Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent
http://www.100md.com
《干细胞学杂志》
a Department of Chemical Engineering and Applied Chemistry;
b Institute of Biomaterials and Biomedical Engineering;
c Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Key Words. Colony forming unit-osteoblast ? Expansion ? Osteoprogenitor ? Dexamethasone ? Self-renewal
Peter W. Zandstra, Ph.D., Institute of Biomaterials and Biomedical Engineering, Room 407, Roseburgh Building, 4 Taddle Creek Road, Toronto, Ontario, M5S 3G9, Canada. Telephone: 416-978-8888; Fax: 416-978-4317; e-mail: peter.zandstra@utoronto.ca
ABSTRACT
Osteogenic cells are an integral part of the dynamic tissue-remodeling process in bone, and as such are potential tools for tissue engineering and cell-based therapies for skeletal pathologies. Osteoblast activities including bone matrix synthesis and mineralization are regulated by various local and systemic factors. Osteoblasts express alkaline phosphatase (ALP) and synthesize type I collagen and noncollagenous proteins such as osteopontin, osteonectin, osteocalcin, bone sialoprotein, and bone morphogenetic proteins . In addition, they respond to osteotropic hormones, including parathyroid hormone (PTH), prostaglandin E2, 1,25 dihydroxyvitamin D3, and glucocorticoids such as corticosterone and dexamethasone (dex) .
Primary bone or bone marrow chicken-, human-, mouse-, and rat-derived cell cultures have been used for studies on the metabolism and hormonal responses of osteogenic cells in vitro . Like those of the fetal or newborn rat calvaria (RC) , these osteogenic cells have the capacity to form discrete three-dimensional bone nodules with the histological, ultrastructural, and immunohistochemical characteristics of woven bone when grown in the presence of ascorbic acid and organic phosphate . These nodules are thought to represent the end stage of the proliferation-differentiation sequence of individual osteoprogenitor cells present in the input cell population. Limiting dilution and clonal analysis have shown that bone nodules can arise from a single progenitor and thus comprise a colony forming unit-osteogenic (CFU-O) assay , with ~0.3% of the RC cell population capable of nodule formation under standard culture conditions . Currently, however, the unambiguous identification and isolation of osteoprogenitor cells within mixed bone cell populations is a problem, as no unique morphological or biochemical criteria are known by which to define the cells. The CFU-O assay thus represents a powerful way to interrogate the effect of exogenous and endogenous parameters on in vitro bone development.
Nodule formation in vitro can be regulated by hormonal additives to the culture medium, such as the synthetic glucocorticoid dex, which increases nodule numbers in a dose-dependent manner . Evidence suggests that the circulating levels of glucocorticoids in humans and animals are similar to the concentrations that have maximal stimulatory effects on in vitro osteoblast responses, i.e., 10-7–10-8 M . In the RC system, the subpopulation of progenitor cells requiring dex to be recruited into or differentiate along the osteoblast lineage are considered more primitive than those that undergo differentiation in control medium . Dex also appears to recruit or promote maturation of more primitive human osteoprogenitors . It has been suggested that dex may regulate osteoblast differentiation positively or negatively, depending on the differentiation stage of the cells .
Previously, we examined the ability of dex to increase the proliferative capacity and differentiation potential of CFU-O capable of forming bone nodules through several passages . We have now extended the analysis to longer times and compared bone progenitor expansion at low versus high initiating cell densities, the latter reported to maintain mineralized nodule formation for longer times in the rat bone marrow system . We have found that CFU-O expansion is dex and cell density dependent, with a greater than 10,000-fold expansion observed under high-density conditions. Our results suggest that osteoprogenitor self-renewal, recruitment, and differentiation in response to dex is cell density dependent, with the hormone differentially influencing the proliferative capacity or onset of differentiation of a subpopulation of cells. The data also support the concept that progenitors at different stages of osteoblast differentiation exhibit selective responsiveness to hormone actions . Evidence of extensive bone progenitor self-renewal under multiple conditions emphasizes the role that cell-cell interactions or community effects play in revealing osteogenic potential and ultimately the ability to control bone development for therapeutic application.
MATERIALS AND METHODS
Dexamethasone Supplementation Affects the Long-Term Growth of Osteoprogenitor Cells
To examine the long-term growth and self-renewal capacity of osteoprogenitors within the heterogeneous RC cell population, cultures were maintained in exponential growth by subculturing the cells at a 1:6 to 1:3 ratio every 3 days. In addition, two cell densities were investigated to examine the impact of the microcellular environment on the long-term self-renewal capabilities of progenitor cells. Cells cultured with this protocol (Fig. 1A) at standard density (Fig. 2A, 2B) and high density (Fig. 2C, 2D) proliferated extensively. Calculation of the CPDL demonstrated that cells in both treatment conditions grew with a constant and similar doubling time during the exponential growth phase at standard (Fig. 2B) and high (Fig. 2D) density, as demonstrated by linear regression of the population doubling with subculture (Table 1). In some experiments, growth crisis was observed in vehicle-treated cultures after ~10–20 or ~30 population doublings at standard or high density, respectively; in dex, however, exponential growth continued until culture termination (Table 1). Cell morphology (polygonal) was similar in vehicle and dex treatment during exponential growth, but as cultures approached crisis, cells assumed a more fibroblastic and then typically senescent shape (data not shown).
Figure 2. Population growth characteristics of RC cells. Primary RC cells were seeded at (A) standard (~1,250 cells/cm2 in 35-mm dish) or (C) high (~12,500 cells/cm2 60-mm dish) density, expanded in vehicle () or dex () and split at a 1:6-1:3 ratio every 3 days. The CPDL achieved is plotted for the cultures initiated at standard (B) or high (D) density, respectively, with the linear regression of the curves overlaid (line). A representative example for each initiating density of three independent experiments is shown.
Table 1. Summary of growth and osteoblast proliferation and differentiation capacities of triplicate experiments completed at two seeding densities
Dexamethasone Supplementation and Density Affect the Maintenance of the Differentiation Capacity of Osteoprogenitor Cells
At each passage, the presence and number of CFU-O were assayed by replating the expanded cell populations (vehicle- or dex-treated) into either vehicle or dex conditions to track progenitor self-renewal and the differentiation or recruitment effects of dex. CFU-O was defined as cells giving rise to colonies with cuboidal cells that were ALP positive and containing mineralized matrix (von Kossa positive), i.e., bone nodules (Fig. 1B). Treated cultures initiated at standard density and expanded in vehicle (Fig. 3A) or dex (Fig. 3B) generated detectable bone nodules for 5–8 subcultures in both vehicle and dex assay conditions. Based on the assumption that one osteoprogenitor cell gives rise to one nodule , a theoretical yield of bone nodules was calculated based on input progenitor numbers (as observed in CFU-O assay) and the dilution factor of each subculture. To emphasize graphically that progenitors self-renewed, the calculated yield was overlaid (lines) on the observed yield of nodules (Fig. 3A, 3B). The number of CFU-O did not exceed the theoretical yield in the expanded populations assayed in vehicle (Fig. 3A, 3B). In contrast, CFU-O number was significantly (p < 0.05; 2-tail z-test) higher than the theoretical yield when cells were assayed in dex on days 6 and 9 for populations expanded in vehicle and for all time points beyond 3 days (with the exception of day 12 and day 30 where the increase failed to reach statistical significance) for populations expanded in dex (Fig. 3A, 3B). Thus, self-renewal was observed for 4 (vehicle) or more than 12 (dex) population doublings for treated cells. Additionally, during the time that cells were capable of self-renewal, a population of CFU-O was recruited by dex in both culture treatments (p < 0.05; 2-tail t-test), yielding an ~2–20- or ~2–25-fold increase in CFU-O output in dex versus vehicle assay conditions (Fig. 3A, 3B).
Figure 3. CFU-O number in sequentially passaged RC cells. The number of mineralized nodules after 21 days in vehicle (hatched) or dex (open) assay conditions from cell populations initiated at standard density and treated during exponential growth with vehicle (A) or dex (B). A representative experiment of three separate experiments is plotted and the mean number of nodules ± standard deviation of triplicate wells per assay condition is shown. Overlaid lines represent the expected osteoprogenitor dilution curve if progenitors did not proliferate in vehicle () or dex (). The significance (p < 0.05; t-test assuming unequal variances, 2-tail) of the separation between nodule formation in the dex assay following expansion in both conditions and the osteoprogenitor dilution curve is indicated (*). Nodule outputs of the vehicle and dex assay are significantly different (p < 0.05, t-test, 2-tail) until day 9 and until day 18 (with the exception of day 12) for vehicle- or dex-treated expansion cultures. Nd indicates that nodules were not detected.
To further quantify the osteoprogenitor pool with time, the fold change in CFU-O number was calculated at each subculture, relative to the number of CFU-O detected on the first subculture (day 3). Mineralized nodules were detectable for a limited number of passages under both cultivation conditions, but the numbers differed between conditions. In populations initiated at standard density that were passaged and assayed in vehicle (Fig. 4A, hatched bars), CFU-O steadily declined in number and could not be detected after 12 doublings (day 15). When the same population was assayed in dex (Fig. 4A, open bars), CFU-O number first increased then decreased to a plateau value prior to a loss of detection after 15 doublings (day 21). Strikingly, in populations initiated at standard density but passaged in dex (Fig. 4B), CFU-O numbers were higher and were maintained longer in both vehicle and dex assay conditions, with maximal numbers in dex assay conditions (Table 1). In cultures initiated at high density, nodules were detected for a greater number of subcultures for nearly all conditions (Fig. 4C, 4D; Table 1). Cells expanded in vehicle at high density demonstrated a higher level of self-renewal and progenitor maintenance over cells expanded at standard density, assayed in both vehicle (p < 0.05; 2-tail z-test; to day 9) and dex (to day 15, except day 9) conditions (Fig. 4A, 4C). Dramatically, high-density expansion in dex significantly (p < 0.05) amplified and sustained the CFU-O number measured in vehicle and dex assay conditions until just prior to or until culture termination (14–18 doublings) (Fig. 4D; Table 1). The CFU-O amplification was greater than 104-fold in the dex assay condition, significantly higher than levels previously reported.
Figure 4. Fold expansion of CFU-O with successive passaging. Capacity for nodule production calculated as the fold expansion of mineralized nodules relative to day 3 (or day 0) nodule output for treatment with vehicle (A, C) or dex (B, D) when cells were initiated at standard (A, B) or high (C, D) density. CFU-O number was assayed in vehicle (hatched) or dex (open) conditions. A representative experiment at each density from three independent experiments is shown with the fold expansion ± standard deviation of the calculation based on population growth and nodule frequencies. Nd indicates that nodules were not detected; * indicates that vehicle and dex assay results for each expansion condition were significantly different (p < 0.05; z-test, 2-tail). A comparison of the fold expansion of nodules during standard and high-density culture showed significant differences (p < 0.05; z-test, 2-tail) to day 9 or day 15 (with the exception of day 9) in vehicle-treated cultures assayed in vehicle and dex, respectively, and differences to day 18 and day 24 for dex-treated cultures assayed in vehicle or dex.
Populations initiated at high density and expanded in dex exhibited similar CFU-O numbers in dex versus vehicle assay conditions for 5–7 passages (data not shown). Further examination of the steadily increasing nodule number that occurred with dex treatment was done by calculating the cumulative nodule doubling level (CNDL) in the vehicle and dex CFU-O assay and comparing it with the growth of the CPDL (Fig. 5). Replicate experiments showed that production of nodules occurred at an indistinguishable rate from the overall population growth, and thus neither progenitor cells nor the bulk fibroblastic cells exhibited a clear growth advantage.
Figure 5. Comparison of nodule and population expansion in dex cultures initiated at high density. A plot of the CNDL in the vehicle () or dex () CFU-O assay and the CPDL is shown for one representative experiment. A reference line for a 1:1 ratio of CNDL:CPDL is shown.
Separation of Expression of the Osteoblastic Phenotype of Expanded Cells and Capacity for Nodule Formation
To examine the proportion of the population exhibiting markers of the osteoblastic phenotype, the high-density culture was characterized in terms of surface expression of ALP and PTH1R by flow cytometry at alternating subcultures (Fig. 6). There was a clear separation between nodule formation, ALP and/or PTH1R expression, and population growth in both expansion conditions. Both vehicle- and dex-expanded cultures stopped producing nodules during a time of stable cell expansion. CFU-O capacity was lost without an associated loss in ALP/PTH1R expression or population growth, and changes in measured marker expression were not coupled to population growth changes. In vehicle, an approximate 60:40 split of ALP+:ALP- cells was seen (Fig. 6A), while in dex, an inverse 40:60 distribution was seen (Fig. 6B). Cells expressing ALP remained fairly stable over 16–17 doublings (day 24) in both vehicle and dex conditions (Fig. 6A, 6B), although populations expanded in vehicle demonstrated a reduced capacity for nodule formation in comparison to their dex-treated counterpart. The ALP+PTH1R+ population appeared in a cyclic fashion over time; however, changes in expression did not correlate to nodule production. At the time that 80%–100% of the total nodules had formed under vehicle conditions, cells assayed in dex had produced only 1%–10% of their total nodule capacity (days 12–18). The largest changes in ALP expression occurred after nodule formation was no longer observed, while ALP+PTH1R+ expression varied over the duration of culture. In the control population, the number of cells expressing ALP steadily declined following 16 doublings (day 24), and the dex population exhibited a reduced number of ALP+ cells prior to a later decline.
Figure 6. Surface expression of ALP and PTH1R was monitored by flow cytometry during expansion of high-density cultures. The percentage of ALP positive cells () or ALP and PTH1R double positive cells () is shown for vehicle (A) and dex (B) expanded cells (right axis) for one representative experiment. A cumulative representation of the total nodule formation from the CFU-O assays in vehicle () or dex () is also overlaid (left axis). NS indicates that there was no sample available.
DISCUSSION
K.A.P. gratefully acknowledges student fellowship support from Ontario Graduate Scholarships in Science and Technology. This work was supported by operating grants from Canadian Institutes of Health Research (J.E.A., MT-12390), Premier’s Research Excellence Awards (P.W.Z.), the Stem Cell Network (J.E.A.; P.W.Z.), and Natural Sciences and Engineering Research Council of Canada (P.W.Z.).
REFERENCES
Bellows CG, Aubin JE, Heersche JN. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner 1991;14:27–40.
Rodan GA, Noda M. Gene expression in osteoblastic cells. Crit Rev Eukaryot Gene Expr 1991;1:85–98.
Nagata T, Bellows CG, Kasugai S et al. Biosynthesis of bone proteins in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. Biochem J 1991;274:513–520.
Rickard DJ, Sullivan TA, Shenker BJ et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev Biol 1994;161:218–228.
Wada Y, Kataoka H, Yokose S et al. Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells. Bone 1998;22:479–485.
Aubin JE, Tertinegg I, Ber R et al. Consistent patterns of changing hormone responsiveness during continuous culture of cloned rat calvaria cells. J Bone Miner Res 1988;3:333–339.
Beresford JN, Joyner CJ, Devlin C et al. The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro. Arch Oral Biol 1994;39:941–947.
Lian JB, Shalhoub V, Aslam F et al. Species-specific glucocorticoid and 1,25-dihydroxyvitamin d responsiveness in mouse MC3T3-E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression. Endocrinology 1997;138:2117–2127.
Cheng SL, Yang JW, Rifas L et al. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 1994;134:277–286.
Bellows CG, Ciaccia A, Heersche JN. Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone. Bone 1998;23:119–125.
Bellows CG, Aubin JE, Heersche JN et al. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int 1986;38:143–154.
Gerstenfeld LC, Chipman SD, Glowacki J et al. Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dev Biol 1987;122:49–60.
Falla N, Van Vlasselaer P, Bierkens J et al. Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow. Blood 1993;82:3580–3591.
Nishida S, Endo N, Yamagiwa H et al. Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation. J Bone Miner Metab 1999;17:171–177.
Siggelkow H, Rebenstorff K, Kurre W et al. Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation. J Cell Biochem 1999;75:22–35.
Yagiela JA, Woodbury DM. Enzymatic isolation of osteoblasts from fetal rat calvaria. Anat Rec 1977;188:287–306.
Kadis B, Goodson JM, Offenbacher S et al. Characterization of osteoblast-like cells from fetal rat calvaria. J Dent Res 1980;59:2006–2013.
Aubin JE, Heersche JN, Merrilees MJ et al. Isolation of bone cell clones with differences in growth, hormone responses, and extracellular matrix production. J Cell Biol 1982;92:452–461.
Moskalewski S, Boonekamp PM, Scherft JP. Bone formation by isolated calvarial osteoblasts in syngeneic and allogeneic transplants: light microscopic observations. Am J Anat 1983;167:249–263.
Nefussi JR, Boy-Lefevre ML, Boulekbache H et al. Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion. Differentiation 1985;29:160–168.
Bhargava U, Bar-Lev M, Bellows CG et al. Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells. Bone 1988;9:155–163.
Herbert B, Lecouturier A, Masquelier D et al. Ultrastructure and cytochemical detection of alkaline phosphatase in long-term cultures of osteoblast-like cells from rat calvaria. Calcif Tissue Int 1997;60:216–223.
Aubin JE. Advances in the osteoblast lineage. Biochem Cell Biol 1998;76:899–910.
Bellows CG, Aubin JE. Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev Biol 1989;133:8–13.
Bellows CG, Aubin JE, Heersche JN. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro. Endocrinology 1987;121:1985–1992.
Ishida Y, Heersche JN. Glucocorticoid-induced osteoporosis: both in vivo and in vitro concentrations of glucocorticoids higher than physiological levels attenuate osteoblast differentiation. J Bone Miner Res 1998;13:1822–1826.
Turksen K, Aubin JE. Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. J Cell Biol 1991;114:373–384.
Stewart K, Walsh S, Screen J et al. Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells. J Bone Miner Res 1999;14:1345–1356.
Walsh S, Jefferiss C, Stewart K et al. Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4. Bone 2000;27:185–195.
Walsh S, Jordan GR, Jefferiss C et al. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis. Rheumatology (Oxford) 2001;40:74–83.
Aubin JE. Bone stem cells. J Cell Biochem Suppl 1998;30–31:73–82.
Bellows CG, Heersche JN, Aubin JE. Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone. Dev Biol 1990;140:132–138.
McCulloch CA, Strugurescu M, Hughes F et al. Osteogenic progenitor cells in rat bone marrow stromal populations exhibit self-renewal in culture. Blood 1991;77:1906–1911.
Aubin JE, Turksen K, Heersche JNM. Osteoblastic cell lineage. In: Noda M, ed. Cellular and Molecular Biology of Bone. New York: Academic Press, Inc., 1993:1–45.
Isogai Y, Akatsu T, Ishizuya T et al. Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages. J Bone Miner Res 1996;11:1384–1393.
Rao LG, Ng B, Brunette DM et al. Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C. Endocrinology 1977;100:1233–1241.
Aubin JE. Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interactions in osteoblast differentiation. J Cell Biochem 1999;72:396–410.
Purpura KA, Aubin JE, Zandstra PW. Two-color image analysis discriminates between mineralized and unmineralized bone nodules in vitro. BioTechniques 2003;34:1188–1192.
Lazarus HM, Haynesworth SE, Gerson SL et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557–564.
Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64:278–294.
Jaiswal N, Haynesworth SE, Caplan AI et al. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312.
Digirolamo CM, Stokes D, Colter D et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275–281.
von Lindern M, Zauner W, Mellitzer G et al. The glucocorticoid receptor cooperates with the erythropoietin receptor and c-KIT to enhance and sustain proliferation of erythroid progenitors in vitro. Blood 1999;94:550–559.
Wessely O, Deiner EM, Beug H et al. The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors. EMBO J 1997;16:267–280.
Owen TA, Aronow M, Shalhoub V et al. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990;143:420–430.
Scutt A, Bertram P, Brautigam M. The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: positive and negative effects. Calcif Tissue Int 1996;59:154–162.
Kim CH, Cheng SL, Kim GS. Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of glucocorticoid-induced bone loss. J Endocrinol 1999;162:371–379.
Scutt A, Bertram P. Basic fibroblast growth factor in the presence of dexamethasone stimulates colony formation, expansion, and osteoblastic differentiation by rat bone marrow stromal cells. Calcif Tissue Int 1999;64:69–77.
Stein GS, Lian JB, Gerstenfeld LG et al. The onset and progression of osteoblast differentiation is functionally related to cellular proliferation. Connect Tissue Res 1989;20:3–13.
Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev 1993;14:424–442.
Malaval L, Modrowski D, Gupta AK et al. Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J Cell Physiol 1994;158:555–572.
Gronthos S, Graves SE, Ohta S et al. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84:4164–4173.
Purpura KA, Zandstra PW, Aubin JE. Flourescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations. J Cell Biochem 2003;90:109–120.
Ogiso B, Hughes FJ, Davies JE et al. Fibroblastic regulation of osteoblast function by prostaglandins. Cell Signal 1992;4:627–639.
Ducy P, Zhang R, Geoffroy V et al. Osf2/cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:747–754.
Qu Q, Harkonen PL, Monkkonen J et al. Conditioned medium of estrogen-treated osteoblasts inhibits osteoclast maturation and function in vitro. Bone 1999;25:211–215.(Kelly A. Purpuraa,b, Jane)
b Institute of Biomaterials and Biomedical Engineering;
c Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Key Words. Colony forming unit-osteoblast ? Expansion ? Osteoprogenitor ? Dexamethasone ? Self-renewal
Peter W. Zandstra, Ph.D., Institute of Biomaterials and Biomedical Engineering, Room 407, Roseburgh Building, 4 Taddle Creek Road, Toronto, Ontario, M5S 3G9, Canada. Telephone: 416-978-8888; Fax: 416-978-4317; e-mail: peter.zandstra@utoronto.ca
ABSTRACT
Osteogenic cells are an integral part of the dynamic tissue-remodeling process in bone, and as such are potential tools for tissue engineering and cell-based therapies for skeletal pathologies. Osteoblast activities including bone matrix synthesis and mineralization are regulated by various local and systemic factors. Osteoblasts express alkaline phosphatase (ALP) and synthesize type I collagen and noncollagenous proteins such as osteopontin, osteonectin, osteocalcin, bone sialoprotein, and bone morphogenetic proteins . In addition, they respond to osteotropic hormones, including parathyroid hormone (PTH), prostaglandin E2, 1,25 dihydroxyvitamin D3, and glucocorticoids such as corticosterone and dexamethasone (dex) .
Primary bone or bone marrow chicken-, human-, mouse-, and rat-derived cell cultures have been used for studies on the metabolism and hormonal responses of osteogenic cells in vitro . Like those of the fetal or newborn rat calvaria (RC) , these osteogenic cells have the capacity to form discrete three-dimensional bone nodules with the histological, ultrastructural, and immunohistochemical characteristics of woven bone when grown in the presence of ascorbic acid and organic phosphate . These nodules are thought to represent the end stage of the proliferation-differentiation sequence of individual osteoprogenitor cells present in the input cell population. Limiting dilution and clonal analysis have shown that bone nodules can arise from a single progenitor and thus comprise a colony forming unit-osteogenic (CFU-O) assay , with ~0.3% of the RC cell population capable of nodule formation under standard culture conditions . Currently, however, the unambiguous identification and isolation of osteoprogenitor cells within mixed bone cell populations is a problem, as no unique morphological or biochemical criteria are known by which to define the cells. The CFU-O assay thus represents a powerful way to interrogate the effect of exogenous and endogenous parameters on in vitro bone development.
Nodule formation in vitro can be regulated by hormonal additives to the culture medium, such as the synthetic glucocorticoid dex, which increases nodule numbers in a dose-dependent manner . Evidence suggests that the circulating levels of glucocorticoids in humans and animals are similar to the concentrations that have maximal stimulatory effects on in vitro osteoblast responses, i.e., 10-7–10-8 M . In the RC system, the subpopulation of progenitor cells requiring dex to be recruited into or differentiate along the osteoblast lineage are considered more primitive than those that undergo differentiation in control medium . Dex also appears to recruit or promote maturation of more primitive human osteoprogenitors . It has been suggested that dex may regulate osteoblast differentiation positively or negatively, depending on the differentiation stage of the cells .
Previously, we examined the ability of dex to increase the proliferative capacity and differentiation potential of CFU-O capable of forming bone nodules through several passages . We have now extended the analysis to longer times and compared bone progenitor expansion at low versus high initiating cell densities, the latter reported to maintain mineralized nodule formation for longer times in the rat bone marrow system . We have found that CFU-O expansion is dex and cell density dependent, with a greater than 10,000-fold expansion observed under high-density conditions. Our results suggest that osteoprogenitor self-renewal, recruitment, and differentiation in response to dex is cell density dependent, with the hormone differentially influencing the proliferative capacity or onset of differentiation of a subpopulation of cells. The data also support the concept that progenitors at different stages of osteoblast differentiation exhibit selective responsiveness to hormone actions . Evidence of extensive bone progenitor self-renewal under multiple conditions emphasizes the role that cell-cell interactions or community effects play in revealing osteogenic potential and ultimately the ability to control bone development for therapeutic application.
MATERIALS AND METHODS
Dexamethasone Supplementation Affects the Long-Term Growth of Osteoprogenitor Cells
To examine the long-term growth and self-renewal capacity of osteoprogenitors within the heterogeneous RC cell population, cultures were maintained in exponential growth by subculturing the cells at a 1:6 to 1:3 ratio every 3 days. In addition, two cell densities were investigated to examine the impact of the microcellular environment on the long-term self-renewal capabilities of progenitor cells. Cells cultured with this protocol (Fig. 1A) at standard density (Fig. 2A, 2B) and high density (Fig. 2C, 2D) proliferated extensively. Calculation of the CPDL demonstrated that cells in both treatment conditions grew with a constant and similar doubling time during the exponential growth phase at standard (Fig. 2B) and high (Fig. 2D) density, as demonstrated by linear regression of the population doubling with subculture (Table 1). In some experiments, growth crisis was observed in vehicle-treated cultures after ~10–20 or ~30 population doublings at standard or high density, respectively; in dex, however, exponential growth continued until culture termination (Table 1). Cell morphology (polygonal) was similar in vehicle and dex treatment during exponential growth, but as cultures approached crisis, cells assumed a more fibroblastic and then typically senescent shape (data not shown).
Figure 2. Population growth characteristics of RC cells. Primary RC cells were seeded at (A) standard (~1,250 cells/cm2 in 35-mm dish) or (C) high (~12,500 cells/cm2 60-mm dish) density, expanded in vehicle () or dex () and split at a 1:6-1:3 ratio every 3 days. The CPDL achieved is plotted for the cultures initiated at standard (B) or high (D) density, respectively, with the linear regression of the curves overlaid (line). A representative example for each initiating density of three independent experiments is shown.
Table 1. Summary of growth and osteoblast proliferation and differentiation capacities of triplicate experiments completed at two seeding densities
Dexamethasone Supplementation and Density Affect the Maintenance of the Differentiation Capacity of Osteoprogenitor Cells
At each passage, the presence and number of CFU-O were assayed by replating the expanded cell populations (vehicle- or dex-treated) into either vehicle or dex conditions to track progenitor self-renewal and the differentiation or recruitment effects of dex. CFU-O was defined as cells giving rise to colonies with cuboidal cells that were ALP positive and containing mineralized matrix (von Kossa positive), i.e., bone nodules (Fig. 1B). Treated cultures initiated at standard density and expanded in vehicle (Fig. 3A) or dex (Fig. 3B) generated detectable bone nodules for 5–8 subcultures in both vehicle and dex assay conditions. Based on the assumption that one osteoprogenitor cell gives rise to one nodule , a theoretical yield of bone nodules was calculated based on input progenitor numbers (as observed in CFU-O assay) and the dilution factor of each subculture. To emphasize graphically that progenitors self-renewed, the calculated yield was overlaid (lines) on the observed yield of nodules (Fig. 3A, 3B). The number of CFU-O did not exceed the theoretical yield in the expanded populations assayed in vehicle (Fig. 3A, 3B). In contrast, CFU-O number was significantly (p < 0.05; 2-tail z-test) higher than the theoretical yield when cells were assayed in dex on days 6 and 9 for populations expanded in vehicle and for all time points beyond 3 days (with the exception of day 12 and day 30 where the increase failed to reach statistical significance) for populations expanded in dex (Fig. 3A, 3B). Thus, self-renewal was observed for 4 (vehicle) or more than 12 (dex) population doublings for treated cells. Additionally, during the time that cells were capable of self-renewal, a population of CFU-O was recruited by dex in both culture treatments (p < 0.05; 2-tail t-test), yielding an ~2–20- or ~2–25-fold increase in CFU-O output in dex versus vehicle assay conditions (Fig. 3A, 3B).
Figure 3. CFU-O number in sequentially passaged RC cells. The number of mineralized nodules after 21 days in vehicle (hatched) or dex (open) assay conditions from cell populations initiated at standard density and treated during exponential growth with vehicle (A) or dex (B). A representative experiment of three separate experiments is plotted and the mean number of nodules ± standard deviation of triplicate wells per assay condition is shown. Overlaid lines represent the expected osteoprogenitor dilution curve if progenitors did not proliferate in vehicle () or dex (). The significance (p < 0.05; t-test assuming unequal variances, 2-tail) of the separation between nodule formation in the dex assay following expansion in both conditions and the osteoprogenitor dilution curve is indicated (*). Nodule outputs of the vehicle and dex assay are significantly different (p < 0.05, t-test, 2-tail) until day 9 and until day 18 (with the exception of day 12) for vehicle- or dex-treated expansion cultures. Nd indicates that nodules were not detected.
To further quantify the osteoprogenitor pool with time, the fold change in CFU-O number was calculated at each subculture, relative to the number of CFU-O detected on the first subculture (day 3). Mineralized nodules were detectable for a limited number of passages under both cultivation conditions, but the numbers differed between conditions. In populations initiated at standard density that were passaged and assayed in vehicle (Fig. 4A, hatched bars), CFU-O steadily declined in number and could not be detected after 12 doublings (day 15). When the same population was assayed in dex (Fig. 4A, open bars), CFU-O number first increased then decreased to a plateau value prior to a loss of detection after 15 doublings (day 21). Strikingly, in populations initiated at standard density but passaged in dex (Fig. 4B), CFU-O numbers were higher and were maintained longer in both vehicle and dex assay conditions, with maximal numbers in dex assay conditions (Table 1). In cultures initiated at high density, nodules were detected for a greater number of subcultures for nearly all conditions (Fig. 4C, 4D; Table 1). Cells expanded in vehicle at high density demonstrated a higher level of self-renewal and progenitor maintenance over cells expanded at standard density, assayed in both vehicle (p < 0.05; 2-tail z-test; to day 9) and dex (to day 15, except day 9) conditions (Fig. 4A, 4C). Dramatically, high-density expansion in dex significantly (p < 0.05) amplified and sustained the CFU-O number measured in vehicle and dex assay conditions until just prior to or until culture termination (14–18 doublings) (Fig. 4D; Table 1). The CFU-O amplification was greater than 104-fold in the dex assay condition, significantly higher than levels previously reported.
Figure 4. Fold expansion of CFU-O with successive passaging. Capacity for nodule production calculated as the fold expansion of mineralized nodules relative to day 3 (or day 0) nodule output for treatment with vehicle (A, C) or dex (B, D) when cells were initiated at standard (A, B) or high (C, D) density. CFU-O number was assayed in vehicle (hatched) or dex (open) conditions. A representative experiment at each density from three independent experiments is shown with the fold expansion ± standard deviation of the calculation based on population growth and nodule frequencies. Nd indicates that nodules were not detected; * indicates that vehicle and dex assay results for each expansion condition were significantly different (p < 0.05; z-test, 2-tail). A comparison of the fold expansion of nodules during standard and high-density culture showed significant differences (p < 0.05; z-test, 2-tail) to day 9 or day 15 (with the exception of day 9) in vehicle-treated cultures assayed in vehicle and dex, respectively, and differences to day 18 and day 24 for dex-treated cultures assayed in vehicle or dex.
Populations initiated at high density and expanded in dex exhibited similar CFU-O numbers in dex versus vehicle assay conditions for 5–7 passages (data not shown). Further examination of the steadily increasing nodule number that occurred with dex treatment was done by calculating the cumulative nodule doubling level (CNDL) in the vehicle and dex CFU-O assay and comparing it with the growth of the CPDL (Fig. 5). Replicate experiments showed that production of nodules occurred at an indistinguishable rate from the overall population growth, and thus neither progenitor cells nor the bulk fibroblastic cells exhibited a clear growth advantage.
Figure 5. Comparison of nodule and population expansion in dex cultures initiated at high density. A plot of the CNDL in the vehicle () or dex () CFU-O assay and the CPDL is shown for one representative experiment. A reference line for a 1:1 ratio of CNDL:CPDL is shown.
Separation of Expression of the Osteoblastic Phenotype of Expanded Cells and Capacity for Nodule Formation
To examine the proportion of the population exhibiting markers of the osteoblastic phenotype, the high-density culture was characterized in terms of surface expression of ALP and PTH1R by flow cytometry at alternating subcultures (Fig. 6). There was a clear separation between nodule formation, ALP and/or PTH1R expression, and population growth in both expansion conditions. Both vehicle- and dex-expanded cultures stopped producing nodules during a time of stable cell expansion. CFU-O capacity was lost without an associated loss in ALP/PTH1R expression or population growth, and changes in measured marker expression were not coupled to population growth changes. In vehicle, an approximate 60:40 split of ALP+:ALP- cells was seen (Fig. 6A), while in dex, an inverse 40:60 distribution was seen (Fig. 6B). Cells expressing ALP remained fairly stable over 16–17 doublings (day 24) in both vehicle and dex conditions (Fig. 6A, 6B), although populations expanded in vehicle demonstrated a reduced capacity for nodule formation in comparison to their dex-treated counterpart. The ALP+PTH1R+ population appeared in a cyclic fashion over time; however, changes in expression did not correlate to nodule production. At the time that 80%–100% of the total nodules had formed under vehicle conditions, cells assayed in dex had produced only 1%–10% of their total nodule capacity (days 12–18). The largest changes in ALP expression occurred after nodule formation was no longer observed, while ALP+PTH1R+ expression varied over the duration of culture. In the control population, the number of cells expressing ALP steadily declined following 16 doublings (day 24), and the dex population exhibited a reduced number of ALP+ cells prior to a later decline.
Figure 6. Surface expression of ALP and PTH1R was monitored by flow cytometry during expansion of high-density cultures. The percentage of ALP positive cells () or ALP and PTH1R double positive cells () is shown for vehicle (A) and dex (B) expanded cells (right axis) for one representative experiment. A cumulative representation of the total nodule formation from the CFU-O assays in vehicle () or dex () is also overlaid (left axis). NS indicates that there was no sample available.
DISCUSSION
K.A.P. gratefully acknowledges student fellowship support from Ontario Graduate Scholarships in Science and Technology. This work was supported by operating grants from Canadian Institutes of Health Research (J.E.A., MT-12390), Premier’s Research Excellence Awards (P.W.Z.), the Stem Cell Network (J.E.A.; P.W.Z.), and Natural Sciences and Engineering Research Council of Canada (P.W.Z.).
REFERENCES
Bellows CG, Aubin JE, Heersche JN. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner 1991;14:27–40.
Rodan GA, Noda M. Gene expression in osteoblastic cells. Crit Rev Eukaryot Gene Expr 1991;1:85–98.
Nagata T, Bellows CG, Kasugai S et al. Biosynthesis of bone proteins in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. Biochem J 1991;274:513–520.
Rickard DJ, Sullivan TA, Shenker BJ et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev Biol 1994;161:218–228.
Wada Y, Kataoka H, Yokose S et al. Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells. Bone 1998;22:479–485.
Aubin JE, Tertinegg I, Ber R et al. Consistent patterns of changing hormone responsiveness during continuous culture of cloned rat calvaria cells. J Bone Miner Res 1988;3:333–339.
Beresford JN, Joyner CJ, Devlin C et al. The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro. Arch Oral Biol 1994;39:941–947.
Lian JB, Shalhoub V, Aslam F et al. Species-specific glucocorticoid and 1,25-dihydroxyvitamin d responsiveness in mouse MC3T3-E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression. Endocrinology 1997;138:2117–2127.
Cheng SL, Yang JW, Rifas L et al. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 1994;134:277–286.
Bellows CG, Ciaccia A, Heersche JN. Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone. Bone 1998;23:119–125.
Bellows CG, Aubin JE, Heersche JN et al. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int 1986;38:143–154.
Gerstenfeld LC, Chipman SD, Glowacki J et al. Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dev Biol 1987;122:49–60.
Falla N, Van Vlasselaer P, Bierkens J et al. Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow. Blood 1993;82:3580–3591.
Nishida S, Endo N, Yamagiwa H et al. Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation. J Bone Miner Metab 1999;17:171–177.
Siggelkow H, Rebenstorff K, Kurre W et al. Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation. J Cell Biochem 1999;75:22–35.
Yagiela JA, Woodbury DM. Enzymatic isolation of osteoblasts from fetal rat calvaria. Anat Rec 1977;188:287–306.
Kadis B, Goodson JM, Offenbacher S et al. Characterization of osteoblast-like cells from fetal rat calvaria. J Dent Res 1980;59:2006–2013.
Aubin JE, Heersche JN, Merrilees MJ et al. Isolation of bone cell clones with differences in growth, hormone responses, and extracellular matrix production. J Cell Biol 1982;92:452–461.
Moskalewski S, Boonekamp PM, Scherft JP. Bone formation by isolated calvarial osteoblasts in syngeneic and allogeneic transplants: light microscopic observations. Am J Anat 1983;167:249–263.
Nefussi JR, Boy-Lefevre ML, Boulekbache H et al. Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion. Differentiation 1985;29:160–168.
Bhargava U, Bar-Lev M, Bellows CG et al. Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells. Bone 1988;9:155–163.
Herbert B, Lecouturier A, Masquelier D et al. Ultrastructure and cytochemical detection of alkaline phosphatase in long-term cultures of osteoblast-like cells from rat calvaria. Calcif Tissue Int 1997;60:216–223.
Aubin JE. Advances in the osteoblast lineage. Biochem Cell Biol 1998;76:899–910.
Bellows CG, Aubin JE. Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev Biol 1989;133:8–13.
Bellows CG, Aubin JE, Heersche JN. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro. Endocrinology 1987;121:1985–1992.
Ishida Y, Heersche JN. Glucocorticoid-induced osteoporosis: both in vivo and in vitro concentrations of glucocorticoids higher than physiological levels attenuate osteoblast differentiation. J Bone Miner Res 1998;13:1822–1826.
Turksen K, Aubin JE. Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. J Cell Biol 1991;114:373–384.
Stewart K, Walsh S, Screen J et al. Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells. J Bone Miner Res 1999;14:1345–1356.
Walsh S, Jefferiss C, Stewart K et al. Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4. Bone 2000;27:185–195.
Walsh S, Jordan GR, Jefferiss C et al. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis. Rheumatology (Oxford) 2001;40:74–83.
Aubin JE. Bone stem cells. J Cell Biochem Suppl 1998;30–31:73–82.
Bellows CG, Heersche JN, Aubin JE. Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone. Dev Biol 1990;140:132–138.
McCulloch CA, Strugurescu M, Hughes F et al. Osteogenic progenitor cells in rat bone marrow stromal populations exhibit self-renewal in culture. Blood 1991;77:1906–1911.
Aubin JE, Turksen K, Heersche JNM. Osteoblastic cell lineage. In: Noda M, ed. Cellular and Molecular Biology of Bone. New York: Academic Press, Inc., 1993:1–45.
Isogai Y, Akatsu T, Ishizuya T et al. Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages. J Bone Miner Res 1996;11:1384–1393.
Rao LG, Ng B, Brunette DM et al. Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C. Endocrinology 1977;100:1233–1241.
Aubin JE. Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interactions in osteoblast differentiation. J Cell Biochem 1999;72:396–410.
Purpura KA, Aubin JE, Zandstra PW. Two-color image analysis discriminates between mineralized and unmineralized bone nodules in vitro. BioTechniques 2003;34:1188–1192.
Lazarus HM, Haynesworth SE, Gerson SL et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557–564.
Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64:278–294.
Jaiswal N, Haynesworth SE, Caplan AI et al. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312.
Digirolamo CM, Stokes D, Colter D et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275–281.
von Lindern M, Zauner W, Mellitzer G et al. The glucocorticoid receptor cooperates with the erythropoietin receptor and c-KIT to enhance and sustain proliferation of erythroid progenitors in vitro. Blood 1999;94:550–559.
Wessely O, Deiner EM, Beug H et al. The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors. EMBO J 1997;16:267–280.
Owen TA, Aronow M, Shalhoub V et al. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990;143:420–430.
Scutt A, Bertram P, Brautigam M. The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: positive and negative effects. Calcif Tissue Int 1996;59:154–162.
Kim CH, Cheng SL, Kim GS. Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of glucocorticoid-induced bone loss. J Endocrinol 1999;162:371–379.
Scutt A, Bertram P. Basic fibroblast growth factor in the presence of dexamethasone stimulates colony formation, expansion, and osteoblastic differentiation by rat bone marrow stromal cells. Calcif Tissue Int 1999;64:69–77.
Stein GS, Lian JB, Gerstenfeld LG et al. The onset and progression of osteoblast differentiation is functionally related to cellular proliferation. Connect Tissue Res 1989;20:3–13.
Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev 1993;14:424–442.
Malaval L, Modrowski D, Gupta AK et al. Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J Cell Physiol 1994;158:555–572.
Gronthos S, Graves SE, Ohta S et al. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84:4164–4173.
Purpura KA, Zandstra PW, Aubin JE. Flourescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations. J Cell Biochem 2003;90:109–120.
Ogiso B, Hughes FJ, Davies JE et al. Fibroblastic regulation of osteoblast function by prostaglandins. Cell Signal 1992;4:627–639.
Ducy P, Zhang R, Geoffroy V et al. Osf2/cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:747–754.
Qu Q, Harkonen PL, Monkkonen J et al. Conditioned medium of estrogen-treated osteoblasts inhibits osteoclast maturation and function in vitro. Bone 1999;25:211–215.(Kelly A. Purpuraa,b, Jane)