title: Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-beta1-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-beta1 (TGF-beta1). 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-beta1. Human bone marrow aspirates were initially cultured, under low serum conditions, in collagen pads or gels impregnated with a genetically engineered TGF-beta1 fusion protein bearing an auxiliary von Willebrand's factor-derived collagen-binding domain (TGF-beta1-vWF). Histologic examination of TGF-beta1-vWF-supplemented collagen pads from 8-day cultures revealed the selective survival of a population of mononuclear blastoid cells. The TGF-beta-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.
authors.affiliation: Department of Pediatrics, Childrens Hospital Los Angeles, and the University of Southern California School of Medicine, 90033, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: Gordon
authors.forename: M
authors.initials: M
authors.lastname: Skotzko
authors.forename: R K
authors.initials: RK
authors.lastname: Kundu
authors.forename: B
authors.initials: B
authors.lastname: Han
authors.forename: J
authors.initials: J
authors.lastname: Andrades
authors.forename: M
authors.initials: M
authors.lastname: Nimni
authors.forename: W F
authors.initials: WF
authors.lastname: Anderson
authors.forename: F L
authors.initials: FL
authors.lastname: Hall
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002452:Cell Count; D002469:Cell Separation; D003094:Collagen / Q000502:physiology; D005164:Factor IX / Q000235:genetics / Q000627:therapeutic use*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008808:Mice, Inbred CBA; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000494:pharmacology*
chemical_list: D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D014841:von Willebrand Factor; D005164:Factor IX; D003094:Collagen