1 Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and
Veterinary Science, Adelaide, South Australia, Australia
2 Myeloma and Mesenchymal Research Group, Matthew Robert's Foundation
laboratory, Institute of Medical and Veterinary Science, Adelaide, South
Australia, Australia
3 Department of Orthopaedics and Trauma, Adelaide University, Adelaide, South
Australia, Australia
4 Craniofacial and Skeletal Diseases Branch, National Institute of Dental &
Craniofacial Research, National Institutes of Health, Maryland, USA
5 Department of Orthopaedics, Royal Melbourne Hospital, Melbourne, Victoria,
Australia
6 Stem Cell Laboratory, Peter MacCallum Cancer Institute, East Melbourne,
Victoria, Australia
* Author for correspondence (e-mail: stan.gronthos{at}imvs.sa.gov.au)
Accepted 14 January 2003
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Summary |
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Key words: Bone Marrow Stroma, Mesenchymal Stem Cells, STRO-1, Bone, Cartilage, Adipose, CFU-F
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Introduction |
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A more direct demonstration of marrow stromal cell precursors was provided
by the pioneering studies of Friedenstein and colleagues, who demonstrated the
in vitro growth of adherent colonies of cells morphologically resembling
fibroblasts (CFU-F) derived from explants of BM
(Castro-Malaspina et al., 1980;
Friedenstein et al., 1970
;
Owen, 1988
). A consistent
feature of marrow CFU-F-derived colonies of virtually all species examined is
their considerable heterogeneity in terms of size, morphology, enzyme
histochemistry, proliferation and developmental potential
(Friedenstein et al., 1987
;
Kuznetsov et al., 1997
;
Owen and Friedenstein, 1988
).
These observations are consistent with the hypothesised existence within
marrow stromal tissue of a hierarchy of cellular differentiation supported at
its apex by a small compartment of self-renewing, pluripotent stromal stem
cells, known as bone marrow stromal cells, stromal precursor cells, bone
marrow stromal stem cells and mesenchymal stem cells
(Owen and Friedenstein,
1988
).
The low incidence of clonogenic CFU-F in adult human BM (range
1-20x105 mononuclear cells plated)
(Gronthos and Simmons, 1996)
was a major limitation to their study, a problem compounded until recently by
the paucity of specific antibody reagents to facilitate CFU-F isolation and
enrichment. Monoclonal antibody STRO-1 reacts with an as yet unidentified cell
surface antigen expressed by a minor subpopulation of adult human BM
(Simmons and Torok-Storb,
1991
). Previous studies have shown that STRO-1 is non-reactive
with haematopoietic progenitors, but included within the STRO-1+
population are essentially all detectable clonogenic CFU-Fs
(Simmons and Torok-Storb,
1991
). Moreover, data from this laboratory demonstrate that within
the STRO-1+ fraction in adult human BM are BMSSCs with the capacity
to transfer a functional hematopoietic microenvironment in vitro and for
differentiation into multiple stromal cell types including smooth muscle
cells, adipocytes, osteoblasts and chondrocytes
(Dennis et al., 2002
;
Gronthos et al., 1994
;
Simmons and Torok-Storb,
1991
). However, use of the STRO-1 antibody is not sufficient to
obtain the purity of BMSSCs required to properly study their properties, owing
to the presence of contaminating populations of glycophorin-A-positive
nucleated red cells and a small subset of B-lymphocytes.
Herein, we report the isolation of a highly enriched population of BMSSCs with clonogenic potential from adult human BM, on the basis of the use of STRO-1 in combination with an antibody directed to vascular cell adhesion molecule-1 (VCAM-1/CD106). In addition, we examine aspects of the molecular, cellular and developmental properties of this poorly characterized population of stromal stem cells. The isolation of BMSSCs is a prerequisite for the study of mechanisms that regulate their differentiation and is likely to be of therapeutic importance given the developmental potential of these cells.
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Materials and Methods |
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Primary antibodies
Monoclonal antibodies (MAbs) STRO-1 (mouse IgM)
(Simmons and Torok-Storb,
1991) and the anti-VCAM-1 antibody 6G10 (mouse IgG1)
(Simmons et al., 1992
) were
used as tissue culture supernatants diluted 1:2 and 1:4, respectively.
Isotype-matched controls of irrelevant specificity, 1A6.12, (IgM), 3D3 (IgG1)
and 1D4.5 (IgG2a) (1/4; kindly provided by L. K. Ashman, Medical Science
Building, University of Newcastle, New South Wales, Australia).
-smooth
muscle actin (5 µg/ml, mouse IgG2a; Immunotech), affinity-purified rabbit
antiserum to collagen type I and rabbit immunoglogulin control (1/200;
Chemicon International Inc., Temecula, CA). Anti-Ki-67-FITC or isotype-matched
IgG1-FITC antibody (1/10; Dakopatts A/S, Glostrup, Denmark).
Magnetic-activated cell sorting
Magnetic-activated cell sorting (MACS) was performed as previously
described (Gronthos, 1998; Gronthos and
Simmons, 1995). In brief, approximately 1-3x108
BMMNCs were sequentially incubated with STRO-1 supernatant, anti-IgM-biotin,
streptavidin microbeads and finally streptavidin-FITC (Caltag Laboratories,
Burlingame, CA) before being separated on a Mini MACS magnetic column
(Miltenyi Biotec Inc., Auburn, CA).
Fluorescence-activated cell sorting and limiting dilution assays
The STRO-1+ (FITC labelled) isolated by MACS was incubated with
purified an anti-VCAM-1 antibody 6G10 or isotype control 1B5 for 30 minutes on
ice, washed and incubated with phycoerythrin (PE)-conjugated goat anti-mouse
IgG antibody (1/50; Southern Biotechnology Associates, Birmingham, AL) for an
additional 20 minutes on ice. Cells were sorted using a FACStarPLUS
flow cytometer (Becton Dickinson, Sunnyvale, CA). Limiting dilution assays
were performed with STRO-1BRIGHT/VCAM-1+ sorted cells
seeded at plating densities of 1, 2, 3, 4, 5 and 10 cells per well (96-well
plates) in replicates of 24 wells per plating density, using the automated
cell deposition unit (ACDU) of the flow cytometer. The cells were cultured in
serum-deprived medium in the presence of PDGF-BB and EGF (10 ng/ml) as
previously described (Gronthos and
Simmons, 1995). Colony efficiency assays were performed using
Poisson distribution statistics by determining the number of wells with no
clonogenic growth at day 14 of culture following staining of the cultures with
0.1% (w/v) toluidine blue in 1% paraformaldehyde. Aggregates of >50 cells
were scored as CFU-F-derived colonies and aggregates of >10 and <50
cells were scored as clusters.
Analysis of cell cycle status
The STRO-1+ cells isolated by MACS were incubated with
streptavidin-PE (Caltag; 1:50) for 15 minutes on ice. After washing with PBS,
the cells were fixed for 10 minutes with 70% (v/v) ethanol on ice. Following
washing, the cells were incubated with either anti-Ki-67-FITC or
isotype-matched IgG1-FITC antibody for 45 minutes on ice. The cells
were then washed in PBS prior to flow cytometric analysis.
Immunostaining
STRO-1BRIGHT/VCAM-1+ cells isolated by FACS were
prepared as cytospins and fixed with cold acetone. After washing, the cells
were blocked in 5% goat serum in PBS and then incubated with primary
antibodies and the corresponding control immunoglobulins. The subsequent steps
of immunoperoxidase staining were performed using Vectastain ABC
immunoperoxidase kits for mouse and rabbit IgG, respectively (Vector
Laboratories, Burlingame, CA) according to the manufacturer's
instructions.
Reverse transcriptase polymerase chain reaction analysis
Total cellular RNA was prepared from 2x104
STRO-1bright/VCAM-1+ sorted cells collected as a bulk
population and lysed using an RNAzolB extraction method (Biotecx Lab. Inc.,
Houston, TX) according to the manufacturer's recommendations. RNA isolated
from each subpopulation was then used as a template for cDNA synthesis,
prepared using a First-strand cDNA synthesis kit (Pharmacia Biotech, Uppsala,
Sweden). The expression of various transcripts was assessed by PCR
amplification, using a standard protocol as described previously
(Gronthos et al., 1999).
Primers sets used in this study are shown in
Table 1. Following
amplification, each reaction mixture was analysed by using 1.5% agarose gel
electrophoresis and visualised by ethidium bromide staining. RNA integrity was
assessed by the expression of GAPDH.
|
Differentiation of CFU-F in vitro
We have previously reported the conditions needed for human BM stromal
cells to develop a mineralized bone matrix in vitro as MEM supplemented
with 10% FCS, 100 µM L-ascorbate-2-phosphate, dexamethasone 10-7
M and 3 mM inorganic phosphate (Gronthos
et al., 1994
). Mineral deposits were identified by positive von
Kossa staining. Adipogenesis was induced in the presence of 0.5 mM
methylisobutylmethylxanthine, 0.5 µM hydrocortisone and 60 µM
indomethacin as previously described
(Gimble, 1998
). Oil Red O
staining was used to identify lipid-laden fat cells. Chondrogenic
differentiation was assessed in aggregate cultures treated with 10 ng/ml
TGF-ß3 as described previously
(Pittenger et al., 1999
).
In vivo assay of bone formation
The adherent cells derived from STRO-1BRIGHT/VCAM-1+
cells at passage two-three were trypsinised, mixed with 40 mg
hydroxyapatite/tricalcium phosphate ceramic particles (Zimmer Corporation,
Warsaw, IN) and then implanted into subcutaneous pockets on the dorsal surface
of two-month-old SCID mice as described previously
(Gronthos et al., 2000). These
procedures were performed in accordance with an approved animal protocol
(Adelaide University AEC# M/079/94). Implants were recovered after 6-8 weeks,
fixed in 4% paraformaldehyde for 2 days, then decalcified for a further 10
days in 10% EDTA prior to embedding in paraffin. For histological analysis, 5
µm sections of the implants were prepared and stained with haematoxylin and
eosin. In situ hybridization for the human specific alu sequence was
performed as previously described
(Gronthos et al., 2000
).
Telomerase repeat amplification protocol
Telomerase cell extracts prepared by the method of Kim et al.
(Kim et al., 1994) were
analysed for the presence of telomerase activity as previously described
(Fong et al., 1997
). To
confirm the specificity of the telomerase products 2 µl aliquots of each in
CHAPS lysate was subjected to denaturation and resolved on a non-denaturing
12% polyacryalmide gel, then visualised by staining with SYBR green
fluorescent dye (FMC Bioproducts, OR) as recommended by the manufacturer. The
telomerase repeat amplification protocol (TRAP) products were analysed using a
FluorImager (Molecular Dynamics, Sunnyvale, CA).
Transmission electron microscopy
Approximately 2x104
STRO-1BRIGHT/VCAM-1+ cells were fixed in 2.5%
glutaraldehyde (EM Grade) in cacodylate buffer and then processed for
embedding into resin as previously described
(Gronthos et al., 1994).
Ultrathin sections were examined using a JEOL 1200 EX II (Tokyo, Japan)
transmission electron microscope.
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Results |
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|
|
To further increase the quantity of CFU-F, we made use of previous studies
that examined the expression of a broad range of cell surface molecules on
CFU-F (Simmons et al., 1994).
VCAM-1/CD106 was one of several cell surface molecules that fulfilled the
criteria of minimal reactivity with BMMNC but produced a high yield of CFU-F
following FACS. In accord with these data, dual-color FACS analysis of
MACS-isolated STRO-1+ cells demonstrated VCAM-1 expression by a
minor subpopulation of cells (1.4%±0.3; n=20), which were
characterized by a high level of STRO-1 expression (STRO-1BRIGHT)
(Fig. 1B). By means of
dual-colour cell sorting, CFU-F were found to be restricted to the small
proportion of marrow cells co-expressing both antigens
(STRO-1BRIGHT/VCAM-1+) (data not shown). To determine
the incidence of CFU-F in the STRO-1BRIGHT/VCAM-1+
population, limiting dilution assays were performed. Analysis of the data from
six different marrow samples yielded a mean frequency of approximately one
CFU-F-derived colony (
50 cells) per three
STRO-1BRIGHT/VCAM-1+ cells, whereas the incidence of all
clonogenic CFU-F (measured as the sum of colonies plus clusters) was one per
two STRO-1BRIGHT/VCAM-1+ cells plated
(Fig. 1C), using Poisson
distribution statistics. Notably, of the 50% of wells deposited with single
STRO-1BRIGHT/VCAM-1+ cells that failed to produce clones
(
10 cells), a significant proportion (approximately 30%) contained either
single or small groups of stromal cells that failed to produce colonies or
clusters (<10 cells) in these assay conditions.
Characteristics of freshly isolated CFU-F
STRO-1BRIGHT/VCAM-1+ cells sorted directly from
fresh, marrow aspirates were large cells with heterochromatic nuclei and
prominent nucleoli, agranular cytoplasm and numerous bleb-like projections of
the cell membrane (Fig. 2A).
Ultrastructural analysis demonstrated an extensive array of cytoplasmic
microfilaments and a complex cell surface morphology dominated by the
bleb-like membrane protrusions observed by light microscopy
(Fig. 2B). Weibel-Palade bodies
characteristic of endothelial cells were not detected in the enriched BMSSCs
population. In addition, immunohistochemical staining of cytospin preparations
of the sorted STRO-1BRIGHT/VCAM-1+ marrow cells showed
strong staining in >90% of cells with anti-collagen type I antibody
(Fig. 2C) but failed to show
any reactivity with antibodies to factor-VIII-related antigen, a marker of
vascular endothelial cells, or to CD45, the common leukocyte antigen (data not
shown). A notable feature, however, was the expression of -smooth
muscle actin (
-SMA) in approximately 70% of
STRO-1BRIGHT/VCAM-1+ cells although the intensity of
immunostaining varied somewhat between individual cells
(Fig. 2D).
|
Dual-colour flow cytometric analysis demonstrated that the STRO-1BRIGHT population lacked detectable expression of the Ki-67 antigen, demonstrating that these cells do not divide in vivo (Fig. 2E). In addition, telomerase activity commonly found in stem cell populations of other renewing tissues was also present in the total STRO-1+ population isolated by MACS and in the minor subpopulation of STRO-1BRIGHT/VCAM-1+ FACS-sorted cells (Fig. 2F). Following transfer of the sorted cells in vitro, they rapidly attached and spread assuming a stellate morphology with long processes (Fig. 2G) capable of clonal expansion forming colonies consisting of collagen type I positive fibroblast-like cells (Fig. 2H,I).
An essential feature of stem cell populations in all renewing tissues is a capacity for extensive proliferation. Initial studies to examine the proliferative potential of BMSSCs were performed in which STRO-1BRIGHT/VCAM-1+ cells were cultured in bulk and then serially passaged over the course of a number of weeks in culture. This analysis reproducibly demonstrated a cumulative expansion in the number of adherent stromal cells for over 40 population doublings prior to the onset of cellular senescence (data not shown). Since these data reflect the proliferative activity of a mixed population of BMSSCs, we chose to conduct a more rigorous examination of the growth potential of individual STRO-1BRIGHT/VCAM-1+ BMSSCs obtained by means of the single cell deposition unit of the cell sorter. A total of 35 CFU-F colonies derived from two BM samples were expanded in culture, as above, and analysed for their cumulative production of cells over a number of weeks in culture. There was marked variation in proliferative capacity between individual CFU-F (Fig. 3). The majority of clones (29/35; 83%) exhibited only moderate growth potential that did not persist beyond 20 population doublings. In contrast, a minor proportion of clones (6/35, 17%) demonstrated continued growth, extending beyond 20 population doublings, generating a mean of 3.0x108 cells per BMSSCs (range, 9.8x106 - 5.2x108 cells). At this time, cells derived from highly proliferative clones (>20 population doublings) were analyzed for their expression of STRO-1 and VCAM-1 by flow cytometry. Although VCAM-1 expression was retained by the progeny of the initiating cells, the STRO-1 epitope was expressed by only a minor subpopulation in all clones analysed (range 3.2-15.8%; median 5.3%; n=6).
|
Gene expression profile of CFU-F in vivo and following proliferation
and differentiation in vitro
We first surveyed the pattern of gene expression in freshly isolated
STRO-1BRIGHT/VCAM-1+ BMMNC by means of RT-PCR. There was
no detectable expression of mature bone markers such as osteopontin,
parathyroid hormone-receptor nor osteocalcin. Importantly, this population
also lacked the expression of the essential early bone-cell-specific
transcription factors CBFA1 and osterix
(Fig. 4). A similar analysis of
mRNA transcripts with restricted expression in adipose cells revealed
constitutive expression of only lipoprotein lipase but not other
adipocyte-related genes including, leptin and the early adipocytic
transcription factor, PPAR2 (Fig.
4). Finally, expression analysis of genes restricted to the
chondrocyte lineage demonstrated neither collagen type II nor aggrecan
expression although collagen type X, a marker associated with hypertrophic
chondrocytes, was consistently detected
(Fig. 4).
|
Next, bulk cultures initiated with
STRO-1BRIGHT/VCAM-1+ cells were expanded by passaging
two to three times, at which point aliquots of cells were plated in conditions
previously described to induce osteogenic
(Gronthos et al., 1994),
adipogenic (Gimble, 1998
) and
chondrogenic (Pittenger et al.,
1999
) differentiation of BMSSCs in vitro. Expression of CBFA-1,
osterix and osteopontin were detected by RT-PCR in both induced and
non-induced control cultures (Fig.
4), whereas osteocalcin and PTH-R transcripts were detected within
4-5 weeks of growth in osteoinduction media. Following 2-3 weeks of adipogenic
induction, transcripts were detected for the fat-associated markers,
PPAR
2 and leptin (Fig.
4). Similarly, in aggregate cultures, prolonged exposure to
TGFß3 induced the mRNA expression of the cartilage-associated matrix
components collagen type II and aggrecan by RT-PCR
(Fig. 4).
Developmental potential of BMSSC clones in vitro and in vivo
Comparison of the in vitro developmental potential of 64 highly
proliferative BMSSC clones (20 population doublings), derived from single
STRO-1BRIGHT/VCAM-1+ cells (n=3 marrow
samples), demonstrated the formation of von Kossa positive mineralized
deposits by all clones in vitro after several weeks under osteogenic-induction
conditions (Fig. 5A).
Similarly, 95% of the same BMSSC clones formed clusters of Oil red O-positive
lipid-laden adipocytes when cultured in adipogenic inductive media
(Fig. 5B). A sample of 20 high
proliferative clones were assayed for their chondrogenic potential using the
well established aggregate culture system
(Pittenger et al., 1999) in
the presence of TGFß3. Again, all clones exhibited synthesis
of collagen type II by immunohistochemistry
(Fig. 5C). However, with
continuous subculture (greater than 25 population doublings) the BMSSCs clones
showed either a reduced capacity or an inability to differentiate into all
three stromal cell lineages (data not shown).
|
The capacity to develop a mineralised matrix in vitro, although consistent
with osteogenic differentiation, may nevertheless not predict the capacity of
the cells to produce an organised bone tissue in vivo. In view of this
concern, all 64 highly proliferative BMSSC clones were assayed for their
capacity to develop human bone tissue following ectopic transplantation in
SCID mice, using hydroxyapatite/tricalcium phosphate (HA/TCP) particles as a
carrier vehicle (Gronthos et al.,
2000; Kuznetsov et al.,
1997
). Histiological examination at week 8 showed that all of the
implants contained an extensive network of blood vessels and fibrous tissue
(Fig. 6A). Bone formation was
evident in 35/64 clones (54%), whereas associated haematopoietic
marrow/adipose elements were observed in 11/35 of the bone-producing clones
(31%). Cartilage development, as assessed by immunohistochemical staining with
an antibody to collagen type II, was not detected in any implant. The origin
of the cellular material within the recovered implants was assessed by in situ
hybridization using a probe to the unique human repetitive alu. The
interstitial tissue, bone lining cells and osteocytes within the newly formed
bone were found to positive for the alu sequence, confirming their
human origin (Fig. 6B).
Conversely, neither the endothelium-lining small blood vessels, haematopoietic
cells nor the adipose and muscle tissue surrounding the HA/TCP carrier
demonstrated hybridization with the alu probe and were therefore
presumed to be of murine origin.
|
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Discussion |
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In this report, we describe the isolation of a minor subpopulation of adult
human BMMNC that represent a near homogeneous population of CFU-F. This was
achieved using the CFU-F reactive antibody STRO-1
(Simmons and Torok-Storb,
1991) in combination with an antibody to VCAM-1, a cell adhesion
molecule constitutively expressed by marrow stromal tissue in vitro and in
vivo (Simmons et al., 1992
)
and also by CFU-F (Simmons et al.,
1994
). This approach enabled the resolution of a discrete
subpopulation of STRO-1BRIGHT/VCAM-1+ marrow cells with
a cloning efficiency for CFU-F approaching 50% using Poisson distribution
statistics. This degree of enrichment for CFU-F in the
STRO-1BRIGHT/VCAM-1+ cell fraction (approximately
5,000-fold relative to unfractionated BM) substantially exceeds that
previously reported for any mammalian species including mice and humans
(Castro-Malaspina et al., 1980
;
Simmons and Torok-Storb, 1991
;
Van Vlasselaer et al., 1994
;
Waller et al., 1995
). A
significant factor contributing to the success of this enrichment strategy is
the use of our previously defined serum-deprived culture conditions for the
assay of CFU-F, in which colony growth at low plating densities is stimulated
by the addition of a combination of EGF and PDGF-BB
(Gronthos and Simmons, 1995
).
CFU-F are generally assayed in medium supplemented with FCS as a growth
stimulus. Although this is adequate for CFU-F growth from unfractionated bone
marrow at high plating densities, when more enriched cell populations are
assayed in FCS-containing medium, growth of CFU-F becomes suboptimal,
particularly at limiting cell concentrations
(Gronthos and Simmons, 1995
).
Indeed, in the current study, STRO-1BRIGHT/VCAM-1+ CFU-F
failed to grow in foetal bovine serum supplemented medium at plating densities
<10 cells per well (data not shown), whereas in the serum-deprived assay, a
significant proportion of cells exhibited clonogenic growth even when plated
at 1 cell per well. It is important to note, however, that even under these
highly efficient culture conditions a significant proportion of single
STRO-1BRIGHT/VCAM-1+ cells, although failing to
proliferate sufficiently to qualify as either clusters or colonies,
nevertheless contained stromal cells that either remained as single cells or
underwent one or two divisions only. The nature of the
STRO-1BRIGHT/VCAM-1+ stromal cells that fail to either
proliferate or do so only poorly remains to be determined. One possibility is
that expression of the antigen identified by STRO-1 may encompass not only
stromal progenitors but also partially differentiated stromal cells with
correspondingly reduced growth potential in vitro. Alternatively, these cells
may represent a subpopulation of stromal progenitors that fail to proliferate
in these assay conditions. Nevertheless, the
STRO-1BRIGHT/VCAM-1+ BMMNC represent a virtually pure
population of collagen type I positive, stromal progenitor cells with varying
clonogenic efficiencies.
As a population, STRO-1BRIGHTVCAM-1+ cells have
several important phenotypic characteristics previously attributed to stem
cells in other renewing tissues (Fuchs and
Segre, 2000). Firstly, freshly isolated BMNC-derived
STRO-1BRIGHT cells lacked expression of the Ki-67 antigen and thus
appear to be a non-cycling population in vivo. This is in accord with previous
studies demonstrating the quiescent nature of CFU-F in unfractionated rodent
and human BM (Castro-Malaspina et al.,
1981
; Falla et al.,
1993
). Secondly, STRO-1BRIGHTVCAM-1+ cells
and their progeny constitutively exhibit telomerase activity, a well
documented feature of stem cell populations in renewing tissues that is lost
during normal somatic cell proliferation and differentiation
(Fuchs and Segre, 2000
;
Harle-Bachor and Boukamp,
1996
). Recent studies have shown that human BMSSCs lose telomerase
activity during ex vivo expansion, whereas enforced telomerase activity
greatly enhanced the proliferative life-span and osteogenic potential of
cultured BMSSCs (Shi et al.,
2002
; Simonsen et al.,
2002
). Thirdly, freshly sorted
STRO-1BRIGHTVCAM-1+ cells exhibit an undifferentiated
phenotype as demonstrated by the absence of gene products characteristic of
endothelial cells and mature stromal elements such as osteogenic cells,
adipocytes and chondrocytes within the BM. Most significantly in this regard,
this population lacked detectable expression of transcription factors with
pivotal roles in the early differentiation of bone (CBFA-1, osterix) and
adipose (PPAR
2) tissues and the signature marker of chondrocytes,
collagen type II (Ducy et al.,
1997
; Nakashima et al.,
2002
; Tontonoz et al.,
1994
).
A notable feature of the BM CFU-F population is the considerable
heterogeneity in their potential for differentiation and proliferation. In
accord with the putative stromal stem cell hierarchy of cellular
differentiation, we found that only a proportion of the
STRO-1BRIGHT/VCAM-1+ cell population was capable both of
extensive proliferation in vitro beyond 20 population doublings and of
differentiation into at least five of the cellular components of BM stromal
tissue, namely myelosupportive stroma, smooth muscle cells, osteoblasts,
adipocytes and chondroblasts. Similar observations were previously reported
(Pittenger et al., 1999) for a
population of plastic adherent fibroblast-like cells comprising 0.01-0.001% of
nucleated cells in human BM.
We have previously demonstrated that all CFU-F clones derived from
STRO-1+ human marrow demonstrated the capacity to synthesise a
mineralized bone matrix in vitro, whereas only 48% of clones showed the
capacity to form adipocytes in vitro
(Gronthos et al., 1994). In
the present study, highly proliferative clones, derived from single
STRO-1BRIGHTVCAM-1+ cells, exhibited osteogenic,
chondrogenic and adipogenic cell differentiation in vitro. However, when
assayed in SCID mice for their capacity to generate human bone tissue
following xenogeneic transplantation
(Gronthos et al., 2000
;
Kuznetsov et al., 1997
) only a
little over half of the clones exhibited the potential to form bone in vivo.
Thus the in vitro culture assay, despite evidence of many of the phenotypic
characteristics of differentiated bone cells including expression of the
master control gene CBFA1, does not accurately predict the osteogenic
potential of BMSSCs clones in vivo, in accord with studies on murine BM
stromal cell lines (Satomura et al.,
2000
). Use of the more stringent in vivo assay demonstrated that
osteogenic differentiation is only exhibited by a subpopulation of clones and
is consistent with the findings of Kusnetsov and colleagues
(Kuznetsov et al., 1997
).
In the present study, the data indicate that CFU-F in adult human BM
represent a mixed population of multi-, bi- and uni-potential progenitors at
different stages of differentiation, as initially proposed by Owen and
Friedenstein (Owen and Friedenstein,
1988) and subsequently supported by others
(Kuznetsov et al., 1997
;
Pittenger et al., 1999
).
Future studies designed to subset the
STRO-1BRIGHTVCAM-1+ population may in time reveal a
minor population with properties attributed to pluripotent stem cells. Efforts
to identify BMSSCs in vivo have been hampered by the lack of precise knowledge
regarding their anatomical distribution within the marrow. It has been
suggested that that osteoprogenitors may be associated with the outer surfaces
of the marrow vasculature (Bianco et al.,
2001
). In the present study,
STRO-1BRIGHTVCAM-1+ cells proved to be a homogeneous
population of large collagen type I positive cells lacking phenotypic
characteristics of leukocytes or vascular endothelial cells. Of particular
note is the expression of
-SMA by this population. In adult human BM in
vivo,
-SMA is limited to vascular smooth muscle cells in the media of
arteries, cells lining the abluminal surface of sinuses, pericytes lining
capillaries and occasional flattened cells on the endosteal surface of bone.
Expression of
-SMA is not detected in other marrow stromal elements
such as reticular cells within haemopoietic cords, adipocytes or vascular
endothelial cells (Galmiche et al.,
1993
). Collectively these observations suggest two possibilities
for the identity and anatomical location of stromal progenitors in the BM:
vascular smooth muscle cells/pericytes and endosteal cells. Furthermore,
accumulating data suggest that vascular pericytes may also fulfil the role of
multipotential mesenchymal progenitors
(Doherty et al., 1998
;
Schor et al., 1995
).
The biological properties of the BMSSCs described herein should be viewed
in the context of recent reports that demonstrate that the inherent
developmental potential of stem cells derived from various mammalian tissues
may be more similar than previously suspected
(Azizi et al., 1998;
Bjornson et al., 1999
;
Gussoni et al., 1999
). This
suggests that the developmental plasticity of stem cells is dictated by the
local tissue microenvironment in which they lodge, and it would therefore be
of great interest to examine whether the population of candidate stem cells
described herein can regenerate tissues other than the marrow stroma and
associated skeletal tissues (Prockop,
1997
). It is conceivable therefore that the properties exhibited
by the STRO-1BRIGHTVCAM-1+ population may, in time, be
useful for a range of novel cellular therapies that extends beyond their more
obvious use in the treatment of disorders of the haemopoietic and skeletal
systems.
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