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Department of Molecular and Medical Genetics, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8

(Requests for offprints should be addressed to J E Aubin; Email: jane.aubin{at}utoronto.ca)

(S Zhang is now at Division of Genetic Services, Department of Pathology and Molecular Medicine, Kingston General Hospital, Queen’s University, Kingston, ON, Canada K7 L 2 V7)

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) inhibits osteogenesis while stimulating adipogenesis in vitro. We hypothesized that 1,25(OH)₂D₃ redirects the fate of osteoblast/adipocyte bipotential progenitors and other potential progenitors towards adipogenesis, a process possibly underlying the pathogenesis of osteopenic diseases such as osteoporosis. We therefore tested the global effects of 1,25(OH)₂D₃ on the recruitment of mesenchymal progenitors including osteogenic, chondrogenic, adipogenic and myogenic lineages (colony forming cell (CFC)-osteoblast (CFC-O), CFC-chondrocyte (CFC-C), CFC-adipocyte (CFC-A), and CFC-myoblast (CFC-M) respectively) in rat calvaria (RC) cell populations using gene expression profiling of single cell-derived colonies. Based on expression of lineage specific transcripts, 86% of single cell-derived colonies in untreated cultures simultaneously co-expressed transcripts of two, three, or four of the mesenchymal lineages tested. The distribution of mesenchymal progenitors in 1,25(OH)₂D₃-treated cultures was significantly changed compared with the control group, i.e. CFC-O were reduced (from 6 to 0%) and CFC-O/A bipotential (0 to 8.2%), CFC-C (4 to 10.2%) and CFC-Fibroblast (CFC-F) (4 to 16%) were increased. 1,25(OH)₂D₃ did not affect the frequency of tri- or tetra-lineage colonies. Single lineage CFC-A colonies were not detected in either the control or 1,25(OH)₂D₃ treatment group under the conditions tested.Since the parietal bones used for cell isolation derive from neuroectoderm, we also analyzed for expression of the neural markers nestin and ß3 tubulin in these colonies. Surprisingly, 90% (45 of 50) of the colonies in the control group expressed neural markers, a frequency not changed by 1,25(OH)₂D₃ treatment. The current studies demonstrate the global and developmental stage-specific effects of 1,25(OH)₂D₃ on mesenchymal lineage progenitors, and suggest that the effects of 1,25(OH)₂D₃ on osteogenesis and adipogenesis in RC populations are mediated, at least in part, by increased recruitment of CFC-O/A, but not CFC-A type precursors.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
It is well known that fetal rat calvaria (RC) tissue contains osteoprogenitors that, under appropriate culture conditions, differentiate into mature osteoblasts that form mineralized bone nodules resembling woven bone (Bellows et al. 1986, Bhargava et al. 1988). However, RC cell populations in culture are a heterogeneous mixture of cell types including different differentiation stages of osteoblasts, chondrocytes and adipocytes (Rifas et al. 1982, Bellows et al. 1989, Aubin 1998, Bellows & Heersche 2001). The fact that the clonally-derived RC cell line RCJ 3.1 is capable of differentiating into cartilage, fat, muscle and bone indicates that mesenchymal stem or multipotential progenitor cells are also present in RC cell populations (Grigoriadis et al. 1988, 1990). The reciprocal relationship seen in bone mass and marrow adipose tissue under certain pathological conditions also suggests the presence of a common osteoblast/adipocyte bipotential progenitor in adult marrow (Meunier et al. 1971, Wang et al. 1977, Kawai et al. 1985, Beresford et al. 1992). One study estimated the frequency of bipotential osteo-adipoprogenitors at ~5% in RC cell populations (Bellows & Heersche 2001), but the frequency of various monopotential versus bi- or multipotential cells in RC populations remains unknown.

The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) has diverse effects on many cell lineages, including osteoblasts where it affects proliferation, apoptosis, specific bone protein expression as well as mineralization (reviewed in van Driel et al. 2004). In the RC cell model, 1,25(OH)₂D₃ inhibits bone nodule formation while concomitantly increasing the number of adipocyte foci (Bellows et al. 1994). However, it is not yet clear whether 1,25(OH)₂D₃ acts separately to inhibit committed osteoprogenitors and stimulate adipocyte precursors and/or whether 1,25(OH)₂D₃ induces an adipogenic fate in bipotential osteoadipoprogenitors or both. To address this issue, and dissect the presence and responsiveness of other multi-lineage mesenchymal precursors in RC cell populations, we analyzed gene expression profiles in untreated and 1,25(OH)₂D₃-treated single cell-derived RC colonies.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Cell culture and isolation of single cell-derived colonies

All procedures were approved by the University of Toronto Animal Care Committee. Fetal (E20–21) rat calvaria cells were harvested by serial enzymatic digestion as previously described (Bellows et al. 1986). Cells recovered from the last four of five enzymatic digestions were cultured overnight in T-75 flasks containing -MEM, 10% fetal bovine serum (FBS) and antibiotics (0.1 mg/ml penicillin G, 50 µg/ml gentamycin sulfate, 0.25 µg/ml fungizone), harvested with 0.2% trypsin, re-plated in 10-cm tissue culture dishes at a density of 10⁴/cm² and incubated at 37° C, 5% CO₂ for another 5 days. Medium was changed every 2 or 3 days. Cells were recovered by trypsinization, washed and re-suspended in Hank’s Balanced Salt Solution containing 2% FBS and 1 mM HEPES.

Single cell-derived RC cell colonies were obtained by sorting single cells into individual wells of 96-well cell culture dishes by fluorescence activated cell sorting (FACS) using a MoFlow equipped with an automatic cell deposition unit (ACDU) (Cytomation, Fort Collins, CO, USA). The cells were cultured in -MEM containing 10% FBS, antibiotics, ascorbic acid (50 µg/ml), ß-glycerophosphate (10 mM) and dexamethasone (10^–8 M) at 37°C in a 5% CO₂ humidified incubator for 4 weeks. Parallel cultures were treated in the same medium but with 10^–8 M 1,25(OH)₂D₃ (Sigma Co.).

At the end of 4 weeks, each well was examined by phase contrast microscopy; wells with identifiable colonies were marked and cells were lyzed with 350 µl RLT budffer (RNeasy, Qiagen) and stored at –80° C for RNA analysis.

RNA isolation, reverse transcription and real time PCR

Harvested colonies were randomly selected, total RNA was extracted with an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions, and samples were further digested with DNase (Qiagen). First strand cDNA was synthesized with SuperScript II reverse transcriptase and oligo-dT primer according to the manufacturer’s instructions (Invitrogen Inc.), and further purified and precipitated according to the protocol of Liss (2002). Real time PCR was performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems Inc., Foster City, CA, USA) with SYBR Green PCR Master Mix (Applied Biosystems) as buffer and source of fluorescence. A total of 50 cycles of amplification was used for all transcripts. Primer sequences, concentrations and annealing temperatures are listed in Table 1 for individual genes. An example of real-time PCR amplification of lineage-specific transcripts is shown in Fig. 1.

View larger version (20K): [in this window] [in a new window]   Figure 1 An example of real-time PCR amplification of lineage-specific transcripts (amplification of BSP in this figure; PCR was performed in triplicate for each transcript in each colony). (Left panels) Colony with BSP expression. (A) Amplification curve, showing exponential increase of fluorescence at certain cycle numbers (X-axis). (B) Dissociation curve in the same analysis. Under dissociation conditions, a drastic change of fluorescence within a narrow temperature range (X-axis) was seen, indicating amplification of gene-specific transcript. (Right panels) Colony without BSP expression. (C) No amplification curve, only background fluorescence, was detected. (D) Under dissociation conditions, fluorescence was emitted randomly over a wide range of temperatures and no single fluorescence peak was detected.

Immunocytochemistry

For immunocytochemical analysis of nestin and ß3 tubulin, single cells were inoculated and cultured as described above. At times as indicated, cells were fixed in fresh 4% paraformaldehyde, washed with PBS and incubated with antibodies against nestin (1:400, BD Biosciences, San Jose, CA, USA) or ß3 tubulin (1:500, Covance Inc., Princeton, NJ, USA), washed, then incubated with FITC-conjugated donkey anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Secondary antibody alone served as a negative control. After immunostaining, cell nuclei were counterstained with Hoechst 33258.

Statistical analysis

Comparison of the distribution of different types of progenitors in control and 1,25(OH)₂D₃ treatment groups was performed using a hypergeometric algorithm developed by Adams and Skopek, a generalization of Fisher’s ‘exact’ test for tables with more than two rows and two columns (Adams & Skopek 1987, Cariello et al. 1994). A two-tail Chi-square test for multiple rows was employed to compare individual lineages in different categories (single lineage, bi-lineage etc.) using a GraphPad Instat software package (GraphPad Software Inc., San Diego, CA, USA).

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Colony forming efficiency

In each experiment, fifteen 96-well culture dishes were used (total ~3000 wells inoculated at 1 cell/well). In the control group, approximately 9% of wells had colonies (4.7 and 13.3% in two independent experiments), while in the 1,25(OH)₂D₃ treatment group, approximately 8.7% (8.1 and 9.3%) of wells had colonies. There was no statistical difference in the colony forming effciency between the control and the 1,25(OH)₂D₃ treatment groups.

The frequency of mesenchymal lineage progenitors in RC cell populations

Amongst 134 and 75 independent colonies collected from the two groups, 60 colonies from each group were randomly selected for analysis by real-time PCR. From these, a total of 50 control and 49 1,25(OH)₂D₃-treated colonies were selected for lineage-specific expression profiling based on their similar level of expression of the housekeeping gene (ribosomal protein L32; detected by threshold cycle (Ct) value). The frequency of the different types of colonies from the control and 1,25(OH)₂D₃ treatment groups is summarized below and shown in Table 2 (the Ct values for expression of particular transcripts in individual colonies are available upon request).

Expression of neural markers in single cell-derived RC cell colonies

Given the high proportion of multi-lineage colonies observed and the fact that the parietal bone used for RC cell isolation derives from neuroectoderm, we next asked whether neural markers (nestin and ß3 tubulin) were expressed in any of the colonies isolated. Strikingly, 45 (90%) colonies in the control group expressed nestin and/or ß3 tubulin (34 expressed both markers; Table 2), and this frequency was not changed by 1,25(OH)₂D₃ treatment (42 expressed nestin and/or ß3 tubulin; 24 expressed both). Neural marker expression was seen across all mesenchymal colony types, and 1,25(OH)₂D₃ did not change the distribution (P > 0.05; Table 2). Amongst colonies designated CFC-F on the basis of the absence of all mesenchymal lineages tested, 1 of 2 in the control and 3 of 8 in the 1,25(OH)₂D₃ treatment groups had expressed neural markers.

To test whether nestin and ß3 tubulin protein expression occurred, 22 colonies from parallel wells as used for mRNA analysis were immunostained; neither nestin nor ß3 tubulin was detected in any of the colonies (data not shown).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
1,25(OH)₂D₃ regulates the development and differentiation of many cell types including monocyte-macrophages (Lindmark & Siegbahn 2002), osteoclasts (Suda et al. 1997), osteoblasts (Ishida et al. 1993, Bellows et al. 1994, van Driel et al. 2004), adipocytes (Kelly & Gimble 1998) and chondrocytes (Grigoriadis et al. 1989). These cell lineages co-exist under physiological and pathological conditions in bone, bone marrow and some other tissue compartments (Caplan 1991, Dorheim et al. 1993, Friedenstein 1995, Aubin 1998), but a comprehensive analysis of the ability of 1,25(OH)₂D₃ simultaneously to recruit or alter the fate of multiple mesenchymal lineage precursors has not previously been carried out. By using gene expression analysis of single cell-derived colonies, we have achieved new insights into the types of progenitors present in RC cell populations and the ability of 1,25(OH)₂D₃ to alter their fate choices.

Consistent with our previous estimates by limiting dilution cloning (Aubin et al. 1982, Grigoriadis et al. 1988, 1990) and osteoprogenitor frequency analysis (Bellows & Aubin 1989), ~ 8% of primary RC cells are capable of colony formation, suggesting that the majority of the population comprises relatively mature cells lacking colony forming capacity. Concomitantly, a large proportion (86%) of colonies are multipotential (at least bi- but also tri- and tetra-potential) on the basis of simultaneous expression of markers for multiple mesenchymal lineages. These data support the presence of a mesenchymal cell hierarchy in primary RC cell populations, with a preponderance of multipotential precursors amongst the colony-forming cells. A mesenchymal cell lineage hierarchy was suggested earlier in stromal tissues and clonally derived RC cell populations (Grigoriadis et al. 1988, 1990, Owen & Friedenstein 1988, Friedenstein 1995). Notably, however, we have found that the presence of each type of precursor was not evenly distributed in the RC cell analysis, e.g. about one third of precursors were of the CFC-O/A/C type. This might indicate a ‘default’ lineage selection under the osteogenic and 1,25(OH)₂D₃ treatment conditions used here, in keeping with the view that extrinsic factors can alter stem cell fate determination (Shah et al. 1994, 1996, Morrison et al. 1995).

In the current studies, RC colonies were defined based on mRNA expression patterns, not on functional criteria such as capacity of a single colony to give rise to more than one differentiated mesenchymal cell type after re-plating under appropriate culture conditions. We showed previously by clonal analyses that such functionally multipotential RC cells exist, but the strategy used to expand cells for testing also caused spontaneous immortalization of the isolated colonies (Grigoriadis et al. 1988, 1990). It remains a challenge to prove directly and without expansion/immortalization that single colonies expressing transcripts of multiple mesenchymal lineages are indeed primed for differentiation along these multiple mesenchymal lineages, since for most of the mesenchymal lineages the only reproducible differentiation culture conditions require cells to achieve high density in colonies (e.g. osteogenesis; Malaval et al. 1999) or require high initial plating cell density (e.g. chondrogenesis; Grigoriadis et al. 1988, Denker et al. 1999). However, evidence from hemopoietic cells suggests a dynamic development process of ‘promiscuous expression’ or ‘lineage primed’ progenitors that may contribute to flexibility and accessibility of multiple fate choices in particular progenitor populations (Miyamoto et al. 2002). Multilineage gene expression has also been detected in human bone marrow mesenchymal stem cells (Seshi et al. 2003). It is also worth noting, given the lineage markers we used, that expression of mature lineage markers in progenitors does not appear to affect their developmental potential; for example, transplanted hemopoietic stem cells expressing the myeloid lysozyme gene repopulate all hempoietic lineages (Ye et al. 2003). Such data have led to a ‘lineage priming’ model for stem cell differentiation, i.e. genes indicating multi-lineage potential are promiscuously expressed preceding commitment to a single lineage (Hu et al. 1997, Enver & Greaves 1998, Seshi et al. 2000, 2003, Miyamoto et al. 2002, Woodbury et al. 2002). Our data obtained from single RC cell-derived progenitors are consistent with such a model. The hierarchical distribution amongst colony types (tetra-, tri-, bi- and mono-lineage) in RC cell populations suggests that when individual multipotential mesenchymal stem or progenitor cells differentiate to more committed progenitors, the genes responsible for other non-selected lineages are extinguished. Similar to hemopoietic stem cells (Ye et al. 2003), in a primed stage, individual RC progenitor cells may express genes encoding lineage exclusive functions (e.g. BSP, adipsin, collagen II, etc), but remain in an apparent state of indecision.

Interestingly, the proportion of tri- and tetra-lineage colonies was not changed after 1,25(OH)₂D₃ treatment, suggesting developmental stage-specific effects 1,25(OH)₂D₃of on RC mesenchymal progenitors. The developmental stage-dependent recruitment of 1,25(OH)₂D₃ on different precursors may contribute to the biphasic effects of 1,25(OH)₂D₃ described previously on adipocyte-osteoblast differentiation (Owen et al. 1991, Ishida et al. 1993, Kelly & Gimble 1998, Atmani et al. 2002). However, the frequency of other progenitor cell types is affected. For example, the frequency of CFC-C was increased after 1,25(OH)₂D₃ treatment, suggesting a stimulatory effect of 1,25(OH)₂D₃ on chondrogenesis.1,25(OH)₂D₃ also decreased the frequency of osteoblast/chondrocyte progenitors (from 20% to 6%; P = 0.03 by one-tail Chi-square test). Although the statistical robustness of the observation is relatively low, the increased CFC-C and decreased CFC-O/C frequency suggests that 1,25(OH)₂D₃ stimulates chondrogenesis by recruitment from the CFC-O/C progenitor pool. However, we cannot rule out an effect of 1,25(OH)₂D₃ on other pathways that may contribute to the CFC-C pool.

The frequency of osteoblast/adipocyte bipotential progenitors has been predicted to be ~5% in primary RC cell populations (Bellows & Heersche 2001). Such cells are detectable at somewhat higher frequency by histological assessment of single cell-derived colonies in very low density cultures after 1,25(OH)₂D₃ treatment, where about a quarter of total colonies (25.2 ± 10%) are stained for lipid (Oil red O) and for the presence of the osteoblast marker alkaline phosphatase, whereas only alkaline phosphatase-positive colonies are detectable in the control group (authors’ unpublished data). Consistent with previous studies in high density cultures (Ishida et al. 1993, Bellows et al. 1994, Bellows & Heersche 2001), 1,25(OH)₂D₃ treatment resulted in loss of CFC-O, indicating an inhibitory effect of 1,25(OH)₂D₃ on committed osteoprogenitors in RC cell cultures. Data such as these have contributed to the hypothesis that the fates of osteoblast versus adipocyte are reciprocally related in RC and stromal cell populations (reviewed in Nuttall & Gimble 2000, Aubin & Trifftt 2003, Gimble & Nuttall 2004). Notably, however, the loss of CFC-O was not accompanied by an increase in CFC-A frequency in our studies, but instead by a significant increase in frequency of CFC-O/A bipotential progenitors (0% to 8%), suggesting that the reciprocal effects of 1,25(OH)₂D₃ on osteogenesis and adipogenesis are mediated by the enhanced recruitment of CFC-O/A. Because 1,25(OH)₂D₃ did not affect the frequencies of tri- and tetrapotential colonies, a dedifferentiation process of CFC-O to CFC-O/A may underlie the decreased CFC-O and increased CFC-O/A frequency observed.

Some progenitors were identified as CFC-Fs in the current study based on absence of expression of any of the mesenchymal lineage markers tested. Such cells may represent a very primitive mesenchymal progenitor or they may belong to lineages for which markers were not tested. In either case, 1,25(OH)₂D₃ significantly increased the frequency of CFC-Fs (P = 0.04), suggesting that 1,25(OH)₂D₃ either re-directs some progenitors towards other mesenchymal lineages or dedifferentiates progenitors to a more primitive stem cell stage. Dedifferentiation was suggested to be responsible for the observation that highly differentiated adipocytes revert to a less differentiated, more proliferative fibroblastic precursor and then to an osteogenic phenotype under certain culture conditions (Park et al. 1999). Based on CFC-Fs and the other colony types observed, we propose that 1,25(OH)₂D₃ alters the fate choice of mesenchymal lineage precursors at several bifurcation points as summarized in Fig. 2.

View larger version (7K): [in this window] [in a new window]   Figure 2 Proposed mechanisms of the effects of 1,25(OH)2D3 on the recruitment of mesenchymal lineage precursors. Open up and down arrows indicate respectively a stimulatory or inhibitory effect of 1,25(OH)2D3 on progenitors observed in the current study. The proposed stimulatory effect of 1,25(OH)2D3 on dedifferentiation of CFC-O/C/A/M or other progenitors to CFC-F or redirection to other mesenchymal lineages is based on the evidence of increased CFC-F frequency. CFC-O/A/M, O/M, A/M, C/M, CFC-A and CFC-M were not observed in the current culture conditions and are not shown in the diagram. CFC, colony forming cell; O, osteoblast; C, chondrocyte; A, adipocyte; M, myocyte; F, fibroblast lineage; MSC, mesenchymal stem cell.

In conclusion, single RC cell-derived colonies define a mesenchymal cell lineage hierarchy in which 1,25(OH)₂D₃ alters fate choices in a developmental stage-specific manner.

	Acknowledgements

 
The authors thank Usha Bhargava for technical support and Dr S A M Taylor for discussion of the manuscript.

	Funding

This work was supported by grants from the Canadian Institutes of Health Research (MT12390) and the Stem Cell Network of Centres of Excellence to J E A. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received in final form 1 February 2006
Accepted 20 February 2006

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