Several lines of evidence suggest that gene traniduction into pluripotent stem cells is inefficient One of the reasons which may account for the difficulties is the quiescent status of stem cells. Since fetal progenitors are known to have higher protiferative potential than adult progenitors, we investigated the efficiency of retrovinl gene transfer into fetal liver progenitors and the subsequent expression of the transgene using both hi vitro and in vivo assays. CD34+4CD38-Lin cells were isolated by cell sorting and infected using the following protocol: 2 days of uqmdoillure followed by 3 days of co-culture with the murine cell line A7.21, which was derived from Psi Crip and produces defective retroviruses encoding modified version of E. Coti b-galactosidase (MFC nlsLacZ). Cells were cultured in the presence of FCS and EL3, DA GM-CSF, KL ±FIk2/Flt3 ligand. X-gal staining was performed to detect the cells expressing the transgene ($-gal+). After infection, β-gal+ clonogenic cells were found in 4-27% and 6-19% of HPP- and LPP-CFC respectively, indicating the successful gene transduction into clonogenic progenitor cells. Genetically modified fetal liver cells were then injected into the human fetal bone marrow or human fetal thymus previously engrafted in SCID mice. Human bone marrow grafts were analyzed 4-7 weeks post-injection. High levels of reconstitution by donor-derived cells were observed in ate CD33+ myeloid-<45-78%). CD 19+ B ccll-<9-57%), and CD34+ progenitor- (13-73%) populations, as shown by HLA typing of marrow cells. Clonogenic assays reveakd that up to 20% of HPPCFC. LPP-CFC, CFU-GM as well as BFU-E recovered from SCID-hu mice expressed β-gal. In cytospin preparations of total marrow cell, both myeloid and lympnoid cells were found to express β-gal. However, the percentages of β~ga!+ cells in total marrow cells were extremely low, in contrast to the high levels of reconstitution by β-gal+ cells in the progenitor compartments. Human tbymic grafts were analyzed 6 to 16 weeks post-injection. 0.1-10% of thymocytes expressed β-gal in cytospin preparations. Collectively these results demonstrate that highly purified CD34++CD38-Lin- fetal liver progenitors are successfully gene transferred by retroviral vectors under the conditions used. The observation that β-gah- cells were found in T cells, B cells, myeloid cells and clonogenic progenitors after in vivo reconstitution strongly suggests that the gene transduction occurs in the primitive, pluripotent progenitors.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1996|