Capture and Expansion of Bone Marrow-Derived Mesenchymal Progenitor Cells with a Transforming Growth Factor- β 1–von Willebrand's Factor Fusion Protein for Retrovirus-Mediated Delivery of Coagulation Factor IX

Abstract

Mesenchymal stem cells give rise to the progenitors of many differentiated phenotypes, including osteocytes, chrondocytes, myocytes, adipocytes, fibroblasts, and marrow stromal cells, which are capable of self-renewal and undergo expansion in the presence of transforming growth factor-β1 (TGF-β1). The present study was designed to test the concept that mesenchymal progenitor cells could be selected and expanded by virtue of their intrinsic physiologic responses to TGF-β1. Human bone marrow aspirates were initially cultured, under low serum conditions, in collagen pads or gels impregnated with a genetically engineered TGF-β1 fusion protein bearing an auxiliary von Willebrand's factor-derived collagen-binding domain (TGF-β1-vWF). Histologic examination of TGF-β1-vWF-supplemented collagen pads from 8-day cultures revealed the selective survival of a population of mononuclear blastoid cells. Thee TGF-β-responsive cells were expanded to form stromal/fibroblastic colonies by serum reconstitution, and further to form osteogenic colonies upon supplementation with osteoinductive factors. In comparative studies, both marrow-derived progenitor cells and mature stromal cells were transduced with a retroviral vector bearing a human factor IX construct. Both the transduced progenitor cells and mature stromal cells expressed the factor IX transgene at levels comparable to those reported for human fibroblasts. Transplantation of murine progenitor cells bearing the human factor IX vector into syngeneic B6CBA mice resulted in detectable circulating levels of the human factor IX antigen. Taken together, these data demonstrate a novel physiologic approach for the selection of mesenchymal precursor cells followed by mitotic expansion, transduction, and transplantation of these progenitor cells with retroviral vectors bearing therapeutic genes.

Overview summary

In this study, transforming growth factor-β-responsive human, rat, and mouse mesenchymal progenitor cells were selected from bone marrow aspirates under serum-restricted conditions, expanded upon reconstitution with 10% fetal bovine serum, and stimulated to undergo cytodifferentiation by osteoinductive factors, indicating their mesenchymal origin. Proliferative mesenchymal precursor cells, as well as stromal cells, were amenable to retrovirus-mediated gene transfer, expressing a human factor IX transgene at levels that were comparable to those of transduced human fibroblasts. Transplantation of factor IX-vector transduced mesenchymal progenitor cells into recipient mice resulted in detectable circulating levels of human factor IX protein. Taken together, these finding demonstrate the potential utility of mesenchymal progenitor cells for the development of ex vivo gene therapy protocols for hemophilia B and other genetic disorders.