High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

Abstract

Objectives: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. Methods: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38–CD34++lineage– (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (ΔNGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 µg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10–21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. Results: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.

Feasibility of Retroviral Vector-Mediated in utero Gene Transfer to the Fetal Rabbit

“…It is known that fetal development results in rapid liver growth [42] , and therefore its size, accessibility, and straightforward localization in the fetal rabbits makes this organ a good target for gene transfer. Moreover, it has been recently shown that fetal liver stem cells can undergo effi cient retroviral vector-mediated gene transfer ex vivo [43] . Likewise, a recent report has also demonstrated that lentiviral vector transduction effi ciency and transgene expression into hepatocytes in vivo is signifi cantly enhanced at early developmental stages [44] .…”

Efficient engraftment of in utero transplanted mice with retrovirally transduced hematopoietic stem cells

“…29,30 In addition, experimental studies with the ovine model showed the possibility of collecting fetal liver cells in sufficient numbers for the autologous transplantation of the animals. 31 The combined use of ex vivo HSC gene therapy and IUT may improve the therapeutic potential of both strategies used as individual therapies, thus facilitating the treatment of diseases diagnosed during fetal development.…”

Megakaryocyte Growth and Development Factor Is a Potent Growth Factor for Primitive Hematopoietic Progenitors in the Human Fetus

“…Administration of MGDF has also been shown to increase the mobilization of progenitors and stem cells (40 -42). Gene therapy performed on hematopoietic stem cells isolated from fetal and/or neonatal blood is being studied as a means to treat a number of inherited disorders of the hematopoi-1055 GROWTH OF FETAL PROGENITORS WITH MGDF etic system (43)(44)(45)(46)(47). Mobilization of stem cells by MGDF administration may increase the yield of stem cells that can be harvested for therapy and returned to the fetal or neonatal circulation.…”