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HEMATOPOIESIS
From the Department of Maternal and Fetal Medicine,
Institute of Reproductive and Developmental Biology; and the
Departments of Haematology and Immunology, Imperial College School of
Medicine, London, United Kingdom.
Human mesenchymal stem/progenitor cells (MSCs) have been identified
in adult bone marrow, but little is known about their presence during
fetal life. MSCs were isolated and characterized in first-trimester
fetal blood, liver, and bone marrow. When 106 fetal blood
nucleated cells (median gestational age, 10+2 weeks
[10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the
mean number (± SEM) of adherent fibroblastlike colonies was 8.2 ± 0.6/106 nucleated cells (69.6 ± 10/µL fetal
blood). Frequency declined with advancing gestation. Fetal blood MSCs
could be expanded for at least 20 passages with a mean cumulative
population doubling of 50.3 ± 4.5. In their undifferentiated state,
fetal blood MSCs were CD29+, CD44+,
SH2+, SH3+, and SH4+; produced
prolyl-4-hydroxylase, In addition to hematopoietic stem cells, it is now
clear that adult human bone marrow contains a rare population of
mesenchymal stem/progenitor cells (MSCs) (0.01% to 0.001%).
Morphologically, MSCs in their undifferentiated state are spindle
shaped and resemble fibroblasts. They can be extensively expanded in
vitro and, when cultured under specific permissive conditions, retain
their ability to differentiate into multiple lineages including bone,
cartilage, tendon, muscle, nerve, and stromal
cells.1-7
MSCs are of great therapeutic potential because of their ability to
self-renew and differentiate into multiple tissues.8 Adult
bone marrow-derived MSCs engraft in numerous organs and differentiate
along tissue-specific lineages when transplanted into fetal
sheep.9 They enhance engraftment of donor hematopoietic cells after cotransplantation in animal models,10-12 and
they migrate into areas of muscle degeneration to undergo myogenic
differentiation in immunodeficient mice.13 In humans, MSCs
have been used to regenerate the marrow microenvironment after
myeloablative therapy.14 Furthermore, Horwitz et
al15 have shown that allogeneic bone marrow transplantation
in children with osteogenesis imperfecta allowed engraftment of
functional donor MSCs, resulting in increased bone marrow density.
Recent work has shown that mesenchymal progenitors are also present at
low frequencies in adult peripheral blood16 and in term
cord blood.17 To date little is known about their presence in early fetal life. MSCs are closely associated with hematopoietic stem cells in adult bone marrow, while first-trimester fetal blood contains significant numbers of hematopoietic progenitors18 and SCID-repopulating cells,19 and fetal liver and bone
marrow are well-known sites of active hematopoiesis during
ontogeny.20 Our aim was to isolate and characterize MSCs
early in gestation, which would promote understanding of the ontogeny
of these cells and possibly open new avenues for in utero cellular and
gene therapy. In this study we show that MSCs can be isolated from
human first-trimester fetal blood, liver, and bone marrow, readily
expanded, and induced to differentiate in vitro.
Samples
First-trimester blood samples (median gestational age,
10+2 weeks [10 weeks, 2 days]; range,
7+6 to 14+0 weeks; n = 34) were collected by
cardiocentesis under ultrasound guidance under general anesthesia
before clinically indicated termination of pregnancy using a
siliconized 20-gauge, 15-cm needle (Cook, Herts, United
Kingdom). Cytospun cells from blood samples of less than 11 weeks' gestation and blood films from samples of more than 11 weeks'
gestation were stained with Leishman stain, and differential cell
counts were performed. Second-trimester blood samples (median
gestational age, 17+5; range, 14+5 to
25+4; n = 7) were collected by ultrasound-guided fetal
blood sampling during clinically indicated termination of pregnancy
from the cord or during clinically indicated fetal blood sampling from the umbilical vein for rapid karyotyping (results all euploid). Third-trimester blood samples (median gestational age,
39+1; range, 38+0 to 40+2; n = 5)
were obtained from the umbilical cord at delivery from uncomplicated pregnancies.
Bone marrow samples were obtained from 4 fetuses (median gestational
age, 13+5 weeks; range, 11+2 to
14+3). Single-cell suspensions of fetal bone marrow were
prepared by flushing the bone marrow cells out of the humeri and femurs using a syringe and 22-gauge needle into Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich, United Kingdom) supplemented with 10% fetal bovine serum (FBS) (Stem Cells Technology, Vancouver BC,
Canada), 2 mM L-glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin (Gibco BRL, Life Technologies, Paisley, United Kingdom). Fetal liver samples were taken from 4 fetuses (median gestational age,
11+6 weeks; range, 10+0 to 14+3
weeks). Single-cell suspensions were prepared by mincing the organ
through a 70-µm nylon filter (Becton Dickinson, United Kingdom), and
cells were resuspended as for the bone marrow samples.
Isolation and culture of fetal blood, liver, and bone marrow
mesenchymal stem/progenitor cells
To expand the cells through successive passages, they were plated at 104 cells/cm2, grown to near confluence, and harvested with the same protocol. Growth curves were assessed by counting the number of adherent cells daily for 16 days, and the number of population doublings was determined by counting the number of adherent cells at the start and end of each passage. To isolate individual colonies, nucleated cells were plated in a 100-mm dish at a density of 12 000/cm2 and were collected by cloning cylinders (Sigma-Aldrich) and expanded. Cultured fetal MSCs were recloned by single-cell sorting using FACS Vantage (Becton Dickinson). Immunocytochemistry of cultured fetal mesenchymal stem/progenitor cells Monolayer and single colony-derived adherent cells (at the second and tenth passages in culture) from first-trimester fetal blood (n = 10), liver (n = 8), and bone marrow (n = 8) were analyzed by immunocytochemistry. Cytospin preparations and growing cells in double-chamber slides (Nunc) were fixed in equal volumes of methanol and acetone for 1 minute at room temperature, washed in TBST (Tris-buffered saline containing Tween; Dako, Bucks, United Kingdom), incubated with 3% hydrogen-peroxide (H2O2; Sigma-Aldrich), and blocked with 10% normal goat or rabbit serum at room temperature. Washed slides were incubated with the following primary antibodies: mouse anti-human macrophage, CD68 mAb (clone PG-M1; Dako), peroxidase-conjugated rabbit anti-human von Willebrand factor polyclonal Ab (Dako), mouse anti-human prolyl-4-hydroxylase mAb (clone 5B5; Dako), rabbit anti-human myoglobin mAb (clone A0324; Dako), mouse anti- -smooth muscle actin mAb (clone 1A4;
Sigma-Aldrich), mouse anti-human VCAM-1/vascular cell adhesion
molecule-1 mAb (CD106; clone BB1G-V1; R&D Systems Europe), mouse
anti-human fibronectin mAb (clone IST-4; Sigma-Aldrich), mouse
anti-collagen type 1 mAb (clone COL-1; Sigma-Aldrich), mouse
antivimentin mAb (clone V9; Sigma-Aldrich), and mouse antilaminin (clone LAM-89; Sigma-Aldrich) at 1: 50 dilution with Dako antibody diluent (Dako) for 30 minutes at room temperature. Antimouse or antirabbit peroxidase-conjugated immunoglobulin G antibodies
(Sigma-Aldrich) were used as secondary antibodies at 1:100 dilution,
and 3,3'-diaminobenzidine in chromogen solution (Dako) was applied to
the slides, which were counterstained with 0.1% Mayer hematoxylin
solution (Sigma-Aldrich).
FACS analysis of cultured fetal mesenchymal stem/progenitor cells Monolayer and single colony-derived adherent cells (at the second and tenth passages in culture) from first-trimester fetal blood (n = 10), liver (n = 8), and bone marrow (n = 8) were trypsinized and stained with anti-CD34-fluorescein isothiocyanate (FITC), CD45-FITC, HLA-DR-phycoerythrin (PE; Becton Dickinson), CD14-FITC, CD105 (endoglin)-PE, CD44 (H-CAM)-FITC CD29 (integrin 1
chain)-PE, CD31 (PECAM-1)-FITC (Serotec, Oxford, United Kingdom),
SH2, SH3, and SH4 mAbs (Osiris Therapeutics, Baltimore, MD) and were
analyzed by FACScalibur flow cytometry (Becton Dickinson).
Coculture of fetal blood mesenchymal stem/progenitor cells and hematopoietic cells After irradiation (8000 cGy), 2.0 × 105 monolayer-derived adherent cells from first-trimester fetal blood (n = 3) were seeded in 12-well plates (Nunc). After 24 hours, 5 × 104 cord blood CD34+ cells, isolated with anti-CD34 antibodies (QBEND/10 mouse immunoglobulin G; Miltenyi Biotec) conjugated with microbeads and eluted through MiniMACS columns, were resuspended in long-term culture medium (Myelocult; Stem Cell Technologies), including 10 6 M hydrocortisone (Stem Cell
Technologies), and were seeded over the adherent cells in the absence
of exogenous growth factors. The fetal stroma-CD34+ cell
cocultures were cultured at 37°C with 5% CO2 for 6 and 8 weeks. At weekly intervals, nonadherent cells in the collected half-volume of the culture medium were assayed for colony-forming cells
by colony assays in standard methylcellulose culture (Methocult; Stem
Cell Technologies). Control experiments were also performed by
culturing cord blood CD34+ cells either in the absence of a
stromal layer and growth factors or over a murine stromal cell line
(M210-B421; kindly donated by Dr C. Eaves, Terry Fox
Laboratories, Vancouver, BC, Canada).
Adipogenic, osteogenic, and chondrogenic differentiation The differentiation ability of MSCs was assessed in single colonies (at the second and tenth passages) collected by cloning cylinders from primary cultures of first-trimester fetal blood (n = 6), liver (n = 4), and bone marrow (n = 4) and in colonies derived from single cells sorted from expanded MSCs at the 2nd passage from first-trimester fetal blood (n = 4), liver (n = 4), and bone marrow (n = 4).Adipogenic differentiation was assessed by incubation with DMEM with 10% FBS supplemented with 0.5 µM hydrocortisone, 0.5 µM isobutyl methylxanthine, and 60 µM indomethacin (Sigma-Aldrich) for 2 weeks as previously described.22 Cells were stained with fresh Oil-red-O solution (Sigma-Aldrich). The percentage of adipocytes was assayed by counting 50 to 100 cells in multiple fields. Osteogenic differentiation was assessed by incubating the cells with
DMEM with 10% FBS supplemented with 10 Serum-free chondrogenic medium containing DMEM supplemented with 10 ng/mL transforming growth factor- Human foreskin fibroblasts (kindly provided by Dr F. Hills, Imperial College School of Medicine, London, United Kingdom) were cultured in DMEM containing 2 mM L-glutamine, 50 µg/mL streptomycin, 50 U/mL penicillin, and 10% FBS and were used as controls for all the differentiation experiments. Statistics Results are expressed as mean ± SEM, and statistical comparisons were performed using the Student t test.
Adherent, fibroblastlike cells in first-trimester fetal blood, liver, and bone marrow The mean nucleated cell concentration was significantly higher in the first trimester (74.3 ± 9.6 × 109/L) than in the second (21.2 ± 4.5 × 109/L; P = .006) and third (19.2 ± 3.2 × 109/L; P = .0007) trimesters. In first trimester, fetal blood erythroblasts were the predominant cells, accounting for 96.3% ± 2.6% of the nucleated cells, whereas blasts and neutrophils accounted for 2.6% ± 1.4% and 0.7% ± 0.03%, respectively. We also consistently noted a small population of cells with large cytoplasm, pseudopodia, and nucleoli that were present in all the first-trimester fetal samples (0.4% ± 0.04%) (Figure 1A). These cells, which produced prolyl-4-hydroxylase and fibronectin as detected by immunocytochemistry, were not seen in any of the second-trimester or term cord blood samples.
When fetal blood nucleated cells were cultured at a density of 105 cells/cm2 in simple medium containing only 10% FBS with no additional growth factors, all first-trimester samples rapidly formed a monolayer of adherent cells that covered the well surface and, when trypsinized (Figure 1D), appeared identical to those in Figure 1A. Nucleated cells plated at a lower density (12 000/cm2) formed individual colonies consisting of several dozen to a few hundred spindle-shaped fibroblastic cells by day 3. After 8 to 10 days of culture, large colonies (more than 1000 cells) and smaller colonies (fewer than 300 cells) were seen. Figure 1B-C shows the development of a first-trimester fetal blood adherent cell colony from the initial stage of proliferation on day 3 through consecutive stages of cell proliferation; more than 1000 cells became confluent after 8 days. The frequency of adherent colonies in first-trimester blood was 8.2 ± 0.6/106 nucleated cells (equivalent to 69.6 ± 10 colonies/100 µL fetal blood). Adherent fibroblastlike colonies could not be isolated from second- and third-trimester fetal blood samples cultured under identical conditions. However, when mononuclear cells from second- and third-trimester samples were enriched by single-density centrifugation and plated at a 25-fold higher concentration (2.5 × 106 cells/mL) than were used for first-trimester blood, small numbers of colonies of adherent cells did develop. Colonies were smaller than those derived from first-trimester blood (fewer than 100 cells/colony) and were present at lower frequencies: 1.3/106 cells plated (second trimester, n = 1) and 0.35/106 cells (term cord blood, n = 4) versus first-trimester fetal blood (8.2/106 cells). We also investigated whether similar cells were present in fetal liver and bone marrow during the first trimester. When cell suspensions from first-trimester fetal liver and bone marrow were prepared and cultured under conditions identical to those for first-trimester fetal blood (10% FBS with no additional growth factors), adherent cells were seen whose morphologic appearance was identical to that of fetal blood (Figure 1E-L). Frequencies of adherent, fibroblastlike colonies in first-trimester liver and bone marrow were 11.3 ± 2.0/106 nucleated cells and 12.6 ± 3.6/106 nucleated cells, respectively. Growth characteristics of first-trimester fetal blood, liver, and bone marrow mesenchymal stem/progenitor cells The growth kinetics of single colony-derived fetal blood adherent cells (n = 3) was measured at passage 2 (Figure 2). Cells were allowed to divide for 16 days, duplicate cultures were harvested daily, and cell counts were taken. Growth curves depicted an initial lag phase of 2 days, followed by a log phase in which cells divided at exponential rates for 3 to 6 days. The log phase was followed by a plateau phase. Fetal blood adherent cells could be readily expanded in vitro by successive cycles of trypsinization, seeding, and culture every 5 days for 20 passages. The mean cumulative number of population doublings for fetal blood-derived colonies was 50.3 ± 4.5. Cells that had undergone up to 20 passages displayed no visible changes either in terms of their morphology by light microscopy, their forward and side scatter properties on flow cytometry, or their growth patterns. Similar growth patterns were observed with single colonies derived from first-trimester fetal liver and fetal bone marrow adherent cells (Figure 2). The mean cumulative number of population doublings of fetal liver and bone marrow adherent colonies was 52.2 ± 5.3 and 49.5 ± 3.5, respectively.
Immunophenotype of first-trimester fetal blood, liver, and bone marrow mesenchymal stem/progenitor cells The immunophenotype of monolayer and single colony-derived adherent cells from first-trimester fetal blood, liver, and bone marrow was determined by flow cytometry and in situ immunocytochemistry using the antibodies listed in Table 1. The staining pattern of the monolayer-derived cells was identical to that seen for the single colony-derived cells. As shown in Table 1, fetal blood adherent cells stained positively for prolyl-4-hydroxylase;-smooth muscle actin; the hyaluronate receptor, CD44; VCAM-1, a
member of the immunoglobulin superfamily; endoglin (CD105); 1
integrin (CD29); SH2; SH3; SH4; laminin; fibronectin; and
vimentin. Significantly, fetal blood adherent cells were all
CD45 (Figure 3A),
consistent with a nonhematopoietic origin and confirming that
hematopoietic cells had been depleted from the cultures. In line with
this finding, no macrophages (CD14+ cells; Figure 3A) could
be detected in the cultures. Finally, fetal blood adherent cells were
all CD34 and CD31(PECAM-1) (Figure 3A).
Cytoplasmic von Willebrand factor (vWF) expression was not detected,
indicating that they were not endothelial cells. Adherent cells
isolated from first-trimester fetal liver and bone marrow showed a
similar immunophenotype (Table 1, Figure 3B-C). The immunophenotypic
profile of fetal blood, liver, and bone marrow adherent cells did not
change after 10 passages in culture.
Immunophenotypic characterization of these cells allowed us to use fluorescence-activated cell sorting (FACS) analysis to determine the frequency of MSCs in first-trimester fetal blood by staining freshly collected blood samples (n = 3; median, GA 9+5 weeks; range, 9+3 to 10+2) with SH-3, an antibody that recognizes a cell surface epitope present on human mesenchymal progenitor cells.24,25 A frequency of 0.33% ± 0.05% SH-3+ cells was found (equivalent to 0.18 ± 0.02 × 106 SH-3+ cells/mL), almost exactly matching the percentage of MSCs identified on morphologic grounds after Leishman staining of the cytospins of first-trimester blood (0.4% ± 0.04%). Because the number of colony-forming cells in first-trimester blood was only 8.2 ± 0.6/106 cells (0.0008% of nucleated cells), this indicates that colony-forming mesenchymal cells represent 0.2% of circulating mesenchymal cells during the first trimester. Ability of fetal blood mesenchymal stem/progenitor cells to support long-term hematopoiesis To determine whether fetal blood MSCs were capable of supporting the proliferation and differentiation of hematopoietic stem-progenitor cells, second-passage MSCs (n = 3; median gestational age, 9+4 weeks) were irradiated and seeded in 12-well plates to form a semiconfluent monolayer. The next day, human CD34+ cord blood cells (5 × 104 CD34+ cells/well) were overlaid and maintained in culture for up to 8 weeks in long-term culture (LTC) medium. As controls, umbilical cord blood CD34+ cells were overlaid on wells with a murine stromal cell line layer (M210B4) or were cultured in the absence of a stromal layer. Similar numbers of typical cobblestone areas, representing foci of hematopoiesis, were observed after 3 weeks culture on both fetal MSCs and M210B4 cultures, whereas no sign of hematopoiesis was observed when CD34+ cells were cultured in the absence of any stromal feeder. Granulocyte macrophage-colony-forming unit (CFU-GM) assays were carried out on the cells contained in the nonadherent fraction of the cultures at weekly intervals and at the time of termination of the cultures. No difference was detected in the number of CFU-GM progenitors generated from CD34+ cells cocultured with the fetal MSC or the M210B4 cell line at any time point (Figure 4). By contrast, CD34+ cord blood cells grown in LTC medium in the absence of stromal cells and growth factors produced no clonogenic progenitors after 2 weeks of culture (Figure 4).
Differentiation of fetal blood, liver and bone marrow mesenchymal stem/progenitor cells into adipocytes, osteocytes, and chondrocytes The differentiation potential of MSCs from first-trimester fetal blood, liver, and bone marrow was tested by culturing cells under conditions that favored adipogenic, osteogenic, and chondrogenic differentiation of adult MSCs.22,23 The first of two approaches we used was to use cloning cylinders to isolate individual colonies from second-passage MSCs. With this approach, adipogenic differentiation from single adherent colonies from first-trimester fetal blood samples (n = 6) was apparent after 2 weeks of incubation with hydrocortisone, indomethacin, and hydroxybutylmethylxanthine by the accumulation of Oil-red-O staining lipid-rich vacuoles: 77.3% ± 2.9% of cells from fetal blood MSCs differentiated into adipocytes (Figure 5A). A similar proportion of cells from fetal liver MSCs (75.2% ± 6.5%; n = 4) and fetal bone marrow MSC (85.2% ± 4.3%; n = 4) differentiated into adipocytes (Figure 5D,G). By contrast, when MSCs from the same fetal blood colonies that differentiated into adipocytes in adipogenic medium were cultured for 2 weeks with dexamethasone,-glycerophosphate, and ascorbic acid to induce osteogenic
differentiation, they formed aggregates and calcium deposits consistent
with bone formation (n = 6 experiments; Figure 5B). MSCs from
first-trimester fetal liver and bone marrow colonies also underwent
progressive osteogenic differentiation when cultured under identical
conditions (Figure 5E,H). MSCs from the same fetal blood colonies that
differentiated into adipocytes and osteocytes also underwent
chondrogenic differentiation, forming chondrocytelike lacunae
visualized in histologic sections after 3 weeks of culture in
serum-free chondrogenic medium and an extracellular matrix rich in
types 1 and 2 collagens, together with the accumulation of anionic
(toluidine blue) and sulfated (Sufranin O) proteoglycans (n = 6
experiments; Figure 5C). First-trimester fetal liver and bone marrow
single colony-derived MSCs also underwent chondrogenic differentiation
(Figure 5F,I) when cultured under identical conditions. Finally, we
found that even late-passage (eighth passage) fetal MSCs from
first-trimester fetal blood, liver, or bone marrow retained the ability
to differentiate into all 3 lineages.
To investigate this further, we used a second approach to assess the differentiation potential of individual MSCs from first-trimester fetal blood, liver, and bone marrow. Using single cell sorting of second-passage MSCs by flow cytometry, individual colonies derived from single FACS-sorted cells were grown in 96-well plates; cells derived from these clones were then subcultured in adipogenic, osteogenic, or chondrogenic media. Cloning efficiency varied from 10% to 17% (n = 3 experiments), and 25% to 30% of the colonies expanded fairly rapidly to more than 1000 cells over a 2-week period; these rapidly growing colonies were used for subsequent differentiation experiments. Such cells (derived from first-trimester blood, marrow, and liver) all retained the ability to differentiate into multiple lineages. Interestingly, however, we found that though single colonies derived from first-trimester fetal blood or liver underwent osteogenic and chondrogenic differentiation, they appeared to lose their capacity for adipogenic differentiation (0 of 4 colonies). By contrast, 50% of the colonies derived from single cells from first-trimester fetal bone marrow retained the capacity for differentiation in all the lineages (adipogenic, osteogenic and chondrogenic).
Although it is known that adult bone marrow-derived MSCs can be rapidly expanded in vitro, migrate, and differentiate into multiple tissues in vivo, there is little information about the characteristics and functional properties of MSCs in the fetus. We report the isolation and characterization of MSCs from first-trimester human fetal blood, liver, and bone marrow. These 3 anatomic sites are likely sources of fetal MSCs for several reasons. First, it is clear that in adult bone marrow, mesenchymal cells provide signals for differentiation and proliferation of hematopoietic stem cells and their progeny through direct cell-cell interactions26 and secretion of hematopoietic growth factors and cytokines.27-29 Second, during human ontogeny, it has been demonstrated that proliferation and differentiation of hematopoietic stem-progenitor cells occurs in a number of histologically distinct microenvironments (yolk sac,30 ventral aorta,31 fetal liver, thymus, spleen, and bone marrow20) and that stem cells migrate from one anatomic site to another through the circulation.32-34 Finally, mesenchymal cells have been shown to colocalize with foci of hematopoiesis early in ontogeny.36 Studies of fixed preparations of human fetal long bones before and
immediately after the onset of hematopoiesis show that early in
gestation (6-8.5 weeks), long bone rudiments contain mainly
chondrocytes, osteoblasts, osteoclast precursors, and CD68+
monocyte-macrophages.35 After this, at 8 to 10 weeks'
gestation, at or just before hematopoietic colonization of the bone
marrow, vascular cells, including CD34+ endothelial cells
and When fetal samples older than 13 weeks of gestation were cultured at a plating density of 106 nucleated cells, we could identify MSCs in fetal liver and bone marrow but not in fetal blood samples. However, small numbers of MSCs could be cultured from second- and third-trimester blood samples when the mononuclear cells were plated at a 25-fold higher density. Their frequencies were 1.3/106 mononuclear cells in the second trimester and 0.35/106 mononuclear cells in term cord blood, thereby confirming the work of others17 showing that it is possible to demonstrate such cells at term but that they are present at an extremely low frequency. These data indicate that the frequency of circulating MSCs decreases substantially with gestational age; we speculate that this might be related to their migration from one hematopoietic site to another during ontogeny, ultimately to colonize bone marrow. Lines of evidence indicate that the fetal MSCs we have identified in
first-trimester fetal blood, liver, and bone marrow are similar to
those present in adult bone marrow. First, their morphology and
immunophenotype are similar to those of adult marrow MSCs, and they are
clearly nonhematopoietic and nonendothelial There are several explanations for the discrepant results obtained using cloning cylinders compared to single-cell sorting. One is that although cultures of fetal MSCs are morphologically and phenotypically homogenous, they might contain cell populations with distinct differentiation potential, as has been observed in adult MSCs.5,40 Alternatively, if the proportion of stem cells with multilineage differentiation potential is very low, then a large number of single cell-derived colonies would have to be isolated and investigated to identify those with stem cell properties. In addition, our results are likely to reflect different methods of MSC colony growth and expansion; in particular, cell-cell contact may play a role in the expansion and multilineage differentiation of fetal MSCs. It is interesting that adult MSCs, which have been extensively cultured (passages 8 to 14), have also been reported to lose their capacity for adipogenic differentiation while osteogenic differentiation is preserved.40 Finally, it is possible that in the intense replicative stress of single sorted cells, culture conditions for adipogenic differentiation might have been suboptimal. The function of fetal MSCs in the human fetus remains to be determined, though they are highly likely to play a role in the establishment and maintenance of hematopoietic stem and progenitor cells. Cotransplantation of adult MSCs has been shown to enhance engraftment of hematopoietic stem cells in a fetal sheep model,10-12 suggesting that in utero cotransplantation of fetal MSCs may also result in accelerated hematopoietic cell engraftment. First-trimester fetal blood represents a potentially harvestable source of autologous MSCs amenable to genetic manipulation and reinfusion in ongoing pregnancies. Indeed, fetal blood has been sampled in ongoing pregnancies from as early as 12 weeks,41 and, with advances in embryofetoscopic cannulation techniques,42 may become obtainable even earlier. We are currently assessing the possible use of fetal blood MSCs for in utero gene therapy for genetic disorders. In conclusion, a population of MSCs was isolated from fetal blood, liver, and bone marrow in the first-trimester of pregnancy. These cells possess morphologic, immunophenotypic, and functional characteristics similar to those of adult-derived MSCs. Fetal MSCs might provide useful models for investigation of mesenchymal cell differentiation and regulation of hematopoiesis during ontogeny.
Submitted January 4, 2001; accepted June 22, 2001.
Supported in part by project grants from Wellbeing.
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 Campagnoli, Department of Maternal and Fetal Medicine, Institute of Reproductive and Developmental Biology, Imperial College School of Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, United Kingdom; e-mail: c.campagnoli{at}ic.ac.uk.
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Abstract
Introduction
, n = 3; fetal liver, 
, n = 3; and fetal
bone marrow, 
, n = 3.
).
consistently CD45