Abstract
Pleuripotent Adipose Stromal Cells (ASCs) have the capacity to differentiate into mesodermal lineage cells such as adipocytes, myocytes, osteocytes and chondrocytes. In addition, the ASCs can trans-differentiate in vitro into hepatogenic, neurogenic, endothelial and endocrine cells. Since hASCs are relatively easy to isolate in large quantity and have high proliferative potential, they provide a potential source of stem/progenitor cells for autologous cell therapy for the regeneration of injured tissue. The goal of this study is to isolate and engraft adipose stromal cells (ASCs) to generate new tissue capable of secreting therapeutic plasma proteins. In particular, can one engraft genetically modified ASCs in a recipient that secrete coagulation factors for the treatment of hemophilia? Hemophilia A and B result from too low levels of plasma Factor VIII or FIX activity, respectively. The aim of the project is to generate hASC derived tissue capable of secreting FVIII or FIX to support hemostasis in hemophilic mice. Unlike murine ASCs, undifferentiated human ASCs express FVIII at low but significant levels (0.03 ug/ml per 105 cells in 48 hours; ∼30% of normal human plasma FVIII level). ASCs were transfected with a plasmid expressing a modified B-domainless FVIII cDNA. Following differentiation into preadipocytes, the level of FVIII secretion increased 3-fold. In preliminary studies, expression of FIX is detectable in hASCs and the level increases following differentiation into preadipocytes. These results indicate that ASCs and preadipocytes are capable of gamma carboxylation. FIX expression was further increased following infection with a lentivirus carrying a FIX expression cassette. Since HGF has been shown to elicit a potent proangiogenic response (Cai et al., in press), hASCs predifferentated into preadipocytes were mixed in hydrogel containing HGF and injected subcutaneously into SCID mice. Oil red O staining cells were recovered in a cellular nodule at the injection site 3 weeks post-transplantation. Preliminary studies indicate that co-injection of endothelial progenitor cells (EPCs) with ASCs promotes the development of more highly vascularized nodules. In summary, hASCs and hASC-derived preadipocytes cultured ex vivo express low levels of coagulation Factors VIII and IX. These levels can be increased following transfection with FVIII or FIX expression vectors. Furthermore, transplantation of hASCs cultured in adipogenic media results in the formation of preadipocyte/adipocyte containing nodules. These results suggest it may be possible to generate tissue derived from genetically modified hASCs capable of secreting coagulation factors for the treatment of hemophilia.
A Monte Carlo framework for managing biological variability in manufacture of autologous cell therapy from mesenchymal stromal cells therapies
