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1994 84: 4164-4173
The STRO-1+ fraction of adult human bone marrow contains the
osteogenic precursors
S Gronthos, SE Graves, S Ohta and PJ Simmons
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The STRO-l+ Fraction of Adult Human Bone Marrow Contains the
Osteogenic Precursors
By S. Gronthos, S.E. Graves, S. Ohta, and P.J. Simmons
The monoclonalantibody STRO-1 identifies clonogenic bone
marrow stromal cell progenitors (fibroblast colony-forming
units [CFU-F]) in adult human bone marrow. These STROl+
CFU-F have previously been shown t o give rise t o cells
with thephenotype of fibroblasts, adipocytes, and smooth
muscle cells.In this study, the osteogenic potential of CFUF derived from the STRO-l+ fraction of adult human bone
marrow was determined. CFU-F were isolated from normal
bone marrow aspirates by fluorescence activated cell sorting, based on their expression of the STRO-1 antigen. Osteogenic differentiation was assessed by the induction of
alkaline phosphatase expression,
by theformation of a mineralized matrix (hydroxyapatite), and by the production of
the bone-specific protein osteocalcin. STRO-l+ cells were
mol/
cultured in the presence of dexamethasone(DEX;
L), ascorbic acid 2-phosphate(ASC-2P; 100 pmol/L), and inorganic phosphate (PO,,;2.9 mmol/L). After 2 weeks of culture, greater than 90% of the cells in eachCFU-F colony
stained positive for alkalinephosphatase using a monoclonal antibody specific for bone and liver alkaline phosphatase. Alkaline phosphatase activity was confirmed by histochemistry. A mineralized matrix developed in the CFU-F
cultures, after 4 weeks of culture in the presence of DEX,
ASC-2P. and PO,. Mineralization was confirmed by both
light and electron microscopy. The mineral was identified
as hydroxyapatite by electron dispersive x-ray microanalysis
and by x-ray diffraction analysis. In replicate cultures, osteocalcin release was
shown after exposure ofthe cells t o 1.25dihydroxyvitamin DJ (10" mol/L) both by radioimmunoassay and Northern blot analysis. This work provides direct
evidence that adult human bone marrow-derived CFU-F are
capable of differentiating into functional osteoblasts and
that osteoprogenitors are present in the STRO-l+ population.
0 1994 by The AmericanSociety of Hematology.
I
tive potential and therefore represent self-maintaining populations within the BM. Alternatively, does there exist a population of stromal cell precursors with the capacity to give
rise to each of the aforementioned stromal elements and is
thus responsible for the turnover of marrow stromal tissue
under steady-state and perturbed situations?
Putative stromal cell precursors have been identified in a
number of species, including humans, by their ability to form
colonies of cells morphologically resembling fibroblasts
when single-cell suspensions of BM mononuclear cells are
explanted at appropriate densities in liquid culture."-" The
clonogenic progenitor responsible for colony formation under these conditions is referred to as fibroblast colony-forming unit (CFU-F)." After their initial description by Friedenstein,I4 subsequent studies of the C m - F population in
the murine system showed heterogeneous developmental potentials after ectopic transplantation of individual CFU-F
colonies. A minor proportion of CFU-F were found to develop into marrow organs containing the full spectrum of
stromal cell types, including bone cells, whereas the remainder of colonies generated stromal organs comprising only
bone cells or soft connective tissue.I4 Based on these and
other data, OwenT5proposed the stromal stem cell hypothesis
in which, by analogy with the hematopoietic system, there
exists a hierarchy of cellular differentiation supported at its
apex by a small compartment of self-renewing, multipotential stromal stem cells.
Although formal proof of the existence of stromal stem
cells is lacking, this hypothesis has nevertheless provided a
useful conceptual basis from which to investigate the developmental potential of marrow stromal tissue. The work of
Frieden~teinl~
was the first to show the osteogenic potential
in the mouse. Subsequent in vitro studies have shown the
presence of cells with the phenotype of bone cells in both
rat and mouse BM cultures when cultured under conditions
permissive for osteogenic development (L-ascorbate, dexamethasone, and P-glycer~l-phosphate).'~-'~
More recently, a
cloned murine cell line from the marrow stroma was established that displays osteogenic characteristics, providing direct evidence that a single stromal progenitor can differenti-
N VIVO, HEMATOPOIETIC cells develop in intimate
contiguity with a phenotypically and probably functionally diverse population of mesenchymal, connective tissue
type cells that, collectively, represent the stromal tissue of
the bone marrow (BM).'.* A considerable body of evidence
derived from studies in vivo andin vitro shows that the
hematopoietic microenvironment (HM) of the hematopoietic
organs is mediated in large part by this heterogeneous population of stromal cells that endow these organs withthe
unique capacity to support hematopoietic cell development.'
Cell types comprising the stromal tissue of the BM include
vascular endothelial cells, smooth muscle cells, reticular
cells, adipocytes, and osteoblast^.^" This diversity of stromal
elements has complicated attempts to elucidate the role of
each cellular component in the support of hematopoiesis.
Moreover, the origin and developmental relationship between each component of the marrow stroma remains to be
determined. Studies in rodents and in humans clearly show
that marrow stromal tissue is capable of extensive regeneration after a variety of insults ranging from radiation to mechanical di~ruption.~"~
These data raise the question as to
whether each of the various stromal cell types has proliferaFrom the Matthew Roberts Laboratory,Leukaemia Research Unit,
Hanson Centre for Cancer Research, IMVS, Adelaide; and the Department of Orthopaedic Surgery and Trauma, Royal Adelaide Hospital, Adelaide, South Australia.
Submitted April 25, 1994; accepted August 23, 1994.
Supported in part by grants from
the National Health and Medical
Research Council and the Anti-Cancer Foundation of the Universities of South Australia.
Address reprint requests to P.J. Simmons, PhD, Matthew Roberts
Laboratory, Leukaemia Research Unit, Hanson Centre for Cancer
Research, IMVS, PO Box 14, Rundle Mall, Adelaide 5000, SA, Australia.
The publication costsof this article were defrayedin part by page
chargepayment, This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8412-0029$3.00/0
4164
Blood, Vol 84, No 12 (December 15). 1994: pp 4164-4173
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OSTEOGENICPOTENTIAL OF STRO-1' BM CFU-F
ate into osteoblast-like cells." In human studies, the ectopic
transplantation of BM cells in diffusion chambers in nude
mice has failed to provide evidenceof the existence of cells
with osteogenicpotential in normal adult humanmarrow?',zz
However, the formation of bone and cartilage in diffusion
chambers containing marrow from young children has been
re~orted.2~ More
recently, cultured adult human BM cells
placed into ceramic cubes have been shown to form bone
m i ~ e . 2Thus
~ far,
(but not cartilage) when implanted in nude
the use of in vivo assays to measure the osteogenic potential
of human marrow cells is clearly still at a developmental
stage. Other studies have examined the osteogenic potential
of cultured humanBM stromal cells bythe in vitro induction
and detection of characteristic bone cell markers, including
collagen type 1 synthesis, alkaline phosphatase expression,
osteocalcin production, and bone mineral
f o n n a t i ~ n .Col~~'~~
lectively, these studies show the presence of cells with an
osteoblast-like phenotype in human BM cultures. However,
giventhe heterogeneityof thestromalcellpopulationin
whole BM, the question arises as to whether these cells are
the osteogenic
already committed bone cells and whether
phenotype can be induced in a more primitive stromal cell
precursor population.
In the present study, we examine the possibility of establishing a reproducible and defined in vitro culture system to
directly show the osteogenic
potentialof C m - F isolated
from adult human BM. To avoid the culture of whole BM
that could contain cells already committed to the osteogenic
lineage, human BM C m - F were purified using the mouse
monoclonal antibody (MoAb) STRO-1.31 BM mononuclear
cells sorted on the basis of STRO-1 expression are capable
of establishing an adherent stromal layer invitro, consisting
of a number of phenotypically distinct stromal cell types,
including fibroblasts, smooth muscle cells, anda d i p o ~ y t e s . ~ '
We show here that in allBM samples examined, the MoAb
STRO- 1 identifies cells with osteogenic potential as assessed
by the development of cells that exhibit three independent
markers of differentiated bone cells: alkaline phosphatase
expression; 1,25-dihydroxyvitamin D,-dependent induction
of the bone-specific protein, osteocalcin; and production of
a mineralized matrix (hydroxyapatite).
MATERIALS AND METHODS
Subjects. Fresh BM aspirates were obtained (after obtaining informed consent) from the iliac crest and/or the sternum of normal
adultvolunteers,accordingtoproceduresapproved
by theethics
committee at the Royal Adelaide Hospital. BM mononuclear cells
(BMMNCs) were obtained by centrifugation over Ficoll (1.077 g/
mL, Lymphoprep; Nycomed,Oslo, Norway) at 400g for30 minutes.
The BMMNCs were washed twice in Hanks' Buffered Saline Solution (HBSS) supplemented with 5% fetal calf serum (FCS; batch
593;GIBCO BRL, Victoria,Australia)and
10 mmoVLHEPES,
pH7.35(GIBCO-BRL) (HHF),beforeimmunolabelingandflow
cytometry.
Bone chips from the tibia were obtained during routine knee replacementsfromtheDepartmentofOrthopaedicSurgeryand
Trauma at the Royal Adelaide Hospital. Explants of trabecular bone
were cultured as described previously?* Cells growing out of the
explants displaya characteristic osteoblastic phenotypein culture."
Primary human bone cells were used asa positive control for osteocalcin mRNA.
4165
Fluorescence-activated cell sorting (FACS). This procedure has
been reported previously." Briefly, BMMNCs (1 to 3 X lo7 cells)
were pelleted in 5 mL polypropylene tubes (Falcon; Becton Dickinson, LinkonPark,NJ)andresuspendedin200
pL ofsaturating
concentrationsof the mouse IgM MoAb STRO-l for 45 minutes at
4°C or withanisotype-matchedMoAbof
irrelevantspecificity,
1A6.12 (donated by Dr L.K. Ashman, Department of Hematology,
IMVS, Adelaide, Australia). Thecells were were then washed twice
in HHF at 4"C, before the addition of 200 pL of a 1150 dilution of
phycoerythrin(PE)-conjugatedgoatantimouseIgMp-chain-specific (Southern Biotechnology Associates, Birmingham, AL). The
cells were incubated for 45 minutes at 4°C and then washed twice
in HHF beforebeingsortedusing
a FACSkfLus flowcytometer
(Becton Dickinson, Sunnyvale, CA). Cells were maintained at 4°C
throughout the cell sorting procedures.STRO-l+ cells were defined
as those exhibiting a level of fluorescence greater than 99% of that
obtained with the the isotype-matched control antibody, 1A6.12.
In vitro formation of bone mineral. This method is a modification of two different pr~ccedures,"~'~
adapted for human BM cells.
a
STRO-l+ cells(2 X lo4/ T-25cultureflask)wereculturedin
modification of Eagle's medium (a-MEM; Flow Laboratories, Irvine, Scotland) supplemented with
20% FCS, L-glutamine (2 mmoY
L), 0-mercaptoethanol (5 X lo-' mom) andwith or without Lascorbic acid 2-phosphate (100 pmovL, ASC-2P; Novachem, Melbourne,Australia)at 37°Cin 5% COz.After 7 days, the culture
medium was switched to a"EM supplemented with 10% FCS, Lglutamine(2 mmol/L), dexamethasonesodiumphosphate(DEX;
lo-' mol& David Bull Laboratories, Sydney, Australia), ASC-2P
(100 pmoVL), KH2P04(1.8 mmoVL; BDH Chemicals, Poole, UK)
to give a final phosphate concentration of 2.9 mmoVL, and HEPES
(20 mmoVL) for those cultures initiated with ASC-2P. Control cultures without ASC-2P were maintained in
a-MEM supplemented
with 10% FCS and L-glutamine
(2 mmoVL). The media was changed
twice a week for varying periods up to 6 weeks.
Alkaline phosphatase acriviry. Two-week-old BM cultures
grown under osteogenic inductive conditions (ascorbic acid 2-phosphate [ASC-SP], DEX, and inorganic phosphate [PO,i]) and control
cultures, wereincubatedwitheither
a mouseMoAbspecific for
human liver and bone alkaline phosphatase, B4-78 (Developmental
Studies Hybridoma Bank, Universityof Iowa, Iowa City, IA)," for
45 minutes at 4°C. Nonspecific staining was assessed by incubating
cells under identical conditions with an isotype-matched MoAb of
irrelevant specificity, 3D3 (generously provided by Dr L.K.Ashman). Cultures were then washed twice in RPM1 before being fixed
in 2% paraformaldehyde for 20 minutes at 4°C.Afterfixation, a
Vectastain ABC kit for mouse IgG (PK-4010; Vector Laboratories,
Burlingame,CA)and a peroxidasesubstratekit AEC(SK-4200;
Vector Laboratories) were used to localize specifically bound antibodyaccordingtothemanufacturer'srecommendations.Alkaline
phosphatase activity was confirmedby histochemistry performed on
replicate cultures using a Sigma (St Louis, MO) in vitro diagnostic
kit, 85L-2, according to the manufacturer's recommendations. After
immunostaining and histochemical staining, the cells were counter
stained with Mayer's hematoxylin and examined and photographed
using an Olympus IMT-2 inverted light microscope (Olympus Optical, Ltd, Tokyo, Japan). Photographs were taken using Kodak Technical PAN black and white film (Eastman Kodak, Rochester, NY).
Von Kossa staining of bone-derived mineral. Mineralized BM
cultures and controls were gently teased off as single layers using
a plasticpipette.Thecelllayerswerefixedin95%ethanolfor
60 minutes before being infiltrated and embedded in 50% glycol
methacrylate (Tokyo Kasei, Tokyo, Japan) and 50% methyl methacrylate (BDH Chemicals) over 1 week at room temperature. Transverse sections(5 pm) of thecell layers were cut using
a glass knife on
a Jung K motorized sledge microtome (Reichert-Jung, Heidelburg,
Germany).Von Kossa stainingwasperformedaccordingtothe
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41 66
Fig 1. lmmunoperoxidase staining withthe MoAb 84-78 depicting
alkaline phosphatase activity on BM CFU-F cultured for 2 weeks in
the presence of either FCS alone (A) or ASC-2P. DEX, and PO,! (B).
Thecultureswerecounter-stainedwithhaematoxylin.Cultures
treated with FCS alone showed a heterogeneous staining patternof
alkaline phoshatase activity.A proportion of cells (25%)in each colony were found to express alkaline phosphatase (arrow; original
magnification x2001 (A). In the cultures treated with ASC-2P. DEX,
and PO,,, greater than 90% of the cells in each colony were found
to express alkaline phosphatase (original magnification x200) (B).
Alkaline phosphatase activity could notbe detected when the same
cells were incubated in the presence of t h e negative control MoAb,
3D3 (original magnification x2001 (C).
GRONTHOS ET AL
method of Pearse." Sections were washed twice in distilled water
and then stained in 5% aqueous AgN03 (May & Baker Laboratory
Products, West Footscray. Victoria, Australia) for 60 minutes under
UV light. After staining with AgN03. the sections were placed
in
5% sodium thiosulphate (BDH Chemicals) for
1 minute. The sections were counterstained with Mayer's hematoxylin and eosin and
mounted in Uvinert aqueous mountant (BDH Chemicals).Von Kossa
staining was analyzed using an Olympus SZ-PT light microscope.
Photographsweretakenusing
Kodak Technical PAN black and
white film.
Detection qf osteocalcin by radioimmrrnonssay (RIA). This
method is a modification of that described by Lajeunesse et a13' and
was adapted for serum-deprived conditions. Six-week-old mineralized BM culturesandcontrolswere
washed twice in phosphatebuffered saline (PBS) and then switched to serum-deprived medium
as previously described." supplemented with CaCI2-2Hz0 (2 mmoll
L; Sigma). KHIP04 ( I .8 mmolL). menadione sodium bisulfite (IO-'
molL; vitamin K?; Sigma),andcalcitriol (IO" mol& 1,25-dihydroxyvitamin D,: donatedby F. Hoffmann-LaRoche Ltd. Basel,
Switzerland) for 48 hours at 37°C in 5%- CO?. After incubation, the
media was removed and the osteocalcin concentration
in the medium
was measured by a specific RIA (Department of Endocrinology of
theIMVS,Adelaide,SouthAustralia)usinganantibody
raised
against bovine osteocalcin (>90% homologous to human osteocalcin). In preliminary experiments, media supplemented with 1 % FCS
contained approximately 3 ng of osteocalcin permilliliter, in contrast
to serum-deprived media, which contained less
than the detection
limit of the assay of 0.4ng of osteocalcin per milliliter. Background
levels of osteocalcin detected in serum-deprived media alone were
compared with theosteocalcinlevelsdetected
in thecultures (ttest). The results were expressed as the mean values from duplicate
cultures of nanograms of osteocalcin per 10' cells per 48 hours of
induction with I ,25-dihydroxyvitamin D,.
Detection qfosteocalcin mRNA by Northern blot unnlysis. Total
RNA from the same BM cultures used for RIA and from primary
human bone cultures was extracted by the acid guanidinium-phenolchloroform method as described previously.2x Denatured total RNA
(20 pg) was separated by 1 %- agarose gel electrophoresis and transferred to a nylon membrane (Shleicher& Schuell, Dassel, Germany).
After UV cross-linking and prehybridization, the blots were hybridized in 50% formamide to a syntheticoligonucleotideprobefor
(S-CCACTCGTCACAGTCCGGATTGAGhuman
osteocalcin
CTCACACACCTCCCT-3'),complementarytothe
mRNA sequence coding for amino acids 20-32 of mature
osteocalcin"' at 42°C
with (y-"P)-ATP (Bresatec,
for 16 hours. The probe was end-labeled
Adelaide, Australia). Washes were performed in 2 x sodium saline
citrate (SSC) at room temperature, followed by washings at
42°C
for I hour. The membrane was exposed to Kodak X-Omat film at
a Cronexintensifyingscreen (du Pont,
-70°C for72hourswith
Wilington, DE). The integrity of the RNA analyzed was confirmed
by ethidium bromide staining.
Transmission electron microscopy (TEM). Six-week-old mineralized BM cultures were washed in 0.05 m o l L sodium cacodylate
buffer and then fixed in 2.5% glutaraldehyde (EM grade) in 0.05
m o l L sodium cacodylate buffer for 2 hours at room temperature.
After washing with 0.05 m o l L cacodylate buffer, the cultures were
postfixed with 2% osmium tetroxide (VIII;BDH Chemicals) in cacodylate buffer for I hour at roomtemperatureand then washed in
cacodylate buffer. After this procedure. the cultures were dehydrated
with graded alcohol (70%. 9096, and 100% ethanol solutions) for
two IS-minute changes for each alcohol grade. Epoxy resin (TAAB
Laboratories, Berkshire, UK) was then used to infiltrate the cultures
overnight at 37°C. Polymerizationofthe resin wasperformed at
a
60°C for 24 hours under vacuum. Ultrathin sections were cut on
LKB 8800 Ultrotome I1 (Bromma, Sweden) and mounted on copper
11
grids.Sectionswere
then examinedusing a JEOL1200EX
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OSTEOGENIC POTENTIAL OF STRO-1'
BM CFU-F
41 67
Fig2.
The mineral formed by
human BM CFU-F cultured for 4
weeks in the presence of ASC-PP,
DEX, and PO,, was shown to be
Von Kossa positive (original m a g
nification x 1001 (Al. Cultureswere
counter-stainedwith hematoxylin.
A light microscopicview of the adherent layers of replicate cultures
shows the development of mineral
deposits (large arrow) and the appearance of clusters of adipocytes
(small arrow) (original magnification x2001 (B). Deposits of mineral
were neverobserved in the B M
CFU-F cultures grown in the presence of FCS alone(original magnification x2001 (C).
(Tokyo, Japan) transmission electron microscope. Photographs were
taken using ILFORD EM Technical film (ILFORD Pty Ltd. Mobberley, UK). Mineral deposits were analyzed using a Tracor-Northern
Series I1 EDX System(EDX:EnergyDispersive x-ray microanausing a camera
lyzer). Electron diffraction analysis was performed
length of 100 cm on the mineralized bone cultures and on a commerciallyavailablehydroxyapatitestandard(hydroxyapatitetype
1:
Sigma).
RESULTS
Induction of alkaline phosphatase expression on RM
CFU-F. STRO-I + and STRO-I cells derived from
BMMNCs from 3 different individuals wereisolated by
FACS and cultured under standard CFU-F conditions, in the
presence or absence of ASC-2P. All cultures derived from
STRO-I - cells failed to produce any CFU-F after 2 weeks
in culture, in accord with previous observations,>' and were
therefore discarded. In the STRO-I' cultures, the CFU-F
were allowed to develop for 7 days, at which time the cultures were supplemented with a combination of ASC-2P,
DEX. and POJifor those cultures initiated with ASC-2P. The
addition ofDEXat
the start of culture caused a marked
decrease in the proliferation of CFU-F that was evident by
day 7 of culture. However, delayed addition of DEX did not
result in the inhibition of stromal cell proliferation (data not
shown). For this reason, the addition of DEX was therefore
performed 7 days after culture initiation.
BM CFU-F colonies were analyzed for their expression
of alkaline phosphatase, a well-documented marker of bone
~
cell differentiation, by immunoperoxidase staining using the
MoAb B4-78, which identifies the bone- and liver-specific
form of the enzyme" (Fig I). Alkaline phosphatase activity
was confirmed by histochemistry performed on replicate cultures (data not shown). After 2 weeks of culture, all of the
CFU-F colonies grown in the presence of ASC-2P, DEX,
and PO,, were found to express alkaline phosphatase (Fig
1 B). Moreover, withinindividual
colonies. consistently
greater than 90% of the cells exhibited alkaline phosphatase.
In contrast, the CFU-F cultured in the presence of FCS alone
showed a heterogeneous staining pattern, with typically
525% of the cells in each colony showing positivity for
alkaline phosphatase (FigIA).To
examine which factor
was responsible for inducing alkaline phosphatase activity,
STRO- I + cells were grownin the presence of ASC-2P. DEX,
and PO.,, and in multiple combinations of the three. DEX
wasfoundtobe
responsible for theincreased expression
of alkaline phosphatase on CFU-F colonies in all groups
examined when compared with CFU-F grown in FCS alone
(data not shown).
STRO-I' BMMNCs can he induced to ,f.m mineral.
The adherent layers of BM cultures after 4 weeks in osteogenic growth conditions (ASC-2P. DEX, and PO,) displayed
large areas of mineralizedmaterialthat stained positively
with the Von Kossa technique (Fig 2A). Adipocyteswere
also observed in the same cultures throughout the adherent
layers, as seen by light microscopy under phase contrast(Fig
2B). Mineral deposits were not found in the control cultures
with FCS alone (Fig 2C).
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GRONTHOS ET AL
41 68
I
-.- - ".".
"
,
L
"
"
-
"
"
~
-
__
-
.-"
.-__
A view of the whole culture flasks under dark field illumination shows the extent of mineralization after 6 weeks of
induction with ASC-2P. DEX, andPOli (Fig 3A). Transverse
sections of the adherent layers from 6-week-old cultures
showed an extensive multilayered growth pattern that was
subsequently confirmed by electron microscopic examination (see Fig 5 below). A similar pattern of growth was
seen in cultures grown in FCS alone (Fig 4B). However,
mineralization was only evident in cultures supplemented
with ASC-2P, DEX, and POli, as shown by positive staining
with the Von Kossa reaction (Fig 4A). Human foreskin fibroblasts cultured under identical conditions for 6 weeks
failed to produce a mineralized matrix (data not shown).
Ultrastructural examination of the cultures by TEM
showed that the mineral was associated with collagen fibrils
in the extracellular matrix of the adherent layers (Fig 5A
and B). EDX was used to evaluate the nature of the mineral
in these cultures. The calcium to phosphorus (Ca/P) ratio in
the mineralized cultures was 1.76 5 0.10 (n = 3; Fig 5C),
which was consistent with the Ca/P ratio of hydroxyapatite
in vivo (1.67). X-ray diffraction studies also showed that the
crystal structure of the mineral deposits in the induced cultures (ASC-2P, DEX, and PO4,)was identical to the crystal
*
~
Fig 3. Representative photographs (dark field) of
cultureflasks depictingthe confluent adherent layers
of B M CFU-F cultured for 6 weeks in the presence of
ASC-2P. DEX, or PO,, (A) or cultured in the presence
of FCS alone (B).Descrete areas of mineral deposits
(arrow) were observed throughout the adherent layers of the cultures treated with ASCQP, DEX, and
POli (Al. No mineral formation was observedinthose
cultures treated with FCS alone (B).
diffraction patterns obtained from the hydroxyapatite standard (Fig 5D).
To determine the incidence of CFU-F with osteogenic
potential, we isolated 21 CFU-F clones by limiting dilution
in standard CFU-F growth conditions. Day-7 colonies were
subsequently cultured for 4 weeks in the presence of DEX,
ASC-2P, and PO,. After this procedure, the colonies were
examined for the formation of a mineralized matrix. All of
the 21 CFU-F clones cultured in the presence of DEX, ASC2P, and PO4i were shown tobe positive for Von Kossa
staining. In addition, adipocytic formation was observed in
only a proportion (48%) of the colonies examined (data not
shown).
Osteocalcin production by BM CFU-F. Six-week-old
mineralized BM cultures and controls were washed in PBS
and then placed under serum-deprived conditions. The biosynthesis of osteocalcin was stimulated for 48 hours by the
addition of 1,25-dihydroxyvitamin D3 ( I ,25-Vit D3). Samples were then taken from the medium of all the cultures
and analyzed for the presence of osteocalcin by RIA. Varying levels of osteocalcin (2.6 to 19.8 ng/lOh ceIls/48 hours
I .25-Vit D3 ) were detected in the mineralized BM cultures,
butnot in control cultures grown in the presence ofFCS
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POTENTIAL
OSTEOGENIC
41
OF STRO-1' BM CFU-F
69
A
Fig 4. Von Kossa staining of transverse sections
of the adherent layers from cultured BM CFU-F
grown for 6 weeks in the presence of ASC-2P. DEX,
and PO,i (original magnification x2001 (A) or in the
presence of FCS alone (original magnification x2001
(B). The sections were counter-stained with hematoxylin and eosin. Areas of Von Kossa-positive mineral deposits (arrow) were observed throughout the
adherent layers of the culturestreated with ACS4P.
DEX, and PO,, (A). No Von Kossa-positive mineral
could be detected in the adherent layers of the control cultures treated with FCS alone (B).
alone (Table l ) . In accord with this data, Northern blot analysis of RNA extracted from the mineralized BM stromal cell
cultures and from primary human bone cultures showed the
presence of osteocalcin mRNA after stimulation of the cells
for 48 hours of incubation with1,25-Vit D, (Fig 6). No
osteocalcin mRNA was detected in the control BM cultures
(FCS alone) stimulated with 1.25-Vit D3.
DISCUSSION
Our data show that osteoprogenitors in human adult BM
express the STRO-I antigen. Although there is no single,
definitive marker of cells of the osteogenic lineage, the cells
produced under the culture conditions described in this report
exhibit three widely accepted characteristics of bone cells,
ie, high expression of alkaline phosphatase?" production of
a mineralized (hydroxyapatite) matrix, and 1,25-VitD3-dependent synthesis of the bone cell specific protein, osteocalcin. The generation of osteoblast-like cells with these phenotypic and functional characteristics was dependent on culture
of the sorted STRO-I + precursor cells in the presence of L-
ascorbate, the glucocorticoid DEX, and a source of inorganic
phosphate, in accord with other studies.'".'x
Previous studies have determined that ascorbate is essential for the survival of human osteoblasts in vitro.?' L-ascorbate helps facilitate bone cellproliferation and differentiation
by increasing the total protein, collagen synthesis, and alkaline phosphatase
In present
the
study, L-ascorbate, which has a half-life of 7 hours in culture, was replaced
by the long-acting derivative of L-ascorbate (ASC-2P),
which has a half-life of 7 days in culture.*l Interestingly,
alkaline phosphatase expression was not affected when human BM CFU-F weregrown in the presence of ASC-2P
when compared with the control cultures without ASC-2P.
The role of glucocorticoids in regulating osteogenic differentiation in vitro has not been precisely defined.A number of
studies have shown that although DEX can induce terminal
differentiation of osteogenic cells in cultures, the presence
of this steroid isnotan absolute requirement for in vitro
o s t e ~ g e n e s i s . " . ~It~ should
.~~
benotedthatmanyofthese
reports are based on the use of rodent systems of the bone
formation assay, the relevance of which as an in vitro model
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4170
GRONTHOS ET AL
R."
C
of bone cell development has recently been questioned.33In
human studies, DEX has been shown to be required for the
differentiation of osteoblast-like cells present in BM stromal
cultures.3" In vitro, DEX was shown to inhibit the 1,25-Vit
D3-induced expression of osteocalcin in human BM cells3"
and human bone cells." In contrast, DEX has been found to
Fig 5. TEM examination of theadherent layers of
BM CFU-F cultured for 6 weeks in the presence of
ASC-2P. DEX, and PO,, depicts the formation
of mineral-like material (hydroxyapatitecrystals; arrow) in
the extracellular spaces of the adherent layers (original magnificationx3.000) (A). Examination of theextracellular matrix showed the presence of deposits
of hydroxyapatite-like crystals (large arrow) in association with a network of collagen fibrils(small
~25,000) (B). EDX
arrow)(originalmagnification
analysis of similar mineral deposits, obtained from
three different BM CFU-F cultures, showed major
peaks for calcium (Ca) and phosphorous (P) (C), with
a mean Ca/P ratio of 1.71 ? 0.1 (n = 31. The x-ray
diffraction pattern of the mineral
deposits, in the
same cultures (Dl, was found t o be identicalto that
of the hydroxyapatitestandard.
increase the expression of mRNA for many genes, including
those encoding for alkaline phosphatase and various protooncogenes, such as c-fos,"' that are associated with osteogenic differentiation.4050In the present study. the addition
of DEX to the culture medium increased the expression of
alkaline phosphatase on CFU-F colonies.
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OSTEOGENICPOTENTIALOFSTRO-1'
4171
BM CFU-F
Table 1. Measurement of Osteocalcin Release
in Human BM CFU-F Cultures
Culture Conditions
BM Sample
Control INS)
1
2
0.3 2 0.3
0.2 2 0.2
0.8 -r 0.6
3
ASC-2P
+ DEX + PO,;'
19.8 2 6.9
9.7 2 1.8
2.6 2 1.1
Values are the mean ? SE nanograms of osteocalcin per 10' cells
per 48 hours of 1.25-Vit D3 treatment. Adult human CFU-F cultured
for 6 weeks in the presence of either ASC-PP, DEX, and POIi or in the
presence of FCS alone (control) were treated with 1.25-Vit D3 for 48
hours in serum-deprived conditions. Samples of the supernatants
from replicate cultures were analyzed for the presence of osteocalcin
by RIA. Osteocalcin levels in each sample were compared with the
background levels of osteocalcin detected in the serum-deprived medium alone. Significant levels of osteocalcin were only detected in
those cultures treated with ASC-2P, DEX, and PO,,.
Abbreviation: NS, not significant.
P < .05 (t-test).
Synthesis by bone cells in vitro of a mineralized matrix
has previously been shown to be dependent on supplementation of their growth mediumwith a source of inorganic
phosphate." In this study, inorganic phosphate was used in
place of &glycerophosphate, whichis a nonphysiological
organic phosphate substrate of alkaline phosphatase.'' The
presence of 0-glycerophoshate in animal studies has been
shown to cause ectopic mineralization in cultures of fetal rat
parietal cells and skinfibroblasts, which does not occur using
inorganic phosphate.'."'' In parallel studies, human foreskin
fibroblasts cultured in the presence of ASC-2P, DEX, and
PO4, for a period of 6 weeks were found to be Von Kossa
negative.
Purified STRO-I + human BM cells were cultured under
standard CFU-F growth conditions in the presence of ASC2P. Day-7 CFU-F cultures were then switched to osteogenic
inductive conditions (ASC-2P, PO4,, and DEX). After 2
weeks, greater than 90% of the cells in all of the CFU-F
colonies were shown to express alkaline phoshatase. Previous studies have shown that alkaline phosphatase is involved
with the induction of hydroxyapatite deposition in collagen
matrices in vivo." The results of the present study showed
that the induction of alkaline phosphatase expression preceded the appearence ofVon Kossa-positive mineral by 7
to 14 days in the induced cultures. TEM analysis of the
adherent layers showed the presence of deposits of hydroxyapatite-like crystals in association with the collagen fibrils
in thematrix.EDX analysis showed that the mineral was
composed of calcium (Ca) and phosphorus (P), consistent
with the C d P ratio of hydroxyapatite crystals in vivo." In
addition, the crystal structure of the mineral was found to
be identical to the crystal structure of the hydroxyapatite
standard, as determined by x-ray diffraction analysis. The
presence of mineral deposits, which stained positive for the
Von Kossa reaction, was shown in all CFU-F colonies isolated by limiting dilution. This finding is in contrast to the
findings in studies of rodent BM that reported that only a
proportion of CFU-F were capable of developing an osteogenic phenotype.4"'x This discrepancy may be attributed to
the presence of accessory cells in the relatively crude BM
preparations used in these studies,"x.sx whichmay affect the
expression of the osteogenic phenotype of CFU-F in vitro.
In addition, the absence of DEXJXand ascorbate5xin the
culture medium may lead to an underestimation of the incidence of osteogenic precursors in rodent BM.
Osteocalcin is an osteoblast-specific proteins0 andwas
therefore used in this study as a marker of osteogenic differentiation. Cultured primaryhumanbone
cells havebeen
shown to synthesize osteocalcin in the presence of 1,25-Vit
D 32.fln.hl In the present study, comparable levels of osteocalcin were found in the culture medium of human BM cultures
induced for 6 weeks under osteogenic conditions (ASC-2P,
DEX, and PO,,) and stimulated with 1 ,2S-VitD1for 48 hours.
The 1,25-VitD3-stimulated induction of osteocalcin mRNA
was confirmed by Northern bot analysis.
This work provides direct evidence that purified human
BM CFU-F underdefined in vitro culture conditions are
capable of osteogenic differentiation. These findings also
indicate thatthe osteogenic precursors are found in the
STRO-1' population of human BM. However,thisstudy
does not exclude the possibility of the presence of primitive
osteogenic precursors in the STRO-I- fraction of the BM.
A
- 28s
- 18s
4- 0.6 kb
B
1
2
3
4
- 28s
- 18s
kL:.
.*L.IP
.L.
Fig 6. A representative autoradiograph of a formaldehyde-agarose gel electrophoresis of RNA from human BMCFU-F cultured for
6 weeks in the presence of either ASC-LP, DEX, and POu (lane 1) or
FCS alone (lane 2) and stimulated with V i D3for 48 hours; primary
human bone cells cultured for 6 weeks in the presence of ASC-2P.
with VitD3induction for48 hours (lane 3) or without VitD1 induction
(lane 4). Descrete bands, characteristic of osteocalcin mRNA were
observed in lanes 1 and 3, after hybridization with a specific '*Plabeled oligo-nucleotide probe(A). The integrity of the RNA analyzed
was confirmed by ethidium bromide staining(B).
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4172
GRONTHOS ET AL
In our study, CFU-F could not be grown from the STRO1- fraction of BM. Previous studies have shown that the
STRO-l' population contains cells with the potential to develop into a number of distinct stromal cell types, including
fibroblasts, smooth muscle cells, and adip~cytes.~'
The present work therefore extends the range of cell types into which
STRO- 1 can develop. At a clonal level, approximately 50%
of the CFU-F examined showed the capacity to differentiate
along both the osteogenic and adipocytic cell lineages. It
will be of interest to determine whether, in adult BM, all
four stromal elements arise from a common multipotential
stromal precursor population as previously reported by othe r ~ .The
~ ~answer
* ~ ~to this question must await the development of techniques to genetically mark putative stromal stem
cells and to follow their progeny.
In conclusion, this study shows that osteoprogenitors in
human BM are restricted to a subpopulation of cells that
express the STRO-1 antigen. These data provide an important basis from which to attempt further enrichment and
characterization of osteoprogenitor cells, in particular to investigate more stringently their requirements for growth and
differentiation and to identify culture conditions that lead to
the expansion of their number in vitro, with a view toward
potential clinical application of these cells.
+
ACKNOWLEDGMENT
This study was approved by the Human Ethics Committee of the
Royal Adelaide Hospital, South Australia. Our thanks and appreciationto R. Moore for the preparation of culture samples for Von
Kossa staining, to B. Dixon and K. Smith for EDX and x-ray diffraction analysis of mineralized matrix, and to C. Hann and his staff for
the detection of osteocalcin by RIA. We thank Drs C.A. Juttner and
L.B. To for provision of the BM aspirates used throughout the course
of this work.
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