1 Department of Anatomy & Cell Biology, University of Toronto, Toronto,
Ontario, M5S 1A8, Canada
2 Department of Molecular & Medical Genetics, University of Toronto,
Toronto, Ontario, M5S 1A8, Canada
* Present address: INSERM Unit 403, Hôpital E. Herriot, Lyon, 69437,
France
Author for correspondence (e-mail:
jane.aubin{at}utoronto.ca)
Accepted 21 January 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Osteoprogenitors, Osteoblasts, Differentiation, PCR, Replica plating
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Earlier, while investigating changes in levels of expression of the
osteoblast-related macromolecules in RC colonies classified morphologically as
comprising early or more mature osteoblasts versus ones designated
`fibroblastic', we found evidence to suggest that developmental stages more
primitive than those recognizable by the presence of cuboidal cells and prior
to upregulation of any of the known osteoblast markers were accessible for
analysis in this model (Liu et al.,
1994). Our solution to the problem of identifying the low
frequency primitive osteoprogenitors was to use replica plating on dishes
plated at low density and sampled early in the development of colonies.
Replica plating has been found to allow screening of large numbers of
individual mammalian cell clones for the phenotype of interest while still
maintaining a master copy of the colonies; use of polyester cloth, in
particular, provides high fidelity copies for a variety of cell types
(Esko, 1989
;
Raetz et al., 1982
). Since
cell number was limited in each colony sampled, we applied global
amplification or poly(A)-PCR (Aubin et al.,
2002
; Brady et al.,
1995
; Brady and Iscove,
1993
) to determine simultaneous expression profiles of the mRNAs
of interest. These included both the osteoblast-related mRNAs and mRNAs for
potential regulatory molecules. We now report that this approach not only
allows molecular fingerprinting of definitive primitive osteoprogenitors but
also reveals novel transient developmental stages of such cells as they
progress through their differentiation sequence.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Replica plating
Replica plating was done essentially as described
(Esko, 1989;
Raetz et al., 1982
) but with
minor modifications. Briefly, a disc of 1 µm pore size polyester cloth
(HD7-1; B&SH Thompson, Scarborough, ON) was floated above the cells and
weighted down by a monolayer of 4 mm glass beads; replica cloths were placed
on cells at day 1 or day 4 and were removed on day 5 or day 11, respectively,
and transferred into a new dish. The master dish and polyester disc were each
rinsed with PBS and fed with supplemented medium as above. The replica disc
was incubated at 37°C; whereas, the master dish was incubated at either
25°C or 30°C to stall the proliferative and differentiation activities
of colonies. The medium was changed every 2-3 days.
In preliminary experiments to determine the transfer efficiency and fidelity of transfer for colonies of fetal RC cells, replica cloths were fixed on day 25 with 10% neutral buffered formalin and stained with the von Kossa technique. Also on day 25, master dishes were transferred from the lower temperature incubators to a 37°C incubator. After 14 days at 37°C, these master dishes were fixed and stained for the presence of bone nodules as described above. For statistical analysis (Fig. 1), the data were expressed as means and standard deviations.
|
Osteoblastic colony isolation and cDNA preparation
After two weeks at 37°C, the replica cloth was fixed in 10% neutral
buffered formalin and stained with the von Kossa technique to identify bone
nodules. The master dish was transferred to 37°C for 5-9 hours. After this
time, the replica disc was matched up with the master dish to localize
primitive osteoblast colonies, which were then marked. In addition, single
isolated osteoblast colonies containing plump cuboidal cells with
unmineralized osteoid or mature osteoblast colonies containing mineralizing
osteoid were marked and collected from dishes that were not cultured with
polyester discs. Dishes were rinsed with PBS and a cloning ring was placed
around the marked colonies. The cells from each colony were released with
0.01% trypsin (when matrix lacked mineral) or a 1:1 mixture of 0.01% trypsin
and collagenase (when osteoid was mineralizing), and the enzyme(s) neutralized
after cell release by adding -MEM containing 15% FBS. Total RNA was
extracted using a mini-guanidine thiocyanate method as described previously
and cDNA was synthesized by oligo(dT) priming, poly(A)-tailed, and amplified
by PCR with oligo(dT) primer (Brady and
Iscove, 1993
; Liu et al.,
1994
). The 108 colonies reported here were collected from 12
independent cell isolations and replica plating experiments.
Southern blots and hybridization
Amplified cDNA (5 µl) was run on 1.5% agarose gels, transferred onto 0.2
µm pore size nylon membrane (ICN, Costa Mesa, CA), and immobilized by
baking at 80°C for 2 hours. Prehybridization and hybridization were
performed as described (Liu et al.,
1994). After hybridization, the blots were washed at 65°C for
1 hour each in 2x SSC/0.1% SDS and in 0.5x SSC/0.1% SDS. The blots
were then exposed to phosphorimager screens (Molecular Dynamics, Sunnyvale,
CA), and digitized images obtained and quantified with the IPLab Gel program
(Signal Analytics Corp., Vienna, VA). After quantifying the data, the signal
intensity for each probe was standardized against total cDNA (see below). For
preparation of comparative histograms of relative expression profiles, we next
determined maximal expression value for each message; the largest value was
divided by 5 to obtain five ranges of values or categories. Samples were then
given a rank of 1, 2, 3, 4, or 5 depending on where their values fell within
each range or were given a rank of 0 if the intensity of signal was not
detectable. For statistical analysis of relative expression levels
(Fig. 4), the actual
standardized expression levels for each probe in each sample were used to
calculate means and standard deviations within categories of populations.
Levels of statistical significance were calculated by the Welch
t-test.
|
Labeled probes were used at an activity of 106 cpm/ml. cDNA
probes were labeled with [32P]dCTP using an oligolabeling kit
(Pharmacia, Uppsala, Sweden). Total cDNA probe was prepared as described
(Sambrook et al., 1989) from
poly(A)+ mRNA isolated (Auffray and
Rougeon, 1980
) from mass populations of fetal RC cells grown in
the presence of dexamethasone in which bone nodules were beginning to
mineralize. For optimal detection of poly(A)-PCR-amplified cDNA, the probes
required sequences at or close to the extreme 3' ends of the native
transcripts. Labeled probes for the cDNAs listed above were generated as
described previously (Liu et al.,
1994
). The rat cDNA probes used were for COLL-I
[(Genovese et al., 1984
) a
gift of D. Rowe, Farmington, CT], bone/liver/kidney ALP
[(Noda and Rodan, 1987
) gift
of G. A. Rodan, West Point, PA], OPN (a gift of R. Mukherjee, Montreal, PQ),
BSP and OCN [prepared by generating specific probes by PCR, screening an
osteoblast library and then confirming isolated cDNAs by sequencing (see also
Liu et al., 1994
)]. Rat
PDGF-R
[(Lee et al.,
1990
) a gift of R. R. Reed, Baltimore, MD] was a 400 bp cDNA
PstI fragment obtained by digesting full length cDNA with
HindIII to remove 6 kb of the 5' region and religating the
sticky ends. Mouse FGF-R1 [(Mansukhani et
al., 1990
) a gift of C. Basilico, New York, NY] was a 400 bp cDNA
HincII-PstI fragment. Rat PTHrP
[(Yasuda et al., 1989
) a gift
of D. Goltzman, Montreal, PQ] was a 700 bp cDNA SmaI fragment, and
rat PTH/PTHrP receptor [PTH1R (Abou-Samra
et al., 1992
) a gift of G. V. Segre, Boston, MA] was a 800 bp cDNA
HindIII-XbaI fragment.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By using optimal conditions and selecting for fingerprinting only colonies in which cells had a robust appearance, and ones discrete/well separated from and so not contaminated by cells from other colony types, we were able to isolate over 80 definitive osteoprogenitor colonies. Visual inspection indicated that, on average, the osteoprogenitors on the stalled master dishes were relatively quiescent and had undergone only 0-1 additional population doublings; a few which had a senescent or dying appearance were discarded from the analysis. All osteoprogenitor colonies selected for analysis comprised cells with a pleiomorphic morphology indistinguishable from non-bone colonies on the same dishes, indicating that none had yet acquired the cuboidal shape characteristic of differentiated osteoblasts. That cells were well recovered prior to mRNA isolation is indicated by the fact that they were relatively mitotically active after 5 hours and 9 hours from dishes originally stalled at 30°C and 25°C, respectively (not shown). Estimated colony size judged by microscopic viewing on a grid ranged from 500 to 3000 cells per colony, colony size did not directly correlate with any specific phenotypes observed by fingerprinting.
Global amplification poly(A)-PCR
Previously, we used poly(A)-PCR to establish molecular profiles for
osteoblastic colonies and single cells that had differentiated to different
extents as recognized morphologically (i.e. early, intermediate, and mature
osteoblast) (Liu et al., 1997;
Liu et al., 1994
) and we
included a few such advanced colonies here. These (labeled OB and Mature OB;
24 colonies total) were subjected to the same poly(A)-PCR manipulations and
Southern blotting as colonies identified by replica plating (labeled
OP/Pre-OB; 84 colonies total) (Fig.
2). A total cDNA probe was used as a control to assess first
strand synthesis and amplification amongst the 108 colonies
(Fig. 2); this proved
relatively constant and its signal strength was used to standardize relative
abundance of all other messages for comparison amongst colonies. Of all
messages probed, OPN was most uniformly and abundantly present in virtually
all colonies analyzed. PTHrP, PTH1R, FGF-R1 and PDGF-R
mRNAs were also
detectable in virtually all colony types but their abundance was generally
lower and heterogeneous from one colony to another
(Fig. 2). Consistent with our
previous data, most of the mature osteoblast colonies expressed all markers,
but to various extents; a few osteoprogenitor colonies also expressed all
markers to some degree, but most a much more restricted repertoire and to
limited levels (Fig. 2).
|
Immature progenitor colonies or more mature osteoblastic colonies analyzed
in the Southern blot shown in Fig.
2 are in random order, reflective only of the order of processing.
To determine whether immature progenitors could be placed in a rank order of
more primitive or less primitive cells, we developed a paradigm for their
comparison. Relative expression levels of all messages in all colonies sampled
were determined by normalization of their signal strengths against that for
total cDNA; colonies in which total cDNA was not well detected were eliminated
from our subsequent analyses. As described in Materials and Methods, the
remaining colony samples were then assigned categories of relative expression
from low (0-1) to high (5). This classification smoothed out the small but not
the large variations in expression levels such that overall patterns of gene
expression became more obvious. Based on numerous previous studies and
techniques, it is known that ALP expression rises as osteoblasts mature and
then declines as osteoid becomes heavily mineralized, while OCN expression is
acquired latest and essentially is diagnostic of post-proliferative
osteoblasts (Malaval et al.,
1999). Therefore, we rank-ordered colonies manually based on ALP
and OCN expression (Fig. 3),
which imposed an order for all other genes probed: the mature end (extreme
right; COLL-I+/ALP+/OCN+), followed in order
to the left by colonies expressing fewer and fewer markers in the order
COLL-I+/ALP+ colonies, then
COLL-I+/ALP- colonies, then
COLL-I-/ALP- (i.e. colonies expressing no osteoblastic
markers are on the extreme left). Among the COLL-I+/ALP-
colonies, surprisingly a few were BSP+. Since a few
COLL-I-/ALP- colonies were also BSP+, we
grouped all these BSP+ colonies together at the
COLL-I+/ALP-COLL-I-/ALP- border.
By using categories established in Fig.
3, we also calculated the means and standard deviations of
corrected (standardized against total cDNA) expression levels for each probe
within categories of populations and looked for statistically significant
changes during the osteogenic differentiation sequence
(Fig. 4).
|
These analyses allowed us to discern whether other unexpected patterns
existed, for example, in the hormone and growth factor receptors (Figs
3,
4). First, there is a
recognizable cohort of cells captured by the replica and considered very
primitive (left; no osteoblast-specific mRNAs expressed); these are not
quiescent since they express low-intermediate levels of mRNAs for OPN, PTHrP,
PTH1R, FGF-R1 and PDGF-R. Second, a group of relatively primitive
(expressing no other osteoblast-associated mRNAs) progenitors transiently
expressing BSP emerges as a distinct developmental stage. Third, during
osteoprogenitor differentiation, BSP mRNA expression undergoes a second
significant upregulation prior to that of OCN, and relatively early after
upregulation of ALP (Fig. 3).
Fourth, FGF-R1 and PTH1R mRNA is significantly upregulated prior to that of
PDGF-R
and earlier than upregulation of ALP; a second significant
upregulation occurs late in the differentiation sequence as cells acquire
other features of differentiated osteoblasts including OCN. Fifth,
PDGF-R
mRNA is maintained at a relatively constant level until it is
upregulated similarly to FGF-R1 and osteoblast-associated markers late in the
differentiation sequence. Sixth, amongst the more mature cells in the lineage
(expressing all osteoblast-associated markers) PTHrP, PTH1R, FGF-R1 and
PDGF-R
mRNAs tended also to decrease in relative concert with the
osteoblast-specific markers (Fig.
3). Seventh, on average, BSP expression remained higher than did
other osteoblast markers in the most mature colonies in the analysis.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The replica technique we applied to the RC population worked with the
fidelity (95%) required for the studies undertaken (i.e. to identify
retrospectively osteoprogenitors early in their developmental lifetime). The
replicas were made in low density cultures from discrete colonies well
separated from other contaminating cell and colony types and early in their
developmental sequence while progenitor cells were still proliferative. The
progenitor cells stalled at lower than physiological temperature on the master
dishes were essentially quiescent or very slowly proliferating, but were able
to resume their proliferation and differentiation capabilities ultimately to
form mineralized bone nodules when transferred back to 37°C. To date, we
have used this replica approach to identify and isolate over 80
osteoprogenitor and preosteoblast colonies, all of which comprised cells with
pleiomorphic morphology and none of which displayed the cuboidal morphology of
differentiated osteoblasts depositing and mineralizing osteoid. Our reason for
selection of large numbers of colonies was to confirm the validity of the
approach, that is, each phenotype was expected to be sampled more than once,
and to acquire information on the differentiation process itself (i.e. whether
cells traversed a continuum of changing expression profiles or traversed the
differentiation sequence in quantal leaps). Our data suggest that both
patterns are characteristic of and define the osteoblast developmental process
(see below).
Previously, we showed that the technique of poly(A)-PCR was a powerful and
discriminating tool to establish molecular profiles for osteoblastic colonies
and single cells that had differentiated to different extents as recognized
morphologically (i.e. early, intermediate and mature osteoblast)
(Liu et al., 1994). When mRNA
expression in the replica colonies analyzed was quantified by this technique
and co-expression profiles of a variety of messages for bone-related
macromolecules and growth factor receptors determined, we found marked
differences amongst colonies. Elsewhere we have discussed heterogeneity of
marker expression amongst single cells at the same developmental stages in
terms of a stochastic process contributing to osteoblast plasticity or
heterogeneity (Liu et al.,
1997
; Liu et al.,
1994
). In the present analysis to seek transition points and
landmarks representative of cells as much more or less primitive, we have
analyzed expression across larger developmental boundaries and averaged
amongst small cohorts of sibling cells (colonies) in which single cell
stochastic variations would be averaged and thus minimized as a contribution
to the profiles achieved. A few colonies displayed features consistent with
their having already reached a preosteoblast or early osteoblast phenotype,
i.e., simultaneously expressing all osteoblast-associated markers analyzed
(PTH1R, COLL-I, ALP, OPN, BSP, and OCN), as did the few morphologically
defined later stage osteoblast colonies used here for comparison and
validation. In marked contrast, other replica colonies expressed none of these
mRNAs to a detectable levels, while others expressed different combinations of
these messages. Expression levels for all markers analyzed with the exception
of OPN, which was relatively uniformly and highly expressed in virtually all
colonies (see below), covered a range of expression levels from undetectable
(off) to detectable (on) but very low to intermediate to very high. This
suggests that to some extent, and at least as represented by those osteoblast
markers analyzed, there is a quantal change (off-on), but that once a marker
is on/acquired its expression as populations of osteoprogenitor cells
differentiate represents a continuum from detectable expression through
gradually higher expression until mature osteoblasts express most markers
analyzed at very high levels.
The rank order profile (Fig.
3) was generated and based on temporal expression of ALP
(expression increases with osteoblast maturation and then decreases with
osteoid mineralization) and OCN (expressed by post-proliferative osteoblasts)
as observed in several previous studies of the bone nodule model (for reviews,
see Rodan and Noda, 1991;
Aubin and Liu, 1996
;
Stein et al., 1996
) and
detailed by Malaval et al. (Malaval et
al., 1999
), where the kinetics of osteoprogenitor and osteoblast
differentiation in vitro was correlated with the expression of several bone
matrix proteins. Based on ALP, OCN and other markers, previously the
proliferation-differentiation sequence for osteoprogenitors has been
categorized mainly into three stages comprising proliferation, matrix
production and matrix maturation and mineralization
(Stein et al., 1996
). In
keeping with these previous analyses, we found a sequential upregulation of
expression of bone-related macromolecules beginning with COLL-I, followed by
ALP and then BSP, and finally by OCN (for reviews, see
Aubin and Liu, 1996
;
Rodan and Noda, 1991
;
Stein et al., 1996
). That
virtually all early osteoprogenitor and preosteoblast colonies expressed
moderate to high levels of OPN is not surprising since, as outlined above,
these colonies had high numbers of cycling cells; OPN is known to be high in
proliferating cells, and it was originally cloned as a cell-cycle-related
molecule induced in cells in vitro by tumor promoters and growth factors
(Nomura et al., 1988
;
Smith and Denhardt, 1987
).
Since we performed the low density and replica plating technique in the
presence of dexamethasone, we expected and did capture both the more primitive
and more mature osteoprogenitors defined earlier by flow cytometry on the
basis of absence (primitive, dex-requiring) or presence (more mature,
dex-independent) of ALP expression
(Turksen and Aubin, 1991
).
However, novel information was also obtained. The results support our
observations made earlier at the single cell level in which we detected the
presence of mRNAs for ALP, COLL-I, OPN, BSP, and/or OCN in a few cells in
fibroblastic colonies and confirm the hypothesis for which we had no
definitive proof then that these were probably representative of committed
osteoblast lineage cells that had not yet acquired the morphological
characteristics of the lineage, rather than cells with leaky or promiscuous
expression of mRNAs (Liu et al.,
1994
). It is also notable that all these markers can be and are
expressed in cells that have not yet assumed the cuboidal shape of
preosteoblasts and osteoblasts; while these studies have not addressed the
presence of deposited protein, they do suggest the possibility that none of
these, including the RGD-containing BSP and OPN, might be directly responsible
for the cuboidal shape determination characteristic of mature osteoblastic
cells, and vice versa that overt cuboidal shape is not itself required for
detectable transcription levels of these particular genes.
Strikingly, our data also point towards a much more complex series of
transitions during this developmental sequence than the three stages commonly
described and suggest a minimum of seven transitions based on the markers
assessed here. For example, this analysis of primitive progenitor cells
revealed a small group of colonies expressing low but detectable levels of
BSP; a few of these colonies also expressed COLL-I, but no other
osteoblast-associated molecules. We (Liu
et al., 1994) and others
(Bianco et al., 1991
;
Bianco et al., 1993
;
Chen et al., 1991
) have shown
that BSP is upregulated as osteoblasts mature in vitro and in vivo at sites of
de novo bone formation. While it is generally considered a relatively late
stage marker, expressed concomitantly with OCN and just prior to
mineralization, where it has been proposed to seed hydroxyapatite crystal
formation (Hunter and Goldberg,
1993
), we have established that it is upregulated prior to OCN and
well before detectable mineralization can be observed
(Liu et al., 1994
). We have
now detected transient BSP expression even earlier, prior to the onset of
COLL-I and ALP expression and osteogenesis. We have also previously shown the
presence of primitive
BrdU+(cycling)/ALP-/BSP+ cells by double
label immunocytochemistry in clonogenic bone nodules in vitro
(Malaval et al., 1999
)
indicating that the mRNA results reported here are meaningful also at protein
translational level. We have further found evidence for a similar primitive
transiently BSP-expressing osteoprogenitor in the sutures of developing rat
calvaria (Candeliere et al.,
2001
). It is interesting to speculate that this early and
apparently transient expression of BSP may reflect its role as a cell adhesion
molecule through its RGD recognition site for integrins, notably the
vß3 vitronectin receptor
(Oldberg et al., 1988
); BSP
has also been reported to mediate osteoblast cell attachment in vitro
(Mintz et al., 1993
). This
role for BSP may be functionally separate from its role during its second
round of upregulation during the later stages of the differentiation sequence
when osteoblasts are actively synthesizing other matrix molecules and
depositing osteoid. Late during this second developmental window, i.e., when
matrix is mineralizing and cells achieve osteocyte and/or lining cell status,
it is also interesting that BSP expression remains on average higher than that
of other bone matrix molecules and ALP.
While levels of PTHrP remained relatively constant from very primitive to
more mature stages, PTH1R, FGF-R1 and PDGF-R mRNAs followed the same
trends as the bone matrix molecule messages, i.e., underwent a significant
increase as osteoblasts matured. However, it is also notable that the most
primitive colonies expressed all the receptors analysed at easily detectable
levels and prior to upregulation of any of the known osteoblast-associated
molecules. PTH1R, FGF-R1 and PDGF-R
play roles in normal skeletal
development. Analysis of knockout mice has shown that FGF-R1 is essential for
cell proliferation and axial patterning in mouse development
(Deng et al., 1994
) and
mutations in FGF-R1 are associated with skeletal abnormalities in humans
(Muenke et al., 1994
). In
homozygous PDGF-R
null mouse mutants, mesenchymal cell proliferation is
affected (Bowen-Pope et al.,
1991
), resulting in growth retardation and deficiencies in
mesodermal structures (Schatteman et al.,
1992
). While early mesodermal cells are affected, cells traversing
the osteoblast lineage specifically also respond to these growth factors.
Growing evidence supports the hypothesis that FGF has considerable anabolic
effects on bone (Dunstan et al.,
1999
; Kawaguchi et al.,
2001
; Liang et al.,
1999
; Zhang et al.,
2002
). PDGF has also been shown to enhance proliferation of
osteoblastic cells, but its effects on differentiation have been inconsistent
in different models in vitro, although in general inhibition is observed
(reviewed by Canalis et al.,
1992
). While these studies have shown that osteoblastic
populations respond to FGF and PDGF, there is little information on which
subpopulations of osteoblastic cells express the receptors and whether
receptor numbers change as a function of differentiation stage. Our results
show that mRNAs for both growth factor receptors are expressed continuously
through the lineage from very early progenitor to mature osteoblast and that
both tend to peak as osteoblasts reach maturity and then decline concomitant
with downregulation of the osteoblast-specific messages. However, they are
differentially regulated in the earlier progenitors in the lineage, which may
allow for important regulatory differences early in development. Our finding
that FGF-R1 goes through the first of its two stages of upregulation
relatively early in the differentiation sequence and prior to upregulation of
ALP suggests that amongst target populations for FGF spanning multiple
developmental stages, FGF may have a special role in very early committed and
proliferatively active osteoprogenitors. The second special target stage for
both receptors that peak at late developmental stages concomitant with other
markers such as BSP and OCN appears to be the matrix synthesizing mature
osteoblast cell. There is also a trend towards more rapid downregulation of
FGF-R1 than PDGF-R
in mineralization phase
(Fig. 3), suggesting that the
latter may play a unique role at this terminal differentiation stage. These
data provide evidence that the growth factor receptors can be used as
additional markers for osteoprogenitors at different developmental stages and
provide some clues as to target genes for their activities.
Many discrepancies exist in earlier determinations of when during
osteogenic developmental PTHrP and PTH1R are expressed (reviewed by
Aubin and Liu, 1996). In this
study, we found that PTHrP is continuously expressed at a relatively constant
level throughout the lineage in keeping with most observations
(Kartsogiannis et al., 1997
;
Suda et al., 1996
) and is not
specifically localized to immature osteoblasts as indicated by others
(Karmali et al., 1992
). Our
results also show that mRNA for PTH1R is expressed continuously through the
lineage from early progenitor to mature osteoblast and that it tends to peak
as osteoblasts reach maturity and then declines concomitantly with
downregulation of osteoblast-specific messages. These observations are
different from studies reporting that the highest number of receptors is on
relatively undifferentiated osteoblasts, with relatively few on the terminally
differentiated mature osteoblast (Rouleau
et al., 1988
), but are in agreement with other studies in which
PTH1R has been demonstrated to be high in the mature osteoblast population
(Bos et al., 1996
;
Suda et al., 1996
), and with
earlier in situ studies showing the receptor to be present on osteoblasts and
its immediate precursors (Lee et al.,
1994
; Silve et al.,
1982
). The fact that we find both molecules expressed already in
immature progenitors through to more mature osteoblasts support the idea of a
widespread autocrine/paracrine function for these molecules during osteoblast
development (Lanske and Kronenberg,
1998
; Suda et al.,
1996
).
Clearly, the combined approach of single colony isolation and poly(A)-PCR
offers a means by which to determine a molecular fingerprint of normal (i.e.
non-established, non-transformed) primitive osteoprogenitors through to
differentiating osteoblasts and mature osteoblasts. The observations suggest
that within the osteoblast differentiation sequence both discrete stages and a
continuum of changing marker expression levels occur with much variation in
expression for any given marker. We have identified novel developmental stages
not previously known and characterized by expression of known
osteoblast-associated markers such as BSP; we also predict that more stages
may be uncovered as more genes are added to this analysis either through
analysis as done here or through use of these amplified pools on DNA
microarrays. We have also demonstrated that the mRNA expression for certain
hormone and growth factor receptors is modulated during osteoblast
differentiation and the sequential expression of different receptors appears
to provide markers for cells earlier in the lineage than those already
expressing ALP and helps to shed light on the cellular targets mediating the
diverse effects of overexpression or under-expression of these families of
molecules. Moreover, these gene expression relationships have allowed us to
statistically examine the developmental flexibility of clonal stem or
progenitor cell differentiation (Madras et
al., 2002). Finally, the poly(A)-PCR approach used has allowed us
to generate cDNA libraries of multiple osteogenic stages, spanning primitive
osteoprogenitor to mature osteoblast, from which we are now isolating novel
differentiation stage-specific osteoblast lineage genes
(Candeliere et al., 1999
).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abou-Samra, A. B., Juppner, H., Force, T., Freeman, M. W., Kong, X. F., Schipani, E., Urena, P., Richards, J., Bonventre, J. V., Potts, J. T., Jr et al. (1992). Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc. Natl. Acad. Sci. USA 89,2732 -2736.[Abstract]
Aubin, J. E. and Liu, F. (1996). The osteoblast lineage. In Principles of Bone Biology (ed. J. P. Bilezikian L. G. Raisz and G. A. Rodan), pp. 51-67. San Diego: Academic Press.
Aubin, J. E. and Triffitt, J. (2002). Mesenchymal stem cells and the osteoblast lineage. In Principles of Bone Biology, 2nd edn (ed. J. P. Bilezikian, L. G. Raisz and G. A. Rodan), pp. 59-81. New York, NY: Academic Press.
Aubin, J. E., Turksen, K. and Heersche, J. N. M. (1993). Osteoblastic cell lineage. In Cellular and Molecular Biology of Bone (ed. M. Noda), pp.1 -45. New York: Academic Press.
Aubin, J. E., Liu, F. and Candeliere, G. A. (2002). Single cell PCR methods for studying stem cells and progenitors. Methods Mol. Biol. 185,403 -415.[Medline]
Auffray, C. and Rougeon, F. (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107,303 -314.[Abstract]
Bellows, C. G. and Aubin, J. E. (1989). Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev. Biol. 133, 8-13.[Medline]
Bellows, C. G., Aubin, J. E., Heersche, J. N. M. and Antosz, M. E. (1986). Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif. Tissue Int. 38,143 -154.[Medline]
Bianco, P., Fisher, L. W., Young, M. F., Termine, J. D. and Robey, P. G. (1991). Expression of bone sialoprotein (BSP) in developing human tissues. Calcif. Tissue Int. 49,421 -426.[Medline]
Bianco, P., Riminucci, M., Bonucci, E., Termine, J. D. and
Robey, P. G. (1993). Bone sialoprotein (BSP) secretion and
osteoblast differentiation: Relationship to bromodeoxyuridine incorporation,
alkaline phosphatase, and matrix deposition. J. Histochem.
Cytochem. 41,183
-191.
Bos, M. P., van der Meer, J. M., Feyen, J. H. and Herrmann-Erlee, M. P. (1996). Expression of the parathyroid hormone receptor and correlation with other osteoblastic parameters in fetal rat osteoblasts. Calcif. Tissue Int. 58, 95-100.[CrossRef][Medline]
Bowen-Pope, D. F., van Koppen, A. and Schatteman, G. (1991). Is PDGF really important? Testing the hypothesis. Trends Genet. 7,413 -418.[Medline]
Brady, G. and Iscove, N. N. (1993). Construction of cDNA libraries from single cells. Methods Enzymol. 225,611 -623.[Medline]
Brady, G., Billia, F., Knox, J., Hoang, T., Kirsch, I. R., Voura, E., Hawley, R. G., Cumming, R., Buchwald, M., Siminovitch, K. et al. (1995). Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Curr. Biol. 5,909 -922.[Medline]
Canalis, E., Varghese, S., McCarthy, T. L. and Centrella, M. (1992). Role of platelet derived growth factor in bone cell function. Growth Regul. 2, 151-155.[Medline]
Candeliere, G. A., Rao, Y., Floh, A., Sandler, S. D. and Aubin,
J. E. (1999). cDNA fingerprinting of osteoprogenitor cells to
isolate differentiation stage-specific genes. Nucleic Acids
Res. 27,1079
-1083.
Candeliere, G. A., Liu, F. and Aubin, J. E. (2001). Individual osteoblasts in the developing calvaria express different gene repertoires. Bone 28,351 -361.[CrossRef][Medline]
Chen, J., Zhang, Q., McCulloch, C. A. G. and Sodek, J. (1991). Immunohistochemical localization of bone sialoprotein in fetal porcine bone tissues: Comparisons with secreted phosphoprotein I (SPP-1, osteopontin) and SPARC (osteonectin). Histochem. J. 23,281 -289.[Medline]
Deng, C.-X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., Ornitz, D. M. and Leder, P. (1994). Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 8,3045 -3057.[Abstract]
Dunstan, C. R., Boyce, R., Boyce, B. F., Garrett, I. R., Izbicka, E., Burgess, W. H. and Mundy, G. R. (1999). Systemic administration of acidic fibroblast growth factor (FGF-1) prevents bone loss and increases new bone formation in ovariectomized rats. J. Bone Miner. Res. 14,953 -959.[Medline]
Esko, J. D. (1989). Replica plating of animal cells. Methods Cell Biol. 32,387 -422.[Medline]
Genovese, C., Rowe, D. and Kream, B. (1984). Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23,6210 -6216.[Medline]
Hunter, G. K. and Goldberg, H. A. (1993).
Nucleation of hydroxyapatite by bone sialoprotein. Proc. Natl.
Acad. Sci. USA 90,8562
-8565.
Karmali, R., Schiffmann, S. N., Vanderwinden, J. M., Hendy, G. N., Nys-DeWolf, N., Corvilain, J., Bergmann, P. and Vanderhaeghen, J. J. (1992). Expression of mRNA of parathyroid hormone-related peptide in fetal bones of the rat. Cell Tissue Res. 270,597 -600.[Medline]
Kartsogiannis, V., Moseley, J., McKelvie, B., Chou, S. T., Hards, D. K., Ng, K. W., Martin, T. J. and Zhou, H. (1997). Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model. Bone 21,385 -392.[CrossRef][Medline]
Kawaguchi, H., Nakamura, K., Tabata, Y., Ikada, Y., Aoyama, I.,
Anzai, J., Nakamura, T., Hiyama, Y. and Tamura, M. (2001).
Acceleration of fracture healing in nonhuman primates by fibroblast growth
factor-2. J. Clin. Endocrinol. Metab.
86,875
-880.
Lanske, B. and Kronenberg, H. M. (1998). Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor. Crit. Rev. Eukaryot. Gene Exp. 8, 297-320.[Medline]
Lee, C., Gardella, T. J., Abou-Samra, A. B., Nussbaum, S. R., Segre, G. V., Potts, J. T., Jr, Kronenberg, H. M. and Juppner, H. (1994). Role of the extracellular regions of the parathyroid hormone (PTH)/PTH-related peptide receptor in hormone binding. Endocrinology 135,1488 -1495.[Abstract]
Lee, K.-H., Bowen-Hope, D. F. and Reed, R. R. (1990). Isolation and characterization of the a platelet-derived growth factor receptor from rat olfactory epithelium. Mol. Cell. Biol. 10,2237 -2246.[Medline]
Liang, H., Pun, S. and Wronski, T. J. (1999).
Bone anabolic effects of basic fibroblast growth factor in ovariectomized
rats. Endocrinology 140,5780
-5788.
Liu, F., Malaval, L., Gupta, A. and Aubin, J. E. (1994). Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: Polymerase chain reaction and immunocytochemical studies at the single cell level. Dev. Biol. 166,220 -234.[CrossRef][Medline]
Liu, F., Malaval, L. and Aubin, J. E. (1997). The mature osteoblast phenotype is characterized by extensive palsticity. Exp. Cell. Res. 232,97 -105.[CrossRef][Medline]
Madras, N., Gibbs, A. L., Zhou, Y., Zandstra, P. W. and Aubin,
J. E. (2002). Modeling stem cell development by retrospective
analysis of gene expression profiles in single progenitor-derived colonies.
Stem Cells 20,230
-240.
Malaval, L., Liu, F., Roche, P. and Aubin, J. E. (1999). Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro. J. Cell. Biochem. 74,616 -627.[CrossRef][Medline]
Mansukhani, A., Moscatelli, D., Talarico, D., Levytska, V. and Basilico, C. (1990). A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF. Proc. Natl. Acad. Sci. USA 87,4378 -4382.[Abstract]
Mintz, K. P., Grzesik, W. J., Midura, R. J., Robey, P. G., Termine, J. D. and Fisher, L. W. (1993). Purification and fragmentation of nondenatured bone sialoprotein: Evidence for a cryptic, RGD-resistant cell attachment domain. J. Bone Miner. Res. 8,985 -995.[Medline]
Muenke, M., Schell, U., Hehr, A., Robin, N. H., Losken, H. W., Schinzel, A., Pulleyn, L. J., Rutland, P., Reardon, W., Malcolm, S. et al. (1994). A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat. Genet. 8, 269-274.[Medline]
Noda, M. and Rodan, G. A. (1987). Type ß transforming growth factor (TGFß) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat sarcoma cells. J. Cell. Physiol. 133,426 -437.[Medline]
Nomura, S., Wills, A. J., Edwards, D. R., Heath, J. K. and Hogan, B. L. M. (1988). Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization. J. Cell Biol. 106,441 -450.[Abstract]
Oldberg, A., Franzen, A. and Heinegard, D.
(1988). The primary structure of a cell-binding bone
sialoprotein. J. Biol. Chem.
263,19430
-19432.
Raetz, C. R., Wermuth, M. M., McIntyre, T. M., Esko, J. D. and Wing, D. C. (1982). Somatic cell cloning in polyester stacks. Proc. Natl. Acad. Sci. USA 79,3223 -3227.[Abstract]
Rodan, G. A. and Noda, M. (1991). Gene expression in osteoblastic cells. Crit. Rev. Eukaryot. Gene Exp. 1,85 -98.[Medline]
Rouleau, M. F., Mitchell, J. and Goltzman, D. (1988). In vivo distribution of parathyroid hormone receptors in bone: evidence that a predominant osseous target cell is not the mature osteoblast. Endocrinology 123,187 -191.[Abstract]
Sambrook, J., Frisch, E. F. and Maniatis, T. (1989). Molecular Cloning, A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schatteman, G. C., Morrison-Graham, K., van Koppen, A., Weston, J. A. and Bowen-Pope, D. F. (1992). Regulation and role of PDGF receptor a-subunit expression during embryogenesis. Development 115,123 -131.[Abstract]
Silve, C. M., Hradek, G. T., Jones, A. L. and Arnaud, C. D. (1982). Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization. J. Cell Biol. 94,379 -386.[Abstract]
Smith, J. H. and Denhardt, D. T. (1987). Molecular cloning of a tumor promoter-inducible mRNA found in JB6 mouse epidermal cells: Induction is stable at high, but not at low, cell densities. J. Cell. Biochem. 34,13 -22.[Medline]
Stein, G. S., Lian, J. B., Stein, J. L., van Wijnen, A. J., Frenkel, B. and Montecino, M. (1996). Mechanisms regulating osteoblast proliferation and differentiation. In Principles of Bone Biology (ed. J. P. Bilezikian L. G. Raisz and G. A. Rodan), pp.69 -86. San Diego: Academic Press.
Suda, N., Gillespie, M. T., Traianedes, K., Zhou, H., Ho, P. W., Hards, D. K., Allan, E. H., Martin, T. J. and Moseley, J. M. (1996). Expression of parathyroid hormone-related protein in cells of osteoblast lineage. J. Cell. Physiol. 166,94 -104.[CrossRef][Medline]
Turksen, K. and Aubin, J. E. (1991). Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. J. Cell Biol. 114,373 -384.[Abstract]
Yasuda, T., Banville, D., Rabbani, S. A., Hendy, G. N. and Goltzman, D. (1989). Rat parathyroid hormone-like peptide: comparison with the human homologue and expression in malignant and normal tissue. Mol. Endocrinol. 3, 518-525.[Abstract]
Zhang, X., Sobue, T. and Hurley, M. M. (2002). FGF-2 increases colony formation, PTH receptor, and IGF-1 mRNA in mouse marrow stromal cells. Biochem. Biophys. Res. Commun. 290,526 -531.[CrossRef][Medline]