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HEMATOPOIESIS
From the Department of Clinical Oncology, Institute of
Medical Science, University of Tokyo, Japan, and the Division of
Hematology, Department of Internal Medicine, Keio University, Tokyo,
Japan.
The CD34 antigen serves as an important marker for primitive
hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question
as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study
confirm developmental changes in CD34 expression on murine HSC. In
fetuses and neonates, CD34 was expressed on
Lin CD34 is a glycoprotein expressed on hematopoietic
cells, vascular endothelium, and embryonic fibroblasts.1-3
Based on in vitro studies identifying cells with the capacity to
differentiate into various hematopoietic lineages in clonal
culture4 and to generate hematopoietic progenitor cells
(HPC) in long-term culture,5 CD34 has served as
the most important marker for primitive hematopoietic cells.
However, despite the widespread clinical use of CD34 antibodies for
enumeration and isolation of human primitive hematopoietic cells in
therapeutic hematopoietic stem cell (HSC) transplantation and gene
therapy, it has remained an open question as to whether or not all HSC
express CD34. Krause and colleagues6,7 and Morel and
coworkers8 reported that murine adult bone marrow (BM)
CD34+ cells were capable of long-term hematopoietic
reconstitution and allow survival of lethally irradiated mice.
Conversely, Osawa and associates9 reported that
CD34low/ A great deal of progress has been made in characterizing murine fetal
HSC. It has been shown that all the stem cell activity required to
reconstitute adult hematopoiesis resides in the
AA4.1+c-Kit+CD34+ population at 14 days postcoitum (dpc) fetal liver cells and that the
AA4.1+c-Kit+CD34 We investigated CD34 expression on HSC in the murine fetus, neonate,
and adult at various ages, using competitive long-term reconstitution
analyses. Our data showed that HSC from the fetus express CD34 at late
gestational stages and continue to express it in neonates and younger
adults, but that this expression decreases with aging. Second
transplantation experiments showed that the decrease in CD34 expression
on HSC occurs with a similar time course, even in older
recipients engrafted with neonatal
Lin Mice
Antibodies
Cell preparation The BM cells were flushed from femurs and tibiae of fetal, neonatal, and adult mice. Liver and spleen cells were obtained by rubbing tissue between 2 pieces of glass and repeated pipetting. Cell suspensions were then filtered through a sterile 40-µm Cell Strainer (No. 2340; Falcon, Lincoln Park, NJ), stained with biotinylated antilineage markers, and enriched for cells not expressing the lineage markers (Lin ), using
streptavidin-conjugated magnetic beads (PerSeptive Biosystems, Framingham, MA). Lin cells were then stained with
fluorescein isothiocyanate (FITC)-anti-CD34 and phycoerythrin
(PE)-anti-c-Kit, and sorting was performed on a FACS Vantage (Becton
Dickinson, Mountain View, CA).26,27 In second
transplantation experiments, Lin cells prepared by a
method using immunomagnetic beads coated with sheep antirat IgG (Dynal
AS, Oslo, Norway)28,29 were stained with biotinylated
anti-Ly-5.2, followed by FITC-anti-CD34, PE-anti-c-Kit, and
PE-cyanine 5-succinimidylester-streptavidin.
Transplantation and analysis of recipients Varying numbers of sorted cells from Ly-5.2 mice were injected into sublethally irradiated Ly-5.1 mice together with 1 × 105 unfractionated BM cells from Ly-5.1 mice. In a preliminary experiment, we determined that 1 × 105 BM cells was the minimum dose of cells required for more than 95% recipient survival. Eight to 10 weeks after transplantation, peripheral blood (PB) was collected from the tail veins of the recipient mice. Red blood cells were removed, and the nucleated PB cells were stained with FITC-anti-Ly-5.2 and PE-antimyeloid cells (Mac-1 and Gr-1), anti-B lymphocytes (B220), or anti-T lymphocytes (Thy-1), and analyzed on a FACScan (Becton Dickinson). The mice in which donor-derived (Ly-5.2+) cells made up more than 1% of all B220+, Thy-1+, and Mac-1+/Ga-1+ cells in PB were scored as positive for successful reconstitution. Stable chimerism was maintained for over 6 months in all engrafted mice, although the reconstitution of T lymphocytes was slightly late compared with that of B lymphocytes or myeloid cells. The secondary transplants into Ly-5.1 mice were carried out using Ly-5.2 cells sorted from BM cells of the primary recipients (Ly-5.1 mice) 4 and 16 weeks after the first transplantation of Ly-5.2 mouse HSC.Assay for colony-forming cells Clonal cell culture was done in triplicate, as described.30,31 Briefly, 1 mL culture mixture containing 2.5 × 102 cells sorted from BM cells of Ly-5.2 mice at various developmental stages, -modified Eagle medium (Flow
Laboratories, Rockville, MD), 1.2% methylcellulose (Shinetsu Chemical,
Tokyo, Japan), 30% fetal bovine serum (Hyclone Laboratories, Logan,
UT), 1% deionized fraction V bovine serum albumin (Sigma Chemical, St.
Louis, MO), 104 M mercaptoethanol (Eastman Organic
Chemicals, Rochester, NY), 100 ng/mL rat stem cell factor (SCF; Amgen,
Thousand Oaks, CA) and human interleukin (IL)-6 (Tosoh, Kanagawa,
Japan), 20 ng/mL mouse IL-3 (Kirin Brewery, Tokyo, Japan) and human
thrombopoietin (Tpo) (Kirin), 2 U/mL of human erythropoietin (Epo)
(Kirin), and 10 ng/mL of human granulocyte colony-stimulating factor
(G-CSF) (Kirin) was plated in each 35-mm suspension culture dish (No. 171099; Nunc, Naperville, IL), which was incubated at 37°C
in a humidified atmosphere flushed with 5% CO2 in air.
Colony types were determined on days 7 to 14 of incubation by in situ
observation using an inverted microscope and according to the criteria
described.30,32 Abbreviations for the colony types are as
follows: GM, granulocyte and/or macrophage colonies; E, erythroid
bursts; MK, megakaryocyte colonies; and Mix, mixed
hematopoietic colonies.
CD34 expression on HSC in neonates Most stem cell activity resides in the Linc-Kit+ cell fraction in the murine
fetus19,20 and adult,33 although a small population of c-Kit dormant HSC in adult BM has been
reported.10 Therefore, we first examined CD34 expression
on Linc-Kit+ HSC in neonatal BM cells of
Ly-5.2 mice. Figure 1A shows a flow cytometric analysis of c-Kit and CD34 expression on Lin
BM cells of a murine neonate. Although most of the
Linc-Kit cells (57.2%) did not express
CD34, Linc-Kit+ cells (42.8%) revealed
various levels of CD34 expression. The Linc-Kit+ cells were fractionated into 3 subsets on the basis of CD34 expression; CD34 (6.6%, the
average of 4 mice), CD34low (58.3%), and
CD34high (35.1%) (R1, 2, and 3, respectively, in Figure
1A). Cells from CD34, CD34low, and
CD34high fractions of Ly-5.2 mouse BM cells were
injected into Ly-5.1 recipients together with 1 × 105
unfractionated BM cells of Ly-5.1 mice. Eight to 10 weeks later, PB of
the recipients was analyzed for Ly-5.2-expressing
Gr-1+/Mac-1+ myeloid cells, B220+ B
lymphocytes, and Thy-1+ T lymphocytes. Figure 1B shows the
results of transplantation experiments. All 8 mice transplanted with
1 × 103
Linc-Kit+CD34high cells and 2 of
4 mice transplanted with 1 × 102
Linc-Kit+CD34high cells had
Ly-5.2+ myeloid and lymphoid cells in the PB.
Figure 1C shows a representative PB profile of a mouse transplanted
with 1 × 103
Linc-Kit+CD34high cells (R3),
where 84.8% of Gr-1+/Mac-1+ cells, 98.4% of
B220+ cells, and 41.3% of Thy-1+ cells were
Ly-5.2+. Although the proportion of
Ly-5.2+ cells depended on the number of cells injected into
the recipient mice, all the mice had a higher proportion of
Ly-5.2+ cells in B220+ B lymphocytes than in
other lineages.
In the transplantation of
Lin We then examined CD34 expression on HSC existing in neonatal liver and
spleen. As shown in Figure 2, although
the proportion of CD34high cells in
Lin
CD34 expression on HSC in fetus Next, CD34 expression on HSC in fetal liver at 14, 16 and 18 dpc was analyzed (Figure 2). Although the proportion of CD34high cells in Linc-Kit+ cells
was larger in 14 and 16 dpc fetal than in neonatal livers, Linc-Kit+ cells of 18 dpc fetal liver had a
distribution of CD34-expressing cells similar to that in neonatal liver
(CD34high cells, 31.9%, 28.6%, 17.1%;
CD34low cells, 53.9%, 52.8%, 72.1%; and
CD34 cells, 14.2%, 18.6%, 10.8%, in 14, 16 and 18 dpc
fetal livers, respectively). In transplantation of the 3 fractions from
14, 16, and 18 dpc fetal liver cells, all 7 mice transplanted with over
1 × 102
Linc-Kit+CD34high cells had
Ly-5.2+ myeloid and lymphoid cells at levels that depended
on the number of cells injected. In mice injected with
Linc-Kit+CD34low cells, only one
recipient receiving 2 × 104 cells from 18 dpc fetal
liver showed engraftment of Ly-5.2+ cells, and 6 with less
than 1 × 104 cells did not. No mouse had
Ly-5.2+ PB leukocytes among the 8 mice transplanted with
1 × 102 to 2 × 104
Linc-Kit+CD34cells. We also
carried out the transplantation using 18 dpc BM cells, in which CD34
expression in Linc-Kit+ cells revealed a
similar distribution to that of neonatal BM cells (CD34high
cells, 49.4%; CD34low cells, 42.6%; and
CD34 cells, 8.0%). In the mouse injected with
3 × 103
Linc-Kit+CD34high BM cells, 60%
of myeloid cells, 84% of B lymphocytes, and 37% of T lymphocytes
expressed Ly-5.2. The mouse injected with 3 × 103
Linc-Kit+CD34low or
Linc-Kit+CD34 cells had no
Ly-5.2+ PB leukocytes. These results indicate that most
stem cell activity in the fetus at late gestational stages, as well as
in neonates, resides in the CD34-expressing cell fraction.
Change of CD34 expression on HSC with aging Competitive long-term reconstitution assays were done using BM cells sorted from mice of various ages, the objective being to examine developmental changes in CD34 expression on HSC. CD34 and c-Kit expression in BM cells of 1-, 4-, 8-, and 16-week-old mice are shown in Figure 3. Although the proportion of CD34 cells in BM
Linc-Kit+ cells showed no remarkable changes
during aging, CD34low cells decreased and
CD34high cells increased in BM of mice over 4 weeks of age.
When transplanted with 1 × 103
Linc-Kit+CD34high,
CD34low, and CD34 cells obtained from
1-week-old mouse BM cells, 8 of 8, 5 of 8, and 0 of 8 mice,
respectively, showed successful engraftment, indicating that HSC were
most enriched in
Linc-Kit+CD34high
fraction. The recipients transplanted with
Linc-Kit+CD34high and
Linc-Kit+CD34low cells sorted
from 4-week-old mouse BM cells revealed a similar engraftment rate.
Transplantation using 8-week-old mouse BM cells showed that, although
most HSC still expressed CD34, they were more enriched in
Linc-Kit+CD34low cells than in
Linc-Kit+CD34high cells. In
contrast to results with BM cells obtained from mice younger than 8 weeks, HSC were found in
Linc-Kit+CD34 in addition to
Linc-Kit+CD34low cell fractions
in BM from 10- to 16-week-old mice. When 1 × 105
Ly-5.2+Linc-Kit+CD34high
cells in 10- to 16-week-old mouse BM, which was the largest population in Linc-Kit+ cells, were transplanted into 4 recipients, none was successfully engrafted with Ly-5.2+
cells. By contrast, only 1 to 5 × 102
Ly-5.2+Linc-Kit+CD34cells
could repopulate in 5 of 8 recipients. Because the number of
Linc-Kit+CD34 cells was only
one twentieth that of
Linc-Kit+CD34high cells in 10- to
16-week-old mouse BM, the result indicates that most hematopoietic
repopulating ability is present in
Linc-Kit+CD34 cell fraction in
10- to 16-week-old mice. Thus, CD34 expression on murine HSC
decreases with aging.
Second transplantation of donor-derived HSC from the primary recipients To confirm that adult Linc-Kit+CD34 HSC are the
progeny of neonatal CD34-expressing HSC, we carried out a second
transplantation of Ly-5.2+ cells sorted from BM of Ly-5.1
primary recipients who were transplanted with 1 × 103 BM
Linc-Kit+CD34high cells from
Ly-5.2 neonates. CD34 expression on
Ly-5.2+Linc-Kit+ BM cells of the
primary recipients 4 and 16 weeks after the first transplantation of
Linc-Kit+CD34high cells from
Ly-5.2 neonatal BM was similar to that for 4- and 16-week-old mice,
respectively (Figures 4A and
5A, and compare Figure 3). We then
sorted
Ly-5.2+Linc-Kit+CD34high,
CD34low, and CD34 cells from the primary
recipients and transplanted them into Ly-5.1 secondary
recipients. As shown in Figure 4B, most of the stem cell
activity of Ly-5.2+ cells resided in the CD34-expressing
cell fraction in BM cells of the primary recipients 4 weeks after the
first transplantation. Figure 4C shows a representative PB profile of a
second recipient transplanted with 2 × 103
Ly-5.2+Linc-Kit+CD34high
cells (R3) from a first recipient. As shown in Figure 5B,
however, the stem cell activity was found in the
Linc-Kit+CD34 cell fraction in
BM cells of the primary recipient 16 weeks after transplantation. A
representative PB profile of a mouse transplanted with
2 × 103
Ly-5.2+Linc-Kit+CD34
cells (R1) in the primary recipient BM 16 weeks after
transplantation is shown in Figure 5C. These results indicate that
neonatal Linc-Kit+CD34high HSC
generate Linc-Kit+CD34 HSC, and
that the decrease in CD34 expression on HSC of young donors occurs even
in aged congeneic recipients with a time course similar to that seen in
the donor.
CD34 expression on colony-forming cells Finally, we examined CD34 expression on murine HPC, using a methylcellulose clonal culture assay. Linc-Kit+CD34high,
CD34low, and CD34 cells
(2.5 × 102 cells) sorted from BM cells of mice at
various developmental stages (18 dpc to 15 weeks old) were cultured in
the presence of SCF, IL-3, IL-6, G-CSF, Epo, and Tpo. As shown in Table
1, Linc-Kit+CD34high cells produced
the largest number of hematopoietic colonies, whereas no colonies were
generated from Linc-Kit+CD34
cells at any developmental stage, indicating that HPC, unlike HSC, continue to express CD34 throughout development.
Despite the clinical importance of CD34 antigen as a marker for
primitive hematopoietic cells for HSC transplantation or gene therapy, it has been controversial whether or not all HSC
express CD34 in mice or humans. We here demonstrated developmental
changes in CD34 expression on murine HSC. In fetal, neonatal, and
younger adult hematopoietic tissues, most of
Lin The function of CD34 in hematopoiesis has been elusive, although
potential adhesive functions of CD34 have been
reported.34,35 Recently, 2 groups of investigators
reported on hematopoiesis in CD34-deficient mice.36,37 One
group noted a decreased number of HPC in 10.5 dpc yolk sac, 14.5 dpc
fetal liver, adult BM, spleen, and PB, and a poor response of adult HPC
to cytokines, which suggested the involvement of CD34 in fetal and
adult hematopoiesis.36 The present observation that HPC,
unlike HSC, expressed CD34 throughout murine development from fetus to
adult is consistent with their results. However, neither group
discussed the biologic activity of long-term repopulating HSC of
CD34-deficient mice. Therefore, the function of CD34 on HSC still
remains unclear. An analysis of differences in biologic activities
between neonatal
Lin We found a difference in differentiation potential between neonatal
Lin We found no difference in proliferation potentials between neonatal
Lin Aside from the evidence for developmental changes in CD34 expression on HSC, the present study has important implications for the clinical application of HSC. Because CD34-enrichment procedures are used to prevent graft-versus-host disease or for the purging of tumor/leukemic cells in therapeutic HSC transplantation, and are also used as a target cell population for gene therapy, it is an extremely crucial issue whether human HSC express CD34. Based on the present findings, we consider that more attention should be directed to age of the donor in discussing this issue. In addition, the present findings also suggest that fetal and neonatal HSC have characteristics different from those of adult HSC. Accordingly, more detailed characterization of umbilical cord blood HSC may contribute to further development of cord blood transplantation, which is now increasingly used as an alternative to BM transplantation.
Submitted July 8, 1999; accepted August 26, 2000.
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, Department of Clinical Oncology, 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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H. S. Radomska, D. A. Gonzalez, Y. Okuno, H. Iwasaki, A. Nagy, K. Akashi, D. G. Tenen, and C. S. Huettner Transgenic targeting with regulatory elements of the human CD34 gene Blood, December 15, 2002; 100(13): 4410 - 4419. [Abstract] [Full Text] [PDF]
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 M. R. Mehrabi, N. Serbecic, F. Tamaddon, C. Kaun, K. Huber, R. Pacher, T. Wild, G. Mall, J. Wojta, and H.-D Glogar Clinical and experimental evidence of prostaglandin E1-induced angiogenesis in the myocardium of patients with ischemic heart disease Cardiovasc Res, November 1, 2002; 56(2): 214 - 224. [Abstract] [Full Text] [PDF]
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 M. C. Marty, F. Alliot, J. Rutin, R. Fritz, D. Trisler, and B. Pessac The myelin basic protein gene is expressed in differentiated blood cell lineages and in hemopoietic progenitors PNAS, June 25, 2002; 99(13): 8856 - 8861. [Abstract] [Full Text] [PDF]
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T. Graf Differentiation plasticity of hematopoietic cells Blood, May 1, 2002; 99(9): 3089 - 3101. [Full Text] [PDF]
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D. Bryder, V. Ramsfjell, I. Dybedal, K. Theilgaard-Monch, C.-M. Hogerkorp, J. Adolfsson, O. J. Borge, and S. E. W. Jacobsen Self-Renewal of Multipotent Long-Term Repopulating Hematopoietic Stem Cells Is Negatively Regulated by FAS and Tumor Necrosis Factor Receptor Activation J. Exp. Med., October 1, 2001; 194(7): 941 - 952. [Abstract] [Full Text] [PDF]
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T. C. C. Kerre, G. De Smet, M. De Smedt, F. Offner, J. De Bosscher, J. Plum, and B. Vandekerckhove Both CD34+38+ and CD34+38- Cells Home Specifically to the Bone Marrow of NOD/LtSZ scid/scid Mice but Show Different Kinetics in Expansion J. Immunol., October 1, 2001; 167(7): 3692 - 3698. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
]
1 × 103 [
] and 5 × 103 cells
[
]) in BM of neonatal mice (Ly-5.2). (C) A representative PB
profile of a recipient mouse (Ly-5.1) engrafted with
1 × 103
Lin
] and
2 × 104 cells [
]) in Ly-5.2 neonatal liver,
neonatal spleen, and fetal liver cells (14-18 dpc). The averages of the
proportions of the 3 fractions in Lin
]) sorted from the BM of
1-, 4-, 8-, and 16-week-old mice (Ly-5.2). The averages of the
proportions of the 3 fractions in Lin
