Fetal liver-derived CD34+, KDR+ hematopoietic stem cells: gene transduction with an HIV-based lentiviral vector

Abstract

Gene therapy is a potentially attractive option for treatment of HIV infection as well as for many hematopoietic disorders. To realize this goal, it is critical that hematopoietic stem cells with the highest self-renewal capacity and long-term engraftment capabilities be utilized as cell targets for therapeutic gene transfer. Previous studies have sought to identify the most primitive hematopoietic cells from sources such as umbilical cord blood (UCB), bone marrow (BM), peripheral blood (PB), and mobilized peripheral blood (MPB). As the fetal liver is the major site of hematopoiesis during embryogenesis, it is likely that a significant number of primitive hematopoietic cells exist in this site. Present studies have sought to identify primitive KOR-expressing CD34 stem cells in human fetal liver using direct mAb staining and to isolate these cells by FACS. Results have shown that approximately 1% of CD34+ cells isolated from 16-23 gestational-week old human fetal livers were positive for the KDR marker. This represents a significantly higher proportion compared to the level of 0.1-0.5% CD34, KDR double positive cells that has been found in other sources (UCB, PB, MPB, BM). It was previously shown that stem cells retain long-term hematopoiesis when cultured in the presence of mesenchymal stem cell (MSC) stromal cell layers. Accordingly, the fetal liver derived CD34+/KDR+ stem cells were cultured in the presence of MSC. This has allowed maintenance of a very small numbers ofCD34+/KDR+ cells (1000 cells) in culture and subsequently expand them to 100-fold. These findings provide a means of maintaining low numbers ofCD34+/KDR+ cells and expanding them to a working population for future experimentation. In vitro maintained CD34+/KDR+ cells were subjected to gene transduction with an HIV-1 based lentiviral vector. Although MSC did not serve to significantly increase transduction efficiencies of CD34+/KDR- progenitors, the progenitors cultured in the presence of MSC were relatively more refractory to the apparent toxicity effects of high-titer lentiviral vector exposure. In comparative studies, it was found that the transducibility of CD34+/KDR+ primitive hematopoietic cells is equivalent to that of CD34+/KDR- progenitors (approximately 86% EGFP expression as analyzed by FACS). Results presented here demonstrate the existence of CD34+, KDR+ cells in fetal liver at significantly higher levels than other sources. These cells could possibly serve as key cell targets for achieving sustained gene transduction with lentiviral vectors for current AIDS gene therapy strategies.