Transduction of Human CD34+ Cells That Mediate Long-Term Engraftment of NOD/SCID Mice by HIV Vectors

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Science  29 Jan 1999:
Vol. 283, Issue 5402, pp. 682-686
DOI: 10.1126/science.283.5402.682


Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable, long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation.

Human HSCs are an attractive target for gene therapy of inherited hematopoietic disorders as well as other acquired disorders because these cells have the ability to regenerate the entire hematopoietic system. A number of in vitro assays have been established to detect pluripotent human hematopoietic cells (1). However, these in vitro assays are unable to evaluate the long-term in vivo repopulating capacity that is a hallmark of HSCs. The NOD/SCID mouse (2) has been used to evaluate human HSCs in vivo (3). CD34+ primitive cells that have the capacity to initiate long-term multilineage engraftment in these mice have been operationally defined as SCID-repopulating cells (SRCs) (4).

Retroviral vectors, derived from oncoretroviruses such as the murine leukemia virus (MLV), have been the most widely used vectors for gene transfer because the vector genome integrates into the chromosomes of target cells, resulting in stable expression of transgenes (5). Although retroviral vectors have proven efficient for transducing mouse HSCs, this finding has not been easily translatable to large animals or humans (6). This lack of success possibly reflects the quiescent nature of human HSCs and the requirement of cell division for retroviral integration. Although retroviral transduction is efficient for human hematopoietic progenitor cells that have been stimulated to divide by cytokines, exposure to cytokines can lead to differentiation of the HSCs, possible loss of homing abilities, and probable reductions in long-term repopulating capacity (7).

We and others have developed HIV vectors that can transduce nondividing cells (8, 9). The HIV vector was pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) to ensure a broad host range and facilitate concentration of virus to high titers. HIV vectors mediate efficient and stable transduction of postmitotic cells in brain, liver, muscle, and retina (8,10). Therefore, HIV vectors may facilitate the transduction of quiescent human HSCs. In this study, we evaluated whether HIV vectors could transfer genes into human CD34+ cells that provide for long-term repopulation of NOD/SCID mice.

CD34+ cells were isolated from human umbilical cord blood and maintained in serum-free medium before transduction (11). To minimize cycling and to maintain the in vivo repopulating capability, we transduced CD34+ cells by means of a simple protocol in the absence of any exogenous cytokines. CD34+ cells were transduced for 5 hours with VSV-G–pseudotyped HIV vector that contained the green fluorescent protein (GFP) gene under the control of the internal cytomegalovirus (CMV) promoter at a multiplicity of infection (MOI) of 60 or 300 (12). For comparison, VSV-G–pseudotyped MLV vector containing the same CMV-GFP expression cassette was used.

Transduction efficiencies were first assessed by in vitro assays. A portion of transduced CD34+ cells was cultured for 5 days in serum-free medium containing recombinant human stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 (13). Under these conditions, about 60% of the cells retained the CD34+ phenotype while cells were expanded about 18-fold. CD34+ cells transduced with either HIV or MLV vector showed comparable numbers of GFP+ cells at an MOI of 60 and 300 [mean ± SE: 35 ± 5% and 54 ± 8% (HIV vector) and 33 ± 16% and 50 ± 22% (MLV vector) for MOI of 60 and 300, respectively], as determined by flow cytometry. A fivefold increase in MOI yielded a 1.5-fold increase in transduction efficiency for either HIV or MLV vector, suggesting that a higher MOI would provide little gain in transduction efficiency. To determine the transduction efficiency of colony-forming cell (CFC) progenitors, we plated transduced CD34+ cells in methylcellulose with cytokines (14). GFP+ CFC colonies, including burst-forming unit–erythroid (BFU-E), colony-forming unit–granulocyte, macrophage (CFU-GM), CFU–granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), and high proliferative potential–CFC (HPP-CFC) colonies, were scored by fluorescence microscopy at days 14 and 21. The number of GFP+ CFC colonies transduced with the HIV vector was 12-fold higher at an MOI of 60 (12 ± 1%) and eightfold higher at an MOI of 300 (17 ± 3%) than that of GFP+ CFC colonies transduced with the MLV vector. These results indicate that the HIV vector was more efficient than the MLV vector for transduction of CD34+ progenitors that generate CFCs. No adverse effect of transduction on cell viability and proliferation was observed in these in vitro assays.

To assess the transduction efficiency of SRCs in the CD34+cell population, we transplanted transduced CD34+ cells into sublethally irradiated NOD/SCID mice (15). Mice were serially bled from 7 to 22 weeks after transplantation to monitor GFP+ human cells in the peripheral blood (PB). The percentage of GFP+ human cells in the PB was determined by two-color flow cytometry (16). Most mice showed high levels of human cells in the PB, and the majority (≥95%) of these human cells were CD19+ B cells, reflecting the dominance of B cell lymphopoiesis in this model (17). Representative results shown in Fig. 1demonstrate that CD34+ cells transduced with the HIV vector gave rise to GFP+ human cells in the PB of engrafted mice and that the proportion of GFP+ human cells remained roughly constant until the animals were killed. GFP+ human cells were present in the PB for up to 22 weeks (see Table 1), the longest engraftment time period analyzed, demonstrating sustained production of human cells with integrated vector. In contrast, none of the mice (n = 6) transplanted with MLV vector–transduced CD34+ cells had detectable GFP+ human cells in the PB (Fig. 1), although these mice had numbers of human cells in the PB similar to those of mice transplanted with HIV vector–transduced CD34+ cells.

Figure 1

Maintenance of GFP+ human cells in the PB of NOD/SCID mice transplanted with HIV vector–transduced CD34+ cells. Mononuclear cells were isolated from mice at indicated times after transplantation. The percentages of GFP+ cells in human PB cells were assessed by two-color flow cytometry with an antibody to human CD45 (leukocyte common antigen). Representative results from four mice transplanted with HIV vector–transduced CD34+ cells and all mice (n = 6) transplanted with MLV vector–transduced CD34+ cells are shown. ▪, mouse number 84, HIV (MOI 60); •, mouse number 95, HIV (MOI 60); □, mouse number 10, HIV (MOI 300); ○, mouse number 92; ▾, all MLV (MOI 60 and 300)

Table 1

HIV, but not MLV, vectors can transduce human CD34+ cells that give rise to lymphoid and myeloid lineages in engrafted NOD/SCID mice.

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GFP expression in human myeloid and lymphoid lineages in the bone marrow (BM) and spleen of engrafted mice was evaluated at various time points after transplantation (16). High levels of human cell engraftment were evident in the BM of most of the mice (seeTable 1). Representative flow cytometry results of BM cells from a mouse transplanted with HIV vector–transduced CD34+ cells are shown in Fig. 2. About 27% of human (CD45+) cells in the BM expressed the GFP gene. GFP expression was detected in CD19+ B cells, the predominant population, and CD14+ myeloid cells. In addition to differentiated human cells, GFP+ CD34+ cells were detected, suggesting that immature GFP+ cells were maintained in the BM. The results from three separate experiments are summarized in Table 1. These results document the presence of GFP+ human cells in the BM (range 1 to 27%), spleen (range 3 to 22%), and PB (range 2 to 20%) of all mice (n = 14) transplanted with HIV vector–transduced CD34+ cells. There was no substantial difference between an MOI of 60 and 300. On the other hand, no GFP+ human cells were detected in all mice (n = 6) transplanted with MLV vector–transduced CD34+ cells. In addition, polymerase chain reaction (PCR) analysis of genomic DNA from BM cells revealed the presence of the GFP gene only in the BM cells from mice transplanted with HIV vector–transduced CD34+ cells (Fig. 3A).

Figure 2

GFP expression in human lymphoid and myeloid cells from the BM of NOD/SCID mice transplanted with HIV vector–transduced CD34+ cells. Representative flow cytometric analyses of BM cells from mice transplanted with mock- or HIV vector–transduced CD34+ cells (mouse number 95) are shown. Both mice had similar levels of human cell engraftment. Presented values are the percentages of total human cells.

Figure 3

GFP gene detection and expression in BM cells and CFC colonies derived from engrafted NOD/SCID mice. (A) PCR analysis of BM cells from engrafted mice to determine the presence of the GFP gene. Genomic DNA isolated from BM cells of NOD/SCID mice transplanted with CD34+ cells infected with either HIV or MLV vector was analyzed by PCR with primers that amplified a 417-bp fragment of the GFP gene. The presence of DNA was confirmed by PCR with primers that amplified a 307-bp fragment of human β-globin gene. The number of the mouse analyzed is indicated above each lane. M, size markers. (B) PCR analysis of CFC colonies to determine the presence of the GFP gene. Individual BFU-E and CFU-GM colonies derived from BM cells of engrafted NOD/SCID mice were analyzed by PCR as described above. Representative results obtained from mouse number 86 are shown. GFP+ (+) and GFP (−) colonies were determined by fluorescence microscopy. (C) Expression of GFP in CFC colonies derived from BM cells of mice transplanted with HIV vector–transduced CD34+ cells. CFC colonies derived from BM cells of engrafted mice were analyzed by fluorescence microscopy. GFP+ BFU-E, CFU-GM, and HPP-CFC colonies are shown. Original magnifications were ×25 for BFU-E and ×12.5 for CFU-GM and HPP-CFC.

To determine the percentages of GFP+ myeloid and erythroid progenitors in the BM of engrafted mice, we performed CFC assays in methylcellulose cultures that only support outgrowth of human progenitors (14). As shown in Fig. 3C, multiple lineages of human CFC colonies, including BFU-E, CFU-GM, and HPP-CFC colonies, derived from BM cells of mice transplanted with HIV vector–transduced CD34+ cells expressed GFP (range 3 to 77%) (see Table 1). The presence of the GFP gene in the GFP+ CFC colonies was verified by PCR analysis of randomly selected colonies (Fig. 3B). In contrast, no GFP+ CFC colonies were detected from mice transplanted with MLV vector–transduced CD34+ cells, further confirming the flow cytometric analysis of PB, BM, and spleen cells.

To address whether HIV vector integration correlated with GFP expression, we randomly isolated individual GFP CFC colonies and analyzed them by PCR for the presence of the GFP gene (18). Although there were some GFP colonies containing the GFP gene (Fig. 3B), the majority of GFPcolonies did not contain the GFP gene (see Table 1). Thus, a large proportion of CFCs transduced with the HIV vector did express the GFP at a level detectable by fluorescence microscopy. On the basis of the results from fluorescence microscopy and PCR analysis of CFC colonies, the average percentage of transduction of CFC colonies from mice transplanted with HIV vector–transduced CD34+ cells was 38 ± 7% (range 12 to 74%) at an MOI of 60 and 28 ± 13% (range 4 to 90%) at an MOI of 300 (Table 1). These results, together with data from PB longitudinal studies, demonstrated that the CMV promoter in the HIV vector can function properly in differentiated myeloid, erythroid, and lymphoid human cells with little silencing. The lack of expression of GFP in CFC colonies containing the GFP gene may have resulted from positional effects of the proviral integration site. Alternatively, these colonies expressed GFP at levels not detectable by fluorescence microscopy. HIV vector–mediated transduction had no adverse effect on human cell engraftment in NOD/SCID mice or colony-forming ability of progenitor cells as compared with mock transduction.

On the basis of human transplantation studies, human HSCs are known to be included in the CD34+ cell population. A number of groups have provided strong evidence that the LinCD34+CD38 cell subpopulation contains SRCs, and it has been proposed that this subpopulation contains HSCs (3). It has also been shown that SRCs were transduced by retroviral vectors only when cytokine prestimulation was used (19). In contrast, our results demonstrate that HIV vectors can mediate efficient transduction of SRCs without cytokine prestimulation. Although several groups have recently shown, by in vitro assays, that CD34+ cells can be transduced (20), we have established here the ability of HIV vectors to transduce human hematopoietic cells capable of long-term repopulation in vivo. Increases in MOI did not improve the transduction efficiency of SRCs, suggesting that there is a subpopulation of SRCs that is refractory for transduction with HIV vectors under the conditions we used. Recent studies have shown that the LinCD34 cell population also has long-term repopulating capacity and may be a precursor of LinCD34+ HSCs (21). Therefore, it would be of interest to determine whether HIV vectors can transduce these LinCD34 cells and possibly confirm the proposed role of these cells in the hierarchy of the hematopoietic system. Finally, a potential problem for the application of HIV vectors to human studies is safety. In this regard, recent improvements, including self-inactivating vectors, packaging constructs eliminating all accessory genes, and inducible packaging cell lines, could further minimize the risk (22). The use of HIV vectors provides a previously unexplored basis for the study of hematopoiesis and for human gene therapy with the use of HSCs.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: verma{at}


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