 86% GFP+), indicating selective proliferation of transduced cells. Rapid decline in the number of cells expressing GFP was noticed upon withdrawal of CID, indicating reversible activation of mpl signaling in the transduced cells. In contrast, transduced human HSC and CLP cultured without CID, proliferated poorly and differentiated rapidly; viable cells were lost by day 22 of culture. To study whether cells stimulated by CID remained immunophenotypically and functionally primitive after dividing, cell divisions were tracked by labeling with the membrane dye PKH26. Proliferation index of transduced HSC was consistently higher in the presence of CID than in its absence. Human CD34+ cells that had undergone 3 divisions in the presence of CID and in absence of any cytokines, maintained a primitive CD34+ immunophenotype in vitro. In contrast, cells that divided in the absence of CID lost CD34 expression. Cells cultured ± CID were isolated after 3 divisions (based on PKH26 staining) and transplanted in equal numbers (40,000 cell/mouse) into sublethally ablated NOD/SCIDb2m −/− mice to assess function. Only CID-expanded cells were able to engraft, producing B lymphoid and myeloid progeny. Bone marrow harvested from the engrafted animals (N=3) contained clonogenic human myelo-erythroid progenitors (confirmed by Alu PCR of human specific CFU); cells cultured without CID failed to engraft. These studies show that CID-induced mpl signaling expands functionally primitive multipotent, engrafting human progenitors. This is a potential approach to selectively and reversibly expand transduced primitive human progenitors for use in cell therapy.

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