Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34+ cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45+ cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.

© 1998 by The American Society of Hematology.

GENE TRANSFER INTO pluripotent hematopoietic stem cells (PHSCs) is one of the most promising alternatives for the curative treatment of a variety of inherited and acquired disorders of blood cells. In murine syngeneic bone marrow (BM) transplantation models, a significant proportion of cells participating in long-term engraftment of lethally irradiated mice can be reproducibly and stably transduced ex vivo by the current generation of retroviral vectors.1-3 However, transfer of this technology to humans, nonhuman primates, and other large outbred animals has been much less successful.4-13 The reasons for this discrepancy are uncertain, but probably reflect incomplete understanding of culture conditions required to maintain the integrity and functionality of the PHSC, an inability of the current generation of murine retroviral vectors to transduce quiescent cells,14,15 and a deficiency of receptors on the PHSC surface for the commonly used amphotropic retroviral envelope.16 17 

In animal models and human trials, high levels of gene transfer to clonogenic progenitor cells and long-term culture-initiating cells (LTC-ICs) in vitro have not been predictive of long-term reconstitution. The development of efficient protocols for PHSC gene transfer has therefore been limited by the failure of in vitro surrogate progenitor assays to represent the repopulating cell fractions of the human hematopoietic system. To address this problem, alternative assay systems have been developed that test the ability of human hematopoietic cells to engraft immunodeficient mice.18-24 In one such model, which is based on the engraftment of severe combined immunodeficiency disease (SCID) and nonobese diabetic/SCID (NOD/SCID) mice, a novel population of human hematopoietic cells, defined as SCID-repopulating cells (SRCs), have been shown to be capable of extensive proliferation and multilineage (lymphoid and myeloid) differentiation in vivo.25Furthermore, this activity is highly enriched in CD34+CD38 fractions, and based on the kinetics of engraftment, represents a more primitive cell population than most LTC-ICs and CFCs.23,26-28 However, ex vivo manipulation of these cells has also been shown to be detrimental to their functionality in terms of repopulation,29 and the levels of gene transfer using murine retroviruses (amphotropic and gibbon ape leukemia virus (GALV) pseudotypes) have been consistently very low.23 Engraftment of pluripotent populations in the beige-nude–X-linked immunodeficiency (bg/nu/xid) model has produced similar findings.24 30 These studies reflect more closely the situation found in large animal studies and human gene therapy trials and indicate the use of this surrogate in vivo assay system for preclinical development of novel vector systems for stem cell gene transfer and optimization of clinically applicable transduction protocols.

In this study, we have evaluated the efficiency of gene transfer to primitive human hematopoietic cells using a GALV-pseudotyped murine retroviral vector, and optimized ex vivo transduction conditions. We show here that these cells retain their ability to repopulate NOD/SCID mice and can be transduced relatively efficiently.

Recombinant human cytokines and growth factors.

Stem cell factor (SCF), interleukin (IL)-3, IL-6, Flt3-Ligand (Flt3-L), and anti-transforming growth factor (TGF)β1 antibodies were obtained from R & D Systems Inc (Minneapolis, MN). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant human granulocyte colony-stimulating factor (G-CSF) were from Amgen (Thousand Oaks, CA).

Purification of hematopoietic CD34+ cells.

Human BM was obtained under local anesthesia from the iliac crest of healthy adult volunteers after informed consent and ethical approval. Samples of umbilical cord blood (CB) were obtained from discarded placental and umbilical tissues by drainage of the blood into sterile collection bags. The BM and CB samples were diluted 1:3 in phosphate-buffered saline (PBS) and enriched for mononuclear cells by density gradient over Ficoll-Paque (1.077 g/mL; Seromed, Berlin, Germany). The BM-derived low density cell fraction was subjected to one cycle of plastic adherence (1 to 2 hours) before the isolation of CD34+ cells. CD34+ cells were isolated by superparamagnetic microbeads selection using the miniMACS system according to the manufacturer’s instructions (Miltenyi Biotec, Inc, Gladbach, Germany). The purity of the cell population ranged between 75% and 97% CD34+ cells as estimated by fluorescence-activated cell sorting (FACS) analysis using either a phycoerythrin (PE)- or a fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody against the human CD34 antigen (anti-hematopoietic-progenitor-cell-antigen-2[anti-HPCA-2], Becton Dickinson; San Jose, CA).

Transduction of human hematopoietic CD34+ cells.

For the transduction of human CD34+ cells, retroviral supernatant was harvested from 80% confluent PG-13 monolayers after 12 to 16 hours cultivation in serum-free X-VIVO10 medium (Boehringer Ingelheim; Heidelberg, Germany) supplemented with 1% bovine serum albumin (BSA; Stem Cell Technologies, Vancouver, Canada), 2 mmol/L L-glutamine and 1% penicillin/streptomycin, filtered (0.45 μm) and kept frozen at −80°C until use. The CD34+-enriched target cell population was prestimulated at a cell concentration of 1 × 105 cells/mL for 20 hours in serum-free X-VIVO10 supplemented with 1% BSA, 2 mmol/L L-glutamine and 1% penicillin/streptomycin in the presence of IL-3 (20 ng/mL), IL-6 (100 U/mL), SCF (50 ng/mL), Flt3-L (100 ng/mL), and anti-TGFβ1 (100 ng/mL). After prestimulation, transduction was performed for 3 consecutive days by replacing half of the cell culture medium with a GALV-pseudotyped LNSN retroviral supernatant (titer: 1 to 5 × 106 ) supplemented with the cytokine combination mentioned above and protamine sulfate to a final concentration of 4 μg/mL. Cells were spun at 2,500 rpm/minute at 32°C for 90 minutes31 before further incubation at 37°C, 5% CO2 for an additional 2.5 hours. Afterwards, half of the medium was replaced with X-VIVO10 containing IL-3, IL-6, SCF, TGFβ1, Flt3-L, and anti-TGFβ at the concentrations mentioned above. In some experiments, plates were precoated with the recombinant fibronectin-fragment CH296 (Boehringer Ingelheim). At day 5, cells were harvested, washed, counted, and analyzed for expression of the low-affinity nerve growth factor receptor (LNGFR) and CD34 by flow cytometry.

Flow cytometric analysis of transduced cells.

After transduction, cells were washed twice in PBS containing 1% heat inactivated fetal calf serum (FCS) and 0.1% sodium azide. To assess for LNGFR expression, cells were incubated with an unconjugated mouse antihuman LNGFR antibody (Boehringer Mannheim, Mannheim, Germany), which was detected with a goat antimouse F(ab)-fluorescein isothiocyanate (FITC) (Dianova; Hamburg, Germany). Alternatively, a biotinylated primary LNGFR antibody (kindly provided by Dr S. Seeber, Boehringer Mannheim, Penzberg, Germany) was used and subsequently detected with PE-conjugated streptavidin (Dianova). For the flow cytometric analysis of engrafted NOD/SCID mice, directly conjugated antibodies against human cell surface antigens were purchased from Becton Dickinson (Oxford, UK) (CD19-FITC, CD34-FITC, CD38-PE, CD45-PerCP) or DAKO, Ltd (High Wycombe, UK) (CD2-PE, CD3-FITC, CD13-PE). A total of 1 × 106 cells obtained from the BM of injected mice were incubated for 30 minutes on ice with saturating amounts of antibodies in staining buffer (PBS, 5% FCS, 0.01% sodium azide). A sample from each mouse was also stained with directly conjugated isotype-matched control antibodies (Becton Dickinson). After incubation, cells were washed three times and fixed in 1% paraformaldehyde. Flow cytometric analysis was performed on a FACScan or FACSCalibur using the CellQuest software package (Becton Dickinson). In all experiments, isotype controls were used to set the quadrant markers such that the quadrant defining negative PE and FITC fluorescence contained at least 97% of the isotype control cells. The engrafted human cells were detected by CD45 positivity and the expression of the lineage markers. LNGFR expression was determined on the CD45-gated population.

Progenitor cell assays.

For clonogenic assays, transduced cells were plated in 35-mm dishes containing 0.35% agar, 25% FCS (Hyclone; Erembodegem-Aalst, Belgium), 50 ng/mL SCF, 20 ng/mL IL 3, 10 ng/mL G-CSF, and 10 ng/mL GM-CSF in McCoy’s medium (Life Technologies; Gaithersburg, MD). Cultures were incubated at 37°C in a 5% CO2 humidified atmosphere and colonies were enumerated after 10 to 15 days. For LTC-IC assay, 500 or 1,000 cells were seeded on a preestablished monolayer of the murine FBMD-1 cell line32 (kindly provided by R.E. Ploemacher, Rotterdam, The Netherlands) in MyeloCult (Stem Cell Technologies) containing 20 ng/mL IL-3, 100 U/mL IL-6, and 50 ng/mL SCF. Cultures were incubated for 5 weeks at 37°C, 5% CO2 with weekly changes of half of the medium. At the end of the 5-week LTC-IC assay period, the nonadherent and adherent fractions were harvested and assayed for the content of hematopoietic progenitors by plating 100,000 hematopoietic cells in clonogenic assays, as described above and scored 14 days later. LNGFR-positive colonies were detected by immunostaining techniques (manuscript in preparation). Similarly, 2 × 105 cells derived from the BM of engrafted cells 6 weeks after injection were analyzed for the presence of human hematopoietic progenitors. Cells were plated in Methocult (Stem Cell Technologies), supplemented with Iscove’s modified Dulbecco’s medium (IMDM), 30% FCS, human growth factors (25 ng/mL SCF, 10 U/mL IL-3, 9 U/mL GM-CSF, 2 U/mL erythropoietin [Epo]; all R&D Systems), 2 mmol/L L-glutamine, and 50 μmol/L 2-mercapto-ethanol, resulting in 0.9% final concentration of methylcellulose. The cultures were incubated in a fully humidified atmosphere at 5% CO2.

NOD/SCID mouse reconstitution assay.

The NOD/LtSz-scid/scid (NOD/ SCID) mice (original stocks kindly provided by John E. Dick, Hospital for Sick Children, Toronto, Canada) were housed in sterile microisolator cages in a laminar flow caging system (Thoren, Hazleton, PA) and supplied with sterile food, acidified water, and bedding. All manipulations were conducted in a laminar flow hood. Transduced CD34+ cells were injected intravenously via the tail vein of 6- to 8-week-old mice, which had been sublethally irradiated with 325 cGy (137Cs source). Mice were killed by CO2 inhalation 6 weeks after injection and BM cells were harvested for flow cytometric analysis and growth of hematopoietic progenitors.

Polymerase chain reaction (PCR) for human LNGFR.

The presence of LNGFR provirus in secondary colonies was determined using the primers 5′-TGTGTGAGCCCTGCCTGGAC, beginning at position 300 in exon 2 and 5′-CGAGCCCTCTGGGGGTGTGG position 725 in exon 4 of the LNGFR gene. The amplification cycle was 30 seconds at 94°C, 1 minute at 66°C, and 1 minute at 72°C, with a final elongation step of 10 minutes at 72°C. After 30 cycles, the specific 425-bp product was detected by Southern blotting.

Optimized transduction of CD34+cells using LNGFR expression.

On the basis of previous studies suggesting that retroviral vectors generated on the PG13 packaging cell line may have advantages over amphotropic vectors for transduction of human hematopoietic cells,13,33-35 a GALV-pseudotyped LNSN retroviral vector was used in all experiments. The LNSN construct, which contains the full-length low-affinity receptor for human nerve growth factor (LNGFR) under the transcriptional control of the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR),36 was selected for these studies because LNGFR expression allows rapid evaluation of gene transfer by flow cytometric analysis and immunocytochemistry.36-39 To optimize gene transfer to human CD34+ cell populations, a detailed study of several parameters that could improve efficiency was performed (manuscript in preparation). The optimized transduction protocol included prestimulation of the CD34+ cells for 20 hours in serum-free medium (X-VIVO10) supplemented with 1% BSA, 2 mmol/L L-glutamine, IL-3, IL-6, SCF, FLT3-L, and anti-TGFβ1. Thereafter, cells were transduced under identical conditions for 4 hours on 3 consecutive days and included a spinoculation step (2,500 rpm/min, 32°C, 90 minutes).31 At the end of the transduction period, a twofold to threefold expansion in total cell numbers was observed, with half of the cells retaining expression of the CD34 cell surface antigen (35% to 46% for BM cells and 57% to 72% for CB cells). Transduction efficiency was determined by flow cytometric analysis of LNGFR expression on day 5. Representative profiles of transduced BM and CB-derived CD34+ cells are shown (Fig 1). In a series of 10 independent experiments, transduction efficiencies of 70% or higher were achieved (Fig 2). Best transduction efficiency was achieved when plates coated with the recombinant human fibronectin fragment, CH-269, were used in conjuction with spinoculation (97.4% LNGFR positive cells; Table 1), confirming the observations of others.40-43 In addition, between 53% and 95% of the cells coexpressed CD34 and LNGFR (Fig 2). LNGFR expression on mock-transduced cells was negligible (Fig 1, central panels).

Fig. 1.

LNGFR expression on BM- and CB-derived CD34+ cells after gene transfer with a GALV-pseudotyped LNSN retroviral vector. Flow cytometric analysis of two representative transduction experiments into CD34+ cells derived from BM (A) and umbilical CB (B). The left panels show the isotype controls for nonspecific IgG1 staining. The CD34 and LNGFR expression on mock-transduced cells and LNSN-transduced cells is shown at the central and right panels, respectively.

Fig. 1.

LNGFR expression on BM- and CB-derived CD34+ cells after gene transfer with a GALV-pseudotyped LNSN retroviral vector. Flow cytometric analysis of two representative transduction experiments into CD34+ cells derived from BM (A) and umbilical CB (B). The left panels show the isotype controls for nonspecific IgG1 staining. The CD34 and LNGFR expression on mock-transduced cells and LNSN-transduced cells is shown at the central and right panels, respectively.

Close modal
Fig. 2.

Summary of gene transfer efficiencies into BM- and CB-derived CD34+ cells. Gene transfer efficiencies into hematopoietic cells (total cells), CD34+ cells, or CFC were estimated by FACS (total cells and CD34+) or immunostaining techniques (CFC). Data from LNSN-transduced cells (circles) and mock transduced cells (squares) are presented. Black symbols: BM-derived CD34+ cells, open symbols: CB-derived CD34+ cells. Each symbol represents a single experiment.

Fig. 2.

Summary of gene transfer efficiencies into BM- and CB-derived CD34+ cells. Gene transfer efficiencies into hematopoietic cells (total cells), CD34+ cells, or CFC were estimated by FACS (total cells and CD34+) or immunostaining techniques (CFC). Data from LNSN-transduced cells (circles) and mock transduced cells (squares) are presented. Black symbols: BM-derived CD34+ cells, open symbols: CB-derived CD34+ cells. Each symbol represents a single experiment.

Close modal
Table 1.

Efficiency of Gene Transfer Into NOD/SCID Repopulating Human Hematopoietic Cells

Experiment Efficiency of Gene Transfer No. of Transplanted Cells Human Cell Engraftment (% CD45+ cells)Gene-Marked Cells Derived From the BM of NOD/SCID Mice (% of all CD45+ cells) LNGFR PCR Second Colonies (LNGFR+/total colonies)
(% LNGFR+ cells) (% CD34+LNGFR+/CD34+ cells)
BM 3  80.8  79.6 1.25 × 106 1.2  6.0 
   2.5 × 106 0.3  
   2.5 × 106 0  0  
BM 5  83.1 78.6  3.0 × 106 5.1  5.6 
   4.2 × 106 0  0  
CB 1  70.8 74.2  1.25 × 106 9.5  16.9  2/10 
   1.25 × 106 12.8  16.7  1/13 
   1.25 × 106 9.0  25.3  1/13 
CB 3  83.3  81.3  1.3 × 106 0  
   1.3 × 106 3.6  13.7  1/20 
CB 4  84.2  80.8  0.5 × 106 0  
   0.5 × 106 0  0  
CB 5-150 97.4  98.0  3.0 × 106 33.3  56.6 9/20  
   3.0 × 106 0.9 15.1 
Experiment Efficiency of Gene Transfer No. of Transplanted Cells Human Cell Engraftment (% CD45+ cells)Gene-Marked Cells Derived From the BM of NOD/SCID Mice (% of all CD45+ cells) LNGFR PCR Second Colonies (LNGFR+/total colonies)
(% LNGFR+ cells) (% CD34+LNGFR+/CD34+ cells)
BM 3  80.8  79.6 1.25 × 106 1.2  6.0 
   2.5 × 106 0.3  
   2.5 × 106 0  0  
BM 5  83.1 78.6  3.0 × 106 5.1  5.6 
   4.2 × 106 0  0  
CB 1  70.8 74.2  1.25 × 106 9.5  16.9  2/10 
   1.25 × 106 12.8  16.7  1/13 
   1.25 × 106 9.0  25.3  1/13 
CB 3  83.3  81.3  1.3 × 106 0  
   1.3 × 106 3.6  13.7  1/20 
CB 4  84.2  80.8  0.5 × 106 0  
   0.5 × 106 0  0  
CB 5-150 97.4  98.0  3.0 × 106 33.3  56.6 9/20  
   3.0 × 106 0.9 15.1 

Abbreviations: BM, bone marrow; CB, umbilical cord blood.

F0-150

Transduction was performed on CH-296–coated plate.

Gene transfer into CFCs and LTC-ICs.

Immunocytochemical detection of LNGFR expression in hematopoietic colonies indicated an efficiency of gene transfer to colony-forming progenitors (CFCs) of 63.4% ± 11.7% (range, 49.0% to 84.0%) (Fig 2). Absence of background staining in mock-transduced preparations confirmed the specificity of detection (not shown). No difference in the efficiency of CFC transduction was observed between CD34+ cells derived from BM (63.6% ± 10.1%) or from CB (63.1% ± 13.4%). To assess gene transfer into more primitive progenitors, transduced cells were maintained under long-term culture conditions on FBMD-1 cells.32 Of colonies derived from progenitor cells removed from the culture after 5 weeks, 23.6% ± 4.3% (range, 19.3 to 28.0%) expressed LNGFR by immunostaining.

Gene transfer into SRCs.

To test for transduction of primitive human cells with repopulating ability, hematopoietic cells (derived from BM or CB) were injected into the tail vein of sublethally irradiated NOD/SCID mice after transduction under the optimized conditions outlined above. Cells recovered from mouse femurs 6 weeks after engraftment were analyzed for the percentage of human cells (CD45 expression) and expression of the LNGFR marker gene by flow cytometry and for the level of gene transfer to CFCs by PCR. Results are summarized in Table 1. After transplantation of between 0.5 and 4.2 × 106 cells, nine of 14 animals showed detectable levels (0.3% to 33.3%) of human cell engraftment measured by flow cytometric detection of CD45 expression (Figs 3A and4). Multilineage engraftment determined by surface immunophenotype (CD19, CD13, CD2), and CFC profile (not shown) was observed in all nine animals (Fig 3B). In five animals, no CD45+ cells were detectable, although engraftment at levels below those measurable in these studies is possible.

Fig. 3.

Multilineage engraftment of human hematopoietic cells into NOD/SCID mice. Sublethally irradiated NOD/SCID mice were injected with CB-derived CD34+ cells transduced for 3 days under serum-free conditions (CB#1 in Fig 2). (A) FACS analysis of hematopoietic cells obtained from the femur of animals injected with human CB-derived CD34+ cells (solid line) or control animals (dotted line). The percentage of human cells was calculated from the number of CD45+ cells found in the femur of the animals after 6 weeks. (B) Multilineage engraftment of human cells. The CD45+ cells shown in (A) were further analyzed for the presence of myeloid cells (CD13), lymphoid cells (CD19, CD2), and immature progenitor cells (CD34, CD38). The percentage of each cell population is shown in the upper right corner of each quadrant.

Fig. 3.

Multilineage engraftment of human hematopoietic cells into NOD/SCID mice. Sublethally irradiated NOD/SCID mice were injected with CB-derived CD34+ cells transduced for 3 days under serum-free conditions (CB#1 in Fig 2). (A) FACS analysis of hematopoietic cells obtained from the femur of animals injected with human CB-derived CD34+ cells (solid line) or control animals (dotted line). The percentage of human cells was calculated from the number of CD45+ cells found in the femur of the animals after 6 weeks. (B) Multilineage engraftment of human cells. The CD45+ cells shown in (A) were further analyzed for the presence of myeloid cells (CD13), lymphoid cells (CD19, CD2), and immature progenitor cells (CD34, CD38). The percentage of each cell population is shown in the upper right corner of each quadrant.

Close modal
Fig. 4.

LNGFR expression on human CD45+ cells obtained from the BM of NOD/SCID mice. NOD/SCID were sublethally irradiated and injected with LNSN-transduced CB-derived CD34+ cells (panels C through J) or mock-transduced cells (panels A and B). Six weeks later, BM cells were obtained from the femur of these animals and analyzed for the presence of human cells (CD45+) and LNGFR expression by FACS. FACS data from mice engrafted with cells derived from CB#1 (panels C through H) or CB#5 (panels I and J) is shown. The left panels show the CD45+expression versus the F(ab)-FITC isotype control. The right panels show the CD45 and LNGFR expression on LNSN-transduced (D through J) or mock-transduced (B) cells. Fluorescence intensities are displayed in logarithmic scale.

Fig. 4.

LNGFR expression on human CD45+ cells obtained from the BM of NOD/SCID mice. NOD/SCID were sublethally irradiated and injected with LNSN-transduced CB-derived CD34+ cells (panels C through J) or mock-transduced cells (panels A and B). Six weeks later, BM cells were obtained from the femur of these animals and analyzed for the presence of human cells (CD45+) and LNGFR expression by FACS. FACS data from mice engrafted with cells derived from CB#1 (panels C through H) or CB#5 (panels I and J) is shown. The left panels show the CD45+expression versus the F(ab)-FITC isotype control. The right panels show the CD45 and LNGFR expression on LNSN-transduced (D through J) or mock-transduced (B) cells. Fluorescence intensities are displayed in logarithmic scale.

Close modal

Efficiency of gene transfer to the input population of CD34+ cells ranged from 70.8% to 97.4%, as measured by flow cytometric detection of LNGFR expression. Of the CD34+cells remaining at this time after the transduction procedure, 74.2% to 98.0% also expressed the LNGFR marker gene (Table 1). Six weeks after transplantation, LNGFR+ cells were detected in the CD45+ cell population at levels ranging from 5.6% to 56.6% in eight of nine engrafted animals (Figs 4 and5). In those animals successfully engrafted with CB-derived cells (six of nine), 13.7% to 56.6% of CD45% cells were positive for LNGFR expression (Table 1). LNGFR+ cells were not detectable in animals engrafted with mock-transduced CB-derived CD34+ cells (Fig 4A and B), indicating that the positive signals obtained were derived from successfully transduced cells. Confirmation of gene transfer to SRCs was obtained by PCR amplification of a transgene-specific sequence in genomic DNA extracted from secondary colonies (colony-forming unit–granulocyte-macrophage [CFU-GM] and burst-forming unit erythroid [BFU-E]). In five of five samples, proviral DNA was detected in individual colonies at rates comparable to that predicted by LNGFR expression (Table 1, Fig 6).

Fig. 5.

Frequency of human cell engraftment and LNGFR expression in NOD/SCID mice injected with transduced BM- or CB-derived CD34+ cells. BM- or CB-derived CD34+ cells were transduced with a GALV-pseudotyped LNSN retroviral vector and injected into sublethally irradiated NOD/SCID mice. The left panel shows the percentage of human cell engraftment (CD45+cells) 6 weeks after injection. The right panel shows the LNGFR expression in CD45+ cells obtained from the BM of engrafted animals. Each symbol represents one mouse.

Fig. 5.

Frequency of human cell engraftment and LNGFR expression in NOD/SCID mice injected with transduced BM- or CB-derived CD34+ cells. BM- or CB-derived CD34+ cells were transduced with a GALV-pseudotyped LNSN retroviral vector and injected into sublethally irradiated NOD/SCID mice. The left panel shows the percentage of human cell engraftment (CD45+cells) 6 weeks after injection. The right panel shows the LNGFR expression in CD45+ cells obtained from the BM of engrafted animals. Each symbol represents one mouse.

Close modal
Fig. 6.

Detection of proviral genome in secondary colonies derived from engrafted NOD/SCID mice. Secondary colonies were established with cells obtained from the BM of engrafted NOD/SCID animals 6 weeks after injection. The presence of LNSN proviral DNA in hematopoietic colonies derived from three mice injected with CB#1 (A), one mouse injected with CB#3 (B), and one mouse injected with CB#5 (C) was assessed by a sensitive LNGFR-specific PCR. The 425-bp–specific LNGFR product was detected by Southern blotting. Amplification of a sequence from the human mannose binding protein (MBP) gene was used to control for the presence of DNA.63 

Fig. 6.

Detection of proviral genome in secondary colonies derived from engrafted NOD/SCID mice. Secondary colonies were established with cells obtained from the BM of engrafted NOD/SCID animals 6 weeks after injection. The presence of LNSN proviral DNA in hematopoietic colonies derived from three mice injected with CB#1 (A), one mouse injected with CB#3 (B), and one mouse injected with CB#5 (C) was assessed by a sensitive LNGFR-specific PCR. The 425-bp–specific LNGFR product was detected by Southern blotting. Amplification of a sequence from the human mannose binding protein (MBP) gene was used to control for the presence of DNA.63 

Close modal

Efficient gene transfer to human PHSCs has been limited by incomplete understanding of their biologic properties and by deficiencies of vector systems and ex vivo transduction conditions. These confounding factors are reflected in data from clinical trials and from studies in which transduced cells are engrafted in immunodeficient mice.4,6,9,10-12,23,24,44 The NOD/SCID model system has been shown to support the engraftment and retention of primitive human hematopoietic cells with the potential for extensive proliferation and multilineage differentiation.23,25-27,45,46 Unlike the majority of LTC-ICs, which are incapable of repopulation, SRCs are found exclusively in the CD34+CD38- cell fraction at a calculated frequency of approximately 1 in 600 in CB and BM and are therefore phenotypically and functionally distinct.26 Furthermore, kinetic experiments indicate that engraftment of SRCs is followed by a large expansion of LTC-ICs in vivo, suggesting that these are derived from a more primitive cell.27 Although both CFCs and LTC-ICs are readily transduced, the efficiency of gene transfer to SRCs has generally been very low, and the repopulating potential is markedly compromised by ex vivo culture.13,23,29 Similar findings have been reported for pluripotent cells engrafting bg/nu/xid mice, although there are qualitative and quantitative differences in repopulating cell engraftment patterns in this model compared with that of SRCs.24 30 

Multiple factors probably contribute to the inefficiency of gene transfer to repopulating cells. The majority of CD34+CD38 cells are quiescent15 and therefore refractory to transduction by the current generation of murine retroviral vectors, which require breakdown of the nuclear membrane to achieve entry of the nucleoprotein complex into the nucleus before integration.14 Ram-1, the receptor for the amphotropic retroviral envelope, is expressed at very low levels on CD34+CD38 cells, and virus binding can only be detected after cytokine stimulation.16,17 In contrast, GALV-pseudotyped retroviruses have been shown to mediate higher levels of gene transfer to CD34+ and CD34+CD38cells, although they have not been extensively evaluated in repopulating assay systems.13,33-35,43 Furthermore, current gene transfer protocols require the removal of PHSC from their natural microenvironmental niches and their manipulation ex vivo, conditions, which may alter the integrity and functionality of these cells.29 However, stem cells can also be protected in vitro by culture on feeder cells or stromal monolayers.47,48 At least partially, this function can be replaced by appropriate combinations of cytokines and growth factors. Flt3 ligand (FL), in particular, has been shown to act synergistically with a range of other cytokines to stimulate proliferation and amplification of very primitive (CD34+CD38) hematopoietic cells both in vitro and in vivo.46,49-54 Moreover, FL enhances transduction efficiency of primitive progenitor cells and preserves the ability of transduced cells to repopulate bg/nu/xid mice after prolonged periods of in vitro culture.44 

Ideally, ex vivo manipulation of PHSCs should preserve the intrinsic properties of these cells. On the basis that maintenance or even expansion of the PHSC can be achieved after cultivation of CD34+CD38 CB-derived cells in serum-free medium,46 55 one major objective of this study was to establish gene transfer into repopulating cells under serum-free conditions. In addition, the use of total CD34+ cells as the target population for gene transfer, rather than highly purified subfractions, is compatible with current clinical practice in stem cell transplantation and thus allows for a rapid transfer of the transduction protocol to clinical situations. The final protocol described in this study represents a major step towards the achievment of this goal.

Previous attempts to transduce CD34+ cells under serum-free conditions have shown gene transfer into CFCs at levels ranging from 1% to 29%.12,56 In an effort to improve gene transfer in the absence of serum, we examined the efficiency of gene transfer to CD34+ cells under varying culture conditions (manuscript in preparation). The combination of cytokines (IL-3, IL-6, SCF, and FL) together with anti-TGFβ1 antibodies was chosen to stimulate cell division in primitive populations and to retain repopulating potential over extended culture periods.57-62Under these conditions, high gene transfer rates into CD34+hematopoietic cells were reproducibly achieved, independently of the donor and source of material used (>70% LNGFR+ cells). Significant repopulating potential of transduced cells was also retained over the 5-day ex vivo culture period, and the majority of engrafted NOD/SCID mice also showed high levels of marker gene expression (6% to 57%) in total CD45+ (lymphoid and myeloid) populations. Comparable percentages of provirus-positive CFCs were detected in secondary colony-forming assays. These results are highly suggestive of successful gene transfer to primitive multilineage repopulating cells, although formal proof of this would require the demonstration of common integrants in lymphoid and myeloid lineages.

In this study, we have shown that efficient retrovirus-mediated gene transfer to cells with the capacity to repopulate sublethally irradiated NOD/SCID mice (SRCs) can be achieved by optimization of transduction conditions ex vivo and optimization of the vector. These are the first studies to show efficient gene transfer to the SRC population using GALV-pseudotyped viruses and support the further investigation of this envelope for transduction of human PHSCs. They also reinforce the use of surrogate repopulating assays for testing of novel vector systems and development of clinically applicable gene therapy protocols. Further improvements of cell culture systems and development of vectors that obviate the requirement for cell division are likely to further enhance transduction of human repopulating cell populations.

We are indebted to S. Seeber (Boehringer Mannheim, Penzberg) for the LNSN construct and LNGFR antibodies, R.E. Ploemacher (Erasmus University, Rotterdam, The Netherlands) for the FBMD-1 cell line, T. Tonn (Blood Bank, Frankfurt) for CB samples, and Mike Blundell for technical assistance.

Supported by grants from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie to M.G. and O.G.O. (FKZ: 01KV9558), the Hermann J. Abs Program of the Deutsche Bank AG, the Primary Immunodeficiency Association, Chronic Granulomatous Disease Research Trust, the European Commission (to G.B.), and the Wellcome Trust (to A.J.T.). The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und Kunst.

A.J.S. and G.B. contributed equally to this work.

Address reprint requests to Manuel Grez, PhD, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42, 60596 Frankfurt, Germany; e-mail:grez@em.uni-frankfurt.de.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Hawley
RG
Fong
AZC
Burns
BF
Hawley
TS
Transplantable myeloproliferative disease induced in mice by an interleukin 6 retrovirus.
J Exp Med
176
1992
1149
2
Ding
C
Kume
A
Björgvinsdóttir
H
Hawley
RG
Pech
N
Dinauer
MC
High-level reconstitution of respiratory burst activity in a human X-linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marow cells by retroviral mediated gene transfer of human gp91-phox.
Blood
88
1996
1834
3
Pawliuk
R
Eaves
CJ
Humphries
RK
Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen.
Hum Gene Ther
8
1997
1595
4
Brenner
MK
Hill
DR
Holladay
MS
Heslop
HE
Moen
RC
Buschle
M
Krance
RA
Santana
VM
Anderson
FW
Ihle
JN
Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.
Lancet
342
1993
1134
5
Bodine
DM
Moritz
T
Donahue
RE
Luskey
BD
Kessler
SW
Martin
DI
Orkin
SH
Nienhuis
AW
Williams
DA
Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells.
Blood
82
1993
1975
6
Deisseroth AB
ZU Z
Claxton
D
Hanania
EG
Fu
S
Ellerson
D
Goldberg
L
Thomas
M
Janicek
K
Anderson
WF
Hester
J
Korbling
M
Durett
A
Moen
R
Berenson
R
Heimfeld
S
Hamer
J
Calavert
L
Tibbits
P
Talpez
M
Kantajian
H
Champlin
R
Reading
C
Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML.
Blood
83
1994
3068
7
Kiem
HP
Darovsky
B
von Kalle
C
Goehle
S
Stewart
D
Graham
T
Hackman
R
Appelbaum
FR
Deeg
HJ
Miller
AD
Storb
R
Schuening
FG
Retrovirus-mediated gene transduction into canine peripheral blood repopulating cells.
Blood
83
1994
1467
8
Xu
LC
Karlsson
S
Byrne
ER
Kluepfel-Stahl
S
Kessler
SW
Agricola
BA
Sellers
S
Kirby
M
Dunbar
CE
Brady
RO
Nienhuis
AW
Donahue
RE
Long-term in vivo expression of the human glucocerebrosidase gene in non-human primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.
Proc Natl Acad Sci USA
92
1995
4372
9
Dunbar
CE
Cottler-Fox
M
O’Shaughnessy
JA
Doren
S
Carter
C
Berenson
R
Brown
S
Moen
RC
Greenblat
J
Stewart
FM
Leitman
SF
Wilson
WH
Cowan
K
Young
NS
Nienhuis
AW
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood
85
1995
3048
10
Bordignon
C
Notarangelo
LD
Nobili
N
Ferrari
G
Casorati
G
Panina
P
Mazzolari
E
Maggiori
D
Rossi
C
Servida
P
Ugazio
AG
Mavilio
F
Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients.
Science
270
1995
470
11
Kohn
DB
Winberg
KI
Nolta
JA
Heiss
LN
Lenarsky
C
Crooks
GM
Hanley
ME
Annett
G
Brooks
JS
El-Khoureiy
A
Lawrence
K
Wells
S
Moen
RC
Bastian
J
William-Herman
DE
Elder
M
Wara
D
Bowen
T
Hershfield
MS
Mullen
CA
Blease
RM
Parkman
R
Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.
Nat Med
1
1995
1017
12
Malech
HL
Maples
PB
Whiting-Theobald
N
Linton
GF
Sekhsaria
S
Vowells
SJ
Li
F
Miller
JA
DeCarlo
E
Holland
SM
Leitman
SF
Carter
CS
Butz
RE
Reads
EJ
Fleischer
TA
Schneiderman
RD
Van Epps
DE
Spratt
SK
Maack
CA
Rokovich
JA
Cohen
LK
Gallin
JI
Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease.
Proc Natl Acad Sci USA
94
1997
12133
13
Kiem
HP
Heyward
S
Winkler
A
Potter
J
Allen
JM
Miller
AD
Andrews
RG
Gene transfer into marrow repopulating cells: Comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competititve repopulation assay in baboons.
Blood
90
1997
4638
14
Miller
DG
Adam
MA
Miller
AD
Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.
Mol Cell Biol
10
1990
4239
15
Hao
QL
Thiemann
FT
Petersen
D
Smogorzewska
EM
Crooks
GM
Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population.
Blood
88
1996
3306
16
Crooks
GM
Kohn
DB
Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells.
Blood
82
1993
3290
17
Orlic
D
Girard
LJ
Jordan
CT
Anderson
SM
Cline
AP
Bodine
DM
The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.
Proc Natl Acad Sci USA
93
1996
11097
18
Kamel-Reid
S
Dick
JE
Engraftment of immune-deficient mice with human hematopoietic stem cells.
Science
242
1988
1706
19
McCune
JM
Namikawa
R
Kaneshima
H
Shultz
LD
Lieberman
M
Weissman
IL
The SCID-hu Mouse: Murine model for the analysis of human hematolymphoid differentiation and function.
Science
241
1988
1632
20
Namikawa
R
Weilbaecher
KN
Kaneshima
H
Yee
EJ
McCune
JM
Long-term human hematopoiesis in the SCID-hu mouse.
J Exp Med
172
1990
1055
21
Lapidot
T
Pflumio
F
Doedens
M
Murdoch
B
Williams
DE
Dick
JE
Cytokine stimulation of multilineage hematopoiesis from immature human cells engraftet in SCID mice.
Science
255
1992
1137
22
Nolta
JA
Hanley
MB
Kohn
DB
Sustained human hematopoiesis in immundeficient mice by cotransplantation of marrow stroma expressing human IL-3: Analysis of gene transduction of long-lived progenitors.
Blood
83
1994
3041
23
Larochelle
A
Vormoor
J
Hanenberg
H
Wang
JCY
Bhatia
M
Lapidot
T
Moritz
T
Murdoch
B
Xiao
XL
Kato
I
Williams
DA
Dick
JE
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy.
Nat Med
2
1996
13329
24
Nolta
JA
Dao
MA
Wells
S
Smogorzewska
EM
Kohn
DB
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.
Proc Natl Acad Sci USA
93
1996
2414
25
Vormoor
J
Lapidot
T
Pflumio
F
Risdon
G
Patterson
B
Broxmeyer
HE
Dick
JE
Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.
Blood
83
1994
2489
26
Bhatia
M
Wang
JCY
Kapp
U
Bonnet
D
Dick
JE
Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.
Proc Natl Acad Sci USA
94
1997
5320
27
Cashman
JD
Lapidot
T
Wang
JCY
Doedens
M
Shultz
LD
Lansdorp
P
Dick
JE
Eaves
CJ
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplantated into immunodeficient mice.
Blood
89
1997
4307
28
Hogan
CJ
Shpall
EJ
McNulty
O
McNiece
I
Dick
JE
Shultz
LD
Keller
G
Engraftment and development of human CD34+-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice.
Blood
90
1997
85
29
Gan
OI
Murdoch
B
Larochelle
A
Dick
JE
Differential maintenance of primitive SCID-repopulating cells, clonogenic progenitors, and long term culture initiating cells after incubation on human bone marrow stromal cells.
Blood
90
1997
641
30
Dao
MA
Hannum
CH
Kohn
DB
Nolta
JA
Flt3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral-mediated transduction.
Blood
89
1997
446
31
Kotani
H
Newton
PB
Zhang
S
Chiang
YL
Otto
E
Weaver
L
Blaese
RM
Anderson
WF
McGarrity
GJ
Improved methods of retroviral vector transduction and production for gene therapy.
Hum Gene Ther
5
1994
19
32
Breems
DA
Blockland
EAW
Neben
S
Ploemacher
RE
Frequency analysis of human primitive hematopoietic stem cell subsets using a cobblestone area forming cell assay.
Leukemia
8
1994
1095
33
von Kalle
C
Kiem
HP
Goehle
S
Darovsky
B
Heimfeld
S
Torok-Storb
B
Storb
R
Schuening
FG
Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector.
Blood
84
1994
2890
34
Glimm
H
Kiem
HP
Darovsky
B
Storb
R
Wolf
J
Diehl
V
Mertelsmann
R
von Kalle
C
Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector.
Hum Gene Ther
8
1997
2079
35
Bauer
TR
Miller
AD
Hickstein
DD
Improved transfer of the leukocyte integration CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope.
Blood
86
1995
2379
36
Mavilio
F
Ferrari
G
Rossini
S
Nobili
N
Bonini
C
Casorati
G
Traversari
C
Bordignon
C
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood
83
1994
1988
37
Valtieri
M
Schiro
R
Chelucci
C
Masella
B
Testa
U
Casella
I
Montesoro
E
Mariani
G
Hassan
HJ
Peschle
C
Efficient transfer of selectable and membrane reporter genes in hematopoietic progenitor and stem cells purified from human peripheral blood.
Cancer Res
54
1994
4398
38
Fehse
B
Uhde
A
Fehse
N
Eckert
HG
Clausen
J
Rüger
R
Koch
S
Ostertag
W
Zander
AR
Stockschläder
M
Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low affinity nerve growth fact receptor (ΔLNGFR) gene.
Hum Gene Ther
8
1997
1815
39
Ruggieri
L
Aiuti
A
Salomoni
M
Zappone
E
Ferrari
G
Bordignon
C
Cell-surface marking of CD34+-restricted phenotypes of human hematopoietic progenitor cells by retrovirus-mediated gene transfer.
Hum Gene Ther
8
1997
1611
40
Moritz
T
Patel
VP
Williams
DA
Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.
J Clin Invest
93
1994
1451
41
Hanenberg
H
Xiao
XL
Dillo
D
Hashino
K
Kato
I
Williams
DA
Co-localization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction mammalian cells.
Nat Med
8
1996
876
42
Hannenberg
H
Hashino
K
Konishi
H
Hock
RA
Kato
I
Williams
DA
Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells.
Hum Gene Ther
8
1997
2193
43
Movassagh
M
Desmyter
C
Baillou
C
Chapel-Fernandes
S
Guigon
M
Klatzmann
D
Lemoine
FM
High level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol.
Hum Gene Ther
9
1998
225
44
Dao
MA
Shah
AJ
Crooks
GM
Nolta
JA
Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice.
Blood
91
1998
1243
45
Pflumio
F
Izac
B
Katz
A
Shultz
LD
Vainchenker
W
Coulombel
L
Phenotype and function of human hematopoietic cells engrafting immune-deficient CB17-severe combined immunodeficiency mice and nonobese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells.
Blood
88
1996
3731
46
Conneally
E
Cashman
J
Petzer
A
Eaves
C
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci USA
94
1997
9836
47
Sutherland
HJ
Lansdorp
PM
Henkelman
DH
Eaves
AC
Eaves
CJ
Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stroma cells.
Proc Natl Acad Sci USA
87
1990
3584
48
Breems
AD
Blockland
EAW
Siebel
KE
Mayen
EM
Engels
LJA
Ploemacher
RE
Stroma-contact prevents loss of hematopoietic stem cell quality during ex-vivo expansion of CD34+ mobilized peripheral blood stem cells.
Blood
91
1998
111
49
Shah
AJ
Smogorzewska
EM
Hannum
C
Crooks
GM
Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38- cells and maintains progenitor cells in vitro.
Blood
87
1996
3563
50
Rusten
LS
Lyman
SD
Veiby
OP
Jacobsen
SEW
The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro.
Blood
87
1996
1317
51
Zandstra
PW
Conneally
E
Petzer
AL
Piret
JM
Eaves
CJ
Cytokine manipulation of primitive hematopoietic cell-self-renewal.
Proc Natl Acad Sci USA
94
1997
4698
52
Nordon
RE
Ginsberg
SS
Eaves
CJ
High-resolution cell division tracking demonstrates the Flt3-Ligand-dependence of human marrow CD34+CD38- cell production in vitro.
Br J Haematol
98
1997
528
53
Haylock
DN
Horsfall
MJ
Dowse
TL
Ramshaw
HS
Niutta
S
Protopsaltis
S
Peng
L
Burrell
C
Rappold
I
Buhring
HJ
Simmons
PJ
Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand.
Blood
90
1997
2260
54
Papayannopoulou
T
Nakamoto
B
Andrews
RG
Lyman
SD
Lee
MY
In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor.
Blood
90
1997
620
55
Bhatia
M
Bonnet
D
Kapp
U
Wang
JCY
Murdoch
B
Dick
JE
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med
186
1997
619
56
Sekhar
M
Kotani
H
Doren
S
Agarwal
R
McGarrity
G
Dunbar
CE
Retroviral transduction of CD34-enriched hematopoietic progenitor cells under serum-free conditions.
Hum Gene Ther
7
1996
33
57
Cashman
JD
Eaves
AC
Raines
EW
Ross
R
Eaves
CJ
Mechanisms that regulate the cell cyclus status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-beta.
Blood
75
1990
96
58
Eaves
CJ
Cashman
JD
Kay
RJ
Dougherty
GJ
Otsuka
T
Gaboury
LA
Hogge
DE
Lansdorp
PM
Eaves
AC
Humphries
RK
Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cell swithin the adherent layer.
Blood
78
1991
110
59
Hatzfeld
J
Li
M
Brown
EL
Sookdeo
H
Levesque
J
O’Toole
T
Gurney
C
Clark
SC
Hatzfeld
A
Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor-β1 oligonucleotides.
J Exp Med
174
1991
925
60
Cardoso
AA
Li
ML
Batard
P
Hatzfeld
A
Brown
EL
Levesque
JP
Sookdeo
H
Panterne
B
Sansilvestri
P
Clark
SC
Hatzfeld
J
Release from quiescence of CD34+CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.
Proc Natl Acad Sci USA
90
1993
8707
61
Ploemacher
RE
van Soest
PL
Boudewijn
A
Autocrine transforming growth factor β1 blocks colony formation and progenitor cell generation by hematopoietic stem cells stimulated with steel factor.
Stem Cells
11
1993
336
62
Soma
T
Yu
JM
Dunbar
CE
Maintenance of murine long-term repopulating stem cells in ex vivo culture is affected by modulation of transforming growth factor-β but not macrophage inflammatory protein-1α activities.
Blood
87
1996
4561
63
Porter
CD
Parkar
MH
Collins
MKL
Levinsky
RJ
Kinon
C
Efficient retroviral transduction of human bone marrow progenitor and long-term culture-initiating cells: Partial reconstitution of cells from patients with X-linked chronic granulomatous disease by gp91-phox expression.
Blood
87
1996
3722
Sign in via your Institution