Abstract

One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in LinCD34+ CD38 cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin CD34+ CD38 cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM.

PLURIPOTENT HEMATOPOIETIC stem cells (HSC) are ideal targets for gene therapy because of their capacity to self-renew and reconstitute all lineages of the hematopoietic system after myeloablation and transplantation.1,2 If retroviruses carrying a therapeutic gene can be introduced into the genome of HSC, the progeny of the transduced HSC would all carry the transferred gene.3 Expression of the therapeutic gene at appropriate levels should lead to a permanent correction of the specific hematologic disorder.1,2 The host range for retroviruses is determined by the gp70 glycoprotein of their envelope.2Ecotropic gp70 binds to an amino acid transporter protein.4The gp70 binding site is not conserved on the amino acid transporter of other mammals, hence ecotropic viruses transduce only mouse cells.5 Under appropriate conditions, ecotropic retrovirus transduction of mouse HSC leads to marking of 20% to 60% of the mature circulating blood cells.6-12 

Two different retroviruses are currently being used to transduce mouse and human HSC. The gp70 envelope proteins of amphotropic retroviruses and the Gibbon Ape Leukemia Virus (GaLV) use homologous but distinct phosphate transport proteins as receptors.13-16 The amphotropic and GaLV gp70 binding sites are conserved among most mammals, which has led to the development of amphotropic and GaLV retroviruses as vehicles for gene transfer into human HSC. In contrast to the efficient transduction of mouse HSC with ecotropic retroviruses, transduction of Rhesus monkey and human HSC with amphotropic or GaLV retroviruses has been relatively inefficient, with fewer than 1% of the circulating blood cells containing proviral DNA sequences.17-21 

We have previously reported that enriched populations of mouse and human HSC from bone marrow (BM) contained relatively low levels of amphoR mRNA.22 Using counterflow centrifugal elutriation (CCE), we identified a subpopulation of murine HSC (FR25 Lin c-kitHI) with a low level of amphoR mRNA and a subpopulation (FR35 Lin c-kitHI) with a higher level of amphoR mRNA.23 We simultaneously transduced both subpopulations of HSC with ecotropic and amphotropic retrovirus vectors of similar titers. Our results showed that HSC containing high levels of amphoR mRNA were transduced by amphotropic retroviruses with an efficiency comparable with that achieved with ecotropic viruses. HSC with low levels of amphoR mRNA were transduced 20-fold less efficiently by amphotropic retroviruses compared with ecotropic viruses.22 

In this study, we measured retrovirus receptor mRNA levels in different populations of human and mouse HSC. Based on our previous results, we hypothesized that identification of subpopulations of HSC with high levels of amphoR and/or GaLVR mRNA would lead to improved retrovirus transduction efficiency. We found a high level of amphoR mRNA in the HSC-enriched population from cryopreserved human cord blood. We also found a high level of amphoR mRNA in several HSC populations from the BM of mice treated with granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF).

MATERIALS AND METHODS

Purification of HSC from human fetal liver.

CD34+ fetal liver cells (purity greater than 80%) were purchased from Poietic Technologies, Inc (Germantown, MD). The cells were stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MoAb) that recognized the lineage markers CD3, CD15, and CD20 (Becton Dickinson, San Jose, CA); glycophorin A (Coulter Immunology, Hialeah, FL); and anti–CD34-PECy5 (PECy5 = phycoerythrin cyanogen 5) and anti–CD38-PE MoAbs, which are specific for primitive cell markers (Becton Dickinson). The LinCD34+ CD38+ (FITCPECy5+ and PE+) and Lin CD34+ CD38(FITC PECy5+ and PE) cells were isolated by flow cytometry. Reanalysis of the sorted samples indicated purities of greater than 95%.

Purification of HSC from human cord blood.

Human cord blood was obtained after normal delivery with informed consent and with the approval of the Institutional Review Board of the Indiana University School of Medicine. Samples were provided as fresh and cryopreserved cord blood. Mononuclear cells (MNCs) were isolated by centrifugation on a layer of Lymphocyte Separation Medium (Organon Teknika Corp, Durham, NC) followed by red blood cell lysis in ACK Lysing Buffer (Bio Whittaker, Walkersville, MD) at 4°C for 30 minutes. The cells were then stained with MoAb for flow cytometry as described above. The Lin CD34+CD38 population was defined as the lowest 1% to 2% of the FITC PECy5+ PE cells. The Lin CD34+ CD38+ cell population was isolated from the highest 1% to 2% of the PE-stained FITC PECy5+ PE+ cells.

Purification of HSC from human adult BM.

Human BM (25-mL samples) was purchased from Poietic Technologies. The cells were fractionated by CCE into FR15 (discarded), FR20, FR25, FR30, FR35, and FR40 fractions. The cells in each fraction were stained with MoAb and sorted by flow cytometry as described above.

Retrovirus binding assay.

The virus binding assay was performed exactly as described by Kadan et al24 and Crooks and Kohn.25 Human adult BM MNCs were incubated with media containing amphotropic retrovirus and 8 μg polybrene/mL media at 37°C for 40 minutes. After washing with 10% heat-inactivated goat serum in phosphate-buffered saline (PBS) to remove unbound virus, the cells were incubated with FITC-conjugated 83A25, a rat MoAb specific for gp70, at 4°C for 60 minutes. Control cells were incubated with FITC-conjugated isotype antibodies. The cells were washed with a 10% solution of heat-inactivated newborn calf serum in PBS. Finally, the cells were stained with anti–CD34-PECy5 and anti–CD38-PE MoAbs as described above. The CD34+(PECy5+) cells were analyzed for FITC (virus binding) and PE (CD38 expression) by flow cytometry.

Purification of HSC from mouse yolk sac.

Yolk sac cells were obtained as previously described.26,27Briefly, 9.5-day embryos were dissected from the uterus of timed-mated C57BL/6J female mice. The yolk sacs were dissected free of the embryo, dispersed by drawing through a 23-gauge needle, and cultured for 60 minutes at 37°C in Hank's buffered saline solution (HBSS) containing 0.1% collagenase (Sigma, St Louis, MO) and 20% fetal calf serum (FCS). The yolk sac cells were collected by centrifugation and incubated at 4°C for 20 minutes sequentially with rat anti-mouse FITC-conjugated CD16/CD32 (Pharmingen, San Diego, CA), a MoAb which is specific for FcγII/III receptors that are expressed on natural killer cells, monocytes, macrophages, granulocytes, B-lymphocytes, and most fetal T lymphocytes before expression of CD4, CD8, and TCR; rat anti-mouse FITC-conjugated TER-119 (Pharmingen), specific for red cells; and rat anti-mouse biotin-conjugated CD34-biotin (Pharmingen) followed by streptaviden-conjugated phycoerythrin. The cells were washed, resuspended in HBSS with 5% FCS, and LinCD34+ (FITC PE+) or Lin CD34 (FITCPE) cells were enriched by flow cytometry using a FACStar instrument (Becton Dickinson).

Purification of murine HSC from the BM of mice treated with G-CSF and SCF.

Adult splenectomized mice were injected subcutaneously for 5 consecutive days with 200 μg/kg/d G-CSF and 50 μg/kg/d SCF. BM cells were collected 14 days after the final injection of cytokines. MNCs were isolated by centrifugation on a layer of Lymphocyte Separation Medium (Organon Teknika) and were fractionated by CCE as described previously.28 Briefly, MNCs were separated at flow rates of 15 (FR15, discarded), 25 (FR25), 30 (FR30), and 35 (FR35) mL/min. The Lin+ cells were subtracted by immunobead selection and the Lin cells were sorted based on c-kitHi expression.28 

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of retrovirus receptor mRNA in mouse and human HSC.

Total cellular RNA from enriched mouse and human HSC populations was purified using RNAzol (Tel-Test, Inc, Friendswood, TX) or Trizol (Tel-Test) following the manufacturer's instructions. RNA levels were analyzed by RT-PCR as described previously.29 Briefly, mRNA from different cell populations was reverse transcribed and the cDNAs were amplified using primer pairs specific for mouse and human β2-microglobulin (β2-m), ecoR, amphoR, and GaLVR (Table1). The PCR was performed at 94°C for 1 minute (denaturing), 58°C or 65°C for 1 minute (annealing), and 72°C for 2 minutes (extension) using a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) for 35 cycles. β2-m mRNA was used in a limiting dilution RT-PCR assay to identify a set point on the linear phase of the PCR amplification curve, which was used to quantify the level of mRNA for each retrovirus receptor relative to β2-m.

Table 1.

Primer Sequences Used in the RT-PCR Assay

Mouse β2-m  Sense, 5′ TGC TAT CCA GAA AAC CCC TC 3′ 
 Antisense, 5′ GTC ATG CTT AAC TCT GCA GG 3′ 
 Fragment size, 258 bp  
Mouse amphoR  Sense, 5′ CGG GCG GAA GAC GAG AAG GA 3′  
 Antisense, 5′ GAA GCC ACT GGA CGG TGT GA 3′  
 Fragment size, 309 bp  
Mouse ecoR Sense, 5′ CTG CCT CAA CAC CTA TGA CC 3′  
 Antisense, 5′ TGC TGA CGT GAG AAC TCT CC 3′  
 Fragment size, 363 bp 
Human β2-m  Sense, 5′ CTC GCG CTA CTC TCT CTT TC 3′ 
 Antisense, 5′ CAT GTC TCG ATC CCA CTT AAC 3′ 
 Fragment size, 330 bp  
Human amphoR  Sense, 5′ CGG AAC ATC TTC GTG GCC TG 3′  
 Antisense, 5′ GCT GGT CAT GAG AGA GCC GTG 3′  
 Fragment size, 220 bp  
Human GaLVR Sense, 5′ GTG TGG CAA CTC GTG GCT TC 3′  
 Antisense, 5′ CAG CAA CGG TGC TCC AG 3′  
 Fragment size, 321 bp 
Mouse β2-m  Sense, 5′ TGC TAT CCA GAA AAC CCC TC 3′ 
 Antisense, 5′ GTC ATG CTT AAC TCT GCA GG 3′ 
 Fragment size, 258 bp  
Mouse amphoR  Sense, 5′ CGG GCG GAA GAC GAG AAG GA 3′  
 Antisense, 5′ GAA GCC ACT GGA CGG TGT GA 3′  
 Fragment size, 309 bp  
Mouse ecoR Sense, 5′ CTG CCT CAA CAC CTA TGA CC 3′  
 Antisense, 5′ TGC TGA CGT GAG AAC TCT CC 3′  
 Fragment size, 363 bp 
Human β2-m  Sense, 5′ CTC GCG CTA CTC TCT CTT TC 3′ 
 Antisense, 5′ CAT GTC TCG ATC CCA CTT AAC 3′ 
 Fragment size, 330 bp  
Human amphoR  Sense, 5′ CGG AAC ATC TTC GTG GCC TG 3′  
 Antisense, 5′ GCT GGT CAT GAG AGA GCC GTG 3′  
 Fragment size, 220 bp  
Human GaLVR Sense, 5′ GTG TGG CAA CTC GTG GCT TC 3′  
 Antisense, 5′ CAG CAA CGG TGC TCC AG 3′  
 Fragment size, 321 bp 

The fragments amplified by PCR were resolved by polyacrylamide gel electrophoresis. The specificity of the primer pairs was confirmed by restriction enzyme digestion of the amplified fragment at sites predicted by the DNA sequence. The intensity of the bands was quantified using a Molecular Dynamics Densitometer System (Sunnyvale, CA). HeLa and NIH-3T3 cells are efficiently transduced by amphotropic retroviruses.30,31 We have shown that HeLa and NIH-3T3 cells have higher levels of amphoR and GaLVR mRNA than human or mouse BM HSC, respectively. The levels of amphoR and GaLVR mRNA in HSC-enriched fractions were normalized to the level of amphoR and GaLVR mRNA in HeLa or NIH-3T3 cells by the following formula:

Receptor mRNA inHSCβ2­mmRNA inHSC×β2­mmRNA in HeLa/3T3CellsReceptor mRNA in HeLa/3T3Cells=Relative Level of Receptor mRNA in Human/MouseHSC

The levels of receptor mRNA in the human HSC-enriched Lin CD34+ CD38 fractions were then compared with the levels in the progenitor-enriched Lin CD34+ CD38+ fractions.

RESULTS

Retrovirus binding assay.

MNCs from untreated adult BM were exposed to amphotropic retrovirus and the binding capacity of the CD34+ cells was determined using a FITC-labeled antibody specific for the retrovirus envelope protein gp70. The highest FITC fluorescence intensity was seen on the CD34+ CD38+ cells (Fig1), a population enriched for progenitor cells. There was a relative decrease in retrovirus binding in cells with decreasing CD38 expression and the HSC-enriched CD34+CD38 subpopulation bound less than 10% of the virus bound by CD34+ CD38+ cells.

Fig. 1.

Retrovirus binding to CD34+CD38+ and CD34+ CD38 cells from adult BM. (A) Cells isolated from a Cell Pro CD34 column were incubated with a virus containing medium and stained with anti–CD34 PECy5 for gating and anti–CD38-PE MoAb and isotype-FITC Ab. (B) Cells incubated as above with FITC-conjugated 83A25 MoAb (anti-mouse retrovirus gp70).

Fig. 1.

Retrovirus binding to CD34+CD38+ and CD34+ CD38 cells from adult BM. (A) Cells isolated from a Cell Pro CD34 column were incubated with a virus containing medium and stained with anti–CD34 PECy5 for gating and anti–CD38-PE MoAb and isotype-FITC Ab. (B) Cells incubated as above with FITC-conjugated 83A25 MoAb (anti-mouse retrovirus gp70).

RT-PCR analysis of retrovirus receptor mRNA levels in human HSC.

Human HSC-enriched Lin CD34+CD38 cells from fetal liver and BM confer long-term engraftment in fetal sheep32 and severe combined immunodeficiency (SCID)- hu mice.33 We measured the relative level of retrovirus receptor mRNA in LinCD34+ CD38 HSC and LinCD34+ CD38+ progenitor cells from both of these hematopoietic organs. The level of receptor mRNA in HSC and progenitor cells was compared with the level of receptor mRNA in HeLa cells, which served as the reference standard. AmphoR and GaLVR mRNA levels were uniformly low in HSC-enriched Lin CD34+CD38 and progenitor-enriched LinCD34+ CD38+ cells from fetal liver (Fig2 and Table 2).

Fig. 2.

RT-PCR analysis of amphotropic retrovirus receptor (Amphotropic R) and Gibbon Ape Leukemia Virus receptor (GALV R) mRNA levels in human fetal liver and adult BM. RNA was extracted from unfractionated (UNFR), HSC-enriched LinCD34+ CD38, and progenitor-enriched Lin CD34+ CD38+ fetal liver and adult BM cells. The levels of Amphotropic R (upper bands) and GALV R mRNA (middle bands) were quantified based on the level of β2-microglobulin mRNA (lower bands) in the same sample. These values were then compared with the levels of Amphotropic R or GALV R mRNA in HeLa cells (right column) and compared with the mRNA levels in unfractionated control BM (see Table 2). Adult human BM cells were fractionated by CCE at flow rates (FR) of 20, 25, 30, 35, and 40 mL/min (FR20 to FR40). Samples from five separate experiments were analyzed (see Table 2).

Fig. 2.

RT-PCR analysis of amphotropic retrovirus receptor (Amphotropic R) and Gibbon Ape Leukemia Virus receptor (GALV R) mRNA levels in human fetal liver and adult BM. RNA was extracted from unfractionated (UNFR), HSC-enriched LinCD34+ CD38, and progenitor-enriched Lin CD34+ CD38+ fetal liver and adult BM cells. The levels of Amphotropic R (upper bands) and GALV R mRNA (middle bands) were quantified based on the level of β2-microglobulin mRNA (lower bands) in the same sample. These values were then compared with the levels of Amphotropic R or GALV R mRNA in HeLa cells (right column) and compared with the mRNA levels in unfractionated control BM (see Table 2). Adult human BM cells were fractionated by CCE at flow rates (FR) of 20, 25, 30, 35, and 40 mL/min (FR20 to FR40). Samples from five separate experiments were analyzed (see Table 2).

Table 2.

RT-PCR Analysis of Retrovirus Receptor mRNA in Human HSC

AmphoR mRNA GaLVR mRNA
Fetal liver 
 Lin CD34+ CD38+ (2) 0.31 ± 0.04* 0.38 ± 0.41  
 LinCD34+ CD38 (2)  0.22 ± 0.13 0.28 ± 0.28  
Cord blood  
 LinCD34+ CD38+ (Fresh) (3)  0.38 ± 0.34 0.08 ± 0.04  
 Lin CD34+CD38 (Fresh) (3)  0.13 ± 0.05  0.07 ± 0.03 
 Lin CD34+ CD38+ (Frozen) (3)  1.54 ± 1.61  Not detected 
 Lin CD34+ CD38 (Frozen) (3)  1.55 ± 0.61†‡ Not detected  
Bone marrow  
 Lin CD34+CD38+ (5)  0.20 ± 0.20  0.22 ± 0.22 
 Lin CD34+ CD38 (4) 0.07 ± 0.10  0.15 ± 0.14  
 FR20 LinCD34+ CD38 (5)  0.10 ± 0.10 0.01 ± 0.01  
 FR25 Lin CD34+CD38 (5)  0.03 ± 0.02  0.02 ± 0.02 
 FR30 Lin CD34+ CD38 (5) 0.05 ± 0.06  0.03 ± 0.03  
 FR35 LinCD34+ CD38 (5)  0.07 ± 0.06 0.05 ± 0.04  
 FR40 Lin CD34+CD38 (5)  0.12 ± 0.15  0.10 ± 0.20 
HeLa cells (5)  1.0  1.0 
AmphoR mRNA GaLVR mRNA
Fetal liver 
 Lin CD34+ CD38+ (2) 0.31 ± 0.04* 0.38 ± 0.41  
 LinCD34+ CD38 (2)  0.22 ± 0.13 0.28 ± 0.28  
Cord blood  
 LinCD34+ CD38+ (Fresh) (3)  0.38 ± 0.34 0.08 ± 0.04  
 Lin CD34+CD38 (Fresh) (3)  0.13 ± 0.05  0.07 ± 0.03 
 Lin CD34+ CD38+ (Frozen) (3)  1.54 ± 1.61  Not detected 
 Lin CD34+ CD38 (Frozen) (3)  1.55 ± 0.61†‡ Not detected  
Bone marrow  
 Lin CD34+CD38+ (5)  0.20 ± 0.20  0.22 ± 0.22 
 Lin CD34+ CD38 (4) 0.07 ± 0.10  0.15 ± 0.14  
 FR20 LinCD34+ CD38 (5)  0.10 ± 0.10 0.01 ± 0.01  
 FR25 Lin CD34+CD38 (5)  0.03 ± 0.02  0.02 ± 0.02 
 FR30 Lin CD34+ CD38 (5) 0.05 ± 0.06  0.03 ± 0.03  
 FR35 LinCD34+ CD38 (5)  0.07 ± 0.06 0.05 ± 0.04  
 FR40 Lin CD34+CD38 (5)  0.12 ± 0.15  0.10 ± 0.20 
HeLa cells (5)  1.0  1.0 

The number of samples are shown in parentheses. Receptor mRNA levels were normalized to the level of β2-m mRNA in the same sample. For quantification, the receptor mRNA levels were compared with the level of the receptor mRNA in unfractionated BM.

*

Mean ± SD of the mRNA levels relative to the level in HeLa cells.

P < .005 compared with Lin CD34+CD38 BM cells.

P < .005 compared with fresh LinCD34+ CD38 cord blood cells.

The mean level of amphoR and GaLVR mRNA in unfractionated human BM was 20% and 22%, respectively, of the mean level of amphoR and GaLVR mRNA in five different preparations of HeLa cell RNA (Table 2). We have previously shown that subpopulations of mouse BM HSC separated by CCE vary in the level of amphoR mRNA. We analyzed amphoR and GaLVR mRNA levels in five different human adult BM HSC populations separated on the basis of size using CCE. For each CCE fraction, a progenitor-enriched Lin CD34+CD38+ population and a HSC-enriched LinCD34+ CD38 population was isolated by flow cytometry. Discrete populations of Lin CD34+CD38 BM cells expressing generally low levels of amphoR mRNA compared with HeLa cells could be identified in each of the five experiments (Figure 2). However, the population(s) of Lin CD34+ CD38 BM cells expressing the lowest and highest levels of amphoR mRNA appeared in different CCE fractions in the five separate experiments. Analysis of pooled data from all five experiments showed no significant difference in the level of amphoR mRNA between any of the CCE fractions (Table 2). The levels of GaLVR mRNA in human HSC separated by CCE followed the same general patterns as the amphoR mRNA levels.

Preliminary studies showed strikingly higher levels of amphoR mRNA in both Lin CD34+ CD38+ and Lin CD34+ CD38 cord blood cells that had been cryopreserved before processing. To test this directly, three cord blood samples were divided at the time of collection, one half for immediate RT-PCR analysis and the other half for analysis after a freeze/thaw cycle (Fig3). We found a significant increase (12-fold, P < .005; 22-fold, P < .005) in the level of amphoR mRNA in the cryopreserved LinCD34+ CD38 cells compared with the amphoR mRNA level in Lin CD34+ CD38cells in fresh cord blood and adult BM, respectively (Table 2). We were not able to detect GaLVR mRNA in either the LinCD34+ CD38+ or LinCD34+ CD38 fraction from cryopreserved cord blood.

Fig. 3.

RT-PCR analysis of amphotropic receptor (ampho R) mRNA levels in fresh and frozen human cord blood cells. The levels of mRNA encoding ampho R mRNA and β2-microglobulin mRNA in unfractionated (UF) Lin CD34+ CD38 and Lin CD34+ CD38+ cells were analyzed as in Fig 2. The level of ampho R mRNA in the HSC-enriched Lin CD34+ CD38 fraction from frozen cord blood is significantly increased (P < .005) over the level of ampho R mRNA in the HSC-enriched Lin CD34+ CD38fraction from fresh cord blood (see Table 2).

Fig. 3.

RT-PCR analysis of amphotropic receptor (ampho R) mRNA levels in fresh and frozen human cord blood cells. The levels of mRNA encoding ampho R mRNA and β2-microglobulin mRNA in unfractionated (UF) Lin CD34+ CD38 and Lin CD34+ CD38+ cells were analyzed as in Fig 2. The level of ampho R mRNA in the HSC-enriched Lin CD34+ CD38 fraction from frozen cord blood is significantly increased (P < .005) over the level of ampho R mRNA in the HSC-enriched Lin CD34+ CD38fraction from fresh cord blood (see Table 2).

RT-PCR analysis of retrovirus mRNA levels in mouse yolk sac HSC.

Mouse HSC capable of reconstituting the entire hematopoietic system have been variously identified by ourselves28 and others34-37 as c-kit+ CD34+and/or CD34 cells. The LinCD34+ and Lin CD34 cells from 9.5-day yolk sac were enriched by flow cytometry and their RNA was extracted for RT-PCR analysis (Fig 4). The amphotropic and ecotropic retrovirus receptor (ecoR) mRNA values were compared with the level of expression in NIH-3T3 cells, which can be efficiently transduced with both amphotropic and ecotropic vectors. AmphoR and ecoR mRNAs were present in unfractionated LinCD34+, and Lin CD34 yolk sac cells. The relative level of amphoR and ecoR mRNA in most yolk sac samples was equal to or greater than the level of amphoR and ecoR mRNA in NIH-3T3 cells.

Fig. 4.

RT-PCR analysis of ecotropic retrovirus receptor (Ecotropic R; top panel) and amphotropic retrovirus receptor (Amphotropic R; middle panel) mRNA levels in mouse 9.5-day yolk sac cells. Receptor mRNA levels are shown for two or three separate samples of RNA isolated from unfractionated (Unfr) yolk sac and sorted Lin CD34+ and LinCD34 yolk sac cells. The levels of mRNA encoding Ecotropic R and Amphotropic R can be estimated based on the level of β2-microglobulin mRNA (lower bands) in the same sample. The levels of Ecotropic R and Amphotropic R mRNA in NIH-3T3 cells are also seen (right column).

Fig. 4.

RT-PCR analysis of ecotropic retrovirus receptor (Ecotropic R; top panel) and amphotropic retrovirus receptor (Amphotropic R; middle panel) mRNA levels in mouse 9.5-day yolk sac cells. Receptor mRNA levels are shown for two or three separate samples of RNA isolated from unfractionated (Unfr) yolk sac and sorted Lin CD34+ and LinCD34 yolk sac cells. The levels of mRNA encoding Ecotropic R and Amphotropic R can be estimated based on the level of β2-microglobulin mRNA (lower bands) in the same sample. The levels of Ecotropic R and Amphotropic R mRNA in NIH-3T3 cells are also seen (right column).

RT-PCR analysis of retrovirus mRNA levels in mouse HSC populations from mice treated with G-CSF and SCF.

We have previously shown that five daily injections of G-CSF and SCF into adult splenectomized mice causes a mobilization of HSC into the peripheral blood. Peripheral blood HSC levels decline after cytokine treatment is stopped, but 14 days after treatment we showed a greater than 10-fold increase in the number of HSC in the BM.38 

BM cells collected on day 14 after cytokine treatment were fractionated by CCE into FR25, FR30, and FR35 cell populations. Lincells were sorted by flow cytometry to isolate c-kitHIcells for RNA analysis. These values were compared with those for the same cell fractions from untreated control mouse BM (Fig5). AmphoR mRNA levels in the HSC-enriched FR25, FR30, and FR35 Lin c-kitHi populations from untreated BM were 5%, 12%, and 14%, respectively, of the level of amphoR mRNA in NIH-3T3 cells (Table 3).This level was significantly lower than the amphoR mRNA level in unfractionated control BM cells (P < .01). The level of amphoR mRNA in unfractionated BM collected 14 days after cytokine treatment was nearly equivalent to the level in NIH-3T3 cells and was 1.7-fold higher than the level in unfractionated control BM from untreated mice (P < .01). In contrast to the low level of amphoR mRNA in FR25, FR30, and FR35 Linc-kitHI cells from untreated BM, the mean level of amphoR mRNA in FR25, FR30, and FR35 Lin c-kitHIcells from mice treated with G-CSF and SCF was increased to a level equivalent to that of unfractionated control BM (Table 3). There was no increase in the levels of ecoR mRNA when the elutriated Lin c-kitHI cell fractions from cytokine-treated mice were compared with the values from untreated mouse BM.

Fig. 5.

RT-PCR analysis of amphotropic retrovirus receptor mRNA levels in BM hematopoietic stem cells from untreated mice (top panel), and from mice injected for 5 consecutive days with G-CSF and SCF (bottom panel). BM cells were collected from mice treated with G-CSF and SCF at 14 days after the last cytokine treatment. The cells were fractionated by CCE at flow rates of 25, 30, and 35 mL/min (FR25, FR30, FR35), and Lin c-kitHI cells were isolated from each fraction by flow cytometry. The levels of amphotropic receptor mRNA were quantified based on the level of β2-microglobulin mRNA (not shown) in the same sample. These values were normalized to the level of amphotropic receptor mRNA in NIH-3T3 cells (right column) and compared with the mRNA level in unfractionated BM (see Table 3).

Fig. 5.

RT-PCR analysis of amphotropic retrovirus receptor mRNA levels in BM hematopoietic stem cells from untreated mice (top panel), and from mice injected for 5 consecutive days with G-CSF and SCF (bottom panel). BM cells were collected from mice treated with G-CSF and SCF at 14 days after the last cytokine treatment. The cells were fractionated by CCE at flow rates of 25, 30, and 35 mL/min (FR25, FR30, FR35), and Lin c-kitHI cells were isolated from each fraction by flow cytometry. The levels of amphotropic receptor mRNA were quantified based on the level of β2-microglobulin mRNA (not shown) in the same sample. These values were normalized to the level of amphotropic receptor mRNA in NIH-3T3 cells (right column) and compared with the mRNA level in unfractionated BM (see Table 3).

Table 3.

RT-PCR Analysis of Retrovirus Receptor mRNA in BM HSC From Untreated and G-CSF– and SCF-Treated Mice

amphoR ecoR
Untreated mice  
 Unfractionated (9)  0.49 ± 0.12* 1.16 ± 0.47  
 FR25 Lin c-kitHI (3)  0.05 ± 0.04 0.49 ± 0.25  
 FR30 Lin c-kitHI(2)  0.12 ± 0.04 Not done  
 FR35 Linc-kitHI (7)  0.14 ± 0.02 1.10 ± 0.33 
Cytokine-injected mice 
 Unfractionated (3) 0.86 ± 0.23 1.53 ± 0.51  
 FR25 Linc-kitHI (2)  0.39 ± 0.37  0.61 ± 0.33 
 FR30 Lin c-kitHI (2) 0.30 ± 0.20  0.67 ± 0.35  
 FR35 Linc-kitHI (2)  0.39 ± 0.06  0.81 ± 0.05 
NIH-3T3  1.0  1.0 
amphoR ecoR
Untreated mice  
 Unfractionated (9)  0.49 ± 0.12* 1.16 ± 0.47  
 FR25 Lin c-kitHI (3)  0.05 ± 0.04 0.49 ± 0.25  
 FR30 Lin c-kitHI(2)  0.12 ± 0.04 Not done  
 FR35 Linc-kitHI (7)  0.14 ± 0.02 1.10 ± 0.33 
Cytokine-injected mice 
 Unfractionated (3) 0.86 ± 0.23 1.53 ± 0.51  
 FR25 Linc-kitHI (2)  0.39 ± 0.37  0.61 ± 0.33 
 FR30 Lin c-kitHI (2) 0.30 ± 0.20  0.67 ± 0.35  
 FR35 Linc-kitHI (2)  0.39 ± 0.06  0.81 ± 0.05 
NIH-3T3  1.0  1.0 

The number of samples are shown in parentheses. Receptor mRNA levels were quantified based on the level of β2-m mRNA in the same sample. They were then normalized to the levels in NIH-3T3 receptor mRNA and compared with the level of the receptor mRNA in unfractionated BM.

*

Mean ± SD of the mRNA levels relative to the level in NIH-3T3 cells.

P < .01 compared with unfractionated control BM.

Cytokine-injected mice received 200 μg G-CSF/kg/d and 50 μg SCF/kg/d for 5 days. BM cells were collected 14 days later.

DISCUSSION

Infrequent transduction of canine, primate, and human HSC with amphotropic and GaLV vectors has been shown in gene marking studies.17-20 The inefficient gene transfer in these trials may be the result of low virus titer,2 inability of the retrovirus genome to integrate into the DNA of quiescent HSC,39-44 and/or a low number of amphotropic receptors in HSC. Several earlier studies, including our own, showed that amphotropic retrovirus transduction of HSC from mouse BM was inefficient, with less than 1% of the peripheral blood cells containing a provirus.22,45 In previous studies we have shown low levels of amphoR mRNA in mouse and human HSC. We have also shown that the level of ecotropic receptor mRNA is relatively high in mouse HSC. Under appropriate culture conditions we have observed relatively efficient transduction of mouse HSC using ecotropic retrovirus vectors, with 35% to 60% of the peripheral blood cells containing the provirus. The relatively efficient transduction of HSC with ecotropic retrovirus vectors indicates the cell cycle status of the target HSC permits proviral integration.6-12,39 

We have shown a direct correlation between amphoR mRNA levels and transduction efficiency of mouse HSC22 and human cultured cell lines.31 Other groups have shown that transient expression of the amphotropic receptor gene in cultured cells increases transduction with amphotropic retroviral vectors.45-47Although we cannot exclude the possibility that canine, primate, and human HSC remain quiescent under conditions which allow mouse HSC to cycle, we hypothesize that the inefficient transduction of HSC using amphotropic retrovirus vectors is a result of limiting numbers of amphotropic receptors.

To identify human HSC which might be more efficiently transduced by amphotropic or GaLV vectors, we examined retrovirus receptor mRNA levels in several sources of HSC which have been successfully used for transplantation into humans,19-21 fetal sheep,32 or nonobese diabetic (NOD)/SCID mice.33,48 We found low levels of amphoR and GaLVR mRNA in human fetal liver Lin CD34+CD38 cells equivalent to the low levels found in fractionated and unfractionated BM. This observation is consistent with the findings of a previous study49 which reported low levels of amphoR mRNA and low levels of transduction in unfractionated and HSC-enriched mouse fetal liver cells.

In contrast to fetal liver and adult BM, relatively high levels of amphoR mRNA were detected in cord blood HSC that had been previously cryopreserved. Cryopreserved cord blood has been used successfully for transplantation,50-55 including the repopulation of myeloablated adult recipients. Kohn et al21 have shown that fresh human cord blood HSC can be marked with amphotropic retrovirus vectors containing an adenosine deaminase cDNA. These authors concluded that the low level of marked cells observed in their patients' peripheral blood was caused in part by the fact that the transduced cord blood cells were transplanted into nonmyeloablated recipients. On the basis of our findings, we propose that previously cryopreserved umbilical cord blood HSC are excellent candidates for gene therapy protocols involving myeloablation.21,56,57 

We are extending our investigation of the effect of cryopreservation on amphoR mRNA expression in HSC. One ongoing study involves analysis of receptor mRNA levels in cryopreserved mouse, monkey, and human HSC. In another study, we will specifically test the effect of a brief in vitro exposure of HSC to dimethyl sulfoxide as a possible basis for enhanced amphoR mRNA expression.

In the mouse, we detected relatively high levels of ecoR and amphoR mRNA in 9.5-day unfractionated mouse yolk sac cells and enriched Lin CD34+ and LinCD34 cells. This contrasts with a previous study49 that failed to detect any amphoR mRNA in unfractionated yolk sac cells from days 9.5 through 13.5 of ontogeny. The Lin CD34+ cells from 9.5-day yolk sac can repopulate neonatal mice26,27 and, based on our observation that these cells express both ecoR and amphoR mRNA, we suggest that yolk sac HSC may be excellent targets for gene marking studies, particularly with ecotropic retroviruses, to show the ability of yolk sac HSC to repopulate all of the hematopoietic lineages of adult mice.

We found high levels of amphoR mRNA in BM from mice treated with G-CSF and SCF. This finding may account for the more efficient gene transfer (10% to 30% positive peripheral blood cells) we recently observed with HSC from the peripheral blood or BM of mice, monkeys, and dogs58-60 treated with injections of G-CSF and SCF. This finding is consistent with an earlier observation by Crooks and Kohn25 that addition of interleukin-3 (IL-3), IL-6, and SCF to CD34+ cells in culture results in an increase in amphotropic retrovirus binding. We propose that cytokine treatment of HSC leads to induction of mRNA expression by the gene encoding the amphoR.

In summary, we have identified several subpopulations of murine and human HSC with high levels of amphoR mRNA. From these findings we predict that HSC from cryopreserved human cord blood may be desirable targets for retrovirus transduction. We also predict that HSC from patients treated with cytokines may be improved targets for amphotropic retrovirus transduction.

ACKNOWLEDGMENT

We thank Dr S-I. Nishikawa of Kyoto University (Kyoto, Japan) for providing the anti c-kit MoAb; Dr Paul Tolstoshev of Genetic Therapy, Inc (Gaithersburg, MD) for providing the 83A25 MoAb; and Dr Larry M. Lantz of the National Institute of Allergy and Infectious Diseases, NIH (Bethesda, MD) for conjugating biotin to the c-kit MoAb.

Supported in part by grants from the NIH RO1 HL 54037 and RO1 HL 46416, and a project in PO1 HL 53586 from the NIH (to H.E.B.).

Address reprint requests to Donald Orlic, PhD, Hematopoiesis Section/Laboratory of Gene Transfer, National Human Genome Research Institute/NIH, Bldg 49/Room 3A11, 49 Convent Dr, Bethesda, MD 20892-4442.

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

REFERENCES

1
Anderson
F
Prospects for human gene therapy.
Science
226
1984
401
2
Mulligan
RC
The basic science of gene therapy.
Science
260
1993
926
3
Varmus
H
Retroviruses.
Science
240
1988
1427
4
Albritton
LM
Tseng
L
Scadden
D
Cunningham
JM
A putative murine ecotropic retrovirus receptor encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.
Cell
57
1989
659
5
Albritton
LM
Kim
JW
Tseng
L
Cunningham
JM
Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine viruses.
J Virol
67
1993
2091
6
Dzierzak
EA
Papayannopoulou
TH
Mulligan
RC
Lineage specific expression of a human β-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells.
Nature
331
1988
35
7
Bodine
DM
Karlsson
S
Nienhuis
AW
Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.
Proc Natl Acad Sci USA
86
1989
8897
8
Lim
B
Apperly
JF
Orkin
SH
Williams
DA
Long-term expression of human adenosine deaminase in mice transplanted with retrovirus infected hematopoietic stem cells.
Proc Natl Acad Sci USA
86
1989
8892
9
Jordan
CT
Lemischka
IR
Clonal and systemic analysis of long-term hematopoiesis in the mouse.
Genes Dev
4
1990
220
10
Sorrentino
BP
Brandt
SJ
Bodine
DM
Gottesman
M
Pastan
I
Cline
A
Nienhuis
AW
Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1.
Science
257
1992
99
11
Belmont
JW
MacGregor
GR
Wagner-Smith
K
Fletcher
FA
Moore
KA
Hawkins
D
Villalon
D
Chang
SM-U
Caskey
CT
Expression of adenosine deaminase in murine hematopoietic cells.
Mol Cell Biol
8
1988
5116
12
Wilson
JM
Danos
O
Grossman
M
Raulet
DH
Mulligan
RC
Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells.
Proc Natl Acad Sci USA
87
1990
439
13
O'Hara
B
Johann
SV
Klinger
HP
Blair
DG
Rubinson
H
Dunn
KJ
Sass
P
Vitek
SM
Robins
T
Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus.
Cell Growth Differ
1
1990
119
14
Kavanaugh
MP
Miller
DG
Zhang
W
Law
W
Kozak
SL
Kabat
D
Miller
AD
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.
Proc Natl Acad Sci USA
91
1994
7071
15
Miller
DG
Edwards
RH
Miller
AD
Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus.
Proc Natl Acad Sci USA
91
1994
78
16
van Zeijl
M
Johann
SV
Closs
E
Cunningham
J
Eddy
R
Shows
TB
O'Hara
B
A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family.
Proc Natl Acad Sci USA
91
1994
1168
17
van Beusechem
VW
Kakler
A
Meidt
PJ
Valerio
D
Long-term expression of human adenosine deaminase in Rhesus monkeys transplanted with retrovirus infected bone marrow cells.
Proc Natl Acad Sci USA
89
1992
7640
18
Bodine
DM
Moritz
T
Donahue
RE
Luskey
BD
Kessler
SW
Martin
DIK
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
19
Brenner
MK
Rill
DR
Holladay
MS
Heslop
HE
Moen
RC
Buschle
M
Krance
RA
Santana
VM
Anderson
WF
Ihle
JN
Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.
Lancet
342
1993
1134
20
Dunbar
CE
Cottler-Fox
M
O'Shaughnessy
JA
Doren
S
Carter
C
Berenson
R
Brown
S
Moen
RC
Greenblatt
J
Stewart
FM
Leitman
SF
Wilson
WH
Cowan
K
Young
NS
Neinhuis
AW
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood
85
1995
3048
21
Kohn
DB
Weinberg
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
Williams-Herman
DE
Elder
M
Wara
D
Bowen
T
Hershfield
MS
Mullen
CA
Blaese
RM
Parkman
R
Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.
Nature Med
1
1995
1017
22
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
23
Orlic
D
Bodine
DM
Pluripotent hematopoietic stem cells of low and high density can repopulate W/WV mice.
Exp Hematol
20
1992
1291
24
Kadan
MJ
Sturm
Anderson
WF
Eglitis
MA
Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.
J Virol
66
1992
2281
25
Crooks
GM
Kohn
DB
Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells.
Blood
82
1993
3290
26
Yoder
MC
Papaioannou
VE
Breitfeld
PP
Williams
DA
Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells.
Blood
83
1994
2436
27
Yoder
MC
Cumming
J
Hiatt
K
Mukherjee
P
Williams
DA
A novel method of myeloablation to enhance engraftment of adult bone marrow cells in newborn mice.
Biol Blood Marrow Transplant
2
1996
59
28
Orlic
D
Fischer
R
Nishikawa
S-I
Neinhuis
AW
Bodine
DM
Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor.
Blood
82
1993
762
29
Orlic
D
Anderson
SM
Biesecker
LG
Sorrentino
BP
Bodine
DM
Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.
Proc Natl Acad Sci USA
92
1995
4601
30
Miller
AD
Buttimore
C
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol Cell Biol
6
1986
2895
31
Sabatino
DE
Bao-Khnah
QD
Pyle
LC
Seidel
NE
Girard
LJ
Spratt
SK
Orlic
D
Bodine
DM
Amphotropic and Gibbon Ape Leukemia Virus (GaLV) retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines.
Blood Cells Mol Dis
23
1997
422
32
Zanjani
ED
Flake
AW
Rice
H
Hedrick
M
Tavassoli
M
Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep.
J Clin Invest
93
1994
1051
33
Muench
MO
Roncarolo
MG
Namikawa
R
Phenotypic and functional evidence for the expression of CD4 by hematopoietic stem cells isolated from human fetal liver.
Blood
89
1997
1364
34
Ogawa
M
Matsuzaki
Y
Nishikawa
S
Hayashi
S-I
Kunisada
T
Sudo
T
Kina
T
Nakauchi
H
Nishikawa
S-I
Expression and function of c-kit in hemopoietic progenitor cells.
J Exp Med
174
1991
63
35
Okada
S
Nakauchi
H
Nagayoshi
K
Nishikawa
S
Nishikawa
S-I
Miura
Y
Suda
T
Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule.
Blood
78
1991
1706
36
Randall
TD
Lund
FE
Howard
MC
Weissman
IL
Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.
Blood
87
1996
4057
37
Kobayashi
M
Laver
JH
Kato
T
Miyazaki
H
Ogawa
M
Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3.
Blood
88
1996
429
38
Bodine
DM
Seidel
NE
Orlic
D
Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow.
Blood
88
1996
89
39
Bodine
DM
McDonagh
KT
Seidel
NE
Nienhuis
AW
Survival and retrovirus infection of murine hematopoietic stem cells in vitro: Effects of 5-FU and method of infection.
Exp Hematol
19
1990
206
40
Springett
GM
Moen
RC
Anderson
S
Blaese
RM
Anderson
WF
Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent.
J Virol
63
1989
3865
41
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
42
Hodgson
GS
Bradley
TR
Properties of haemopoietic stem cells surviving 5-fluorouracil treatment: Evidence for a pre-CFU-S cell?
Nature
281
1979
381
43
van Zant
G
Studies of hematopoietic stem cells spared by 5-fluorouracil.
J Exp Med
159
1984
679
44
Harrison
DE
Lerner
C
Most primitive hemopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil.
Blood
78
1991
1237
45
Osborne
WRA
Hock
RA
Kaleko
M
Miller
AD
Long-term expression of human adenosine deaminase in mice after transplantation of bone marrow infected with amphotropic retroviral vectors.
Hum Gene Ther
1
1990
31
46
Lieber
A
Vrancken
PMJ
Kay
MA
Adenovirus-mediated transfer of the amphotropic retrovirus receptor cDNA increases retroviral transduction in cultured cells.
Hum Gene Ther
6
1995
5
47
Yamaguchi
S
Wakimoto
H
Yoshida
Y
Kanegae
Y
Salito
I
Aoyagi
M
Hirakawa
K
Amagasa
T
Hamada
H
Enhancement of retrovirus mediated gene transduction efficiency by transient overexpression of the amphotropic receptor, GLVR-2.
Nucleic Acids Res
23
1995
2080
48
Wang
JCY
Doedens
M
Dick
JE
Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.
Blood
89
1997
3919
49
Richardson
C
Bank
A
Developmental-stage-specific expression and regulation of an amphotropic retroviral receptor in hematopoietic cells.
Mol Cell Biol
16
1996
4240
50
Broxmeyer
HE
Douglas
GW
Hangoc
G
Cooper
S
Bard
J
English
D
Arny
M
Thomas
L
Boyse
EA
Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells.
Proc Natl Acad Sci USA
86
1989
3828
51
Gluckman, E, Broxmeyer HE, Auerbach AD, Friedman H, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J, Boyse EA: Hematopoietic reconstitution in a patient with Fanconi anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321:1174, 1989
52
Kurtzberg
J
Laughlin
M
Graham
ML
Smith
C
Olson
JF
Halperin
EC
Ciocci
G
Carrier
C
Stevens
CE
Rubinstein
P
Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients.
N Engl J Med
335
1996
157
53
Wagner
JE
Rosenthal
J
Sweetman
R
Shu
XO
Davies
SM
Ramsay
NKC
McGlave
PB
Sender
L
Cairo
MC
Successful transplantation of HLA-matched HLA-mismatched umbilical cord blood from unrelated donors: Analysis of engraftment and acute graft-versus-host disease.
Blood
88
1996
795
54
Broxmeyer
HE
Hangoc
G
Cooper
S
Ribeiro
R
Graves
V
Yoder
M
Wagner
J
Vadhan-Raj
S
Benninger
L
Rubinstein
P
Broun
ER
Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.
Proc Natl Acad Sci USA
89
1992
4109
55
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
56
Moritz
T
Keller
DC
Williams
DA
Human cord blood cells as targets for gene transfer: Potential use in gene therapies of severe combined immunodeficiency disease.
J Exp Med
178
1993
529
57
Lu
L
Xiao
M
Clapp
DW
Li
Z-H
Broxmeyer
HE
High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34+++ hematopoietic stem/progenitor cells from human umbilical cord blood.
J Exp Med
178
1993
2089
58
Bodine
DM
Seidel
NE
Gale
MS
Neinhuis
AW
Orlic
D
Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte-colony stimulating factor and stem cell factor.
Blood
84
1994
1482
59
Dunbar
CE
Seidel
NE
Doren
S
Sellers
S
Cline
AP
Metzger
ME
Agricola
BA
Donahue
RE
Bodine
DM
Improved retroviral gene transfer into murine and rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.
Proc Natl Acad Sci USA
93
1996
11871
60
(abstr)
Whitwam
T
Seidel
NE
Haskins
ME
Anderson
SM
Henthorn
PS
Bodine
DM
Puck
JM
Marking of canine hematopoietic progenitors in cytokine stimulated bone marrow with retroviruses containing the IL2RG gene.
Blood
88
1996
275a