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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-01-0025.
HEMATOPOIESIS
From the Division of Research Immunology and Bone
Marrow Transplantation, Children's Hospital of Los Angeles, CA.
The cell surface protein CD34 is frequently used as a marker for
positive selection of human hematopoietic stem/progenitor cells in
research and in transplantation. However, populations of reconstituting
human and murine stem cells that lack cell surface CD34 protein have
been identified. In the current studies, we demonstrate that CD34
expression is reversible on human hematopoietic stem/progenitor cells.
We identified and functionally characterized a population of human
CD45+/CD34 Evidence that CD34 Using another long-term xenograft model, the preimmune fetal sheep
system, it was demonstrated that human
CD34 The current studies demonstrate that a human CD34 Isolation of hematopoietic stem/progenitor cells
Primary transplantation in immunodeficient mice
Secondary transplantation in immunodeficient mice Sublethal conditioning of NOD/SCID mice for secondary transplantation of the human hematopoietic cells from the primary bnx bone marrow was done 2 hours prior to injection of the marrow cell inoculum, by administration of 300 rads using a Shepard Mark IV irradiator with an attenuator to deliver the dose at the slowest possible rate. Faster delivery of the same dosage resulted in significant mortality to this strain. All mice were screened to rule out the presence of murine T and natural killer (NK) cells that can arise due to "leakiness," prior to use. For secondary transplantation, the 2-mL cryovials (Nunc, Naperville, IL) containing the human cells recovered from the primary bnx recipients were thawed, carefully diluted up to 35 mL in Iscove modified Dulbecco medium (IMDM) with 10% fetal calf serum (FCS), DNAse, and heparin as described,16 and incubated overnight in 75-mL flasks (lying flat) with a combination of IL-6 and SCF (50 ng/mL each; R & D Systems, Minneapolis, MN) prior to transplantation. The frozen cells from one primary recipient, initially transplanted with human CD34+/CD38 cells, then verified by RNA and
FACS to contain no human CD34+ cells at the time of harvest
8 to 12 months later, were used to transplant 2 NOD/SCID secondary
recipients, in each set of experiments.
Determination of the presence of human hematopoietic cells in tissues recovered from the mice Bone marrow was flushed from the 4 long bones of the hind legs and used immediately for FACS analysis and cell sorting, or cryopreserved for later analyses. To determine the total human hematopoietic cell content in the bone marrow, the single-cell suspensions were labeled with HLE-1 (antihuman CD45; Becton Dickinson), and then subjected to FACS analysis. In all secondary transplant analyses, samples were gated on human CD45+ cells using antihuman CD45-allophycocyanin (APC; Pharmingen, San Diego, CA) against forward scatter, and the lineages within that gated population were then identified by 3-color analysis. Directly conjugated antibodies used to identify human-specific cell-surface antigens were My9-RD1 (anti-CD33; Coulter, San Francisco, CA), Leu-12 (anti-CD19; Becton Dickinson), Leu-3a (anti-CD4; Becton Dickinson), Leu-2a (anti-CD8; Becton Dickinson), HPCA-1 and HPCA-2 (anti-CD34; Becton Dickinson), and Leu-17 (anti-CD38; Becton Dickinson). Samples were acquired on a Becton Dickinson FACScan and analysis of 10 000 or 100 000 cells acquired from each tissue was done using CellQuest software (Becton Dickinson). The appropriate isotype controls were used in all analyses, and, because each lot of antibody can differ in its cross-reactivity to murine cells, parallel staining and FACS analyses were done on normal human and nontransplanted bnx mouse bone marrow controls, to confirm specificity for human cells.LTCIC assay The LTCICs were grown as described12,17 in 96-well plates on pre-established monolayers of primary human stromal/mesenchymal stem cells. The stromal cells had previously been expanded to result in a relatively homogeneous monolayer of myofibroblastic cells that had less than 1% contaminating CD45+ macrophages, when analyzed by FACS at passage 3, as we have described.13,14 Within 1 week before addition of hematopoietic progenitors, the stromal cultures were trypsinized and irradiated with 20 Gy; then 1000 cells/well were plated in 96-well plates (Corning Costar, Cambridge, MA) in basal bone marrow medium (BBMM, which is IMDM with 30% FCS, 1% bovine serum albumin [BSA; Sigma, St Louis, MO], 100 µM 2-mercaptoethanol, 106 M hydrocortisone [Sigma], 50 U/mL penicillin G, 50 µg/mL streptomycin sulfate, and 2 mM L-glutamine). Human
CD45+ cells from the marrow of bnx or NOD/SCID
mice were plated by FACS onto the stromal monolayers at 100 cells/well.
Cultures were fed once a week by replacing 50% of the medium. Wells
that showed growth at week 6 were collected and plated in
human-specific colony-forming assay.
Human-specific CFU plating To determine the number of clonogenic human hematopoietic progenitors derived from the LTCIC assay, or recovered from the murine bone marrow, cells were plated in human-specific colony-forming unit (CFU) assay as described.13 Prior to plating, the bnx/human bone marrow cells were incubated in IMDM with 20% fetal bovine serum (Omega Scientific, Tarzana, CA) for 4 to 12 hours to remove (by adherence) murine stromal cells and monocyte/macrophages, which secrete murine cytokines and invalidate the specificity for growth of human hematopoietic colonies measured in the assay. The medium used for CFU plating was IMDM with 30% FCS (Omega Scientific), 1% BSA (Sigma), 1.3% methylcellulose (Sigma), 104 M 2-mercaptoethanol, 50 U/mL penicillin G, 50 µg/mL
streptomycin sulfate, 2 mM L-glutamine, 106 M
hydrocortisone, and 10 ng/mL recombinant human IL-3 (rhIL-3; Immunex, Seattle, WA). Recombinant human erythropoietin (Epo; Epoietin
 ; Amgen, Thousand Oaks, CA) was added to a concentration of 2 U/mL
on day 4 of culture, after Epo-dependent murine erythrocyte CFUs (CFU-Es) had died off. Methylcellulose, FCS, and BSA were previously screened to provide maximal
granulocyte-erythrocyte-macrophage-megakaryocyte CFU (CFU-GEMM)
development from human CD34+ cells. Then
5 × 104 and 1 × 105 plastic nonadherent
cells from engrafted and control mice were plated in duplicate in 1 mL
of the medium in gridded culture dishes (Nunc). Colonies were grown in
a fully humidified incubator and enumerated on day 30 as previously
described, because there is poor CFU development from primitive cells
at day 14.18 In this medium, growth of murine colonies,
from nontransplanted control mice, was observed only when stromal cells
had contaminated the CFU dish. Therefore, any plates containing
adherent stromal or fibroblastic colonies were discarded.
RNA and cDNA preparation and testing RNA was isolated from stimulated and nonstimulated cells using RNA STAT-60 (TEL-TEST, Friendswood, TX). Samples were quantitated using a spectrophotometer, and equal amounts of RNA from all samples were subjected to first-strand cDNA synthesis using the Superscript Preamplification System (Gibco BRL, Gaithersburg, MD). For some experiments where human CD45+ cells were not reisolated from the bnx bone marrow, the amount of RNA contributed by the human, as opposed to murine CD45+ cells in the sample was first calculated from FACS analysis. The same amount of RNA was then used from KG-1A cells, human umbilical cord blood (UCB), and bone marrow-positive controls for first-strand synthesis. This precise quantitation was necessary because human CD45+ cells in bnx/hu mice were present at a far lower frequency than in the human control samples. Therefore, a direct comparison of equal amounts of RNA from bnx/hu mouse marrow and human samples could have underestimated the human CD34+ cell frequency in the mice, giving false-negative results in the analyses.Following cDNA amplification from the standardized bnx/hu marrow, reisolated human CD45+ cells from the bnx/hu marrow, and human KG-1a, UCB, or bone marrow control samples, polymerase chain reaction (PCR) was performed for CD34 and GAPDH (used as a loading control) as described.19 Samples were loaded on 2% ethidium bromide-stained gels, electrophoresed, and read with an Eagle Eye reader (Stratagene, La Jolla, CA). Statistical analyses All analyses were done using the Microsoft Excel 5.0 software. Average values are listed with SDs. The significance of each set of values was assessed using the 2-tailed t test assuming equal variance.
Engraftment of primary bnx mice with highly
purified human CD34+/CD38
stem/progenitor cells from 12 different human marrow donors were
transplanted into a total of 28 recipient bnx
mice (Figure 1). The transplanted cells
had high expression of CD34 and very low CD38 expression, as we
have previously described (Figure 2A).11,12
Following a period of long-term engraftment (8-12 months), the mice
were harvested and FACS analysis was done on marrow, spleen, and blood
cells to determine whether they were engrafted with human hematopoietic
cells. Of the 28 mice receiving transplants, 23 mice in this study had
more than 1% engraftment with human hematopoietic cells, as indicated by FACS analysis using an antihuman CD45 antibody. The 23 well-engrafted mice were further evaluated by a FACS immunophenotyping
panel to identify the human T, B, myeloid lineages that had developed, as we have described.11,13,20,21 In addition, the
percentages of human CD34+ and CD38+ cells
within the mouse marrow were evaluated by FACS.
The bnx mice had an average of 5.2 ± 1.1 (SEM) human
CD45+ cells in their marrow (n = 23). The percentage of
the human graft that was composed of CD33+ myeloid
progenitors and cells averaged 47.3% ± 5.2% of the human CD45+ cells (n = 23). There were also significant
percentages of CD8+ (average, 23.8% ± 2.6%) and
CD4+ cells (average, 16.4% ± 2.0%), with fewer
CD19+ (average, 7.8% ± 1.5%) and lin
Although there was no expression of CD34 on the surface of the human CD45+ cells recovered from the long-term engrafted bnx mice, it was possible that there could have been RNA expressed, or CD34 protein that was lying beneath the membrane of the cells and not on the surface, as previously determined by Fackler et al.19 To study this issue, mRNA was prepared and also immunohistochemistry was performed on cytospin preparations of FACS-isolated human CD45+ cells from the bnx marrow, and also the total bone marrow suspensions. No human CD34 protein was detected in the immunohistochemical analyses of marrow cells from bnx mice, whereas human CD34+ cells were readily detectable in the marrow of the control NOD/SCID mice (n = 10 each, data not shown). To further confirm the FACS and immunohistochemical data that indicated
a lack of CD34 protein on the long-term engrafted human hematopoietic
cells, mRNA was prepared from both FACS-isolated human
CD45+ cells, recovered from the bnx marrow, and
from the total bnx/hu bone marrow that contained the
long-term engrafted cells. In the instances where human
CD45+ cells were not reisolated from the bnx
bone marrow, the amount of RNA contributed by the human, as opposed to
murine CD45+ cells in the sample was first calculated from
FACS analysis. The same amount of RNA was then used for the
first-strand cDNA synthesis from KG-1A cells, human UCB, and
marrow-positive controls. This precise quantitation was necessary
because human CD45+ cells in bnx/hu mice were
present at a far lower frequency than in the human control samples.
Therefore, a direct comparison of equal amounts of RNA from
bnx/hu mouse marrow and human samples could have
underestimated the human CD34+ cell frequency in the mice,
giving false-negative results in the analyses. No CD34 mRNA was
detected in the human CD45+ cells recovered from
bnx mice, or in the bnx/hu marrow, in any of the
reverse transcription-PCR (RT-PCR) assays. An example is shown in
Figure 3.
Recovery of clonogenic human progenitors that lack CD34 expression from the marrow of long-term engrafted bnx mice For years we have puzzled over the presence of clonogenic human progenitors and LTCIC in bnx/hu mouse marrow, without expression of CD34 at the cell surface. In the current studies we plated human-specific CFUs and LTCICs from the marrow of 23 long-term engrafted bnx mice, in comparison to the marrow of 11 NOD/SCID mice that had undergone transplantation with similar marrow-derived and purified human hematopoietic cell populations, but harvested after only 2 to 4 months, due to the abbreviated lifespan of the strain.Approximately one fourth of the human graft in the NOD/SCID mice was composed of CD34+ cells, whereas none were detected in the bnx marrow, as determined by FACS analysis of 100 000 cells/sample (Table 1). However, although the human cells engrafted in the marrow of bnx mice lacked CD34 expression, the number of clonogenic cells (day 30 CFUs) and LTCICs per human CD45+ cell engrafted in the marrow of the bnx mice was significantly higher than the number detected in the NOD/SCID marrow (P < .005; Table 1). We observed that the human CFUs plated from the bnx marrow did not begin to develop for the first 10 to 14 days in methylcellulose culture, whereas the human CFUs from the NOD/SCID marrow were already dividing 4 days after plating, at the time of addition of Epo. Because the early forming cells from the NOD/SCID mice were still intact at day 30, we counted all colonies at that point. We have previously observed that cells from 4-hydroperoxycylophosphamide-treated human marrow do not form day 14 CFUs, but do form day 30 CFUs,18 and that these cells are synchronized in the G0 phase of the cell cycle,20 and remain capable of multilineage reconstitution in humans.18 Shah et al have demonstrated that extended LTCICs are primitive, quiescent cells with late, but not early, colony-forming capacity.17 The observation of late, but not early, colony formation from the bnx marrow suggested that the human cells engrafted in the bnx mice at the 8- to 12-month time point might be more primitive than those engrafted in the NOD/SCID mice at the 2- to 4-month time point, or that, at the late time point, the bnx marrow no longer contained committed human progenitors that were readily triggered to divide by the cytokines in the methylcellulose medium. Secondary transplantation to establish the regenerative capacity of
the human CD34 cells in bnx mice suggests that
the human stem and progenitor cells had lost expression of CD34 over
time, but had retained hematopoietic generative capacity. We next
tested this theory further in secondary reconstitution studies.
Sublethally irradiated (300 rads) NOD/SCID mice were used as the
recipients for secondary transplantation of the human hematopoietic
cells from the primary bnx bone marrow.
Our initial secondary transplantation attempts (n = 16 mice receiving transplants from 8 primary transplant donors) did not result in engraftment when the human cells from the bnx marrow were injected into NOD/SCID mice directly after thawing and removal of the dimethyl sulfoxide (DMSO) cryoprotectant. We then learned that an overnight preincubation of the cells from the primary recipients in IL-6 plus SCF prior to injection would greatly enhance the homing and engraftment of the human cells (J. Dick, personal oral communication, February 25, 2000). This may be due to the fact that the combination of IL-6 and SCF up-regulates CXCR4, which enhances stem cell homing.22,23 In subsequent experiments we preincubated the thawed, cryopreserved samples in SCF plus IL-6 (50 ng/mL) overnight, prior to the secondary transplantation, and met with much better success. Two mice each received transplants of cells from 6 primary recipients. One fourth of the marrow from each primary recipient had been used for FACS analysis, RNA preparation, and CFU and LTCIC assays, as described above, at the time of harvest. The remaining (cryopreserved) three fourths of the marrow cells from each primary recipient, averaging 2.8 × 107 total marrow cells with 9.2% human CD45+ cell content, was then thawed and divided between 2 secondary recipients per primary bnx mouse. We did not attempt to reisolate the human cells from the thawed marrow, in case the manipulation lost or altered the content. We used the murine cells from the primary bnx recipients as "carriers" for the human cells, rather than using human mesenchymal stem cell carriers as we have described in our previous studies.13-15,20 We were worried that, due to the recent reports of stem cell plasticity, transplantation of human MSCs might give rise to CD34+ cells and invalidate the secondary assays. In the primary recipients, no human MSCs homed into the murine marrow, as we have previously reported,15 and neither bnx nor NOD/SCID mice given transplants of MSCs alone ever had human CD45+ or CD34+ cells in their blood or marrow. The murine carrier cells from the primary bnx mice were optimal for the current studies because they provided no background in our end-point analyses because all antibodies, primers, and culture assays are strictly optimized for the specific detection of human cells. The transplanted human cells from the bnx marrow generated
multilineage reconstitution in secondary NOD/SCID recipients. Human myeloid, B, and T cells were detected (n = 12, example shown in Figure 4). The most significant
information that we obtained from the secondary transplants, however,
was that CD34+ cells were easily identified in the
secondary recipients. In 6 mice that had more than 5% total human
CD45+ cell engraftment, an average of 14.6% ± 2.5% of
those cells clearly expressed CD34 (Figure 4). These data demonstrate
that CD34 expression is reversible:
CD34+/CD38
Human CD34+/CD38 Until 1996, when the first report on CD34 Our data agree with that of Sato's group, who hypothesized that
up-regulation of CD34 expression in the murine system is an "activation event."10 The "activation" to which
Sato and colleagues refer is not likely to be simply cell cycle
induction, which could be induced by recruiting primitive hematopoietic
stem cells into cycle to reconstitute hematopoiesis in a
5-FU-conditioned animal. Human hematopoietic cells can be identified
that express high levels of CD34, yet are very highly
quiescent,17,24 cannot be readily induced into cell cycle
by cytokines, and lack expression of the marker Ki67, the absence of
which indicates that they are in the G0 phase of the cell
cycle.20,25-27 In the studies where our group induced
highly quiescent CD34+/CD38 The murine and human CD34 promoters have been characterized to
determine which transcription factors act as positive and negative regulators.29-32 Okuno et al35 have
introduced human artificial chromosomes that carry the entire human
CD34 genomic locus into transgenic mice. Human CD34 was expressed in
murine stem cells that were CD34 Fackler et al hypothesized that small numbers of CD34+
cells contaminating the CD34 The current studies demonstrate that human CD34+
stem/progenitor cells can give rise to a population of human
CD34 In summary, we have demonstrated that a portion of the human
stem/progenitor cell compartment loses CD34 expression in the immunodeficient mouse marrow following the primary transplantation. When harvested 8 to 12 months later, the human cells are very quiescent
and are no longer producing progeny into the bloodstream of the mice.
At this point the human CD45+ cells are not expressing
human CD34 protein or mRNA. However, when transplanted into sublethally
irradiated secondary immunodeficient mouse recipients, "activation"
occurred, and the cells were stimulated to produce multilineage
progeny, and either to up-regulate CD34 expression, or to produce
progeny that were CD34+. The current studies provide the
first evidence that expression of CD34 is reversible on engrafting
human hematopoietic stem/progenitor cells that retain the capacity for
multilineage secondary reconstitution. To understand the processes
involved in greater detail, further studies are required to define the
cell cycle characteristics, biology, transduction, and hematopoietic
reconstitution of the CD34
The authors would like to thank Renee Traub-Workman and Miriam Figueroa for talented and devoted care of our immunodeficient mice. Sally Worttman heads our excellent animal facility and Dr Janet Baer provided highly useful veterinary advice.
Submitted January 17, 2002; accepted June 4, 2002.
Prepublished online as Blood First Edition Paper, July 25, 2002; DOI 10.1182/blood-2002-01-0025.
Supported by the National Institutes of Health National Heart, Lung and Blood Institute (SCOR no. 1-P50-HL54850) and the National Institute of Diabetes and Digestive and Kidney Diseases (RO1DK 53041 and RO1DK 61848).
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: Jan A. Nolta, Washington University School of Medicine, Division of Oncology/Stem Cell Biology, 660 S Euclid, Box 8007, St Louis, MO 63110; e-mail: jnolta{at}im.wustl.edu.
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Abstract
Introduction
)CD34(