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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the BMT Program, University of Colorado Health
Sciences Center, Denver, CO.
Ex vivo expanded peripheral blood progenitor cells (PBPCs) have
been proposed as a source of hematopoietic support to decrease or
eliminate the period of neutropenia after high-dose chemotherapy. CD34
cells were selected from rhG-CSF mobilized PBPCs from patients with
breast cancer and were cultured for 10 days in defined media containing
100 ng/mL each of rhSCF, rhG-CSF, and PEG-rhMGDF in 1 L Teflon bags at
20 000 cells/mL. After culture the cells were washed and reinfused on
day 0 of transplantation. On day +1, cohort 1 patients (n = 10) also
received an unexpanded CD34-selected PBPC product. These patients
engrafted neutrophils (absolute neutrophil count, >500/µL) in a
median of 6 (range, 5-14) days. Cohort 2 patients (n = 11), who
received expanded PBPCs only, engrafted neutrophils in a median of 8 (range, 4-16) days. In comparison, the median time to neutrophil
engraftment in a historical control group of patients (n = 100) was 9 days (range, 7-30 days). All surviving patients are now past the
15-month posttransplantation stage with no evidence of late graft
failure. The total number of nucleated cells harvested after expansion
culture was shown to be the best predictor of time to neutrophil
engraftment, with all patients receiving more than
4 × 107 cells/kg, engrafting neutrophils by day 8. No
significant effect on platelet recovery was observed in any patient.
These data demonstrate that PBPCs expanded under the conditions defined
can shorten the time to engraftment of neutrophils compared with
historical controls and that the rate of engraftment is related to the
dose of expanded cells transplanted.
(Blood. 2000;96:3001-3007) Transplantation of mobilized peripheral blood
progenitor cells (PBPCs) after high-dose chemotherapy has resulted in
decreased time to neutrophil and platelet engraftment, compared to the
engraftment resulting from bone marrow transplantation.1,2
However, clinically significant periods of neutropenia and
thrombocytopenia after transplantation of mobilized PBPCs still exist.
Recent studies in normal baboons demonstrated that the use of ex vivo
expanded PBPCs could greatly reduce, and in some animals eliminate,
neutropenia after lethal total body irradiation.3 In these
animal studies,3,4 CD34+ PBPCs were cultured
in defined media (Amgen, Thousand Oaks, CA) containing recombinant
human stem cell factor (rhSCF), recombinant human granulocyte
colony-stimulating factor (rhG-CSF), and recombinant human
megakaryocyte growth and development factor (PEG-rhMGDF) for 10 days in
Teflon (American Fluoroseal, Gaithersburg, MD) bags. Animals that
received expanded PBPCs and posttransplantation growth factor support
engrafted neutrophils by day 3, whereas control animals engrafted
neutrophils on day 13. Animals transplanted with ex vivo expanded cells
that did not receive growth factors after transplantation had
engraftment equivalent to that of animals transplanted with unexpanded
cells. No effect was observed on platelet engraftment with the expanded
cells.3
Ex vivo expansion has also been shown to be an effective strategy for
purging bone marrow harvests or PBPC autografts for patients with
chronic myelogenous leukemia,5,6 acute myelogenous leukemia,7 and non-Hodgkin lymphoma.8 Because
it is extremely difficult to demonstrate the clonogenic growth of
primary breast cancer cells in liquid culture,9 we
postulated that ex vivo expansion might be an effective way to
eradicate or reduce breast cancer cells contained in PBPC autografts.
Numerous clinical studies10-13 have been reported using ex
vivo expanded cells generated under a range of culture conditions that
differ with respect to the growth factor regimens, the culture media,
and, in some studies, the addition of fetal calf serum. The results of
these studies have confirmed the safety of infusion of ex vivo expanded
cells but have shown little, if any, clinical benefit with respect to improved rates of engraftment or purging of tumor cells. The aim of the
current study was to evaluate, in patients with breast cancer receiving
high-dose chemotherapy, the culture conditions that were effective in
reducing the period of neutropenia in lethally irradiated
baboons,3 to determine whether they would provide a
product capable of rapid engraftment and purged of tumor cells.
Patients
Study patients.
The study protocol was approved by the Combined Multiple Institutional
Review Board at the University of Colorado Health Sciences Center, and
all patients gave written informed consent before study enrollment.
Patients with stage II or III breast cancer involving 4 or more
axillary lymph nodes or with stage IV disease were eligible for this
study. Normal vital organ function and a Karnofsky performance status
of 80% or more were enrollment requirements, as previously
described.14
Control patients.
The control group included 100 breast cancer patients treated within 2 years of the study patients, matched for stage of disease, prior
therapy, phase of trial (I vs II), and high-dose regimen. The
comparability of study and control patients is summarized in Table
1. All control patients were mobilized
with rhG-CSF and received systemic rhG-CSF after transplantation until
engraftment (absolute neutrophil count [ANC] > 500/µL).
Mobilization and posttransplant doses of rhG-CSF were identical to
those used in the study patients, described below.
Study design
Collection phase.
Eligible patients received rhG-CSF 10 µg/kg per day by subcutaneous
injection for 9 consecutive days (Figure
1A,B). Leukapheresis began on day 5 of
growth factor administration and was performed daily for 5 consecutive
days. Each leukapheresis processed 2.8 to 4 times the patient's blood
volume (approximately 12 to 18 L) using a Cobe Spectra apheresis device
(Cobe Laboratories, Lakewood, CO). The first 4 leukapheresis products
were CD34-selected using the Isolex-300i device (Nexell, Irvine, CA),
following the manufacturer's recommended procedures, and the selected
cells were cryopreserved. The CD34 selection and cryopreservation
procedures were performed on the day of collection for all products.
Five leukapheresis products were collected for each patient. The first
4 apheresis products were CD34-selected, and the fifth was
cryopreserved as a backup.
Transplantation and follow-up.
Within 2 to 10 days of completion of the leukapheresis procedures,
patients were admitted to the hospital and treated with high-dose
chemotherapy. Study and control patients enrolled on phase II
chemotherapy protocols received cyclophosphamide, 1875 mg/m2 per day, given intravenously over 1 hour on each of 3 days (days Ex vivo expansion On day 10 of treatment, 2 apheresis products were thawed and
placed into ex vivo expansion culture. The cells were diluted to
20 000 cells/mL in 800 mL in defined media (Amgen) supplemented with
100 ng/mL each of rhSCF, PEG-rhMGDF, and rhG-CSF (all growth factors
were supplied by Amgen) and then transferred to Teflon bags (American
Fluoroseal). The bags were incubated at 37°C for 10 days in a 5%
CO2 incubator. On day 0 of high-dose therapy administration (day 10 of ex vivo culture), the cultured cells were harvested using a
cell washer (Cobe), and the media and growth factors were removed
with washing.
Immediately before transplantation, an aliquot of each day 10 PBPC culture was removed for the following analyses: complete blood count and differential, CD34+ cell count, breast cancer cell immunohistochemistry, and microbial culture (aerobic and anaerobic bacteria, fungi, and Mycoplasma). Megakaryocytic cell analysis Cytospins of the day 10 cultures were prepared and stained with anti CD41 for immunohistochemistry, and the megakaryocyte content was enumerated by the collaborating pathologists.Progenitor cell assays Myeloid progenitor cells were evaluated on apheresis products, CD34-selected products, and ex vivo expanded cells by flow analysis of CD34 expression using the ISHAGE analysis procedure.15Breast cancer cell assays A sample from every unmanipulated, CD34+, and cultured PBPC fraction was evaluated for breast cancer. For the CD34-selected fractions, the limited number of cells available precluded extensive evaluation. Mononuclear cells from the unmanipulated fraction and the CD34+ and day 10 cultured cells were washed with phosphate-buffered saline containing 10% fetal bovine serum and adjusted to a concentration of 5.0 × 106 cells/mL. Two hundred microliters of the cell suspension were added to cytocentrifuge chambers, and the cells were centrifuged at 500 rpm for 5 minutes onto silane-coated slides. Ten slides (with a total of 10 × 106 cells) prepared with cells from the apheresis products and the ex vivo expanded cells were stained with a panel of monoclonal antibodies previously shown to be effective for identifying breast tumor cells among hematopoietic cells16 (Bre-3; donated by Dr Roberto Ceriani) using a previously described APAAP technique.17 Immunostained slides were counterstained with hematoxylin and microscopically examined in a blinded fashion. The stained cells were systematically scanned with a conventional light microscope using a low-power (10 ×) objective. Immunoreactive cells identified at scanning magnification were scrutinized at high-power magnification to confirm the presence of malignant histologic features. These features included large cell size, high nuclear/cytoplasmic ratio, active-appearing nuclear chromatin, prominent nucleoli, and cell clustering.Study end points and statistical analyses Primary efficacy end points for this study included the number of days from the first day of infusion of cells (day 0) to neutrophil engraftment (500/µL or more) and the number of days until platelet engraftment (20 000/µL or more), independent of platelet transfusions. Statistical analysis of the time to neutrophil and platelet engraftment of cohorts 1 and 2 compared to the historical group was performed using the Kruskal-Wallis nonparametric analysis of variance from SAS PROC NPAR1WAY. The secondary end points included the magnitude of breast cancer cell depletion and myeloid progenitor cell expansion.
Twenty-one patients with breast cancer were enrolled in this
study. The demographic data and the high-dose chemotherapy regimens the
patients received are summarized in Table 1. Twelve patients were
enrolled on phase II chemotherapy protocols in the adjuvant setting
with either 4 to 9 (n = 3) or 10 or more (n = 7) positive axillary
lymph nodes or inflammatory cancer (n = 2). The remaining patients
with metastatic disease were enrolled on phase II chemotherapy trials
with no evidence of disease (n = 5) or disease responding to
induction therapy (n = 2), or they were enrolled on phase I chemotherapy trials for refractory disease (n = 2). The high-dose chemotherapy regimens for patients enrolled in cohort 1 or 2 are summarized in Table 2. The first 10 patients enrolled into the study were assigned to cohort 1 and received
both expanded and uncultured CD34+ PBPCs. Six of these 10 patients received CCB, 2 patients received CCT, and 2 patients received
TCM. The subsequent 11 patients were assigned to cohort 2 and received
CCB (n = 10) or CCT (n = 1), followed by the expanded
CD34+ PBPCs as their sole hematopoietic support.
Selection and ex vivo culture For cohort 1, the median total number of CD34+ cells collected in 4 apheresis products was 8.4 (range, 2.5 to 23.9) × 106 CD34+ cells/kg. The median number of CD34+ cells/kg in the 2 expanded apheresis products was 4.4 (range, 1.1 to 13.3) × 106 CD34+ cells/kg. The 2 apheresis products used for expansion in cohort 2 contained a median of 4.6 (range, 1.8 to 7.6) × 106 CD34+ cells/kg.After leukapheresis the number of cells available for selection
and expansion were a median of 8 × 1010 total nucleated cells (TNC) and 1.2 × 1011 TNC for cohorts 1 and 2, respectively (Table 3). The median total
CD34+ cells, available for selection, was
3.0 × 108 (range, 1 to 9 × 108) and
3.4 × 108 (range, 1 to 6 × 108) for
cohorts 1 and 2, respectively (Table 4).
Overall the median recovery of CD34+ cells after selection
was 57% with a median purity of 83% CD34+ cells. The
CD34+ cell products were cultured in Teflon (American
Fluoroseal) bags at a cell density of 20 000 cells/mL to a maximum of
10 bags. Cultured cells formed a monolayer of cells on the lower
surface of the bags, as shown in a typical day 10 culture presented in Figure 2. The median TNC harvested after
ex vivo expansion was 4.4 (range, 0.6 to 7) × 109 cells
(Table 3). There was a median 15-fold (range, 1.3 to 37) expansion of
TNC. The median number of CD34+ cells harvested from the
expansion cultures was 3.9 × 108 CD34 cells (Table 4),
with a median expansion of CD34+ cells of 1.8-fold (range,
0.7 to 4.5).
Bags were inspected for visible microbial contamination before harvest by microscopy, and no contamination was observed in any cultures. Microbial analyses were negative for bacteria, fungus, and Mycoplasma for all patients. The average viability of the cultured cells, as measured by trypan blue dye exclusion, was 98% (range, 97%-100%). Immunohistochemistry for CD41 was performed on expanded cells from a
subset of patients, and the median percentage of CD41-expressing cells
was 40%. A typical immunohistochemical stain for CD41 is shown in
Figure 3.
Breast cancer cell detection Seven of the unmanipulated PBPC products from 5 patients had detectable breast cancer cells. After CD34 selection a limited number of cells were available for culture inoculation; thus insufficient cells remained for adequate performance of the immunodetection assay. No tumor cells were detectable in the ex vivo expanded products, where adequate cell numbers were assayed for tumor cells, which was consistent with a lack of clonogenic growth of breast cancer cells in in vitro culture.9 There might have been a reduction in tumor cells during the culture period; however, our inability to analyze sufficient cells from the CD34-selected fractions precluded any evaluation of this issue.Clinical results The median number of expanded TNC reinfused into patients was 72 (range, 12 to 91) × 106 TNC/kg and 52 (range, 7 to 125) × 106 TNC/kg for cohorts 1 and 2, respectively. The median number of expanded CD34+ cells infused for cohort 1 was 3.8 (range, 0.9 to 13.0) × 106 CD34+ cells/kg, and the total CD34+ cells infused (expanded plus unexpanded products) was a median of 8.5 (range, 2.3 to 24.6) × 106 CD34+ cells/kg. Cohort 2 patients received a median of 6.6, with a range of 1.5 to 22.7 × 106, CD34+ cells/kg. There was no toxicity associated with the infusion of expanded PBPCs. Engraftment data for patients in both cohorts are presented in Table 5. The median time to neutrophil engraftment, defined as the time to achieve an ANC of 500/µL or greater for 3 consecutive days, was 6 days and 8 days for cohorts 1 and 2, respectively. The time to achieve a platelet count of 20 000/µL or greater was 18.5 days and 15 days, respectively, for cohorts 1 and 2 and was not significantly different from the time to platelet engraftment of the control patients (15 days). There was no difference in the requirements for red blood cell and platelet transfusions between the 2 cohorts.
Engraftments for the historical group of patients (n = 100) are presented for comparison in Figure 3. The median time to an ANC greater than 500/µL was 9 days, with a range of 7 to 30 days. Neutrophil recovery resulting from the transplantation of the expanded cells resulted in a 3- to 4-day earlier engraftment for a number of the study patients compared with this historical control group (Figure 3), in which the earliest engraftment occurred at day 7. Statistical analysis demonstrated a significant difference in the time to neutrophil engraftment between cohort 1 (P = .02) and cohort 2 (P = .02) and the historical group. No significant difference resulted between cohorts 1 and 2 (P = .55). Long-term follow-up Adjuvant/inflammatory and stage IV patients with no evidence of disease remain in remission at 4 to 10 months of follow-up. Most have received chest wall radiation therapy without clinically significant myelosuppression. One patient in cohort 2 experienced delayed platelet recovery. She had a platelet count of 30 000 on day +40, when the posttransplantation radiation therapy to the chest wall was scheduled to begin. None of the other cohort 2 patients experienced delayed platelet engraftment. Patients with responsive metastatic disease remain in remission, whereas 1 of the 2 patients with refractory disease had a relapse after a short-lived partial response to high-dose therapy. Patients in cohort 2 have continued to maintain peripheral blood counts, and all patients are past the 12-month posttransplantation stage and have maintained durable engraftment. No unusual opportunistic infections were documented in any of the study patients.
These studies have demonstrated that PBPC products from patients
with breast cancer can be cultured ex vivo using these culture conditions and can be safely transplanted after high-dose chemotherapy. Expanded PBPC autografts resulted in more rapid median time to engraftment of neutrophils (P = .02 for cohorts 1 and 2 versus the historical group), with a number of patients engrafting
between days 4 and 6, compared to the earliest time of engraftment of day 7 in our historical controls (n = 100, Figure 4). Analysis of the
engraftment data of study and control patients by chemotherapy regimen
demonstrated that the conditioning regimen did not influence the time
to neutrophil engraftment. Similarly improved rates of neutrophil
engraftment have been reported by Reiffers et al,18 with
the same conditions used in this study to culture the autologous PBPC
fractions of patients with myeloma.18
To date the major predictor of the time to engraftment for patients
receiving nonexpanded PBPCs has been the number of CD34+
cells per kilogram transplanted. Several studies19-21
using nonexpanded products have demonstrated that there is a
correlation between the time to engraftment and the number of CD34
cells per kilogram infused up to a threshold level, above which even a
10-fold higher dose of CD34+ cells does not result in
faster recovery. For neutrophil recovery, doses of CD34+
cells per kilogram from 2 to 2.5 × 106 up to
20 × 106 CD34+ cells/kg result in recovery
of neutrophils at day 7 at the earliest.19-21 We therefore
evaluated the numbers of CD34+ cell per kilogram from the
expanded products reinfused in this study, and a median of
3.8 × 106 CD34 cells/kg and 6.6 × 106
CD34+ cells/kg were transplanted for cohorts 1 and 2, respectively. The total CD34+ cells infused (expanded plus
unexpanded products) for cohort 1 was a median of
8.5 × 106 CD34+ cells/kg. No significant
correlation was obtained between the time to ANC greater than 500/µL
and the dose of total CD34+ cell per kilogram
infused (r2 = 0.24; Figure
5A).
Similarly, the dose of expanded CD34+ cells per kilogram
infused showed little correlation to the time to neutrophil engraftment
(r2 = 0.24). However, comparison of the total
expanded nucleated cells per kilogram to the time to ANC recovery
resulted in a highly significant correlation
(r2 = 0.79; Figure 5B). In our study, the
correlation of the number of CD34+ cells to time to
recovery of ANC was poor (r2 = 0.24); however,
all patients who received a minimum of 40 × 106 TNC per
kilogram engrafted neutrophils in 8 days or less. Patients who received
less than 40 × 106 TNC per kilogram all had slower
neutrophil recovery (9 to 16 days). This level of TNC is lower than the
minimal cell numbers recommended for unmanipulated bone marrow
transplantation (2 × 108 MNC/kg) and suggests that the
expanded cell products contain a higher frequency of cells capable of
providing rapid neutrophil engraftment. Alternatively, ex vivo culture
regenerates these cells, which are killed during the freezing of bone
marrow or PBPCs used for autologous transplantation. These cells may be committed mature neutrophil precursors, which may be killed during freezing along with mature neutrophils. It will be of interest to
evaluate the engraftment potential of ex vivo expanded allogeneic PBPC
products where a direct comparison could be made to bone marrow or
PBPCs that has not been cryopreserved.
The effect of tumor cell depletion was evaluated in all patients. There were 7 phereses (5 patients) with tumor cells in the unmanipulated PBPC fractions. No tumor cells were detected in the expanded products, but the assay did not have the sensitivity to determine whether the CD34+ preculture fraction contained tumor cells. Additionally, because the expansion cultures result in expansion of total cells there may be a dilution effect of tumor cells, and more extensive analysis is needed to determine the exact effects of ex vivo culture on purging of breast cancer cells. Previously reported studies22 have been unable to eradicate breast cancer cells completely with the CD34 selection of bone marrow or PBPC autografts. Preliminary results of this study suggest that expansion may result in important elimination of tumor cells. Because many purging procedures used to date result in a significant loss of normal CD34+ progenitor cells, the expansion procedure may compensate for these losses by expanding the committed progenitors while potentially providing additional tumor depletion benefit. Recent data from Holmberg et al23 have suggested that selection and infusion of CD34+ cells from autologous PBPC grafts result in an increased incidence of cytomegalovirus disease. The patients reported in this study did not experience cytomegalovirus or other opportunistic infections after transplantation. The results of this study raise several issues that remain to be resolved: The effect of the expansion culture on long-term engrafting cells. The fate of stem cells during the expansion cultures has not been determined. Cohort 2 patients who received expanded cells as the sole source of hematopoietic support will provide supportive data on the long-term durability of engraftment. All patients in cohort 2 are now past the 12-month posttransplantation stage and continue to experience durable engraftment. The potential of residual stem cells in the patients (endogenous autologous recovery) to provide the long-term hematopoiesis will confound the issue of the effect of expansion on stem cells, and this may only be answered in a definitive manner by gene marking of the expanded cells and showing marked cells long-term in these patients. At a practical level, whether the long-term hematopoiesis comes from the expanded cells that were infused or from residual stem cells in the patient is less important than the ability of the patients to maintain long-term blood cell production with the high-dose chemotherapy regimens used. Expanded cells from the patients in cohort 2 have also been simultaneously transplanted into NOD/SCID mice24 to assay for human SCID repopulating cells (SRC). These studies have demonstrated the presence of SRC in the expanded products25 and should provide some insight into the primitive nature of the expanded cell population. No significant difference in platelet recovery was observed with
expanded cells. Immunohistochemical analysis of CD41
expression of the expanded cells demonstrates the presence of a large
number of mature megakaryocytes in the expanded cell fraction; however, this is not reflected in the time to platelet recovery. There are
several possible explanations for the lack of effect obtained on
platelet recovery Is it possible to eliminate neutropenia in these patients?
The results presented in this study demonstrate the effect of expanded cells on improving neutrophil engraftment; however, it is possible that
if more CD34+ cells are available for expansion, even more
rapid engraftment may occur. Alternatively, daily reinfusion of
expanded cells on days 0 through 7 may provide mature neutrophils such
that patients never have neutrophil counts lower than 500/µL. Another
possibility is the infusion of expanded cells at day The potential application of expanded cells for cord blood allogeneic transplantation is also being explored at this institution. Initial data suggest that cord blood progenitors, expanded under the same culture conditions used in the current study, enable engraftment of adults with fewer total nucleated cell per kilogram of patient weight than reported with unexpanded cord blood.26,27 In summary, this study demonstrates the potential of ex vivo expanded PBPCs to provide more rapid engraftment than historical controls transplanted with unmanipulated PBPCs after high-dose chemotherapy. Ongoing analysis may provide more insight into the definitive cell types capable of providing rapid neutrophil and platelet engraftment and the growth factor requirements of these cells. Further studies are warranted to define the optimal timing for reinfusion and possible further improvements in the expansion culture conditions.
We thank Dr R. Ceriani for his generous supply of the Bre3 antibody for the breast cancer cell detection assay. We also thank Robert Briddell, Dr Anthony Gringeri, and Dr Graham Molineux (Amgen) for their support in conducting this study. Finally, we thank Nexell Therapeutics for supplying the selection columns for this study.
Submitted July 24, 1999; accepted July 3, 2000.
Supported in part by National Institutes of Health/National Cancer Institute grant RO1-CA61508 (E.J.S.) and by Amgen (I.M.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ian McNiece, BMT Program B-116, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Denver, CO 80262; e-mail: ian.mcniece{at}uchsc.edu.
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© 2000 by The American Society of Hematology.
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 L. T. Huang, C. J. Paredes, E. T. Papoutsakis, and W. M. Miller Gene expression analysis illuminates the transcriptional programs underlying the functional activity of ex vivo-expanded granulocytes Physiol Genomics, September 11, 2007; 31(1): 114 - 125. [Abstract] [Full Text] [PDF]
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 P. J. Real, Y. Cao, R. Wang, Z. Nikolovska-Coleska, J. Sanz-Ortiz, S. Wang, and J. L. Fernandez-Luna Breast Cancer Cells Can Evade Apoptosis-Mediated Selective Killing by a Novel Small Molecule Inhibitor of Bcl-2 Cancer Res., November 1, 2004; 64(21): 7947 - 7953. [Abstract] [Full Text] [PDF]
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 Y. Sasaki, C. T. Jensen, S. Karlsson, and S. E. W. Jacobsen Enforced expression of cyclin D2 enhances the proliferative potential of myeloid progenitors, accelerates in vivo myeloid reconstitution, and promotes rescue of mice from lethal myeloablation Blood, August 15, 2004; 104(4): 986 - 992. [Abstract] [Full Text] [PDF]
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S. J. Szilvassy, T. E. Meyerrose, P. L. Ragland, and B. Grimes Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver Blood, October 1, 2001; 98(7): 2108 - 2115. [Abstract] [Full Text] [PDF]
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O. Kollet, A. Spiegel, A. Peled, I. Petit, T. Byk, R. Hershkoviz, E. Guetta, G. Barkai, A. Nagler, and T. Lapidot Rapid and efficient homing of human CD34+CD38{-}/lowCXCR4+ stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice Blood, May 15, 2001; 97(10): 3283 - 3291. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
if they
had more than 3 pheresis products
