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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4053-4059
Ex Vivo Generation of CD34+ Cells From
CD34 Hematopoietic Cells
By
Yoshihiko Nakamura,
Kiyoshi Ando,
Jamel Chargui,
Hiroshi Kawada,
Tadayuki Sato,
Takashi Tsuji,
Tomomitsu Hotta, and
Shunichi Kato
From the Research Center for Cell Transplantation, Department of
Pediatrics, and the Department of Hematology, Tokai University, School
of Medicine, Kanagawa, Japan; JT Inc, Kanagawa, Japan; and the Unit of
Transplantation and Clinical Immunology, Claude Bernard University,
Pavillon P, Hopital E. Herriot, Lyon, France.
 |
ABSTRACT |
The human Lin CD34 cell population
contains a newly defined class of hematopoietic stem cells that
reconstitute hematopoiesis in xenogeneic transplantation systems. We
therefore developed a culture condition in which these cells were
maintained and then acquired CD34 expression and the ability to produce
colony-forming cells (CFC) and SCID-repopulating cells
(SRCs). A murine bone marrow stromal cell line, HESS-5,
supports the survival and proliferation of
LinCD34 cells in the presence of fetal
calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem
cell factor, granulocyte colony-stimulating factor,
interleukin-3, and interleukin-6. Although LinCD34 cells do not initially form any
hematopoietic colonies in methylcellulose, they do acquire the
colony-forming ability during 7 days of culture, which coincides with
their conversion to a CD34+ phenotype. From 2.2% to
12.1% of the cells became positive for CD34 after culture. The
long-term multilineage repopulating ability of these cultured cells was
also confirmed by transplantation into irradiated NOD/SCID mice. These
results represent the first in vitro demonstration of the precursor of
CD34+ cells in the human CD34 cell
population. Furthermore, the in vitro system we reported here is
expected to open the way to the precise characterization and ex vivo
manipulation of LinCD34 hematopoietic
stem cells.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE MEMBRANE phosphoglycoprotein CD34 has
been proven to be a useful marker of human hematopoietic
stem/progenitor cells, and various types of colony-forming activity in
human bone marrow (BM), cord blood (CB), and mobilized peripheral blood
(PB) are contained in the CD34+ population.1,2
Clinical transplantation studies using enriched CD34+ BM
cells have also indicated the presence of hematopoietic stem cells
(HSCs) with long-term BM reconstitution ability within this fraction.3,4 Based on such evidence, the current acceptance of experimental and clinical strategies for the enrichment of human
HSCs relies on the positive selection of the CD34 antigen. However,
studies on the murine system showed that HSCs with a long-term
reconstitution capacity were present in the lineage markers-negative
(Lin) CD34 cell population rather
than in CD34+ cells.5,6 Osawa et
al5 reported that single murine
Linc-kit+Sca-1+CD34low/
cells transplanted into lethally irradiated mice were able to sustain
long-term multilineage engraftment. As a result, the presence of
another class of HSCs other than CD34+ cells has also been
presumed in the human hematopoietic system.7
Recently, 2 groups demonstrated the possibility of human
CD34 HSCs in xenogenic transplantation experiments.
Zanjani et al8 showed that the transplantation of human
CD34 populations resulted in long-term, multilineage
human cell engraftment in a human/sheep model, and Bhatia et
al9 reported an ex vivo culture system that induced
SCID-repopulating cells' (SRCs) expansion. Although they showed
CD34+ cells to be derived from
LinCD34 cells using in vivo
models, there has yet to be any direct evidence that CD34+
cells are generated from CD34 cells. An in vitro
culture system is thus required for a precise analysis of
LinCD34 cells that produce
CD34+ cells and CFC.
Some murine stromal cell lines have demonstrated a remarkable ability
to support the survival and proliferation of human primitive progenitors in vitro.10-13 We established a murine BM
stromal cell line, HESS-5,14 and showed that it
dramatically and synergistically supported the expansion of human
CD34+38 cells with human cytokines
thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL).13
The current studies were thus designed to assess whether the coculture
system with HESS-5 cells can maintain human
LinCD34 cells. The results
obtained in this study showed this system to be useful in analyzing the
behavior of these cell populations and also demonstrated direct
evidence of CD34+ multilineage stem cells from
LinCD34 cells in vitro.
| |
MATERIALS AND METHODS |
Cell purification.
Umbilical cord blood was obtained from normal full-term deliveries
according to the guidelines approved by the Tokai University Committee
on Clinical Investigation. The cells were processed within 24 hours of
collection. Mononuclear cells (MNCs) were isolated using Ficoll-Hypaque
(specific gravity = 1.077 g/dL) density gradient centrifugation and put through the red blood cell (RBC)-depletion filter (Asahi Medical Co Ltd, Oita, Japan), which made it possible to
pass small-size cells for further enrichment.15 The cells were stained with phycoerythrin (PE)-conjugated antihuman CD2 (39C1.5),
CD3 (UCHT1), CD4 (SK3), CD14 (LeuM3), CD16 (3G8), CD19 (4G7), CD20
(2H7), CD33 (WM53), CD34 (QBEnd10: class II), CD41 (P2), CD56 (N901),
glycophorin A (KC16), and reacted antimouse IgG-conjugated magnetic
beads (Dynabeads; NIHON DYNAL, Tokyo, Japan) and then were depleted
according to the manufacturer's manual. The cells were again stained
with fluorescein isocyanate (FITC)-conjugated antihuman CD45 (T-200),
PE-CD34 (class I:Immu133), and PE-lineage-specific antigens and sorted
as positive for CD45 but negative for 11 lineage-specific antigens
(CD2, CD3, CD4, CD14, CD16, CD19, CD20, CD33, CD41, CD56, and
glycophorin A) and CD34. The cells were sorted on FACSVantage (Becton
Dickinson, Mountain View, CA) equipped with an argon laser tuned to 488 nm.
In vitro culture system.
The hematopoietic-supportive stromal cell line HESS-5 was previously
established from murine BM.14 HESS-5 cells were maintained in minimum essential medium (MEM)- supplemented with
10% horse serum at 37°C under 5% CO2 in humidified
air. The LinCD34 cells were
plated at 4 × 104 to 30 × 104 cells
per 35-mm2 plate onto pre-established irradiated HESS-5
layers in StemProTM-34SFM (GIBCO BRL, Grand Island, NY) supplemented
with StemProTM-34 Nutrient Supplement, 5% fetal calf serum (FCS), and
cytokines. The final concentrations of cytokines were as follows: TPO,
300 ng/mL; FL, 300 ng/mL; stem cell factor (SCF), 300 ng/mL; granulocyte colony-stimulating factor (G-CSF), 10 ng/mL;
interleukin-3 (IL-3), 10 ng/mL; and IL-6, 10 ng/mL. Human IL-3, G-CSF,
SCF, and TPO were a generous gift from the KIRIN Brewery Co Ltd (Tokyo,
Japan). All cells were harvested by vigorous pipetting, washed in
phosphate-buffered saline (PBS), and then used for further analyses.
Flow cytometric immunophenotyping.
Aliquots of cells or cultured cells were suspended in EDTA-BSA-PBS and
incubated with mouse IgG (Inter-Cell Technologies, Hopewell, NJ) to
block any nonspecific binding. The cells were then reacted for 15 minutes with antihuman CD34 (class I:Immu133, class II:QBEnd10, and
classIII:581). Unbound antibodies were removed by 2 washes, and then
the cells were resuspended in EDTA-BSA-PBS. Stained cells were then
passed though a nylon mesh filter and subjected to a 2-color flow
cytometric analysis. Gating on the lymphoid region was used to exclude
stromal cells by size and granularity. The cells labeled with FITC- and
PE-conjugated mouse isotype-matched antibodies were used as a control.
The surface markers of the cells were analyzed by FACSCalibur using
Cell Quest software (Becton Dickinson).
Polymerase chain reaction (PCR) and reverse transcriptase-PCR
(RT-PCR).
To detect whether any CD34+ cells were contaminated in the
sorted samples, the total RNA was prepared from 1 × 105 freshly sorted cells using ISOGEN (Nippon Gene, Toyama,
Japan) and reverse transcribed using oligo dT primer and RAV-2 reverse transcriptase (Takara, Otsu, Japan). To detect human cells in BM from
NOD/SCID mice, high molecular DNA was prepared from whole BM cells by a
series of phenol-chloroform extractions and ethanol precipitation. The
PCR conditions were optimized for each primer set to maintain
amplification in the linear range. The primer pairs were as follows:
CD3416, 5' sequence (5')
5-TAGATTTCACTGAGCAAGAT-3 and 3' sequence (3')
5-CTTGCCCCACCTAGCCGAGT-3; HLA-DPB1,17 (5') 5-GTGAAGCTTTCCCCGCAGAGAATTAC-3 and (3')
5-CACCTGCAGTCACTCACCTCGGCGCTG-3; and glyceraldehyde phosphate
dehydrogenase (GAPDH), (5') 5-GATGACATCAAGAAGGTGGTG-3 and
(3') 5-GCTGTAGCCAAATTCGTTGTC-3. The samples were denatured at
94°C for 3 minutes, followed by amplification rounds consisting of
94°C for 1 minute (denaturing), 55°C to 65°C for 2 minutes (annealing), and 72°C for 3 minutes (extension) for 30 cycles. The
products were separated on a 1.0% agarose gel, transferred to the
membranes, and then hybridized with probes labeled by using ECL
labeling and a Detection System (Amersham, Buckinghamshire, UK). The
sensitivities of RT-PCR and PCR were 105 and
104, respectively.
Clonogenic assay.
The colony-forming unit-cells (CFU-C) assay was performed
as described previously.13 All cultures were performed in
triplicate, and the number of CFU-C was scored at day 14 of culture.
The colony types were determined by in situ observations using an
inverted microscope.
NOD/SCID repopulating cell (SRC) assay.
An SRC assay was performed as described by Hogan et al.18
Eight-week-old NOD/shi-scid/scid (NOD/SCID) mice were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan)
and were maintained in the germ-free animal facility located at the
Tokai University School of Medicine. Purified cell populations at the
indicated dose were transplanted by tail-vein injections into 350 cGy
irradiated mice. The mice were killed 4 to 6 weeks after
transplantation, and the BM from the 2 femurs and 2 tibias of each
mouse were flushed into RPMI1640 containing 10% FCS. The presence of
human hematopoietic cells was determined by detecting the number of
cells positively stained with FITC-conjugated antihuman CD45 in flow
cytometric analyses. The specific subsets of human hematopoietic cells
were quantified by gating on human CD45+ cells and then
assessing staining with antihuman CD14-PE, CD19-PE, CD33-PE, and
CD34-PE. Human hematopoietic cells were also determined by detecting
the human HLA-DPB1 sequence in DNA isolated from BM cells by a PCR
analysis as described above.
Statistical analysis.
A comparison of the mean values of the parameters was performed using
the 2-group paired t-test. The strength of the correlation was
estimated by Pearson's coefficient of correlation.
| |
RESULTS |
Purification of LinCD34 cells.
Although the definition of
LinCD34 cells is an important
issue, no consensus about the best combination of surface antigens or
clones of monoclonal antibodies has yet been made. We herein defined
Lin as 11 different lineage-specific antigens
including CD2, CD3, CD4, CD14, CD16, CD19, CD20, CD33, CD41, CD56, and
glycophorin A.
The filtration of MNCs by newly developed RBC-depletion filter enriched
Lin small-size cells from 0.52% to 10.6%
(~20-fold) in this particular sample15
(Fig 1A and B). We then depleted human cord
blood of MNCs that express CD34 (class II) and 11 different
lineage-specific antigens, CD2, CD3, CD4, CD14, CD16, CD19, CD20, CD33,
CD41, CD56, and glycophorin A by using immunomagnetic beads. The cells
were again stained with FITC-CD45, PE-CD34 (class I), and
PE-lineage-specific antigens and sorted as positive for CD45 and
negative for 11 lineage-specific antigens. More than a 99.8% pure
fraction of LinCD34 cells was
sorted, as shown in Fig1C. The frequency of
LinCD34 cells among all nucleated
CB cells was 0.58% ± 0.36% (mean ± SD, n = 11). In 3 experiments, the sorted LinCD34
cells were relabeled with CD34 class III antibodies that recognize the
different epitopes of the CD34 molecule, and they also showed no
positive events and confirm the lack of CD34 surface expression (data
not shown). In the 11 separate experiments, each involving a different
normal donor, the number of
LinCD34 cells obtained ranged
from 0.4 × 105 to 3.7 × 105 (mean,
1.7±1.2 × 105).
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| Fig 1.
A flow cytometric analysis of lineage-specific antigens
and CD34 expression on human cord blood cells and their purification.
Staining profile of lineage markers and CD34 versus CD45 expression on
human cord blood MNCs before (A) and after filtration (B). (C) Sorted
LinCD34 cells show a more than 99% pure
fraction. (D) Isotype antibody was used as a negative control. (E)
Semiquantitative RT-PCR analysis from
CD34Lin cells. Top lane, human CD34 cDNA
amplification; bottom lane, GAPDH cDNA amplification used to normalize
RT-RNAs; PC, RT-PCR products from KG-1 cells; NC, water;
CD34, sorted CD34 cells;
CD34+, sorted CD34+ cells;
101, 102, 103,
104, and 105, ratio of positive and
negative cell; 105, mean positive and negative cell
ratio is 1:105.
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These fractions of cells were further analyzed to determine their mRNA
expression of CD34 by using a very sensitive semiquantitative RT-PCR,
and it was confirmed that no CD34 mRNA were expressed (Fig 1E). These
results indicate both that the sorted fractions are highly purified
LinCD34 cells that do not express
mRNA of CD34 and that the possibility of CD34+ cell
contamination is impossible.
Induction of CD34+ cells from
LinCD34 cells in vitro.
The viability of the cultured
LinCD34 cells in the stroma-free
condition was reduced by 50% within 4 days of culture
(Fig 2). The proliferation of these cells
was impossible, even in the presence of a variety of human cytokines
reported to support the proliferation of human
CD34+38 stem cells: TPO, FL, SCF, G-CSF,
IL-3, and IL-6.13 This observation also excludes the
possibility of CD34+ cell contamination.
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| Fig 2.
Cell kinetics in stroma-free and stromal culture system.
LinCD34 cells were plated onto
pre-established irradiated HESS-5 stromal cells or no stroma cells in
medium containing 10% FCS and human cytokines: TPO, FL, SCF, G-CSF,
IL-3, and IL-6. The cell number was determined on days 4 and 7.
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Some murine stromal cell lines were reported to support the survival
and proliferation of human stem/progenitor cells.10-13 We
have recently demonstrated that the murine BM stroma cell line, HESS-5,
dramatically supported the in vitro expansion of human CD34+38 cells in combination with human
cytokines.13 We therefore plated LinCD34 cells on HESS-5 cells in
combination with human TPO, FL, SCF, G-CSF, IL-3, and IL-6. After
plating, we observed that some cells were attached to HESS-5 cells.
Therefore, the viability and number of the nonattached cells were
measured. The viability of cells was maintained, and the number of
cells was increased by 2.2- ± 0.8-fold (n = 5) after 7 days in
culture (Fig 2).
We then analyzed the surface expression of CD34 in these cultured
cells. Although they represented only a small proportion of the total
number of cultured cells, a definite expression of CD34 was detected
and is shown in Fig 3. The mean frequency
was 6.0% ± 3.7% (n = 11). The cells expressed CD45, confirming
that they were of human hematopoietic and not of stromal origin. The CD34+ cells were derived from CD45med but not
from CD45high fraction. These results indicate that the
LinCD34 cells can be induced to
differentiate into cells that express CD34 after in vitro cytokine
stimulation.
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| Fig 3.
FACS analysis of CD34 cells after 7 days
of culture on irradiated HESS-5 stroma cells. The cells were evaluated
for their expression of CD34 and CD45. The percentage of
CD34+ cells is indicated in the upper right of the
panel.
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Clonogenic ability of LinCD34
cells before and after culture.
To evaluate the coincidence of CD34 induction on the cell surface with
a generative capacity of LinCD34
cells, CFU-C output was determined before and after culture. As shown
in Fig 4, the number of CFU-C per 1,000 cells was 82.2 ± 41.1 (n = 5) after 7 days of culture,
although they did not initially possess a hematopoietic colony-forming
capability. In some experiments, cultures of up to 10,000 cells before
culture in methylcellulose supplemented with IL-3, SCF, and EPO showed no colonies, even after 28 days of culture. The colony-forming activity
was positively correlated with the extent of CD34 expression among
LinCD34 cells (r = .91).
These data suggest that there is a strong relationship between the
expression of CD34 and the in vitro response to cytokines that drive
myeloid and erythroid differentiation.
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| Fig 4.
The induction of CFUs from
LinCD34 cells in vitro.
LinCD34 cells were isolated from 5 individual samples and cultured for 7 days on HESS-5 stroma cells. One
thousand cells were plated in methylcellulose medium before and after
culture. The colonies were counted 14 days after plating. *The
percentages of CD34+ cells in the
LinCD34 cultured cells at day 7, when the
1,000 cells were plated in the methylcellulose.
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Long-term multilineage reconstitution ability of
LinCD34 cells before and after
culture.
To compare the relative ability of the long-term repopulation of
LinCD34 cells before and after
culture, we examined this cell population regarding its engraftment
ability in NOD/SCID mice. As summarized in
Table 1, 0.4 to 4.6 × 105
cells after 4 days of culture derived from 6 individuals were transplanted into 6 NOD/SCID mice. Mouse BM was analyzed for the presence of human cells after 4 to 6 weeks of transplantation. Four of
6 mice transplanted with LinCD34
cells after 4 days of culture were chimeric. The presence of human
hematopoietic cells in the recipient BM was also confirmed by the
detection of human HLA-DPB1 sequences in PCR analysis of the BM cells'
DNA (data not shown). The presence of 0.8% to 65.4% CD45+
cells represents a total number of at least 8 × 105
to 65.4 × 106 human cells, indicating that SRCs
derived from LinCD34 cells after
4 days of culture have an extensive proliferative capacity. Both the
degree of engraftment and the calculated total number of
CD34+ cells induced during the culture period showed a
positive correlation. On the other hand, when 1 × 105
cells before culture were transplanted into NOD/SCID mice (n = 5),
human CD45+ cells were found in only 1 recipient mouse with
1.3% of chimerism in BM cells, as determined by flow cytometry. A more
sensitive PCR analysis of the HLA-DPB1 gene confirmed the result. These data indicated that SRCs were induced from the
LinCD34 population after 4 days
of in vitro culture.
Human CD45+ cells in mice that were engrafted with cultured
LinCD34cells were further
analyzed to determine the presence of multilineage reconstitution. In
BM cells, 16.2% of CD14+ cells, 8.1% of CD33+
cells, 5.6% of CD19+ cells, and 4.3% of CD34+
cells were detected on mouse no. 1, and 3.5%, 4.3%, 0.5%, and 3.8%
of each lineage of cells were detected on mouse no. 3 in Table 1
(Fig 5). The presence of multiple lineage
of myeloid and lymphoid cells in the engrafted mice indicates that the
SRCs derived from the LinCD34
cell fraction during in vitro culture have an extensive differentiation capacity in vivo and that they are developmentally earlier than CD34+ stem cells in the hierarchy of human hematopoiesis.
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| Fig 5.
The multilineage reconstitution in NOD/SCID mice 6 weeks
after transplantation of cultured LinCD34
cells. Human LinCD34 cells were cultured
on HESS-5 cells for 4 days and transplanted into irradiated NOD/SCID
mice. Mice were killed 6 weeks after transplantation and BMC were
analyzed for human CD45-FITC and lineage markers labeled with PE.
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| |
DISCUSSION |
The presence and characteristics of human CD34 HSCs
have gained attention from both biological and clinical points of
view7-9 since murine HSCs with long-term reconstitution
capacity have been demonstrated to be present in the
Linc-kit+Sca-1+CD34low/
cell population rather than in CD34+ cells.5
Our studies demonstrated the generation of CD34+ cells,
CFC, and SRCs from LinCD34 cells
in vitro. These in vitro data indicate that CD34
cells contain precursor cells of CD34+ HSCs. This
experiment is, therefore, the first demonstration of human
CD34+ induction ex vivo from CD34 HSCs.
When comparing the data of
LinCD34 cells, it is first
necessary to define Lin. However, the consensus
about the best combination of surface antigens or clones of monoclonal
antibodies has not yet been made. Bhatia et al9 used the
combination of CD2, CD3, CD14, CD16, CD19, CD24, CD41, CD56, CD66b, and
glycophorin A, whereas Zanjani et al8 used T-cell
lineage-depleted (CD3, CD4, and CD8) fractions of
CD34 cells. We defined Lin as 11 different lineage-specific antigens, including CD2, CD3, CD4, CD14,
CD16, CD19, CD20, CD33, CD41, CD56, and glycophorin A, which were slightly modified from the former
combination. We used CD20 instead of CD24 as a B-cell lineage marker
and CD33 instead of CD66b as a myeloid lineage marker. We added CD4 to the lineage markers, because it was reported to function as a marker of
primitive hematopoietic cells.19
The next issue involves the possible contamination of our
CD34 cell preparation by CD34+ cells,
because it would compromise the interpretation of the results of
experiments presented herein. We have to be very careful about
contamination below the threshold level when using flow cytometry. To
avoid this, 2-step depletion with magnetic beads and sorting by
fluorescence-activated cell sorting (FACS) was performed and resulted
in a very pure fraction of
linCD34 cells. Although the cells
expressing CD34 cytoplasmically could not be eliminated by the methods
used in this study, the results from the RT-PCR exclude this
possibility. Finally, the inability to proliferate in a stroma-free
condition in the presence of human cytokines, and the absence of CFU-Cs
detected in the LinCD34 cells
provide independent confirmation that they are not contaminated with
CD34+ cells. It also indicates that
LinCD34cells are not able to
respond to the cytokines that support differentiation of CFC in
methylcellulose, as has been previously reported for subpopulations of
CD34 cells.7,9 These data strongly
excluded the possible contamination of our sorted samples by
CD34+ cells.
It is widely accepted that the microenvironment plays an important role
in hematopoiesis in vivo and that stromal cells are the principal
components of the microenvironment.20,21 Indeed, several
cloned stromal cell lines can promote the survival, proliferation, and
differentiation of hematopoietic cells in vitro. We, along with others,
demonstrated the successful maintenance and proliferation of
CD34+CD38 stem/progenitor cells in a
xenogeneic stromal cell coculture system combined with human
cytokines.10-13 Murine stromal cell line, HESS-5, was also
shown to support the survival and differentiation of the
LinCD34 cell population. The
proliferative and cell survival effects have been attributed to the
many cytokines and extracellular matrix molecules produced by the
stroma. Development of the stroma-dependent culture condition of
LinCD34 cells will contribute to
the elucidation of the molecular mechanisms associated with the
commitment and the differentiation of those newly defined stem cells.
To further assess the long-term repopulating ability of cultured cells,
we performed an SRC assay. We detected 1 engraftment of 5 transplantations when 105
LinCD34 cells before culture were
transplanted in NOD/SCID mice, whereas Bhatia et al9
reported more than 80% engraftment in NOD/SCID mice. There are several
possible reasons to explain the difference. We did not administer any
human cytokines to mice after transplantation, whereas they regularly
administered human SCF, IL-3, and GM-CSF. The sorted population of
LinCD34 cells was slightly
different, as mentioned previously. In either case, we detected human
cell engraftment when the cultured cells were transplanted under the
same conditions as the transplantation of cells before culture, which
means that SRC expanded after 4 days of culture. NOD/SCID mice are
known to support high levels of B-lymphocyte production when
CD34+ cells are transplanted.13 However, in our
results shown in Fig 5, only low levels of B lymphocytes were detected.
It is possible that LinCD34 cells
in mice should require longer time than 6 weeks before producing mature
B lymphocytes.
Our present study raises several important questions that need to be
solved to understand further the nature of the
LinCD34 population. We showed
that the LinCD34 population
subdivided into CD45high and CD45med fraction,
and CD34+ cells derived from CD45med fraction
in Figs 1 and 3. This indicates that the
LinCD34 population might be still
heterogeneous; a further fractionation study is now in progress.
Although the coincidence of the CD34 induction and acquisition of the
capability to form CFC and SRCs was shown, a direct relationship
between generation of CFC or SRCs and CD34 induction should thus be
demonstrated by comparing the sorted fraction of CD34+ and
CD34. These issues are now being investigated in our
laboratory (K.A., manuscript submitted).
The findings presented in this study are expected to help the study and
manipulation of these newly defined HSCs. The further establishment of
optimal conditions that can maintain CD34 cells will
thus be useful as a valuable model to study the mechanism of stem cell
differentiation and self-renewal and as a gene transduction system.
| |
ACKNOWLEDGMENT |
The authors thank Hideyuki Matsuzawa for technical assistance, Shizuko
Imai for secretarial work, and the members of Tokai CBSC Study Group
for their assistance. The authors also thank the KIRIN Brewery Co Ltd
for supplying various growth factors.
| |
FOOTNOTES |
Submitted April 16, 1999; accepted July 29, 1999.
Supported in part by the Japan Society for the Promotion of Science
(JSPS) Grant No. JSPS-RFTF97 I 00201 and by a Research Grant of the
Science Frontier Program from the Ministry of Education, Science,
Sports, and Culture of Japan.
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.
Address reprint requests to Kiyoshi Ando, MD, Department of Hematology,
Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa
259-1183, Japan.
| |
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