|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3247-3254
Identification of Human and Mouse Hematopoietic Stem Cell Populations
Expressing High Levels of mRNA Encoding Retrovirus Receptors
By
Donald Orlic,
Laurie J. Girard,
Stacie M. Anderson,
Louise C. Pyle,
Mervin C. Yoder,
Hal E. Broxmeyer, and
David M. Bodine
From the Hematopoiesis Section, Laboratory of Gene Transfer, National
Human Genome Research Institute (NHGRI), National Institutes of Health
(NIH), Bethesda, MD; the Department of Pediatrics, Riley Hospital for
Children; the Departments of Microbiology/Immunology and Medicine; and
the Walther Oncology Center, Indiana University School of Medicine,
Indianapolis, IN.
 |
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 Lin
CD34+ 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.
| |
INTRODUCTION |
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.2
Ecotropic gp70 binds to an amino acid transporter protein.4
The 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 Lin
CD34+ CD38+ (FITC
PECy5+ 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.26,27
Briefly, 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 Fc II/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 Lin
CD34+ (FITC PE+) or
Lin CD34 (FITC
PE) 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
 2-microglobulin (2-m), ecoR, amphoR, and GaLVR (Table 1). 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. 2-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 2-m.
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:
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 (Fig
1), 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.
View larger version (23K):
[in this window]
[in a new window]
| 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 Lin
CD34+ CD38 HSC and Lin
CD34+ 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 Lin
CD34+ CD38+ cells from fetal liver (Fig
2 and Table 2).
View larger version (46K):
[in this window]
[in a new window]
| 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 Lin
CD34+ 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
2-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).
|
|
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 Lin
CD34+ 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 (Fig 3). We found a significant increase
(12-fold, P < .005; 22-fold, P < .005) in the
level of amphoR mRNA in the cryopreserved Lin
CD34+ CD38 cells compared with the amphoR
mRNA level in Lin CD34+ CD38
cells in fresh cord blood and adult BM, respectively (Table 2). We were
not able to detect GaLVR mRNA in either the Lin
CD34+ CD38+ or Lin
CD34+ CD38 fraction from cryopreserved cord
blood.
View larger version (24K):
[in this window]
[in a new window]
| 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 2-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+ CD38
fraction 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 Lin
CD34+ 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 Lin
CD34+, 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.
View larger version (54K):
[in this window]
[in a new window]
| 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 Lin
CD34 yolk sac cells. The levels of mRNA encoding
Ecotropic R and Amphotropic R can be estimated based on the level of
2-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. Lin
cells were sorted by flow cytometry to isolate c-kitHI
cells for RNA analysis. These values were compared with those for the
same cell fractions from untreated control mouse BM (Fig 5). 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 Lin
c-kitHI cells from untreated BM, the mean level of amphoR
mRNA in FR25, FR30, and FR35 Lin c-kitHI
cells 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.
View larger version (18K):
[in this window]
[in a new window]
| 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 2-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).
|
|
| |
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.45-47 Although 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 Lin
CD34 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.
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted December 19, 1997.
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.
| |
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.
| |
REFERENCES |
1.
Anderson F:
Prospects for human gene therapy.
Science
226:401,
1984[Abstract/Free Full Text]
2.
Mulligan RC:
The basic science of gene therapy.
Science
260:926,
1993[Abstract/Free Full Text]
3.
Varmus H:
Retroviruses.
Science
240:1427,
1988[Abstract/Free Full Text]
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
57:659,
1989[Medline]
[Order article via Infotrieve]
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
67:2091,
1993[Abstract/Free Full Text]
6.
Dzierzak EA,
Papayannopoulou TH,
Mulligan RC:
Lineage specific expression of a human -globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells.
Nature
331:35,
1988[Medline]
[Order article via Infotrieve]
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
86:8897,
1989[Abstract/Free Full Text]
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
86:8892,
1989[Abstract/Free Full Text]
9.
Jordan CT,
Lemischka IR:
Clonal and systemic analysis of long-term hematopoiesis in the mouse.
Genes Dev
4:220,
1990[Abstract/Free Full Text]
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
257:99,
1992[Abstract/Free Full Text]
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
8:5116,
1988[Abstract/Free Full Text]
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
87:439,
1990[Abstract/Free Full Text]
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
1:119,
1990[Abstract]
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
91:7071,
1994[Abstract/Free Full Text]
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
91:78,
1994[Abstract/Free Full Text]
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
91:1168,
1994[Abstract/Free Full Text]
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
89:7640,
1992[Abstract/Free Full Text]
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
82:1975,
1993[Abstract/Free Full Text]
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
342:1134,
1993[Medline]
[Order article via Infotrieve]
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
85:3048,
1995[Abstract/Free Full Text]
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
1:1017,
1995[Medline]
[Order article via Infotrieve]
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
93:11097,
1996[Abstract/Free Full Text]
23.
Orlic D,
Bodine DM:
Pluripotent hematopoietic stem cells of low and high density can repopulate W/WV mice.
Exp Hematol
20:1291,
1992[Medline]
[Order article via Infotrieve]
24.
Kadan MJ,
Sturm,
Anderson WF,
Eglitis MA:
Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.
J Virol
66:2281,
1992[Abstract/Free Full Text]
25.
Crooks GM,
Kohn DB:
Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells.
Blood
82:3290,
1993[Abstract/Free Full Text]
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
83:2436,
1994[Abstract/Free Full Text]
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
2:59,
1996 [Medline]
[Order article via Infotrieve]
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
82:762,
1993[Abstract/Free Full Text]
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
92:4601,
1995[Abstract/Free Full Text]
30.
Miller AD,
Buttimore C:
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol Cell Biol
6:2895,
1986[Abstract/Free Full Text]
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
23:422,
1997[Medline]
[Order article via Infotrieve]
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
93:1051,
1994
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
89:1364,
1997[Abstract/Free Full Text]
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
174:63,
1991[Abstract/Free Full Text]
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
78:1706,
1991[Abstract/Free Full Text]
36.
Randall TD,
Lund FE,
Howard MC,
Weissman IL:
Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.
Blood
87:4057,
1996[Abstract/Free Full Text]
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
88:429,
1996[Abstract/Free Full Text]
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
88:89,
1996[Abstract/Free Full Text]
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
19:206,
1990
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
63:3865,
1989[Abstract/Free Full Text]
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
10:4239,
1990[Abstract/Free Full Text]
42.
Hodgson GS,
Bradley TR:
Properties of haemopoietic stem cells surviving 5-fluorouracil treatment: Evidence for a pre-CFU-S cell?
Nature
281:381,
1979[Medline]
[Order article via Infotrieve]
43.
van Zant G:
Studies of hematopoietic stem cells spared by 5-fluorouracil.
J Exp Med
159:679,
1984[Abstract/Free Full Text]
44.
Harrison DE,
Lerner C:
Most primitive hemopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil.
Blood
78:1237,
1991[Abstract/Free Full Text]
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
1:31,
1990[Medline]
[Order article via Infotrieve]
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
6:5,
1995[Medline]
[Order article via Infotrieve]
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
23:2080,
1995[Free Full Text]
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
89:3919,
1997[Abstract/Free Full Text]
49.
Richardson C,
Bank A:
Developmental-stage-specific expression and regulation of an amphotropic retroviral receptor in hematopoietic cells.
Mol Cell Biol
16:4240,
1996[Abstract]
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
86:3828,
1989[Abstract/Free Full Text]
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
335:157,
1996[Abstract/Free Full Text]
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
88:795,
1996[Abstract/Free Full Text]
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
89:4109,
1992[Abstract/Free Full Text]
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
90:8707,
1993[Abstract/Free Full Text]
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
178:529,
1993[Abstract/Free Full Text]
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
178:2089,
1993[Abstract/Free Full Text]
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
84:1482,
1994[Abstract/Free Full Text]
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
93:11871,
1996[Abstract/Free Full Text]
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
88:275a,
1996
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K.-L. Ang, L. Takura Shenje, L. Srinivasan, and M. Galinanes
Repair of the damaged heart by bone marrow cells: from experimental evidence to clinical hope.
Ann. Thorac. Surg.,
October 1, 2006;
82(4):
1549 - 1558.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Lucas, N. E. Seidel, C. D. Porada, J. G. Quigley, S. M. Anderson, H. L. Malech, J. L. Abkowitz, E. D. Zanjani, and D. M. Bodine
Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes
Blood,
July 1, 2005;
106(1):
51 - 58.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Brenner, N. L. Whiting-Theobald, G. F. Linton, K. L. Holmes, M. Anderson-Cohen, P. F. Kelly, E. F. Vanin, A. M. Pilon, D. M. Bodine, M. E. Horwitz, et al.
Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/{beta}2-microglobulin-/- repopulating mobilized human peripheral blood CD34+ cells
Blood,
October 15, 2003;
102(8):
2789 - 2797.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Hematti, S. E. Sellers, B. A. Agricola, M. E. Metzger, R. E. Donahue, and C. E. Dunbar
Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates
Blood,
March 15, 2003;
101(6):
2199 - 2205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Tsai, H. L. Malech, M. R. Kirby, A. P. Hsu, N. E. Seidel, C. D. Porada, E. D. Zanjani, D. M. Bodine, and J. M. Puck
Retroviral transduction of IL2RG into CD34+ cells from X-linked severe combined immunodeficiency patients permits human T- and B-cell development in sheep chimeras
Blood,
June 17, 2002;
100(1):
72 - 79.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Orlic, J. Kajstura, S. Chimenti, F. Limana, I. Jakoniuk, F. Quaini, B. Nadal-Ginard, D. M. Bodine, A. Leri, and P. Anversa
Mobilized bone marrow cells repair the infarcted heart, improving function and survival
PNAS,
August 10, 2001;
(2001)
181177898.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. Beltrami, K. Urbanek, J. Kajstura, S.-M. Yan, N. Finato, R. Bussani, B. Nadal-Ginard, F. Silvestri, A. Leri, C. A. Beltrami, et al.
Evidence That Human Cardiac Myocytes Divide after Myocardial Infarction
N. Engl. J. Med.,
June 7, 2001;
344(23):
1750 - 1757.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. F. Kelly, J. Vandergriff, A. Nathwani, A. W. Nienhuis, and E. F. Vanin
Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein
Blood,
August 15, 2000;
96(4):
1206 - 1214.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. B. Deisseroth
Clinical Trials Involving Multidrug Resistance Transcription Units in Retroviral Vectors
Clin. Cancer Res.,
July 1, 1999;
5(7):
1607 - 1609.
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. C. MacNeill, H. Hanenberg, K. E. Pollok, J. C. M. van der Loo, M. F. A. Bierhuizen, G. Wagemaker, and D. A. Williams
Simultaneous Infection with Retroviruses Pseudotyped with Different Envelope Proteins Bypasses Viral Receptor Interference Associated with Colocalization of gp70 and Target Cells on Fibronectin CH-296
J. Virol.,
May 1, 1999;
73(5):
3960 - 3967.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. Whitwam, M. E. Haskins, P. S. Henthorn, J. N. Kraszewski, S. E. Kleiman, N. E. Seidel, D. M. Bodine, and J. M. Puck
Retroviral Marking of Canine Bone Marrow: Long-Term, High-Level Expression of Human Interleukin-2 Receptor Common Gamma Chain in Canine Lymphocytes
Blood,
September 1, 1998;
92(5):
1565 - 1575.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Orlic, J. Kajstura, S. Chimenti, F. Limana, I. Jakoniuk, F. Quaini, B. Nadal-Ginard, D. M. Bodine, A. Leri, and P. Anversa
Mobilized bone marrow cells repair the infarcted heart, improving function and survival
PNAS,
August 28, 2001;
98(18):
10344 - 10349.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract
