Long-term expression of a retrovirally introduced ?-galactosidase gene in rodent cells implanted in vivo using biodegradable polymer meshes

Abstract

Grafts of various types of cells have been performed using bioresorbable polymer matrices. These synthetic fibers are degraded by hydrolysis into normal metabolic intermediates and induce a number of events that are conductive to healing and/or repair, the most important of which may be angiogenesis. The use of biodegradable meshes to deliver genetically altered cells was studied. A beta-galactosidase gene was inserted into Long-Evans rat bone marrow stromal (BMS) cells or fibroblasts derived from C57BL/6J mouse embryos using the retroviral vector LNL-SLX beta gal. Expression was monitored using X-gal staining. X-gal+ cells from monolayer cultures were seeded onto either polyglycolic acid (PGA) or polyglactin (PGL) biodegradable meshes and grown to confluence. Two types of grafts were performed: (1) embryonic C57BL/6J mouse fibroblasts (EMF) into either nude mice or adult C57BL/6J mice, and (2) Long-Evans rat BMS into Long-Evans rats. Beta-Galactosidase activity was found for up to 152 days for EMF in nude mice, 123 days for EMF in adult C57BL/6J mice, and 90 days for grafts of syngeneic BMS cells into Long-Evans rats. Noninfected cells grafted using the same methods did not stain with X-gal.

Human three-dimensional fibroblast cultures express angiogenic activity

“…These cell types have been considered the major sources of such factors in a woundhealing context. However, although little emphasis has been placed on the possible role of fibroblasts in controlling the wound healing process, preliminary studies have suggested that tissue formed by three-dimensional fibroblast cultures might demonstrate angio-genic activity when placed subcutaneously in the mouse (Naughton et al, 1992).…”

Enhanced maintenance and retroviral transduction of primitive hematopoietic progenitor cells using a novel three-dimensional culture system

“…In this regard, ents. 19,20 The absence of an appropriate microenvironment architecture in monolayer culture systems may be one reason that the addition of cytokines is instrumental cell research. Observations that three-dimensional culture systems can foster stem cell survival and maintain stem in supporting the growth of hematopoietic progenitors in these systems.…”

“…Interestingly, engraftment. 19,20 the TCPB cultures also yielded a population of CD34 + The evidence reviewed indicates that while at present cells that demonstrated a surface phenotype of no suitable system exists that is able to support optimally CD34 + 38 dim Thy-1 dim which is consistent with a popustem cell viability and pluripotency, three-dimensional lation of immature hematopoietic progenitors. 25 devices, either alone or in combination with exogenous Although represented in the BMS cultures, this popufactors or substrates, may offer substantial possibilities lation of cells is less evident than in the cells recovered in advancing these goals.…”

“…[16][17][18] Such systems have also proved useful ( Figure 1). Importantly, TCPB cultures also yielded in achieving gene transduction and long-term gene approximately twice as many CD34 + /CD38 − cells as expression 19 and may facilitate multilineage BMS cultures (P = 0.01, Student's t test). Interestingly, engraftment.…”

Sustained gene expression in transplanted skin fibroblasts in rats

“…When fibroblasts are in a quiescent state the choice of promoter for the transduced gene was a major determinant for long-term in vivo gene expression. 36,37 When dihydrofolate reductase (DHFR) and CMV promoters were compared, long-term expression of ␤-galactosidase was only achieved by DHFR promoter. 36 This difference may reflect inactivation of the CMV viral promoter whereas DHFR, a house-keeping promoter, can still be active in quiescent cells.…”