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HEMATOPOIESIS
From the Divisions of Cellular Therapy and Molecular
Therapy, Advanced Clinical Research Center, and the Department of
Transfusion Medicine, Institute of Medical Science, University of
Tokyo, Tokyo, Japan; the Central Institute for Experimental Animals,
Kawasaki, Japan; the Department of Pediatrics, Nihon University School
of Medicine, Tokyo, Japan; and the Department of Pediatrics, Kyoto
University, Kyoto, Japan.
In this study, cord blood CD34+ cells expressed CD81, a
member of the transmembrane 4 superfamily, and were classified into 3 subpopulations on the basis of their expression levels:
CD34+CD81+,
CD34lowCD81+, and
CD34+CD81high. The lymphohematopoietic activity
of each subpopulation was then examined by using suspension and
clonogenic cultures for hematopoietic potential, coculture with MS-5
cells for B-cell potential, organ cultures of thymus lobes from
nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID)
fetal mice, coculture with stromal cells derived from NOD/SCID
fetal-mouse liver tissue for natural killer (NK) cell and mast cell
potentials, and xenotransplantation into NOD/SCID mice for long-term
repopulating (LTR) ability. CD34+CD81+ cells
represented a heterogeneous population that had all the lymphohematopoietic activities, including NOD/SCID mouse-repopulating ability. CD34lowCD81+ cells were enriched in
erythroid, megakaryocytic, and NK lineage potentials but had lost
T-cell and B-cell potentials and LTR ability. The
CD34+CD81high fraction was depleted of most
lymphohematopoietic potentials except NK cell and mast cell potentials.
Thus, along the differentiation cascade from
CD34+CD81+ lymphohematopoietic stem cells, an
up-regulation of CD81 or a down-regulation of CD34 results in a change
in lymphohematopoietic properties. CD81 may serve as a marker for
defining developmental stages of lymphohematopoietic stem cells.
(Blood. 2001;97:3755-3762) Lymphohematopoietic stem and progenitor cells,
which are common precursors of hematopoietic and lymphocytic cells,
exist through clinical transplantation in humans as well as in
laboratory animals.1-5 The differentiation cascade from
lymphohematopoietic stem and progenitor cells toward a specific lineage
can be defined by the expression of surface markers. In humans,
CD34+ cells are capable of reconstituting human
lymphohematopoiesis in scid mouse bone marrow (BM) in
vivo6,7 and of differentiating into all lineages,
including lymphoid lineages, of blood cells in vitro.8-11
Evaluation of coexpression of a variety of markers with CD34 is useful
for defining the events in a given pathway leading to a specific cell
lineage. It was demonstrated that human lymphoid and myeloid cells can
be derived in vitro from single cells defined by expression of CD34 and
other surface markers.9-11 Other researchers also
delineated a fraction phenotyping lineage marker-negative
(Lin CD81, a member of the tetraspanin or transmembrane 4 superfamily
(TM4SF), is a 26-kd surface protein composed of 4 transmembrane and 2 extracellular domains and having an overall topology shared by other
members of the TM4SF, such as CD19, CD37, CD53, CD63, CD82, and
CD151.13,14 CD81 plays a role in a variety of biologic responses by associating with other proteins; regulating cell adhesion,
migration, proliferation, and differentiation; and changing cell
morphology and signal transductions.15-20 CD81 is
expressed in most human tissues, including blood.21
Studies of CD81 in blood cells, however, have been done mostly by using
lymphoid cells, and the expression of CD81 on lymphohematopoietic stem and progenitor cells is not well understood. In this study, we delineated expression of CD81 on human lymphohematopoietic cells and
revealed a possible role for CD81 (when costained with CD34) in the
development of lymphohematopoietic stem and progenitor cells.
Mice
Cell preparations
Cytokines Recombinant human interleukin 3 (IL-3), thrombopoietin (TPO), and erythropoietin (EPO) were donated by Kirin Brewery (Tokyo, Japan). Recombinant human stem cell factor (SCF) and IL-6 were provided by Amgen (Thousand Oaks, CA). Recombinant human granulocyte colony-stimulating factor (G-CSF) was provided by Chugai Pharmaceutical (Tokyo, Japan). Recombinant human IL-7 and IL-15 were purchased from R&D Systems (Minneapolis, MI). All cytokines were pure recombinant molecules and were used at concentrations that induced an optimal response in cultures of human cells. The concentrations used were 100 ng/mL for SCF and IL-6, 20 ng/mL for IL-3, 2 U/mL for EPO, and 10 ng/mL for TPO, G-CSF, IL-7, and IL-15.Flow cytometry and antibodies For staining, CB MNC were suspended in phosphate-buffered saline (PBS) with 0.1% deionized fraction V bovine serum albumin (BSA) (Sigma Chemical, St Louis, MO) at a concentration of 5 × 105 cells per tube. In all experiments, cell samples were preincubated in 0.1 mL PBS supplemented with 10 µL normal rabbit serum (Funakoshi, Tokyo, Japan) for 30 minutes to block nonspecific binding. After a wash with PBS, cells were stained for 30 minutes on ice with various monoclonal antibodies (mAbs) conjugated by fluorescein isothiocyanate (FITC), phycoerythrin (PE), or phycoerythrin-cyanin 5.1 (PC-5). In some experiments, cells were first stained with a biotin-coated mAb for 30 minutes, washed with PBS, and then stained with PC-5-conjugated streptavidin (Immunotech, Marseille, France). Stained cells were washed with PBS, and expression of cell-surface antigens specific for mAb was determined by using a fluorescence-activated cell-sorter scanner (FACS) flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA).For flow cytometric analysis, FITC-conjugated mouse
immunoglobulin G1 (IgG1) and IgG2a (both used as negative isotype
controls); mouse antihuman CD3 (SK7), CD4 (SK3), CD8 (SK1), CD10
(N8E7), CD13 (L138), CD16 (NKP15), CD19 (4G7), CD45 (2D1), and T-cell receptor (TCR) Alkaline phosphatase-anti-alkaline phosphatase staining Mast cells were detected by means of a reaction with a mouse mAb against human tryptase by using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method (Dako) as described previously.23,24 Briefly, cytocentrifuged samples were fixed with buffered formalin and acetone, washed with Tris-buffered saline (TBS), and preincubated with normal rabbit serum to block the Fc receptors on the cell surface. After 3 washes with TBS, the samples were allowed to react with mouse antihuman tryptase mAb for 30 minutes at room temperature in a humidified chamber. After another 3 washes with TBS, the samples were incubated with rabbit antimouse IgG Ab, washed 3 more times, and then allowed to react with a calf intestinal alkaline phosphatase (AP)-mouse monoclonal anti-AP complex. Finally, AP activity was detected by using naphthol AS-MX phosphate, Fast Red TR, and levamisole to inhibit nonspecific AP activity. The specimens were counterstained with hematoxylin.Cell sorting Cells were sorted on a FACS Vantage sorter (Becton Dickinson) as described previously.25,26 Briefly, CB MNC were passed through a cell strainer (Falcon 2350; Becton Dickinson Labware, Lincoln Park, NJ) to remove cell aggregates. The cells were then stained with PC-5-conjugated CD34 and PE-conjugated CD81 as described above. A lymphocyte-sorting gate was established for both forward and side scattering. To provide a negative control, cells were stained with PC-5-conjugated or PE-conjugated mouse IgG1. Viability of the sorted cells was determined by dye exclusion.Clonal cell cultures Clonal cell cultures were done in triplicate as described previously.25,27 Briefly, 1-mL aliquots of a culture mixture containing 1 × 102 cells, -minimum essential
medium (-MEM; Life Technologies, Grand Island, NY), 1.2%
methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% fetal-calf
serum (FCS) (Hyclone, Logan, UT), 1% deionized fraction V BSA,
10 4 M mercaptoethanol (Sigma), and a cocktail of
cytokines (SCF, IL-6, IL-3, G-CSF, TPO, and EPO) were plated in 35-mm
suspension culture dishes (1710099; Nunc, Naperville, IL). Cultures
were then incubated at 37°C in a humidified atmosphere flushed with 5% carbon dioxide (CO2) in air. Except for megakaryocyte
(Mk) colonies, all aggregates consisting of more than 50 cells were considered colonies. Colony types were determined on days 7 to 14 of
the incubation by in situ observations using an inverted microscope,
according to criteria established by Nakahata and Ogawa28
and Nakahata et al.29 Colonies were considered Mk colonies
if they contained at least 4 Mks.30 In experiments to
analyze constituent cells, individual colonies were removed from the
methylcellulose medium by using a 3-µL Eppendorf (Cologne, Germany)
pipette under direct microscopical visualization and collected in
Eppendorf microcentrifuge tubes containing 0.5 mL PBS. After 2 washes
with PBS, samples were resuspended in 0.2 mL PBS containing 2.5% BSA
and centrifuged in a cytocentrifuge (Shandon Southern, Elliott, IL) at
800 rpm for 5 minutes. To identify cell types, each smear was stained
with May-Grünwald-Giemsa stain.
Suspension cultures Suspension cultures were done as described previously.30 Briefly, 2-mL aliquots of a culture mixture containing 5 × 103 cells,-MEM, 20% FCS, 1%
deionized fraction V BSA, and a cocktail of cytokines (SCF, IL-6, IL-3,
G-CSF, TPO, and EPO) were plated in 12-well tissue-culture plates
(Becton Dickinson) at 37°C in a humidified atmosphere flushed with
5% CO2 in air. The culture medium was refreshed every 4 days with the same mixture. Cells were harvested from each well at
selected times.
Fetal-thymus organ cultures T-cell differentiation was determined in NOD/SCID mouse fetal-thymus organ cultures (FTOC) with modifications as previously reported.31-33 Briefly, thymus lobes were dissected from NOD/SCID mouse fetuses on day 16.5 of gestation. The lobes were depleted of endogenous T-cell progenitors by incubation with 1.35 mM 2'-deoxyguanosine (Sigma) for 5 days and then individually plated with 5 to 10 × 103 cells in 30 µL FTOC medium in Terasaki plates (Sumitomo Bakelite, Tokyo, Japan). The plates were inverted immediately to allow the lobe and cells to make contact at the bottom of a hanging drop. After 48 hours of incubation, each lobe was transferred to filter membranes (Costar, Cambridge, MA; pore size, 8 µm) placed on sponges (Gelfoam, Upjohn, Kalamazoo, MI) that had been soaked in FTOC medium. The lobes were incubated for 4 to 5 weeks, with fresh FTOC medium added every 3 days. At selected times, lobes were pressed gently under a glass coverslip in 0.1 mL PBS containing 0.1% BSA to release cells, and the isolated cells were analyzed by flow cytometry for expression of CD3 and CD4. The FTOC medium used throughout the FTOC experiments consisted of RPMI-1640 medium (Sigma), 20% FCS, antibiotics, MEM nonessential amino acids solution ( × 100; Sigma), and MEM vitamin solution ( × 50; Sigma). FTOC cultures were maintained in 7% CO2 at 37°C in a humidified incubator. In all FTOC experiments, 2'-deoxyguanosine-depleted thymus lobes were incubated according to the same procedures, along with colonized lobes as negative controls.Cocultures on MS-5 stromal cells The B-cell potential of the cells was tested in a cocultivation system on a murine stromal cell line, MS-5 (provided by Dr K. J. Mori, Niigata University, Niigata, Japan), as reported previously.33,34 Briefly, 3 × 103 cells were cultured in-MEM supplemented with 10% FCS, SCF, and G-CSF on
confluently coated MS-5 cells in 12-well culture dishes. The cultures
were incubated at 37°C in a humidified atmosphere flushed with 5%
CO2 in air and were refreshed each week by renewing half of
the medium. After 4 weeks, cells were harvested by using 0.05% trypsin
(Life Technologies) and 0.02% EDTA (Wako, Osaka, Japan) and were
analyzed by flow cytometry for expression of CD10 and CD19.
Cocultures on stromal cells derived from NOD/SCID fetal-mouse livers Natural killer (NK) cell and mast cell potentials were determined by coculturing the cells on stromal cells derived from NOD/SCID fetal-mouse livers. Briefly, livers were dissected from NOD/SCID mouse fetuses on day 14.5 of gestation and torn with fine tweezers to release the cells. The isolated cells were cultured in-MEM containing 10% FCS in a 75-cm2 culture flask
(Falcon 353111; Becton Dickinson Labware) overnight. Nonadherent cells
were removed by replacing the culture medium. After the fetal-liver
stromal cells had become confluent, they were harvested by using
trypsin and EDTA and frozen at  80°C. Before use, the frozen cells
were thawed and plated in 12-well culture dishes at a concentration of
2 × 105 cells/well in 2 mL -MEM medium containing
10% FCS. After the cells were confluent for 2 days of culture, they
were exposed to 50 Gy  -ray irradiation. The cells were
cultured on these stromal cells at a concentration of
5 × 103 cells/well with a culture medium consisting of
-MEM, 10% FCS, 104 M 2-mercaptoethanol, and a
cocktail of cytokines including SCF, IL-7, and IL-15. After 3 to 5 weeks of culture, nonadherent cells were harvested, washed, and stained
with CD45 and CD56, CD16, or CD3. Preliminary experiments revealed that
these nonadherent cells were 100% positive for an antihuman CD45 mAb.
Cells that expressed CD56 but not CD3 were considered NK lineage cells.
Mast cells were identified as the cells stained with antihuman tryptase when the APAAP method was used.
Transplantation into NOD/SCID mice Xenotransplantation of human lymphohematopoietic cells was done with modifications as described previously.7,35 Briefly, 1 × 104 human cells were injected through a tail vein into NOD/SCID mice irradiated with 2.4 Gy (cobalt 60) total-body irradiation. To reduce NK cell activity, the recipient mice were injected intraperitoneally with 400 µL PBS containing 20 µL anti-asialo GM1 antiserum (Wako) immediately before the transplantation. Identical treatments were done on days 11, 22, and 33 after transplantation. Mice were killed in a CO2 chamber 12 to 16 weeks after transplantation, and BM cells were harvested with PBS containing 5% FCS. Cell suspensions were filtered through a cell strainer (pore size, 40 µm; Falcon 352340; Becton Dickinson Labware) to remove clumps and debris and were then processed for flow cytometric analysis.Statistical analysis Data are presented as mean ± SD values. Statistical significance was determined by using the Student t test.
Expression of CD81 on human CB cells We first investigated expression of CD81 and hematopoietic lineage-specific antigens on CB cells. As shown in Figure 1, more than 80% of CD13+ or CD14+ myeloid cells expressed low to medium levels of CD81, whereas all the lymphoid cells, including CD3+ or TCR + T cells, CD19+ B cells, and
CD56+ or CD16+ NK cells were 100% positive for
CD81. The level of expression of CD81 was highest in the
CD19+ B cells. On T and NK cells, the intensity of
expression was somewhat lower, although still high. Erythroid cells
positive for glycophorin A (GPA) were negative for CD81. Thus,
consistent with observations in a previous study,21 we
found that CD81 was highly expressed on B, T, and NK lymphocytes,
weakly or moderately expressed on myeloid cells, and not expressed on
erythroid cells.
We next analyzed coexpression of CD81 on CD34+ cells.
Figure 2B shows a representative flow
cytometric profile for CB MNC. Most of the CB CD34+ cells
expressed CD81 (97.4% ± 3.1%; n = 6). Among them,
85.9% ± 7.9% expressed a medium level and 11.9% ± 6.2% a high
level of CD81. As shown in Figure 2C, BM MNC and CB MNC were similar with respect to the pattern of expression of both markers. According to
the flow cytometric profile for coexpression of CD81, CD34+
cells could be divided into 3 fractions:
CD34+CD81+ (56.1% ± 6.2%),
CD34+CD81high (7.5% ± 4.6%), and
CD34lowCD81+ (21.2% ± 5.1%; fractions R1,
R2, and R3, respectively, in Figure 2B).
Hematopoietic potential of CB cells defined by expression of CD81 and CD34 We created suspension cultures of CD34+CD81+, CD34+CD81high, and CD34lowCD81+ cells sorted from CB MNC to explore their hematopoietic potential. Sorting gates were determined as shown in Figure 2B, and sorted cells were reanalyzed cytometrically to confirm purity (Figures 2D-2F). Five thousand cells in each fraction were cultured with SCF, IL-3, IL-6, G-CSF, TPO, and EPO (Figure 3A). CD34+CD81high cells could not proliferate in the culture. The time-course study revealed that CD34+CD81+ cells reached a proliferative peak on day 16 of culture, whereas CD34lowCD81+ cells did so 4 days earlier. Moreover, the peak number of CD34+CD81+ cells was 2 times as large as that of CD34lowCD81+ cells (Figure 3A). Cytospin preparations of the cultured cells on day 12 showed that the differentiation potential of the CD34+CD81+ and CD34lowCD81+ fractions was quite different (Figure 3B). Most of the cells cultured from the CD34+CD81+ fraction were myeloid cells (86.6% ± 2.1%; n = 4), whereas most of the cells from the CD34lowCD81+ fraction were erythroid cells (86.9% ± 3.3%) and only a few myeloid cells were present (7.3% ± 0.8%). Interestingly, flow cytometric analysis of the cultured cells on day 15 showed that those derived from the CD34+CD81+ population contained a substantial number of CD34+ cells, whereas cells from the CD34lowCD81+ fraction did not (Figure 3C).
To examine the proliferation and differentiation potentials of the
cells in the 3 fractions more closely, methylcellulose clonal cultures
were done with the same cytokine combinations used in the suspension
cultures (Table 1).
CD34+CD81high cells gave rise to no colonies in
6 experiments, whereas CD34+CD81+, and
CD34lowCD81+ cells did produce colonies,
although the numbers and types of colonies were different. The
observations regarding CD34+ cell colonies (as expected by
the estimation from the proportion of each fraction in
CD34+ cells) and colony formation indicated that binding of
CD81 did not affect colony formation.
CD34+CD81+ cells generated the most colonies,
among which were all the types of hematopoietic colonies except for Mk
colonies. Mk colonies were generated only from
CD34lowCD81+ cells, but few myeloid colonies
were produced from that fraction. Erythroid bursts were produced from
both CD34+CD81+ and
CD34lowCD81+ cells. Mixed colonies were also
generated from both fractions, but those derived from
CD34lowCD81+ cells were greatly enriched
(78.3% ± 7.6%; range, 65.2%-87.5%; P < .001 when compared with the CD34+CD81+ fraction).
The cytospin preparations of individual colonies chosen randomly on day
10 of culture (10 colonies for each fraction) showed that the mixed
colonies derived from CD34lowCD81+ cells
contained a higher proportion of erythroid cells and a lower proportion
of myelocytic cells than those from CD34+CD81+
cells (Table 2). Colonies containing Mks
were found more frequently in mixed colonies derived from
CD34lowCD81+ cells. These results indicate that
most of the myeloid and megakaryocytic progenitors were in the
CD34+CD81+ and
CD34lowCD81+cell fractions, respectively, and
that erythroid and multipotential progenitors were shared by both
fractions but that multipotential progenitors in the
CD34lowCD81+ fraction had lost most myeloid
potential.
B-cell potential of cells defined by expression of CD81 and CD34 To examine the potential to commit to a B-cell lineage, 3 × 103 cells from each fraction were plated on MS-5 stromal cells in the presence of SCF and G-CSF.34 CD34+CD81high cells could not grow on MS-5 cells and disappeared within a week. CD34lowCD81+ cells also could not proliferate on MS-5 and disappeared gradually during 2 to 3 weeks. B cells expressing CD19 or CD10 were generated only from the CD34+CD81+ fraction (Figure 4A). Similar results were obtained in 3 independent experiments. Thus, B-lymphoid lineage potential was observed only in the CD34+CD81+ cell population.
T-cell potential of cells defined by expression of CD81 and CD34 T-cell potential was determined in FTOC as reported previously.33 When 5 × 103 CD34+CD81+ cells were cocultured with NOD/SCID fetal-mouse thymus lobes, the number of cells recovered in FTOC 4 weeks later was 7.42 ± 0.38 × 104/lobe (n = 10). Flow cytometric analysis revealed that 75.2% of the cells recovered were CD4+ cells and 9.4% were CD3+ T cells (Figure 4B). On the other hand, thymus lobes cocultured with neither CD34lowCD81+ nor CD34+CD81high cells were enlarged and few cells were recovered in FTOC. To eliminate the possibility that committed T-cell progenitors that could develop into mature T cells in a shorter period existed in the CD34lowCD81+ or CD34+CD81high fractions, we also harvested cells in FTOC on days 14 and 21 of culture. Cells from these 2 fractions did not generate lymphoid cells. Similar results were obtained in 3 independent experiments. Thus, the T-lymphoid potential existed exclusively in the CD34+CD81+ cell fraction.NK cell and mast cell potentials of cells defined by expression of CD81 and CD34 Human NK cells have been reported to be induced in vitro from CD34+ cells in many different culture systems.10,36-38 One study found that a stromal cell line derived from murine fetal-liver tissue supported generation of NK cells from human LinCD34+CD38
cells.38 In our preliminary experiments, CD34+
cells from human CB MNC could generate
CD56+CD3 NK cells when cultured on stromal
cells derived from NOD/SCID fetal-mouse liver with a cytokine cocktail
of SCF, IL-7, and IL-15 for 4 to 6 weeks (data not shown). Human
tryptase-positive mast cells were also found in the cocultures (data
not shown). Using this culture system, we investigated NK cell and mast
cell potentials in the 3 cell fractions. In 5 weeks of coculture with
stromal cells derived from NOD/SCID fetal-mouse liver,
5 × 103 CD34+CD81+,
CD34lowCD81+, and
CD34+CD81high cells yielded 5.17 ± 0.96,
6.11 ± 0.36, and 0.29 ± 0.06 × 105 cells,
respectively. CD34+CD81high cells, which had
low proliferation potential on NOD/SCID fetal-mouse liver stroma,
generated large granular lymphocytes and mast cells that stained with
antihuman tryptase when the APAAP method was used (Figures 5A and
5B). CD34+CD81+
and CD34lowCD81+ cells also generated NK cells
and mast cells in addition to myeloid cells. Flow cytometric analysis
revealed that CD56+CD3 NK cell and
CD56CD3low mast cell fractions were present
in the coculture of CD34+CD81high cells on
day 28 (Figure 5C). These cells were also produced from CD34+CD81high and
CD34+CD81+ fractions, but the most pronounced
production of CD56+CD3 NK cells was
observed in the CD34lowCD81+ population (39%
of total cell recovery).
LTR ability of the cells defined by expression of CD81 and CD34 To examine the LTR ability of the 3 fractions, cells of each fraction were transplanted into NOD/SCID mice (Table 3). When CD34lowCD81+ or CD34+CD81high cells were transplanted into 9 mice (1 × 104 cells in each recipient), none of the mice had a successful engraftment. However, when 1 × 104 CD34+CD81+ cells were transplanted into 13 mice, 9 of the mice had human CD45+ cells in BM 12 to 16 weeks after transplantation. A representative BM profile of a mouse that had successful engraftment with CD34+CD81+ cells is shown in Figure 6. The CD45+ cells contained CD13+ or CD33+ myeloid cells and CD10+ or CD19+ B cells (or both) but no or few CD3+ T cells (Figure 6B and Table 3). The CD45+ cells also contained CD34+ immature cells, most of which coexpressed CD81. This result indicates that LTR hematopoietic stem cells exist in the CD34+CD81+ population.
In this study, we found that CD81 is expressed on the majority of
human blood cells except mature erythrocytes. In particular, human
CD34+ cells from either CB or BM were mostly positive for
CD81. The CD34+ cells could be divided into 3 populations Stem cell activity was evaluated by the ability to reconstitute human lymphohematopoiesis in NOD/SCID mouse BM and was present exclusively in the CD34+CD81+ cell fraction. This may be why CD34+ cells were retained after 15 days of suspension culture of CD34+CD81+ cells. LTR ability was not detected in the CD34lowCD81+ or CD34+CD81high cell fraction. Therefore, down-regulation of CD34 or up-regulation of CD81 on CD34+CD81+ lymphohematopoietic stem cells may indicate loss of stem cell activity. The divergence of lymphoid potential in the development of
lymphohematopoietic stem and progenitor cells has not been well described. We previously showed that three fourths of mouse
Lin Overall, the current study indicates that there is a complex of classes
in the hierarchy of lymphohematopoietic progenitor and stem cells and
that they may have unique types of expression of surface markers
according to their potentials. Galy et al8,12 showed that
human BM CD34+ cells could be fractionated into
lymphohematopoietic stem and progenitor cells with various potentials
by using Thy-1, CD45RA, and CD10. Another study showed that T-lymphoid
potential is quantitatively different among
CD34+Lin In summary, the current study delineated expression of CD81 on human lymphohematopoietic stem and progenitor cells. The ubiquitous expression on these cells indicates the existence of functional roles for CD81 in the development of lymphohematopoiesis. According to our findings, along the differentiation cascade from CD34+CD81+ lymphohematopoietic stem cells, there appear to be pathways to CD34lowCD81+ or CD34+CD81high cells, even if they are indirect. CD34lowCD81+ pathways define the loss of LTR ability, T- and B-cell potentials, and myeloid potentials, whereas CD34+CD81high pathways represent the exclusive commitment to NK cells and mast cells. Thus, CD81 may serve as a marker for separating human lymphohematopoietic stem and progenitor cells in their developmental stages. This research may increase understanding of the characteristics of human lymphohematopoietic stem and progenitor cells and benefit clinical use of fractionated lymphohematopoietic stem and progenitor cell transplantation.
Submitted December 10, 2000; accepted February 7, 2001.
Supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan, and a Grant-in Aid for the High-Tech Research Center from the Japanese Ministry of Education, Science, Sports, and Culture to Nihon University.
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: Kohichiro Tsuji, Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: tsujik{at}ims.u-tokyo.ac.jp.
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Abstract
Introduction
), CD
34lowCD81+ (
), and
CD34+CD81high cells (
) in a time-course
experiment. (B) Photographs of progenies derived from
CD34+CD81+ and
CD34lowCD81+ cells in suspension cultures on
day 12. A tendency for CD34+CD81+ cells to
generate myelocytes and for CD34lowCD81+ cells
to generate erythrocytes is shown (× 400). (C) Low cytometric
profiles of expression of CD81 and CD34 on progenies derived from
CD34+CD81+ and
CD34lowCD81+ cells in suspension cultures on
day 14. CD34+ cells were obtained only from the
CD34+CD81+ fraction.
CD34+CD81+,
CD34lowCD81+, and
CD34+CD81high