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Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-05-1511.
HEMATOPOIESIS
From the Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, PA; the Departments of Hematology-Oncology
and Virology, Istituto Superiore di Sanità, Rome; and the
Department of Obstetrics and Gynecology, Catholic University, Rome,
Italy.
Postnatal CD34+ cells expressing vascular
endothelial growth factor receptor 2 (KDR) generate hematopoietic or
endothelial progeny in different in vitro and in vivo assays.
Hypothetically, CD34+KDR+ cells may comprise
hemangioblasts bipotent for both lineages. This hypothesis is
consistent with 2 series of experiments. In the first series, in
clonogenic culture permissive for hematopoietic and endothelial
cell growth, CD34+KDR+ cells generate large
hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas
CD34+KDR The ontogenic development of
hematopoietic and endothelial lineages is linked to vascular
endothelial growth factor receptor 2 (KDR)/Flk1. Knockout of the
Flk1 gene causes a combined defect of hematopoiesis and
endothelial cell (EC) growth,1,2 possibly mediated by a
defect of the embryonic hemangioblast In human postnatal life, CD34+ cells comprise primitive
hematopoietic5 and endothelial6,7 precursors.
Specifically, the minuscule KDR+ subset of
CD34+ cells is enriched for hematopoietic stem cells
(HSCs)8 and endothelial progenitors9;
conversely, the CD34+KDR We observed that in liquid culture, CD34+KDR+
cells generate hematopoietic and endothelial cells and some cells
expressing both types of markers, suggesting their bilineage
differentiation potential. On this basis, we hypothesized that
postnatal CD34+KDR+ cells comprise
hemangioblasts, which feed into hematopoietic and endothelial precursors.
Cell purification
Cell culture
Liquid culture.
CD34+KDR+ and
CD34+KDR Clonogenic culture.
Three different culture conditions were applied: condition a,
HPCs, including primitive ones (high proliferative potential colony-forming cell [HPP-CFC]); condition b, EC precursors; condition c, progenitors of hematopoietic cells and ECs. In condition a, hematopoietic (Hem) culture involved serum-free methylcellulose medium
supplemented with a multilineage hematopoietic growth factor (HGF)
cocktail8 Secondary culture of split subcolonies.
Colonies were picked up and usually divided in 3 aliquots. The first
one was used for RT-PCR analysis; the second one for selective
hematopoietic cell growth in clonogenic Hem culture in IMDM and FCS/HS,
12.5% each, supplemented with HGFs as in condition a, including
EPO/TPO at saturating concentrations8; the third one for
selective EC growth in medium composed of M199 (Gibco, Gaithersburg,
MD), 100 µg/mL ECGS, 10 U heparin, 5 ng VEGF and bFGF, 18% FCS, and
2% human serum9 in fibronectin-collagen-coated wells.13 Hematopoietic cells and ECs were collected after
4 weeks of subcolony culture.
Sibling cells.
Single CB CD34+KDR+ cells were seeded in flat
wells containing liquid Hem medium (see above) to generate 4-cell
clusters. The 4 siblings were seeded in unicellular semisolid Hem, End,
or Hem-End culture.
Stroma-based 3-month ELTC: assay of ELTC initiating cells and
hemangioblasts.
Primary ELTCs: The ELTC wells were seeded at limiting dilution (1-50 cells/well). After 3 months they were individually harvested, and
adherent cells were depleted and seeded in clonogenic culture to
evaluate the ELTC-initiating cell (ELTC-IC) frequency.8,14 Secondary and tertiary ELTCs: Within the primary ELTCs, a
subset of unicellular wells was scored for cobblestone-area-forming
cells (CAFCs).15 Wells positive for hematopoietic colony
formation (cobblestone area) were harvested, and adherent cells were
depleted by overnight incubation in IMDM containing FCS and HS, 12.5%
each; small aliquots were then diluted in 0.4% trypan blue to
differentially count viable small blastlike cells. Finally, the blasts
were reseeded in secondary limiting-dilution ELTC. Tertiary ELTC was
performed according to the same procedure. At the end of secondary and
tertiary ELTC, we assayed the frequency of ELTC-ICs.8
Blasts from a separate subset of unicellular ELTC wells were seeded in
Hem-End clonogenic culture to evaluate the generation of
hemangioblast colonies.
Cell analysis
IF analysis. Double and triple labeling immunofluorescence was performed to detect CD45, von Willebrand factor (VWF), and VE-cadherin/CD144. Briefly, cells were fixed and permeabilized with Cytofix/Cytoperm solution (Becton Dickinson/PharMingen, San Diego, CA), then rinsed with phosphate-buffered saline (PBS) spotted on a positively charged glass slide (SuperFrost Plus; Menzel-Glaser, Braunschuring, Germany) and air dried. Cells were incubated for 20 minutes at room temperature in PBS containing 10% normal goat serum and then overnight at 4°C with the appropriate primary antibody or antisera. After washing in 0.2% BSA in PBS, cells were incubated for 1 hour at room temperature with the appropriate secondary antibodies and washed. For triple-labeling immunofluorescence, cells were incubated with anti-VWF and anti-VE-cadherin antibody. Cells were then incubated with antimouse FITC-conjugated and antirabbit tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies. After washing, cells were incubated with anti-CD45 allophycocyanin-conjugated antibodies (Becton Dickinson/PharMingen). After washing, slides were mounted on a glass coverslip with Slow fade mounting medium (Molecular Probes) and analyzed using a Leica TCS 4D confocal microscope. No labeling was detected in the absence of primary antibodies and antisera; cross-reactivity was not observed on control cell lines (HUVEC, U937). In each sample, the number of immunoreactive cells was counted in 8 or more nonoverlapping fields, for a total of more than 300 cells. The total number of cells in each field was determined by transmitted light analysis. Primary antibodies and antisera: 3 µg/mL rabbit immunoglobulin G (IgG) antihuman VWF (DAKO, Glostrup, Denmark); 5 µg mouse antihuman CD45 (DAKO); 5 µg mouse IgG antihuman VE-cadherin (Immunotech, Marseilles, France). Secondary antibodies: 4 µg FITC-conjugated goat antimouse IgG (Caltag Laboratories, Burlingame, CA) and 5 µg TRITC-conjugated swine antirabbit IgG (DAKO). LDL uptake assay. Cells from Hem-End colonies were resuspended in fresh medium and seeded onto collagen-fibronectin-coated chamber slides.6 After 3 days of culture, cells were incubated for 4 hours with 2.5 µg Dil Ac-LDL (CellSystems Biotechnologie; Vertrieb GmbH, Katharinen, Germany). Noninternalized Dil Ac-LDL was washed by PBS. Cells were then counterstained to detect CD45 or VE-cadherin expression. Samples were finally fixed with 4% PFA and washed in PBS. Slides were mounted and analyzed as indicated above. Tube formation assay was performed as previously described.17 Immunocytochemistry analysis. Cells were cytospun and fixed with cold 80% ethanol for 10 minutes. After pretreatment with endogenous peroxidase blocking reagent (Laboratory Vision, Fremont, CA) and protein blocking solution (DAKO) for 10 minutes and 30 minutes, respectively, cells were simultaneously incubated with rabbit antihuman VWF polyclonal antibody (DAKO) and mouse antihuman CD45 mAb (DAKO) for 60 minutes at room temperature, followed by incubation with peroxidase-conjugated goat antirabbit (DAKO Envision System) and alkaline-phosphatase-conjugated goat antimouse (DAKO) for 30 minutes at room temperature. The reaction was first developed with fast red (DAKO) and then with 3,3-diamino-benzidine (DAB) chromogen substrate (Vector Laboratories). Cells were counterstained with Mayer hematoxylin, and slides were mounted with a coverslip by aqueous mounting medium (DAKO). Semiquantitative RT-PCR analysis.
RNA was isolated using the Dynabeads mRNA direct kit (Dynal, Oslo,
Norway) and reverse transcribed by Moloney murine leukemia virus
reverse transcriptase (Gibco BRL) with oligo(dT) as primer. RT-PCR was
normalized for
In preliminary studies, we confirmed the capacity of
CD34+KDR+ to generate
hematopoietic8 and endothelial9 progeny.
Thus, CB CD34+ cells labeled by anti-KDR mAb (KDR1 or KDR2)
were separated into KDR+ (approximately 1%) and
KDR
To test this hypothesis, CD34+KDR+ cells from
CB and BM were cloned in semisolid medium permissive for growth of
hematopoietic (Hem), endothelial (End), or mixed hemato-endothelial
(Hem-End) progeny. CB and BM cells, seeded immediately after sorting or after 3 to 14 days of serum-free liquid culture (Figure 1), yielded similar results. In Hem and Hem-End cultures, results were independent of seeding cell level (1-50 cells/well). In End culture, however, colony formation was absent when seeding fewer than 250 cells/well, indicating the requirement of cell-cell interactions in these specific
conditions, as reported.18 We specifically observed that
in Hem culture CD34+KDR+ cells generated only
GM colonies (11.1% ± 1.2%), as reported.8 We also
observed that End culture, seeded with 250 cells/well, generated less
than 10% colonies, composed of either ECs (3.9% ± 0.3%) or
myeloid (4.7% ± 0.6%) progeny. In mixed Hem-End culture, we scored
not only GM clones (12.9% ± 2.5%) but also Hem-End colonies (5.0% ± 0.4%; Figure 2A). These
mixed colonies are large (5-10 × 103 cells/colony) and
are characterized by clusters of round hematopoietic cells combined
with spindlelike cells of apparent EC morphology, which are
interspersed between and interconnected with the myeloid clusters
(Figure 2B). Extensive morphology, immunocytochemistry, immunofluorescence, and RT-PCR analysis, as well as LDL uptake and
tube-formation functional assays, indicated that the Hem-End colonies
comprise myeloid cells and ECs (Figure 2C-G and results not shown).
Myeloid cells, clearly recognized at the morphologic level (Figure 2C),
expressed CD45 and other myeloid markers, but not the EC-specific and
EC-associated markers detailed below (Figure 2D-F). The ECs, often
large (Figure 2C), were identified on the basis of their
immunophenotype, RT-PCR profile, and functional tests (Figure 2D-G).
Specifically, ECs were negative for CD45 and other hematopoietic
markers (CD14, CD1a, and CD41, thus excluding their monocytic,
dendritic, and megakaryocytic nature) while positive for endothelial
markers. These included EC-specific proteins (VE-cadherin/CD144, ECSM2)9,19 and EC-associated markers shared by a subset of hematopoietic cells (ie, VCAM1/CD106 and VWF, also expressed on dendritic and megakaryocytic cells, respectively; TIE2, CD31, and
CD105, also expressed on early hematopoietic precursors).9 Furthermore, to confirm the presence of hematopoietic and endothelial progeny, Hem-End colonies were divided in 2 portions, which were replated in 2 separate cultures selective for either endothelial or
hematopoietic growth. As expected, the cells generated in secondary culture were essentially either endothelial or hematopoietic (Figure 2E-F). Finally, functional studies revealed that the ECs actively incorporate LDL (Figure 2G) and undergo tube formation on Matrigel (not
shown).
Negative controls were represented by
CD34+KDR To assess the clonality of mixed colonies, we performed a second series of experiments at limiting dilution (25, 2.5, and 0.25 CB or BM cells/well). Here again, CD34+KDR+ cells seeded in Hem-End culture generate not only 10% to 15% or less myeloid colonies but also 4% mixed colonies, as evaluated by limiting-dilution Poisson statistics (representative results in Figure 2H). Mixed colonies contained hematopoietic and endothelial cells, based on morphology immunofluorescence, immunocytochemistry, and RT-PCR analysis (not shown). These experiments formally demonstrated the clonality of mixed colonies. A third series of experiments was performed on sibling cells. Single
CD34+KDR+ cells were grown in liquid Hem medium
to generate 4 daughter cells; each sibling was then seeded in a
secondary well containing either Hem-End, Hem, or End semisolid medium
(Figure 3A, left). In 2 representative
experiments, 2% and 3% of the 4-cell clones were composed of
hemangioblast siblings. Specifically, these siblings consistently gave
rise to large hemangioblast colonies in Hem-End medium, whereas they
generated small hematopoietic colonies if seeded in Hem culture (Figure
3A, right). In End medium, one of the hemangioblast siblings generated
a small Hem-End cell cluster (Figure 3A, right). Here again, colony
identification was validated by immunofluorescence and RT-PCR analysis
(Figure 3B-C and data not shown). These results indicate that the
bilineage differentiation gene program of hemangioblasts is flexible
and is modulated by specific culture conditions.
It is apparent that 5% to 6% or fewer
CD34+KDR+ cells function as hemangioblast
progenitors. However, the above results do not imply that the
hemangioblasts have long-term proliferative and self-renewal capacity
and, hence, stem cell activity. To address this issue, we used
stroma-based ELTCs. This assay identifies primitive hematopoietic
cells
Previous studies indicate that HSC-enriched populations in humans8,9 (Figure 1) and mice10,11 comprise precursors for hematopoietic and endothelial lineages. The present findings newly identify the human postnatal hemangioblast in a small subset of CD34+KDR+ cells, which are uniquely endowed with long-term proliferative potential and bilineage differentiation capacity. The frequency of hemangioblasts in CB and adult BM is very
low The relationship between hemangioblasts, primitive HPCs, and primitive endothelial progenitors is a key issue. In early embryonic life, hemangioblasts generate the progenitor cells of hematopoietic and endothelial lineages.1-4 Hypothetically, this hierarchical relationship may persist in postnatal life, and, though exceedingly rare, hemangioblasts may function throughout the lifetime as a reservoir and/or a steady state source of primitive hematopoietic and endothelial cells. Sibling cell studies indicate that postnatal hemangioblasts generate either bilineage or unilineage colonies, depending on the specific culture conditions. The gene program of hemangioblasts may be flexible and modulated by microenvironment stimuli to undergo either bilineage or unilineage differentiation. Accordingly, hemangioblasts may function as unipotent hematopoietic or endothelial progenitor/stem cells if tested in assays permissive for either hematopoietic8 or endothelial9 differentiation, respectively. Preliminary experiments suggest that a subset of
CD34+KDR+ cells is endowed with plasticity for
the generation of mesodermic, ectodermic, and endodermic tissues (eg,
skeletal muscle, neural, and hepatic cells) in diverse assay systems
(eg, murine blastocyst, regenerating skeletal muscle) (results not
shown). Within the CD34+KDR+ population, the
cell subsets endowed with hemangioblast activity and plastic capacity
may partially or totally overlap Finally, hemangioblasts may play a role in cellular mechanisms underlying abnormal hematopoiesis. A hematopoietic cell line with endothelial features was recently isolated from a patient with a transformed myeloproliferative syndrome.20 In chronic myeloid leukemia, the bcr-abl fusion protein is apparently present not only in hematopoietic cells but also in endothelial cells.21 These findings suggest that hemangioblasts may be targeted by the oncogenic hit in selected myeloproliferative disorders.
We thank Dr S. Rafii for providing the endothelial cell subculture medium and A. M. Cerio for skillful technical help. We also thank M. Blasi, M. Fontana, and A. Zito for editorial help.
Submitted May 29, 2002; accepted June 12, 2002.
Prepublished online as Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-05-1511.
Supported in part by National Institutes of Health grant 1R01HL63168.
E.P. and M.V. contributed equally to this manuscript.
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: Cesare Peschle, Kimmel Cancer Center, Room 609, Thomas Jefferson University, 233 S 10th St, Philadelphia, PA 19107-5541; e-mail: cesare.peschle{at}mail.tju.edu.
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
cells do not. Limiting-dilution
analysis indicates that Hem-End colonies are clonally generated by
single hemangioblasts. Sibling cells generated by a hemangioblast,
replated in unicellular culture, produce either hematopoietic or
Hem-End colonies, depending on the specific culture conditions.
Identification of endothelial cells was based on the expression of
VE-cadherin and endothelial markers and with lack of CD45 and
hematopoietic molecules, as evaluated by immunofluorescence,
immunocytochemistry, and reverse transcription-polymerase chain
reaction. Furthermore, endothelial cells were functionally identified
using low-density lipoprotein (LDL) uptake and tube-formation assays.
In the second series, to evaluate the self-renewal capacity of
hemangioblasts, single CD34+KDR+ cells were
grown in 3-month extended long-term culture (ELTC) through 3 serial
culture rounds
that is, blast cells generated in unicellular ELTC were
reseeded for a subsequent round of unicellular ELTC. After 9 months,
10% blasts from tertiary ELTC functioned as hemangioblasts and
generated macroscopic Hem-End colonies in clonogenic culture. These
studies identified postnatal hemangioblasts in a
CD34+KDR+ cell subset, endowed with long-term
proliferative potential and bilineage differentiation capacity.
Although exceedingly rare, hemangioblasts may represent the lifetime
source/reservoir for primitive hematopoietic and endothelial progenitors.
(Blood. 2002;100:3203-3208)
2 microglobulin. Oligonucleotide primer sequences
were: 
,
) vs
CD34+KDR
) cells supplemented with VEGF.
Starting from day 14, the number of small blastlike (
) in 6 independent experiments (mean ± SEM values; cell
number/well is indicated). See "Results." (B) Microscopy analysis
of a representative Hem-End colony (original magnifications, left
× 50; right × 100). (C) Morphology of
granulocytes, a large macrophage, and a putative endothelial cell in
Hem-End colony (original magnification, × 400).
(D) Immunofluorescence analysis of cells in Hem-End colony by double
labeling with anti-CD45 (green) and anti-VWF (red) mAb (original
magnification, × 600). (E-H) Characterization of mixed
Hem-End colonies (representative results). (E,F) RT-PCR analysis of
Hem-End colonies from primary Hem-End culture or secondary
hematopoietic (Hem) and endothelial (End) culture
