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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3928-3940
Human Cord Blood Progenitors Sustain Thymic T-Cell Development and
a Novel Form of Angiogenesis
By
Laura Crisa,
Vincenzo Cirulli,
Kent A. Smith,
Mark H. Ellisman,
Bruce E. Torbett, and
Daniel R. Salomon
From the Departments of Molecular and Experimental Medicine and the
Department of Immunology, The Scripps Research Institute, La Jolla, CA;
and the Department of Pediatrics, The Whittier Institute and the
National Center for Microscopy and Imaging Research, University of
California San Diego, CA.
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ABSTRACT |
There is growing interest in using human umbilical cord blood (CB)
for allogeneic bone marrow transplantation (BMT), particularly in
children. Thus, CB has been identified as a rich source of hematopoietic progenitors of the erythroid, myeloid, and B-cell lineages. Whether CB blood cells engrafting in the BM space also comprise T-cell progenitors capable of trafficking to the thymus and
reconstituting a functional thymopoiesis in young recipients is
presently unknown. Here, we show that CB progenitors, engrafted in the
BM of immunodeficient mice, sustain human thymopoiesis by generating
circulating T-cell progenitors capable of homing to and developing
within a human thymic graft. Surprisingly, development of CB stem cells
in this in vivo model extended to elements of the endothelial cell
lineage, which contributed to the revascularization of transplants and
wound healing. These results demonstrate that human CB stem cell
transplantation can reconstitute thymic-dependent T-cell lymphopoiesis
and show a novel role of CB-derived hematopoietic stem cells in angiogenesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TRANSPLANTATION of hematopoietic stem
cells is currently the treatment for reconstitution of the immune
system in immunodeficiency diseases1,2 or after
myeloablative chemotherapy.3,4 Clinical
experience5,6 and animal models7-11 indicate
that both the proliferative and differentiative capacity of the
transfused stem cells are critical for restoration of host immune
competence, including a functional mature B- and T-cell repertoire.
Human umbilical cord blood (CB) has been recently identified as a rich
source of multipotent hematopoietic progenitors.12-15 As
compared with adult bone marrow (BM), CB progenitors have been shown to
have a higher proliferative and self-renewal
potential,16-19 suggesting a higher capacity for
reconstitution of hematopoiesis. Colony-forming unit (CFU) assays
demonstrated that CB hematopoietic stem cells differentiate along
granulocyte, erythrocyte, monocyte, and megakaryocyte
lineages.20-23 Transfusion of human CB in immunodeficient severe combined immunodeficiency (SCID) mice demonstrated repopulation of the BM with clonogenic progenitors, which support development of
erythroid, myeloid, and B-cell lineages.24-26 Although CB
is a mixture of primitive and more committed hematopoietic progenitors, only the most primitive CD34+CD38 cell
population allows for long-term multilineage engraftment in nonobese
diabetic (NOD)/SCID mice.27 Whether
CD34+CD38 CB stem cells can also
reconstitute T-cell lymphopoiesis in vivo is presently unknown.
Understanding the T-cell developmental potential of CB progenitors is
important to determine whether transplanted patients will recover
T-cell-dependent primary antibody responses and immunity against
environmental pathogens.
Human CB CD34+ cells injected into transplanted fetal
thymus or fetal thymic organ culture can give rise to mature
thymocytes.28-30 Although these studies suggest the
presence of T-cell progenitors, they do not establish whether such
progenitors exist in the CB as terminally committed cells or more
primitive elements. Moreover, these studies cannot address the question
of whether T-cell progenitors traffic from the BM to the thymic
compartment. To address these issues, we developed an in vivo model of
continuous human T-cell lymphopoiesis. In this model, the BM
compartment of NOD/SCID mice is reconstituted with T- and
B-cell-depleted human CB, followed by transplantation of human fetal
thymus under the kidney capsule. Implicit to this model is the
requirement of primitive human CB progenitors to engraft in the BM
compartment and sustain T-cell lymphopoiesis by regulated homing of
their progeny to the thymus. Furthermore, a functional vascular
connection between the mouse tissue and the human thymic graft must
develop to mediate progenitor homing and the exit of mature T cells to
the periphery. Therefore, we extended our studies to investigate the
development of this critical vascular interface at the site of thymic transplants.
Here we report that CB-derived hematopoietic stem cells, engrafted in
the BM of NOD/SCID mice, generated T-cell progenitors capable of homing
to and developing within a human thymic graft transplanted at a distant
site. Human thymopoiesis led to the exit of mature T cells to
peripheral lymphoid compartments. These results demonstrate that human
CB stem cell transplantation supports thymic T-cell development.
Surprisingly, BM reconstitution with CB also led to engraftment of
CB-derived progenitors of the endothelial lineage, which were recruited
to form new blood vessels extending from the thymic implant into the
surrounding mouse tissue. This finding shows that human CB may also
represent a unique reservoir of endothelial progenitors and suggests a
novel mechanism of neovascularization involving the participation of
circulating endothelial stem cells.
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MATERIALS AND METHODS |
Animals.
NOD/LtSz-SCID mice (NOD/SCID) were obtained from our colony
(original founders a kind gift of Dr L. Shultz, Bar Harbor
Laboratories, Bar Harbor, ME). The colony was derived by
cesarean section under specific pathogen-free conditions. Mice were
maintained on an irradiated sterile diet and autoclaved acidified water.
Human CB and thymic tissue.
Sterile human CB was obtained from normal deliveries at the Mary Birch
Hospital for Women (San Diego, CA). Samples were processed within 24 hours. Mononuclear cells were first separated on a Ficoll gradient. CB
cells were depleted of mature T and B cells by negative selection using
an immunomagnetic separation technique. Cells were with anti-CD3
monoclonal antibody (MoAb) (OKT3; American Type Culture Collection
[ATCC], Bethesda, MD) and anti-CD19 MoAb (HD37, Dako, Carpinteria,
CA) for 30 minutes at 4°C followed sequentially by a
biotin-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA),
fluorescein isothiocyanate (FITC)-conjugated streptavidin (GIBCO,
Gaithersburg, MD) and biotin-magnetic beads (Miltenyi Biotec,
Sunnyvale, CA). The cells were applied to a VarioMacs magnetic column
(Miltenyi Biotec, a kind gift of Dr Kevin Mills) and nonadherent cells
collected. This method consistently yielded a cell purity > 95% as
determined by double-color immunostaining using an R-phycoerythrin
(RPE)-conjugated anti-human T-cell receptor (TCR) / (BMA031;
Immunotech, Westbrook, ME) and an FITC-conjugated anti-CD20 MoAbs
(B-Ly1; Dako). Cell preparations contained less than 5% CD20-positive
contaminants, but no detectable TCR/-positive cells. The
proportion of CD34+ progenitors in each CB sample was
finally assessed with an FITC-conjugated anti-CD45 MoAb (Becton
Dickinson, Bedford, MA) and a biotin-conjugated anti-CD34 MoAb
(QBEnd10, Immunotech) followed by Cy-Chrome-streptavidin (Pharmingen,
San Diego, CA). CD34+ cells represented 2% to 5% of total
CB cells after T- and B-cell depletion.
Fetal thymic tissue (18 to 24 weeks of gestation) was obtained from ABR
(Alameda, CA). To deplete the tissue of endogenous thymocytes, the
thymi were minced in  1 mm × 1 mm pieces and cultured in Iscove
medium containing 10% fetal calf serum (FCS), penicillin/streptomycin, and 1.35 mmol/L deoxyguanosine (dGuo) (Sigma, St Louis, MO) for 5 to 6 days.
Generation of human BM and thymus-NOD/SCID chimeras.
NOD/SCID mice (6 to 8 weeks old) were sublethally irradiated (300 cGy)
and injected in the tail vein with T- and B-cell-depleted CB cells.
The number of injected cells was adjusted to deliver 100 to 200 × 103 CD34+ progenitors per mouse. After 4 to 6 weeks, the mice were transplanted under the kidney capsule with the
human thymic fragments (2 to 3 pieces/mouse). At 2 and 4 months
postthymic transplant, the mice were euthanized. Bone marrow cells were
recovered by flushing femurs and tibia with RPMI-10% FCS. The thymic
implants and spleens were excised and either embedded for histologic
analysis or cells were prepared by straining through a 100-µm
stainless steel mesh. Peripheral blood lymphocytes (PBLs) were obtained
by cardiac puncture and separated from red blood cells on a
Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden).
Flow cytometry and cell sorting.
BM, thymic, splenic, and PBL cells were incubated for 15 minutes at
4°C in the presence of polyclonal mouse IgG (50 µg
mL1) and anti-mouse Fc MoAb (2.4G2; Pharmingen).
Lymphocytes were then stained using the following antibodies: FITC
anti-mouse H2kd (SF1-1.1, Pharmingen); RPE anti-human
HLA-A,B,C (G46-2.6; Pharmingen); an FITC anti-human CD45 MoAb (2D1;
Becton Dickinson), an RPE anti-human CD4 MoAb (13B8.2; Immunotech); a
Cy-Chrome anti-human CD8 (RPA-T8; Pharmingen), or an FITC anti-human
CD8 (B9.11; Immunotech); an RPE anti-human TCR TCR/ (BMA031;
Immunotech) and FITC, RPE, or CyChrome species and isotype matching
control antibodies. In preliminary experiments, only minimal
cross-reactivity to mouse determinants was observed for the MoAbs,
whereas the anti-human CD45 and HLA-A,B,C MoAbs showed no
cross-reactivity. Therefore, these antibodies were used to gate for
human lymphocytes. The samples were analyzed with a FACScan (Becton
Dickinson). In some experiments, single- and double-positive thymocytes
were stained with FITC anti-human CD8 and RPE anti-human CD4 MoAbs and
purified by cell sorting (FACStar Plus; Becton Dickinson).
Spleen and thymic graft histology.
Ten-micrometer cryostat tissue sections were fixed in
phosphate-buffered saline (PBS) 2% paraformaldehyde (PFA) for 10 minutes at room temperature. Sections were permeabilized in PBS/0.1%
Triton-X 100 for 5 minutes, blocked first in PBS/50 mmol/L glycine and then in PBS/10% goat or donkey serum for 1 to 2 hours. For
immunohistochemistry, sections were treated for 5 minutes with a 0.05%
H2O2 solution for saturation of the endogenous
peroxidase activity and then probed with a human-specific anti-CD45
MoAb (T29/33; Dako) followed by a biotinylated human-adsorbed goat
anti-mouse IgG Fab2 (Caltag) and Peroxidase-streptavidin (GIBCO).
For the detection of human lymphoid and endothelial cells, thymic
sections were probed with anti-CD45 MoAb and Lissamine-Rhodamine (LSRC)
anti-mouse IgG (Jackson Labs, West Grove, PA) or a combination of a
mouse anti-CD34 MoAb (8G12; Becton Dickinson) and a goat anti-PECAM-1
(platelet endothelial adhesion molecule-1) (polyclonal IgG M-20; Santa
Cruz Biotechnology, Santa Cruz, CA) followed by an LSRC anti-mouse IgG
Fab2 and an FITC anti-goat IgG Fab2 (Jackson Labs). In some experiments, an anti-human-specific von Willebrand Factor (vWF) MoAb (Takara, Shiga, Japan; clone vW1-2) was used. The
anti-HLA-A2 antibody (clone MI2.1) was the kind gift of Dr H. Kaneshima (Systemix, Palo Alto, CA). The secondary reagents were
preadsorbed to eliminate cross-species reactivity. In some experiments,
NOD/SCID mice reconstituted with T- and B-depleted CB cells or human
umbilical vein endothelial cells (HUVECs) labeled with
the vital dye DiI (Molecular Probes, Eugene, OR) were
used as hosts for thymic transplants. In these mice (n = 4), blood vessels developed at the interface with the thymic transplants and
comprising endothelial cells of CB origin (ie, DiI+)
were quantified by morphometric analysis. For this purpose, a total of
160 optical fields (324.8 µm × 216.5 µm) were scored for the
presence of PECAM-1+ vascular profiles containing
DiI+ cells.
For simultaneous detection of human CD45+ and apoptotic
cells, air-dried thymic sections were fixed in PBS 2% PFA for 10 minutes at room temperature. Sections were incubated with
digoxigenin-labeled deoxyuridine triphosphate (dUTP) and terminal
deoxynucleotidyl transferase (TdT) enzyme (Oncor,
Gaithersburg, MD) for 1 hour at 37°C. After blocking the enzymatic
reaction, the sections were incubated with an FITC sheep
antidigoxigenin antiserum (Oncor) for 30 minutes at room temperature.
The sections were blocked overnight in PBS/10% donkey serum and
sequentially probed with the anti-CD45 MoAb and an LSRC donkey
anti-mouse IgG preabsorbed with sheep IgG (Jackson Labs). Sections were
analyzed on a Zeiss Axiovert microscope with a scanning laser confocal
attachment (MRC 1024; BioRad, Hercules, CA).
Polymerase chain reaction (PCR) of Y chromosome-specific DNA.
To identify male BM-derived T cells in the thymic grafts, total or
sorted thymocytes were lysed in 10 mmol/L Tris-HCl, 1.5 mmol/L
MgCl2, 50 mmol/L KCl, 0.5% Tween 20, 100 µg/mL1 Proteinase K (Boehringer Mannheim,
Indianapolis, IN), pH 8.3, for 45 minutes at 56°C. After
inactivation of Proteinase K by heating the samples at 95°C for 10 minutes, an aliquot of each sample (DNA from 10,000 cells) was used for
PCR amplification of human Y chromosome sequences. The Y
chromosome-specific probes were: forward
5'-TGGGCTGGAATGGAAAGGAATCGAAAC-3' and reverse
5'-TCCATTCGATTCCATTTTTTTCGAGAA-3'.31 PCR was
performed for 25 cycles at 95°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute, and a 10-minute final extension at
72°C. PCR products were separated on 1.2% agarose gels and visualized by ethidium bromide.
To investigate the presence of male BM-derived stromal cells at the
site of thymic engraftment, sections were first stained with an
anti-human CD34 MoAb to localize human vessels. Based on this
localization, samples were then scraped from consecutive sections using
a 30 G needle under microscopic visualization.32 Samples
were collected at 3 sites: within the thymic graft, at the interface of
the transplant with the mouse kidney, and within the kidney parenchyma
distant from the graft. The scraped tissue was placed in 50 µL of
lysing buffer, DNA was extracted, and used as template for PCR
amplification of human Y chromosome-specific sequences.
Fluorescence in situ hybridization (FISH).
To simultaneously identify cells bearing human Y chromosome and CD4,
CD8, CD34, or PECAM-1 in tissue sections, we adapted the protocol
described by Gerritsen et al33 for 2-color FISH. Briefly,
10-µm cryostat sections were fixed in acetone for 7 minutes at
 20°C. The sections were blocked for 1 hour at room
temperature in PBS/10% donkey serum and then probed sequentially with
anti-CD4/CD8 MoAbs (OKT4, OKT8), or anti-CD34 MoAb (8G12) followed by
LSRC or CY5 donkey anti-mouse IgG absorbed to sheep IgG. In some
experiments, sections were probed with anti-PECAM-1 followed by a
biotin donkey anti-goat IgG and Cy5 streptavidin. In this case, free
binding sites of the secondary reagent were blocked with sheep IgGs (50 µg/mL1). Sections were fixed for 10 minutes at
room temperature in PBS/1% PFA and dehydrated through graded ethanol
solutions (70%, 80%, and 100%). Thirty microliters of hybridization
mixture (Hybrisol V, Oncor) containing the digoxigenin-labeled Y
chromosome-specific DYZ1 and DYZ2 probes (Oncor) was then applied to
the sections. The slides were sealed with a glass coverslip, heated to
80°C for 10 minutes, and hybridized for 16 hours in a humidified
chamber at 37°C. After washing in 1X sodium citrate buffer (SSC)
for 5 minutes at 72°C, the sections were incubated in the presence
of FITC sheep antidigoxigenin for 30 minutes at room temperature. Sections were washed 3 times and analyzed by confocal microscopy.
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RESULTS |
Engraftment of CB-derived BM progenitors sustains thymopoiesis in
thymic grafts transplanted at a distant site.
To study the capability of CB-derived BM progenitors to sustain human
thymopoiesis in vivo, NOD/SCID chimeras were generated by
reconstitution of the murine BM compartment with human CB depleted of
mature T and B cells, followed by transplantation of human fetal thymus
under the kidney capsule. Control mice received only thymic
transplants. We refer to these two groups of animals as CB/Thy-NOD/SCID
and Thy-NOD/SCID chimeras, respectively. At 4 and 6 months post-CB
reconstitution, the BM and thymocytes from the thymic transplants were
harvested and analyzed by flow cytometry for the presence of human
cells expressing the leukocyte common antigen CD45.
Figure 1a shows human CD45+
cells detected in the BM and thymic grafts of CB/Thy-NOD/SCID chimeras
at 4 months post-CB reconstitution. As many as 80% and 95% of the
lymphoid cells populating the BM and thymic grafts, respectively, were
human. At 6 months, a decrease in the proportion of human cells was
observed in the BM compartment (Fig 1b, %CD45+ cells: 7.7 ± 2.8 at 6 months v 51.3 ± 6.5 at 4 months, mean ± standard error of mean [SEM], n = 11). However,
overall, the percentage of human CD45+ cells in the thymic
transplants remained unchanged (Fig 1b; %CD45+ cells:
55.8 ± 13 at 6 months v 57.3 ± 10.4 at 4 months, mean ± SEM, n = 11). Indeed, by 6 months, the thymic grafts
had grown considerably from the original size (eg, 2
mm3) at the time of implantation to 250 to 500 mm3. Moreover, the comparison of thymic cell numbers at 4 and 6 months (Fig 1c and d) demonstrates an increased number of human
cells. In contrast to the significant growth of thymic tissue in
CB/Thy-NOD/SCID chimeras, the grafts of control Thy-NOD/SCIDs displayed
significantly lower cellularity (CD45+ cells: 3.1 ± 0.8 × 106 v 12.1 ± 4.3 × 106; mean ± SEM; n = 11, P = .02).
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| Fig 1.
Engraftment of human CD45+ lymphoid cells
in the BM compartment and thymic grafts of CB/Thy-NOD/SCID mice. Each
bar represents the proportion (a and b) or absolute numbers (c and d)
of human CD45+ cells detected by FACS analysis of cell
suspensions from the BM (white bars) or thymic grafts (gray bars) of
individual mice at 4 months (a and c) and 6 months (b and d) post-CB
reconstitution. Data in (a and c) were generated in 4 different
experiments and data in (b and d) in 6 different experiments using
different CB donors in each experiment.
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Three-color immunostaining for human CD45, CD4, and CD8 showed that the
thymic grafts 4 months postthymic transplant (eg, 6 months post-CB
reconstitution) comprised thymocytes at both double- and
single-positive stages of maturation (Fig
2a through d). Notably, most thymocytes in the grafts of
CB/Thy-NOD/SCIDs were human (Fig 2a). Overall, they comprised a
significantly higher percentage of CD4 single-positive cells than
thymocytes from Thy-NOD/SCIDs (Fig 2b, d, and e;
%CD4+: 17.9 ± 3.3 v 6.8 ± 1.6, mean ± SEM, P < .01, n = 11). Conversely, in the absence of human
BM, fewer human CD45+ cells were detected in the thymic
grafts and the distribution of thymic subsets was abnormally skewed
toward a CD8 single-positive phenotype (Fig 2c through e).
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| Fig 2.
T-cell development in the thymic grafts of
NOD/SCID-hu chimeras. Three-color flow cytometric analysis of
thymocytes harvested from thymic grafts of a CB/Thy-NOD/SCID at 6 months post-CB reconstitution (a and b) and a control Thy-NOD/SCID
mouse (c and d) stained for human CD45, CD4, and CD8. The staining
specific for CD45 (a and c) identifies a population of human
CD45+ lymphoid cells (Region 1, R1). Gating on R1 shows
that human CD45+ cells from the grafts of
CB/Thy-NOD/SCIDs comprise both CD4/CD8 double- and single-positive
subsets (b), whereas human CD45+ cells from Thy-NOD/SCID
mice harbor mainly CD4 and CD8 single-positive thymocytes (d). The
vertical lines within the histograms (a and c) mark the upper limit of
the fluorescence intensity corresponding to the isotype-control
antibody. In the dot plots (b and d), quadrants were set to comprise
the staining given by control antibodies in the left lower quadrant.
Histograms and dot plots represent the analysis of 10,000 events. (e)
Distribution of human thymic subsets in the grafts of individual
CB/Thy-NOD/SCID ( ) and Thy-NOD/SCIDs ( ) at 6 months post-CB
reconstitution. Horizontal lines in each column mark mean values.
Thymocytes from CB/Thy-NOD/SCID mice comprise a significantly higher
percentage of CD4 single-positive cells than thymocytes from
Thy-NOD/SCID (%CD4+ = 17.9 ± 3.3 v 6.8 ± 1.6, mean ± SEM, P < .01).
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Further phenotypic analysis confirmed that human CD4 and CD8
single-positive thymocytes of either CB reconstituted or control mice
expressed high levels of TCR /, HLA class I, and CD69 (not shown). This phenotype is consistent with that of terminally
differentiated thymocytes, further evidence for a full thymic T-cell
development program. However, thymocytes from Thy-NOD/SCID mice
demonstrated a significantly lower viability as determined by light
scatter analysis (eg, % viable TCRhigh cells = 59 ± 7.7 in Thy-NOD/SCID v 81 ± 4.8 in CB/Thy-NOD/SCID mice [n = 6], mean ± SEM, P < .05) suggesting that development in these control grafts is incomplete and many cells undergo cell death. Finally, flow cytometry of lymphocytes from the NOD/SCID mouse
thymi showed the presence of less than 2% human CD45+
cells, indicating that human T cells developed preferentially in the
human thymic microenvironment.
To study the morphology of thymic grafts, sections from human fetal
thymi before transplantation and after engraftment in vivo were stained
for human CD45 and/or mouse major histocompatibility complex (MHC)
class I antigens. In agreement with previous reports,34 human fetal thymi cultured in the presence of deoxyguanosine were composed mainly of an epithelial network, almost completely devoid of
human CD45+ cells (not shown). After engraftment in mice
reconstituted with human BM, the thymic transplants displayed a defined
cortex and medulla densely repopulated by human CD45+ cells
(Fig 3a). Few
mouse cells were observed within the grafts (Fig 3b, red fluorescence,
arrow). In contrast, grafts from Thy-NOD/SCIDs showed a profoundly
perturbed architecture, with no clear demarcation of cortical and
medullary regions (Fig 3d). Consistent with the lower cellularity of
these transplants, the grafts showed large areas depleted of
lymphocytes or harboring only dispersed human CD45+ cells.
Clusters of mouse cells were often observed within these grafts (Fig
3e, red fluorescence).
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| Fig 3.
Histological analysis of thymic transplants. Cryostat
sections from thymic grafts of CB/Thy-NOD/SCID mice (a, b, c, g, and h)
and control Thy-NOD/SCID mice (d through f) stained by a peroxidase
detection method with an anti-human CD45 MoAb (a and d, dark brown
color), by 2-color immunofluorescence with an anti-human CD45 MoAb (b
and e, green fluorescence), and an anti-mouse MHC class I MoAb (b and
e, red fluorescence) or by a TUNEL assay and propidium iodide (c and f,
green and red fluorescence, respectively). (g and h) Background
staining of isotype control antibodies for the immunohistochemistry and
immunofluorescence techniques, respectively. The preservation of
cortical and medullary regions (a, cx, and m) in the grafts of
CB/Thy-NOD/SCID mice contrast with the perturbed tissue architecture of
the grafts from control Thy-NOD/SCID mice (d). Clusters of mouse cells
(e, arrows) and numerous apoptotic cells organized in rosette-like
clusters (f) can be frequently observed in the grafts of Thy-NOD/SCID
mice. In contrast, rare mouse cells (b, arrow) and sparse apoptotic
events are observed in the grafts of CB/Thy-NOD/SCID mice (c).
Bar in a, d, and g, 254 µm; Bar in b, c, e, and h,
63.5 µm.
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To investigate the occurrence of apoptosis in the developing thymic
grafts, sections of thymic transplants were stained using a
TdT-mediated dUTP nick-end labeling (TUNEL) assay. Thymic transplants from CB/Thy-NOD/SCID chimeras showed few apoptotic cells, mostly scattered in the cortex (Fig 3c, green fluorescence). In contrast, thymic grafts from the control Thy-NOD/SCIDs showed numerous apoptotic cells arranged in rosette-like clusters throughout the sections (Fig
3f, green fluorescence) or grouped in foci of more than 50 cells around
Hassals corpuscles (not shown). A rosette pattern of apoptotic cells
has previously been described in pediatric thymi.35
Two-color immunofluorescence to detect fragmented DNA and human CD45
demonstrated that the apoptotic cells were human (not shown).
Morphometric analysis on more than 30 microscopic fields from sections
cut at 100-µm intervals demonstrated that grafts from control
Thy-NOD/SCIDs harbored a higher number of apoptotic events than those
from mice reconstituted with human CB (number apoptotic cells per
field = 0.99 ± 0.2 in Thy-NOD/SCIDs, n = 3 v 0.3 ± 0.1 in CB/Thy-NOD/SCIDs, n = 3, mean ± standard deviation
[SD], P = .02; size of each field = 249 × 217 µm).
This result supports the hypothesis that a high number of thymocytes developing in the grafts of the Thy-NOD/SCID mice die in the thymus and
is consistent with the increased proportion of nonviable thymocytes shown by our fluorescence-activated cell sorting (FACS) analysis.
Taken together, the data indicate that on reconstitution of a human BM
hematopoietic compartment by CB cells, human thymic grafts transplanted
at a distant site maintain their thymopoietic capability. The data
suggest that CB-derived stem cells generated thymocyte progenitors
capable of homing to and developing within the human thymic grafts.
Detection of BM-derived progenitors in the thymic grafts.
The fact that some human CD45+ cells were detected in
thymic grafts of control Thy-NOD/SCID mice indicated that some lymphoid cells of thymic donor origin had survived the dGuo treatment and may
have contributed to the cellularity of the thymic grafts in the
CB/Thy-NOD/SCIDs. To unequivocally demonstrate the presence of CB
donor-derived thymocyte progenitors in the grafts of CB/Thy-NOD/SCIDs, chimeras were generated with male human CB cells and transplantation of
female fetal thymi. At 4 months postthymic transplant, the CD4/CD8
double- and single-positive subsets from the thymic grafts were sorted
and used for PCR amplification of Y chromosome DNA-specific sequences.
These experiments showed that thymic transplants from CB/Thy-NOD/SCID
mice harbored immature double-positive as well as mature
single-positive thymocytes of CB origin
(Fig 4). Thus, CB-derived thymocyte
progenitors indeed home to the thymic grafts and develop through
intermediates to the most mature single-positive stages. Parallel
experiments using NOD/SCID mice transplanted with CB and thymi
mismatched for HLA-A2, confirmed that as many as 95% of thymocytes
repopulating the thymic transplants were of CB origin, as determined by
FACS analysis for CD3, HLA-A2, CD4, and CD8.
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| Fig 4.
Detection of CB-derived thymocytes in the thymic grafts
of CB/Thy-NOD/SCID mice. CB/Thy-NOD/SCID chimeras were generated by BM
reconstitution with male human CB followed by transplantation of female
thymi. Control Thy-NOD/SCID received human female thymi only. At 6 months post-CB reconstitution, the CD4/CD8 double and single-positive
thymic subsets were purified by cell sorting. DNA was extracted and
assessed for the presence of Y chromosome-specific sequences by PCR.
The resulting products resolved on a 1.2% agarose gel are shown. A
strong band with the predicted size of 150 bp is detected in the BM,
total thymocytes (Total Thys), as well as double and single-positive
thymic subsets of CB/Thy-NOD/SCID mice (SCID no. 1 and no. 2),
indicating the colonization of the grafts by BM-derived thymocyte
progenitors of CB origin. DNA from total thymocytes of Thy-NOD/SCID
mice (SCID controls) is negative. Each lane corresponds to the analysis
of 10,000 cells. Control PCR using DNA extracted from male ( ) and
female ( ) CB cells are shown on the left. The far left lane
represents a 100-bp DNA ladder. The lane on the far right shows a
control using Y chromosome-specific primers and no template DNA.
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Circulating T cells are present in NOD/SCID chimeras reconstituted
with human CB and thymus.
To assess whether reconstitution of human thymopoiesis in the NOD/SCID
chimeras led to repopulation of the periphery by mature T cells,
spleens and peripheral blood were screened for the presence of human
CD45+ TCR /+ T cells by
immunohistochemistry and/or flow cytometry. Human CD45+
cells filling the periarteriolar T-cell-dependent areas of the spleen
were detected in mice reconstituted with human CB (Fig 5a), but not in
control mice (Fig 5b). Flow cytometry of splenocytes demonstrated that
human CD45+ cells represented 7.34% ± 1.6% (mean ± SEM, n = 5) of the total lymphoid cells populating the spleen of
CB/Thy-NOD/SCIDs. Immunofluorescence of either splenocytes or PBL for
human CD45 and TCR / or human CD45, CD4, and CD8 further
demonstrated that the majority of circulating human lymphocytes in
these mice were, in fact, T cells with mature CD4 and CD8 phenotypes
(Fig 5c; %TCR /+ cells within the CD45 subset = 62.3 ± 7.8, n = 9). In contrast, less than 0.5% human T cells were
detected in the spleen or peripheral blood of control Thy-NOD/SCID
mice.
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| Fig 5.
Circulating mature T cells are present in the periphery
of CB/Thy-NOD/SCID chimeras. Splenic sections from a CB/Thy-NOD/SCID
mouse at 6 months post-CB reconstitution (a) and a control Thy-NOD/SCID
mouse (b) stained with an anti-human-specific CD45 MoAb using a
peroxidase detection method. Human CD45+ cells occupying
the periarteriolar areas are observed in the spleen of CB/Thy-NOD/SCID
mouse (a). The brown staining detected in the spleen of the control
Thy-NOD/SCID mouse (b) corresponds to the background levels of staining
obtained with the isotype control antibody (not shown). The staining is
representative of 3 CB/Thy-NOD/SCID and 4 Thy-NOD/SCID chimeras
generated in independent experiments. Percentage of human TCR
 /+ lymphocytes (c) in the periphery of individual
CB/Thy-NOD/SCIDs as detected by flow cytometry of splenocytes or PBL.
The proportion of CD4+ (closed bars) and
CD8+ (open bars) lymphocytes within the human TCR
/+ population is represented. The NOD/SCID chimeras
are numerated as in Fig 1 to allow direct comparison of levels of
reconstitution in the BM and thymic compartments. (d) Confocal
microscopy of a splenic section from a CB/Thy-NOD/SCID mouse
reconstituted with male CB stained by 2-color FISH with human Y
chromosome-specific probes (green fluorescence) and anti-CD4 and CD8
MoAbs (red fluorescence). This staining is representative of 3 CB/Thy-NOD/SCID chimeras generated in independent experiments. The
inset in (d) shows a magnified detail of Y chromosome-positive cells
identified in the spleen. Bar in (a and b) = 125 µm; bar in d = 63.5 µm.
|
|
To directly demonstrate that circulating T cells were BM-derived and
therefore of CB origin, splenic sections from the chimeras reconstituted with male CB and female thymi were analyzed by FISH for
the presence of cells expressing the human Y chromosome and the T-cell
markers CD4 and CD8 (Fig 5d). Numerous cells coexpressing male Y
chromosome (green fluorescence) and CD4/CD8 markers (red fluorescence)
were observed. Notably, not all of the cells expressing the CD4/CD8
markers displayed a positive signal for the Y chromosome. This result
may reflect a limitation of this technique to detect chromosomes on a
single focal plane imposed by the tissue sectioning. Similar results
were obtained in 2 other chimeras. Unlike SCID mice reconstituted with
T- and B-cell-depleted CB followed by transplantation with thymic
tissue, we never detected circulating mature T cells in SCID mice
without thymic transplants, regardless of whether they were
reconstituted with either whole CB (n = 50)36 or purified
CD34+ cells (n = 20) (B.E.T., unpublished
observations). Altogether, these results indicate that
colonization of the thymic grafts by CB-derived thymocyte progenitors
generated a mature T-cell progeny capable of exit and repopulation of
peripheral immune compartments.
Identification of human endothelial cells at the site of the thymic
implant.
Revascularization plays an important role in the engraftment of
transplants. In the case of thymic grafts, the newly formed vasculature
is also a primary component regulating the homing of progenitors and
possibly release of mature T cells from this organ.37
Because these processes of lymphocyte trafficking may be restricted by
species-specific adhesive interactions on the endothelium,38 we next investigated the extent to which
host and/or donor endothelium contributed to the revascularization of
the thymic transplants. For this purpose, sections were stained with an
anti-human CD34 MoAb and an anti-PECAM-1 polyclonal antibody. In
experiments testing the species specificity of these antibodies, we
found that the anti-PECAM-1 antibody cross-reacts with human and mouse
endothelium, whereas the anti-CD34 MoAb is human-specific. Figure 6 shows
a series of microscopic fields from a thymic graft of a CB/Thy-NOD/SCID
chimera. Numerous human blood vessels identified by the coexpression of
CD34 and PECAM-1 (Fig 6a and b; coexpression marked by yellow) were
observed in the subcapsular and interlobular connective tissue (Fig
6a, arrows). Many human vessels were also identified
infiltrating the mouse kidney parenchyma directly adjacent to the graft
(Fig 6b). To determine the distance human vessels could be detected
within the surrounding mouse tissue, we analyzed a series of
consecutive microscopic fields (Fig 6d). Human vessels were detectable
within a range of 700 µm from the edge of the thymic graft. Thymic
transplants of control Thy-NOD/SCID mice demonstrated a similar human
vascular network, indicating that these vessels may form from
endothelial elements contained within the transplant. This human
vascular component provides a mechanism by which human marrow-derived
T-cell progenitors could be effectively targeted to human thymic grafts
at a distant site in our xenogeneic model.
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| Fig 6.
Human blood vessels contribute to the vascularization of
the thymic implant. Confocal microscopy of cryostat sections from the
thymic graft of a CB/Thy-NOD/SCID mouse stained by 2-color
immunofluorescence with an anti-human-specific CD34 MoAb (a, b, and d,
red fluorescence) and an anti-PECAM-1 polyclonal Ab (a, b, and d,
green fluorescence) or with isotype-matched control antibodies (c).
Colocalization of the 2 markers (yellow) identifies numerous human
vascular structures within the thymic parenchyma, as well as in the
subcapsular region of the graft (a, arrows). Human vessels are also
seen to penetrate the mouse kidney at the interface with the thymic
graft (b, interface marked by dots). (d) Composite of 4 consecutive
microscopic fields acquired from a section stained as above showing
numerous human vessels infiltrating the mouse kidney at the interface
with the thymic graft. Bar in a, b, and c = 42 µm; bar in d = 32 µm.
|
|
CB-derived progenitors of the endothelial cell lineage contribute to
the vascularization of thymic transplants and wound healing.
Previous studies have shown that an adherent fraction of human CB cells
can be induced to differentiate in vitro into endothelial cells.39 This observation suggested that human CB may
contain circulating endothelial cells or endothelial progenitors. To
test whether CB-derived cells of the endothelial lineage had engrafted in our NOD/SCID chimeras and participated in the revascularization of
the thymic transplants, frozen sections from female thymi engrafted into mice reconstituted with male CB were screened for the presence of
male cells at the interface of the mouse kidney with the thymic grafts.
This anatomically discrete region contained human CD34+
blood vessels (Fig 6), but lacked detectable human CD45+
lymphoid cells as assessed by confocal microscopy (not shown). The
presence of human Y chromosome-positive cells at this site was first
investigated by a PCR technique, which allows amplification of gene
sequences from tissue sections.31 Tissue samples from thymic graft/kidney interfaces were dissected from frozen sections under microscopic visualization. DNA was extracted and used for PCR of
human Y chromosome-specific sequence (Fig 7). DNA from regions within
the thymic grafts containing thymocytes of BM origin (eg, male) served
as positive controls. In addition, to exclude the possibility of
detecting Y chromosome-positive cells of marrow origin circulating in
the mouse blood stream, DNA from the mouse kidney distant from the
grafts was also used. Figure 7a documents the tissue sampling sites
from a representative tissue section. Human Y chromosome DNA was
detected at the interface of the thymic grafts with the mouse kidney,
as well as within the thymic grafts, but not within the mouse kidney at
sites distant from the graft (Fig 7b). The data indicate that
CB-derived cells are resident at the interface of the mouse kidney with
the thymic implant in a region in which no CD45+ human
cells (eg, leukocytes) are detected.
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| Fig 7.
Detection of CB-derived endothelial cells at the
site of thymic implant. (a) Cryostat section from a human female thymic
transplant engrafted into a NOD/SCID mouse reconstituted with male CB,
stained by hematoxylin/eosin showing the sites of tissue sampling used
for DNA preparation. PCR of these DNA samples for human male Y
chromosome-specific sequences (b) shows the presence of Y chromosome
in tissue within the graft, as well as in the mouse kidney, at the
interface with the graft, but not in the mouse kidney distant from the
thymic transplant. The DNA products amplified from triplicate samples
of the indicated anatomical regions obtained from 3 consecutive
sections are shown. The product from a positive control PCR using male
CB cells () is also shown on the left. The far left lane represents
a 100-bp DNA ladder. The lane on the far right shows a control PCR
using Y chromosome-specific primers and no template DNA. (c through f)
Confocal microscopy of female thymic grafts from CB/Thy-NOD/SCID mice
reconstituted with male CB stained by 2-color FISH using human Y
chromosome-specific probes (green fluorescence) and a human specific
anti-CD34 MoAb (c and d, red fluorescence) or an anti-PECAM-1
polyclonal antibody (e, red fluorescence). (f) Background fluorescence
obtained using the FITC-conjugated antidigoxigenin antibody and
isotype-matched control antibodies for the red fluorescence.
CD34+ cells bearing the male Y chromosome are detected at
the interface of the grafts with the mouse kidney (c, arrowheads), and
in the pericapsular connective tissue (d, arrowheads). The star in (c)
indicates the thymic graft. Y chromosome-bearing cells comprised
within vascular structures, unequivocally identified by the PECAM-1
staining (e, red fluorescence, arrowheads), as well as other
nonendothelial stromal elements (e, arrows) are also observed at the
interface of the grafts with the mouse kidney. Bar in c, d, and f, 63.5 µm; bar in e, 31 µm.
|
|
To determine whether these cells at the graft interface comprised
endothelial cells, we used the in situ fluorescence hybridization assay
described above to identify cells coexpressing the endothelial markers
CD34, PECAM-1, or vWF and human Y chromosome. In 3 of 5 grafts, this
analysis showed the presence of stromal elements coexpressing CD34 or
PECAM-1 and human Y chromosome (Fig 7c through e) or human vWF and
human Y chromosome (Fig 8a and b).
CD34+ cells bearing the Y chromosome were identified at the
interface of the grafts with the mouse kidney (Fig 8a, arrows) and at a subcapsular location (Fig 8b). Figure 7e shows the interface of a graft
with the mouse kidney stained for PECAM-1 (red fluorescence) and Y
chromosome (green fluorescence). Cells coexpressing the 2 markers can
be observed within a blood vessel (arrowheads), indicating the presence
of CB-derived endothelial cells. Other Y chromosome+ cells
negative for PECAM-1 (arrows) are also evident in this region. They
appear as large fibroblast-like cells by light microscopy. Y
chromosome+ endothelial or stromal cells were not observed
within the thymic parenchyma (not shown). The presence of blood vessels
of cord origin was further demonstrated by studying SCID chimeras
transplanted with CB and thymic tissue mismatched for HLA-A2. Figure 8c
and d shows microscopic fields at the thymic-kidney interface of 1 of
such chimeras stained with an anti-human-specific vWF antibody (red
fluorescence) and an anti-HLA-A2 specific MoAb (green fluorescence) identifying cells of thymic origin. As can be observed, blood vessels
of both CB origin (eg, positive for vWF, but negative for HLA-A2,
arrowheads) and thymic origin (eg, positive for both vWF and HLA-A2,
arrows) are present at this site.
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| Fig 8.
Detection of CB-derived blood vessels at the site of
thymic implant in SCID CB/Thy chimeras mismatched for sex or HLA-A2. (a
and b) Confocal microscopy of a thymic graft from a CB/Thy SCID chimera
transplanted with CB and thymus mismatched by sex. Human blood vessels,
here identified by the expression of vWF (red), are shown to comprise Y
chromosome-positive cells of CB origin, as detected by FISH (green,
arrowheads). (c and d) Confocal microscopy of a thymic graft from a
CB/Thy SCID chimera transplanted with CB and thymus mismatched for the
expression of HLA-A2. The transplanted thymic tissue was from an
HLA-A2+ donor, whereas CB cells were from an
HLA-A2 donor. Human blood vessels of thymic origin
(arrows) are identified by the coexpression of vWF (red) and HLA-A2
(green). In addition, a number of human vWF+ blood
vessels lacking expression of HLA-A2 are observed (arrowheads),
indicating the participation of CB cells to the revascularization
of the graft. Bar in b = 42.3 µm; bar in a, c, and d = 63.5 µm.
|
|
These results indicate that unlike the human vessels described in the
control Thy-NOD/SCIDs, which must be of thymic graft origin,
recruitment of CB-derived cells of the endothelial lineage occurred in
the CB/Thy-NOD/SCIDs. Quantitative histology using T-and B-depleted CB
cells labeled with a fluorescent vital dye indicated that 40% to 60%
of PECAM-1+ vascular profiles identified at the interface
with the thymic grafts comprised cells of CB origin. We considered
whether the formation of CB-derived vessels could be attributed to
mature endothelial cells, which might contaminate our CB preparations. Thus, T- and B-depleted CB samples (n = 3) were analyzed by flow cytometry for cells expressing the mature endothelial markers PECAM-1,
E-selectin, and vWF. These experiments showed that nonmyeloid (eg,
CD11b) PECAM-1+ cells,
possibly comprising mature endothelial cells, represented less than
0.5% of T- and B-depleted CB. Purification of this subset by positive
selection on a magnetic column demonstrated that only 5.7% of
CD11b/PECAM-1+ cells expressed vWF (ie,
0.02% of T- and B-cell-depleted CB cells) and could therefore be
unequivocally identified as mature endothelial cells. Thus, at most,
3,000 vWF+ cells were injected per mouse in our
reconstitution experiments. Further experiments in which as many as 2 × 106 HUVEC labeled with a vital dye were injected in
our NOD/SCID model demonstrated that mature endothelial cells do not
efficiently contribute to the development of blood vessels at the site
of thymic transplantation (manuscript in preparation).
Taken together, these experiments indicate that CB-derived endothelial
cells present in the vessels between the mouse kidney and the
transplants were derived from circulating progenitors trafficking into
this site of surgical injury and subsequent healing.
| |
DISCUSSION |
In these studies, we demonstrate that CB hematopoietic progenitors,
engrafted in the BM compartment of immunodeficient hosts, sustain human
T-cell lymphopoiesis by generating circulating progenitors capable of
homing to and developing within a human thymus. Furthermore, we provide
evidence that the in vivo developmental program of CB stem cells is not
limited to blood cell lineages, but also extends to the generation of
endothelial cells, as well as stromal elements, which contribute to the
revascularization of transplants and wound healing. This result
discloses a novel role of CB-derived stem cells in angiogenesis.
Immunodeficient SCID and NOD/SCID mice are valuable in vivo models to
study human hematopoiesis.40-43 In particular, studies have
shown that intravenous injection of human BM or whole CB in these mice
results in the engraftment of primitive hematopoietic stem cells of
myeloid, erythroid, and B-cell lymphoid lineages.24-27 We
show that injection of CB rigorously depleted of mature T and B cells
consistently results in high levels of engraftment of human cells in
the BM compartment. This observation indicates that mature T and/or B
cells from CB are not required for initial engraftment of human stem
cells.44,45
Bone marrow reconstitution was demonstrated up to 6 months post-CB
injection, indicating long-term in vivo self-renewal of the engrafted
stem cells. However, by 6 months, exhaustion of the human BM component
was evident in most of our chimeras. Several factors may explain this
observation. First, it may reflect a limitation of the murine
environment to support human hematopoiesis. Second, this result may
indicate a propensity of primitive CB stem cells to proliferate at high
rate in vitro,15,18 but for a limited number of cycles in
vivo. Third, the purification procedure may deplete the CB of the most
primitive progenitors or this population does not efficiently home to
the BM compartment. Late graft failure after initial engraftment has
been associated with T-cell-depleted allogeneic BM transplantation
(BMT) in human patients.46 To date, only 1 report has
investigated the repopulation capability of purified human
CD34+ progenitors in NOD/SCID mice.26 While
engraftment of CD34+ progenitors could be demonstrated up
to 4 months postreconstitution, further experiments testing for the
persistence of primitive hematopoietic progenitors by serial BM
transplants showed much lower levels of BM engraftment in secondary as
compared with primary recipients after 6 months (eg, 6% to 10% human
cells in secondary recipients v 51% in primary recipients).
These results suggest a decline in the frequency and/or engrafting
capability of primitive progenitors after reconstitution with purified
hematopoietic progenitors from CB.
A key result in our experiments is that engraftment of CB-derived BM
progenitors supported human T lymphopoiesis in animals transplanted
with human thymic grafts. In the presence of BM input, the thymic
transplants increased in cellularity, preserved a relatively normal
architecture, and supported thymocyte survival and development. Moreover, human thymopoiesis led to the production of mature T cells of
CB origin that populated the periphery. These results represent the
first evidence that CB stem cells comprise progenitors of the T-cell
lineage capable of supporting continuous thymopoiesis in vivo from
circulating elements. Recent studies have demonstrated that primitive
hematopoietic progenitors from CB (eg,
CD34+CD38) are required for long-term
engraftment of human cells in the NOD/SCID BM.27 In
contrast, more committed CD34+CD38+ progenitors
appear not to engraft, as they are not detected 4 weeks after
transplantation.27 This suggests that circulating thymic
progenitors, which colonize the thymic grafts in our model, arise from
primitive progenitors in the BM. The requirement of a thymic tissue for
the generation of mature circulating T cells in our chimeras is
consistent with ours as well other previous reports showing a lack of
T-cell lymphopoiesis in SCID mice transplanted with purified
CD34+ CB cells,36,47 BM, or fetal liver
hemopoietic progenitors.48 Our observation has important
implications for the use of CB in clinical transplantation. For
example, it indicates that CB stem cells may successfully reconstitute
pediatric recipients with naive thymus-derived T cells and reestablish
a normal T-cell repertoire after ablative radiotherapy or chemotherapy.
In light of our results and other transplant models,48,49
CB reconstitution in combination with thymus transplantation may also
be envisaged as a strategy to regenerate thymic-derived T-cell
lymphopoiesis in aging, human immunodeficiency virus (HIV)-induced
immunodeficiency, or autoimmune diseases.
T-cell lymphopoiesis in our model was dependent on the successful
trafficking of circulating BM-derived progenitors to the human thymic
graft. The contribution of human vessels to the revascularization of
the thymic grafts in our chimeras may have played an important role in
this process. Indeed, evidence that human hematopoietic cells can only
establish weak adhesive interaction with the mouse endothelium has been
previously reported.38 In contrast with the successful
progenitor cell homing observed in our model, previous in vivo studies
of human thymopoiesis using fragments of human fetal liver and fetal
thymus showed that progenitors from the fetal liver grafts transplanted
under the kidney capsule of SCID mice fail to reconstitute thymopoiesis
in thymic tissue engrafted under the capsule of the opposite
kidney.40 These data indicated a defect of progenitor
homing in that model.
We show that CB-derived cells contribute to the generation of new blood
vessels at sites of graft implantation and wound healing. This result
provides evidence for the existence of a previously unrecognized source
of endothelial progenitors within human CB. That such cells have
characteristics of progenitors is supported by their ability to engraft
and survive in vivo, while retaining the ability to home and develop
into blood vessels at sites of wound healing. These data provide the
first documentation that the developmental potential of CB stem cells
in vivo is not restricted to hematopoietic lineages. The coexistence of
hematopoietic and endothelial progenitors in CB may not be fortuitous.
Much evidence supports a developmental and/or functional relationship
between endothelial cells and hematopoietic precursors during
embryogenesis. Thus, formation of blood vessels progresses
simultaneously with hematopoiesis in the blood islands of the yolk sac;
the center of these islands form primitive blood cells and the outer
cell layers develop into endothelial cells.49,50 Peptides
secreted by the developing thymic epithelium such as thymosin
1 may promote differentiation of immature thymocyte
precursors51 and function as angiogenic factors for
endothelial cells.52 The umbilical cord itself may
represent a unique site of hematopoiesis and endothelial cell
development. Hence, in the mouse embryo, the endothelium of the
proximal portion of umbilical arteries display an unusual rounded
morphology with clusters of CD34+ cells bound to the
luminal surface.53 This pattern is reminiscent of that
described for the paraaortic hematopoietic sites in the avian
embryo.54 It suggests that the umbilical cord may comprise sites in which endothelial and hematopoietic progenitors coexist and be
released into the embryo's circulation.
The formation of new vessels from circulating progenitors contrasts
with the current view that neovascularization of tissues in adult life
occurs only by angiogenesis the local proliferation of preexisting
endothelial cells.55 The evidence we present for a role of
circulating progenitors in neovascularization rather implies mechanisms
of endothelial differentiation similar to those occurring during
vasculogenesis, the process by which preendothelial cells condense to
form new vascular channels in the embryo.55 Interestingly,
a similar mechanism of vessel formation from circulating endothelial
progenitors was recently shown in a model of limb ischemia. In this
study, immunodeficient mice, injected with human CD34+
cells purified from peripheral blood of adults, develop new human blood
vessels in the ischemic tissue.56 The development of
BM-derived endothelial cells has been also documented in dogs
transplanted with allogeneic BM.57 Thus, the presence of
circulating endothelial progenitors and their unique mode of vessel
formation appears not to be limited to fetal life. Indeed, our studies
suggest that reparative/inflammatory processes such as those occurring
at sites of surgical injury and wound healing may provide stimuli
for the mobilization, homing, and/or differentiation of such
endothelial progenitors. Thus, the physiologic role of stem cells
documented to circulate in the CB of newborns and peripheral
blood of adults may not be restricted to hematopoiesis, but also extend
to healing processes such as those following birth trauma, accidental
tissue injury, and surgery.
| |
ACKNOWLEDGMENT |
We thank the medical staff of the Mary Birch Hospital for the
procurement of the CB and Dr Alberto Hayek for instructing us on murine
microsurgery. We also thank Drs Zaverio Ruggeri and Thomas Edgington
for critical reading of this manuscript. This is TSRI manuscript
11495-MEM.
| |
FOOTNOTES |
Submitted January 11, 1999; accepted July 27, 1999.
Supported in part by Grants No. RO1 DK49886-01 (to B.E.T.) and R01
AI42384-01 (to D.R.S.) from the National Institutes of Health (NIH).
The National Center for Microscopy and Imaging Research is supported by
NIH Grant No. RR-04050 (to M.H.E.). L.C. and V.C. were supported by
Career Development Awards from The Juvenile Diabetes Foundation
International. L.C. was also supported in part by a grant from The
Scripps Clinic and Research Foundation, Department of Academic Affairs.
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 Daniel R. Salomon, Department of Molecular
and Experimental Medicine, MEM55, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037; e-mail: dsalomon{at}scripps.edu.
| |
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