Yonago Acta medica 2015;58:23–29

Original Article

Construction of a Luciferase Reporter System to Monitor Osteogenic
Differentiation of Mesenchymal Stem Cells by Using a Mammalian
Artificial Chromosome Vector
Takashi Narai,* Motonobu Katoh,†§ Toshiaki Inoue,†§ Makoto Taniguchi,‡¶ Kanako Kazuki,§‖ Yasuhiro Kazuki,§‖Kenzo Sato,‡ Isamu Kodani,* Kazuo Ryoke* and Mitsuo Oshimura§*Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan, †Division of Human Genome Science, Department of Molecular
and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan, ‡Division of Molecular
Biology, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 6838503, Japan, §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan and ‖ Department of Biomedical
Science, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago, Tottori 683-8503, Japan

ABSTRACT

in Chinese hamster ovary cells (OC-Luc/MAC) andtransferred this into human MSC cells via chromosometransfer.
Results
OC-Luc/MAC in human MSC cells are responsive to positive and negative stimulation by 1 alpha,25-dihydroxyvitamin D3 and DEX in differentiation
stage of MSCs to osteoblasts, reflecting the manner ofphysiological expression.
Conclusion The OC-Luc/MAC reporter system maycontribute not only to monitoring the osteogenic differentiation stage from MSC but also to identify novel
osteogenic drugs.

Background

Mesenchymal stem cells (MSCs) holdpromise for application in adult stem cell-mediated regenerative medicine in bone remodeling and fracture repair. MSCs in vitro can be directed to osteogenic lineage
by dexamethasone (DEX); however, the use of DEX is
not practical in clinical settings because of adverse side
effects such as glucocorticoid-induced osteoporosis.
For identifying substances that facilitate osteogenesis,a monitoring system, which detects the osteogenic differentiation stage of MSCs accurately and easily, is required.
Methods By focusing on the human osteocalcin (OC)gene whose expression profile is described along withosteogenic differentiation, we constructed the luciferase(Luc) reporter gene driven by the enhancer/promoter
sequence of the human OC gene (OC-Luc) utilizing a
mammalian artificial chromosome. Mammalian artificial chromosome is a suitable platform for loading
reporter constructs, because of its stable episomal maintenance in host cells, transferability into any cell and assurance of long-term physiological transgene expression.
We loaded the OC-Luc on a mammalian artificial chromosome vector engineered from mouse chromosome(designated as mouse artificial chromosome, MAC)

Key words

mammalian artificial chromosome; mesenchymal stem cells; osteogenic differentiation; osteocalcin; reporter assay
In the human body, the skeletal system is one of thelargest organs, which undergoes remodeling throughout a person’s lifespan.1 Bone remodeling and fracture
healing processes are thought to be mediated via osteoprogenitors and their ancestors, mesenchymal stem
cells (MSCs).2 In in vitro studies, a protocol using dexamethasone (DEX) is generally accepted for inductionof osteoblastic differentiation of MSCs.3 In a clinicalsetting, however, the use of DEX is not practical becauseof its adverse side effects such as glucocorticoid-induced
osteoporosis.4 For successful treatment of bone-loss disorders, chemical compounds and bioactive substancesthat effectively induce bone formation are required.
In an effort to develop an evaluation system for bone
remodeling, we previously generated transgenic mice that
express the luciferase (Luc) reporter gene under the control of human osteocalcin enhancer/promoter sequences.5
Osteocalcin (OC) is the main non-collagenous hydroxyapatite-binding protein, which is synthesized by osteoblasts, odontoblasts and hypertrophic chondrocytes.6 It
was recognized as a marker of bone formation6 and has

Corresponding author: Motonobu Katoh, PhD
mkatoh@med.tottori-u.ac.jp
Received 2014 December 16
Accepted 2015 January 5
Abbreviations: ALP, alkaline phosphatase; CHO, Chinese hamster ovary; DEX, dexamethasone; DMEM, Dulbecco’s modified
Eagle’s medium; ECM, extracellular matrix; FISH, fluorescencein situ hybridization; FBS, fetal bovine serum; hiMSC, humanimmortalized MSC line; Luc, luciferase; MAC, mouse artificial
chromosome; MMCT, microcell-mediated chromosome transfer; MSC, mesenchymal stem cell; OC, osteocalcin; OPN, osteopontin; VD3, dihydroxyvitamin D3¶Present address: Department of Life Science, Medical ResearchInstitute, Kanazawa Medical University, 1-1 Daigaku Uchinada,
Kanazawa 920-0293, Japan23

T. Narai et al.

been used for detection of osteoblast maturation andosteogenic activation.7 Using in vivo bioluminescenceimaging in the OC-Luc transgenic mice, Luc activity
was induced throughout their skeletons by administration of 1,25-dihydroxyvitamin D3 (VD3), which regulates the expression of a number of genes including OC
in bone cells.8 Furthermore, enhanced bioluminescence
was observed at the forced radius fracture site reflecting
progression of the fracture repair. Although the OC-Luc
transgenic mice would contribute to the final confirmation of drug efficacy selected from hundreds of candidates, an in vitro cell based-assay system is preferable forthe first screening of high content candidate compounds.
Mammalian artificial chromosome vectors havebeen utilized for introduction of genes to recipient cells.9
As for the origin of their chromosomes, there are twokinds of artificial chromosomes, i.e. human artificial
chromosome (HAC) and mouse artificial chromosome(MAC) that have been constructed by chromosome engineering. Since HAC or MAC is maintained as an independent chromosome in recipient cells, physiologicalregulation of the loaded gene is expected.10 Previously,
feasibility of cell lineage-specific expression of a transgene by a HAC vector was demonstrated in a human
MSC line.11 The GFP reporter gene under the regulation of human osteopontin (OPN) promoter loaded on a
HAC vector was transferred to a human MSC line. OPN
is another marker of bone formation, whose expression
is upregulated during maturation of osteoblasts.7 The reporter GFP gene in the MSCs was specifically expressedin response to osteogenic differentiation induction in coordination with the transcription of endogenous OPN. In
order to distinguish the osteoblast differentiation processfrom the subsequently occurred maturation processes
accurately, however, reference reporter system, which
can trace osteoblast differentiation steps, is required.
Our aim is setting up a screening platform to identify compounds that promote or induce differentiationof MSCs towards osteoblasts. To this end, we made areporter construct composed of the Luc gene under the
regulation of OC enhancer/promoter in a MAC vector(OC-Luc/MAC). The MSC lines carrying the OC-Luc/
MAC vector were tested for differentiation ability toosteogenic lineage. The induction or restraint of Luc activity responding to the already-known positive stimulus
via VD3 or negative stimulus via DEX treatment wasalso addressed for validation as monitor cells.

F-12 nutrient mixture (Wako, Osaka, Japan) supplemented with 10 % fetal bovine serum (FBS) (Biowest,
Kansas, MO). The MAC4 carries an EGFP gene,
5’HPRT-loxP and a hygromycin gene.10 A mouse A9
hybrid cell line carrying the MAC4 vector was maintained in Dulbecco’s modified Eagle’s medium (DMEM)(Wako) supplemented with 10% FBS (Biowest). A
human immortalized MSC line (hiMSC)12 was maintained in DMEM (Wako) supplemented with 10% FBS(Biowest). To induce osteogenic differentiation, hiMSCderived hybrid cells were fed with osteogenic differentiation medium (LONZA, Basel, Swiss Confederation).
Cells were maintained at 37 °C with 5% CO2.
Construction of OC-Luc/MAC4 reporter vector

The plasmid pOC-Luc contains firefly Luc gene derived
from pGL3 (Promega, Madison, WI) driven by 10 kb
enhancer/promoter region of the human OC gene.5 The
plasmid v907-3’HPRT/loxP contains the donor loxP siteand latter half of the HPRT gene. The 14.4 kb AseI/SalI
fragment containing the Luc gene expression cassette
was excised from the pOC-Luc plasmid and ligated tothe compatible AseI/SalI site of the v907-3’HPRT/loxP
plasmid, yielding the v907 OC-Luc-ins-3’HPRT/loxP.
Plasmid transfection was carried out by lipofection (Lipofectamine 2000, Invitrogen, Carlsbad, CA). Drug resistant colonies obtained by selection with HAT (SigmaAldrich, St.Louis, MO) were picked up and expanded.
Presence of the sequence on OC-Luc/MAC4 was tested
by PCR amplification.
Assessment of the hOC/Luc transcription activity
in CHO/MAC4 cells

The HAT resistant CHO/MAC4 transfectants wereplated (1 × 106 cells in 6 well cluster). The next day, cells
were harvested and lysed in 100 mL lysis buffer (Reporter
Lysis Buffer, Promega). An aliquot (20 mL) of lysate was
mixed with 50 mL of Luc solution and measured witha luminometer (ALVO MX, Perkin Elmer, Waltham,MA). For OC-Luc stimulation study, the HAT resistant
CHO/MAC4 transfectants were plated (1 × 106 cells
in 35 mm dish). After treatment with 100 nM VD3 orvehicle (DMSO) for 6 h, cells were washed twice withphosphate-buffered saline, harvested and lysed in 100
mL lysis buffer. Lysate (20 mL) was mixed with 50 mL of
Luc substrate. Luc activity was measured by a luminometer (ALVO MX, Perkin Elmer). Statistical analysis was
performed using unpaired Student’s t-test (two-tailed).

MATERIALS AND METHODS
Cell culture

Microcell mediated chromosome transfer

A Chinese hamster ovary (CHO) hybrid cell line containing the MAC4 vector10 was maintained in Ham’s

The OC-Luc/MAC4 vector was introduced into hiMSCs
by microcell-mediated chromosome transfer (MMCT)24

Osteogenic differentitation monitoring cellFig. 1. Construction of the OC-Luc/MAC4 reporter vector in donor CHO cells.
A: A scheme of loading human osteocalcin luciferase (hOC-Luc) into MAC4. MAC4 has an EGFP gene driven by CAG promoter, 5’HPRT
and acceptor loxP site. The OC-Luc construct is composed of 10 kb human osteocalcin enhancer/promoter sequences with 60 bp of
the 5’-untranslated sequences (Oc e/p), a luciferase gene (FLuc), the SV40 late polyadenylation signal (pA), the two copies of insulator
sequences (Ins), 3’HPRT and donor loxP site. The loxP site-specific integration reconstructs the HPRT gene that confers resistance to
HAT selection. The predicted composition after the loxP site-specific integration is presented at the bottom. The arrows indicate PCRprimers for detection of the OC-Luc in MAC4.
B: Presence of the OC-Luc sequences in HAT resistant CHO transfectants was detected by PCR amplification with the primer pairs indicated in A. Size of each amplicon is shown on the right side of the panel. C, CHO/MAC4 genomic DNA; M, size marker; V, vector
DNA (OC/Luc) used for transfection.

as described previously.13, 14 While microcells preparedfrom the A9 donor cells were fused with hiMSCs using
47% polyethylene glycol (molecular weight 1000) (Wako)
and 10% DMSO (Sigma-Aldrich), microcells from theCHO donor cells were fused by fusogenic envelope proteins derived from measles virus.14 Microcell hybrids
were selected with HAT supplement (Sigma-Aldrich) for
2 weeks.

of host cells was counterstained with DAPI (SigmaAldrich). Microscopic images were captured using the
NIS-Elements system (Nikon, Tokyo). Thirty metaphasespreads were observed for each analysis.
Induction of osteogenic differentiation

The hiMSC cells (3 × 104 cells per well) were seeded in
6-well plates with growth medium. The next day, medium was changed to differentiation medium (LONZA)
with or without 10 nM VD3 (Calbiochem, Tokyo). After
1 week of osteogenesis culture, alkaline phosphatase activity (ALP) and luciferase activity were tested.

FISH

Preparation of metaphase chromosome spreads from thehybrid cells and fluorescence in situ hybridization (FISH)
analyses were carried out with standard protocols.13
Digoxigenin-labelled (Roche, Tokyo, Japan) mouse Cot1 DNA (Invitrogen) and biotin-labelled (Roche) hOCLuc DNA were used as probes. Chromosomal DNA

Detection of ALP activity
Histochemical staining for ALP activity was carried out
using a commercially available kit based on naphthol
25

T. Narai et al.
Table 1. Summary of FISH analyses in CHO (MAC4hOC/Luc) cells

AS-MX phosphate and fast red violet B salt (SigmaAldrich). The cultured cells were fixed with citrate
fixative containing 60% acetone for 30 s at room temperature, rinsed twice with deionized water, treated with
ALP staining solution for 1 h, rinsed twice with deionized water and then photographed.
Luciferase assay

Cells were lysed by the addition of 100 mL Reporter
Lysis Buffer (Promega) in culture vessels, followed bya freeze-thaw cycle. Protein concentration of the celllysate was determined by ultraviolet absorption spectrophotometry. Luc activity was measured using the Pikkagene Luciferase Assay System (TOYO B-Net, Tokyo).
Luc activity was normalized to the protein concentrationof the cell lysate.

Clone#

MAC#		
0
1

Integration* Others†

4
10
13
14
17

3
1
0
2
1

1
3
1
2
3

13
13
17
13
12

3
3
2
3
4

*MAC was integrated into a host chromosome(s).
†Ploidy of the host cells was elevated to tetraploid range.

fragment carrying upstream from the human OC gene
was chosen (Fig. 1A). The fragment contains a VD3-responsive element (VDRE), a GAGA motif and a TATA
box.5 Co-transfection of the OC-Luc reporter construct
and the Cre recombinase expression vector pBS185 into
the CHO cells carrying the MAC4 vector yielded HAT
resistant colonies after selection culture. Presence of the
OC-Luc sequence in the HATr cell clones was first tested by PCR amplification of the OC-Luc and MAC4 specific sequences in genomic DNA of the candidate CHO
clones (Fig. 1B). The junction sequence between the OC
promoter and Luc gene was detected in almost all clones
tested. The sequence spanning the 5’half and 3’half of
the HPRT gene was also detected, indicating the correctsite-specific insertion of the OC-Luc construct at theloxP site on the MAC4 vector.
Since the 560 bp minimum promoter of the OC
gene was previously found to exhibit promoter activity

RESULTS
Preparation of the OC-Luc/MAC reporter vector indonor CHO cells

Stable maintenance of vectors in host cells is crucial forgene delivery studies. We chose the MAC4 vector as aplatform for the OC-Luc reporter construct, because of
its stable maintenance in cultured human cells in addition to mouse cells and transchromosomic mice. 15 It
carries the GFP gene driven by CAG promoter, whichallows constitutive expression in various types of cells.
As a regulatory sequence for the monitoring of OC
gene expression, an approximately 10 kb genomic DNA

Fig. 2. Detection of Luc activity in CHO
cells.
A: Luciferase activity was assessed in
the HAT resistant CHO transfectants. Measured activities were normalized to those of parental CHO/
MAC4 cells. *P < 0.01: compared to
the CHO/MAC4 control value.
B: Response of the OC/Luc to VD3
was tested in the HAT resisitant
CHO transfectants. Luciferase activity was measured after 6 h incubation with or without 100 nM VD3.
The measurements were normalized
to those of cells treated with DMSO.
*P < 0.01: compared to the DMSO
control value.
The bar above each column represents
SD determined from three independentmeasurements (A and B).

26

Osteogenic differentitation monitoring cellA

in CHO cells,16 we next addressed the functional maintenance of the OC-Luc reporter activity in CHO cellsby Luc assay (Fig. 2A). Luc activity was substantially
increased in CHO (OC-Luc/MAC4) cells, compared to
the CHO (MAC4) cells in which the OC-Luc construct
is absent. As the measured Luc activity varied among
CHO clones, we arbitrarily chose the 5 clones showing
relatively high Luc activity, and performed FISH analysis to examine the status of the MAC (Table 1). In eachclone, among 20 metaphase spreads tested, over 60%
showed diploid CHO karyotype plus one copy of the
MAC. The retention rate of the MAC in CHO cells washigh enough for use as an MMCT donor.
Since CHO cells were reported to respond to VD3
stimulation through VD3 receptor expression,17 we next
tested the OC-Luc reporter activity in the CHO cells (Fig.
2B). VD3 treatment induced Luc activity that confirmed
the responsiveness of the OC regulatory sequence in
the OC-Luc/Mac4 vector in the CHO background. Two
CHO clones (#4 and #17) were arbitrarily chosen as thedonor cells for the transfer of the OC-Luc/MAC4 vector
to the human MSC cells. From CHO clone #4, the OCLuc/MAC4 vector was once transferred by MMCT tomouse A9 cells that were used as intermediate host cells
because of being highly competent to produce micronuclei.

#4

#17

C

OD

B

Parental MSC

#4

#17

Fig. 3. Detection and characterization of the OC-Luc/MAC4 vector in hiMSC hybrid cells.
A: Representative metaphase spreads of FISH of the hiMSC
hybrid cells. Digoxigenin-labelled mouse Cot-1 DNA (red)was used for detection of the MAC4. Biotin-labelled OC-Luc
DNA (green) was used for detection of the OC-Luc cassette in
the MAC4. Chromosomal DNA in the host hiMSC cell wascounterstained with DAPI (blue). White arrowheads indicate
the OC-Luc/MAC4 vectors and the insets show enlarged images of the OC-Luc/MAC4.
B: ALP staining of the hiMSC hybrid and parental cells. Photographs of the fixed and stained cells on the culture vessel were
presented. Upper panels show the cells cultured for 7 days inthe differentiation induction medium (OD), and lower panels
show the cells cultured for 7 days in the growth medium (C).
Red dye staining indicates ALP activity. Scale bar shows 1
mm.

Transfer of the OC-Luc/MAC4 reporter vector to amesenchymal stem cell line

A mesenchymal stem cell line hiMSC, which retains
differentiation potential to osteogenic lineages12 provided recipient cells for the assessment of the OC-Luc/
MAC4 reporter vector. To utilize the reconstructed
HPRT gene as a selection marker for MMCT, an HPRT–
subline established by 6-thioguanine selection was used.
From the donor cells (A9#4 and CHO#17), MMCT was
carried out to the hiMSC (HPRT–) cells. As a result of
HAT selection culture, five and one HPRTr hybrids wereobtained from the two OC-Luc/MAC4 donor clones #4
and #17, respectively. Retention of the OC-Luc/MAC4
was addressed by FISH analysis (Fig. 3A). Karyotypesof the metaphase spreads were not necessarily uniform
and a few variations were observed (data not shown).
Since the extent of the variation differed among theclones, we chose the two hiMSC clones (#4 and #17)
showing the highest retention score (over 40%) of asingle copy MAC without host chromosome integration.

gene, the hiMSC hybrids were cultured in osteogenic
differentiation medium, which contains DEX, ascorbic
acid and beta-glycerophosphate.3 During osteogenic
differentiation of MSCs, ALP activity is upregulated in
the early stage.7 ALP activity was addressed by histochemical staining assay at day 7 (Fig. 3B). In the hiMSC
hybrid cells, qualitative difference in ALP activity between DEX treated and untreated culture was detected,
indicating that the hiMSC hybrid cells retain osteogenic
differentiation potential.
Next, the hiMSC hybrid cells were tested for their
OC-Luc activity in response to a given stimulation. As
agonistic and antagonistic compounds for the OC transcription, VD3 and DEX were used, respectively (Fig. 4).
While culture for 7 days in differentiation medium with
DEX scarcely induced luciferase activity, addition of

Evaluation of the OC-Luc/MAC4 reporter vector in
the mesenchymal stem cell hybrids

As differentiation potential is a prerequisite for monitoring the transcriptional regulation of the OC-Luc reporter27

T. Narai et al.

VD3 and restrained by DEX. These results suggest that
the OC-Luc reporter construct is faithfully regulated bypositive and negative stimulation during the differentiation of MSCs.
Comparison of VD3 and DEX effects on OC induction was reported in human marrow stromal cells.18
The report demonstrated no apparent induction of OC
mRNA expression by DEX, moderate induction by VD3
and prominent induction by combined application of
DEX and VD3. For the screening of substances using
our OC-Luc reporter cells system, four culture medium
compositions are supposed, i.e. the MSC growth medium with or without VD3 and the osteogenic differentiation medium containing DEX with or without VD3. Detection of OC-Luc activation in the media without VD3
may be read as identifying the substitution for DEX ornovel differentiation inducer. In the media with VD3, itis expected that the synergistic enhancer for VD3 hasbeen found.
In addition to optimizing the composition of culture
medium, control of stem cell fate has been explored in
the aspect of signal transduction by adhesion and mechanosensory stimuli from the extracellular matrix andsubstratum. Integrin-dependent cellular interactions withthe extracellular matrix could control the differentiationof stem cells.23 Another report described that the elasticity of the underlying substratum influences the differentiation of MSCs.24 Moreover, surface characteristics
of substratum, like topography, surface chemistry andsurface energy, are critical parameters for the adhesionof stem cells to biomaterials and for differentiation into
the osteogenic lineage. In the development of new materials for medical implants, these parameters have to betaken into account.25 The OC-Luc/MAC repoter system
may be applicable for evaluation of biophysical signalingfrom culture substratum.
In this study, we established an evaluation system,
which can trace differentiation of MSCs toward osteoblast by monitoring Luc activity under the control of OC
gene regulatory sequences. By utilizing this system, theability of a given chemical and/or physical stimulation
to either facilitate or retard osteoblastic differentiation
could be monitored in a time-dependent manner. Consequently, our approach could offer a qualified tool for
temporal monitoring of osteogenesis in studies dealing
with drug screening and tissue engineering.

Fig. 4. Responsiveness of the OC-Luc activity was tested against
VD3 treatment and osteogenic induction medium containing
DEX. Effect of VD3 addition (10 nM) was tested in culture medium (C) and osteogenic induction medium (OD) at day 7. Data areexpressed by mean values and distribution range from duplicate
experiment.

VD3 in either growth medium or differentiation medium
affected the induction of Luc activity. These results support the previously reported effects of VD3 and DEX for
OC transcriptional regulation in the early stage of osteogenic differentiation of human MSCs.3, 18 It was notedthat combined application of VD3 to the DEX medium
exceeded the extent of activation by VD3 treatment
alone, suggesting a synergistic effect of differentiationinduction and VD3.
The MAC4 vector is equipped with the EGFP gene
under the regulation of CAG promoter (Fig. 1A). GFP
fluorescence was consistently observed regardless of culture medium compositions and added supplements (datanot shown), indicating the utility of GFP as an internal
control for the evaluation by the OC-Luc reporter system.
DISCUSSION
In addition to its role in systemic calcium homeostasis,
VD3 affects cell proliferation, differentiation and function of cells expressing the vitamin D receptor.19 VD3
was reported to enhance the differentiation of hMSCs toosteoblasts, suggesting a potential autocrine/paracrinerole of VD3 in osteoblast differentiation.20 On the contrary, DEX contained in the osteogenic differentiation
medium3 negatively regulates OC transcription throughglucocoSticoid receptor during osteogenic lineage commitment of MSCs.21 It was also reported that glucocoSticoid receptor binding interfered with the OC basal
promoter.22 In the present study, we showed that OCLuc activity in the MSC hybrid cells was stimulated by

Acknowledgments: We thank Professor Hiroyuki Kugoh for his
invaluable advice and critical review of this research.
This study was supported in part by Regional Innovation
Strategy Support Program by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (M.O.).
The authors declare no conflict of interest.

28

Osteogenic differentitation monitoring cell
14 Katoh M, Kazuki Y, Kazuki K, Kajitani N, Takiguchi M,
Nakayama Y, et al. Exploitation of the interaction of measles
virus fusogenic envelope proteins with the surface receptor
CD46 on human cells for microcell-mediated chromosometransfer. BMC Biotechnol. 2010;10:37. PMID: 20444293.
15 Kazuki K, Takehara S, Uno N, Imaoka N, Abe S, Takiguchi M,
et al. Highly stable maintenance of a mouse artificial chromosome in human cells and mice. Biochem Biophys Res Commun. 2013;442:44-50. PMID: 24216103.
16 Takahashi Y, Tsuji S, Kazuki Y, Noguchi M, Arifuku I,
Umebayashi Y, et al. Development of evaluation system forbioactive substances using human artificial chromosome-mediated osteocalcin gene expression. J Biochem.2010;148:29-34.
PMID: 20299329.
17 Dokoh S, Donaldson CA, Marion SL, Pike JW, Haussler MR.
The ovary: a target organ for 1,25-dihydroxyvitamin D3. Endocrinology. 1983;112:200-6. PMID: 6291916.
18 Beresford JN, Joyner CJ, Devlin C, Triffitt JT. The effects ofdexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic
differentiation of human marrow stromal cells in vitro. Arch
Oral Biol. 1994;39:941-7. PMID: 7695507.
19 Geng S, Zhou S, Bi Z, Glowacki J. Vitamin D metabolism inhuman bone marrow stromal (mesenchymal stem) cells. Metabolism. 2013;62:768-77. PMID: 23375059.
20 Liu P, Oyajobi BO, Russell RG, Scutt A. Regulation of osteogenic differentiation of human bone marrow stromalcells: interaction between transforming growth factor-beta
and 1,25(OH)(2) vitamin D(3) In vitro. Calcif Tissue Int.
1999;65:173-80. PMID: 10430653.
21 Leclerc N, Noh T, Khokhar A, Smith E, Frenkel B. Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20-binding enhancer. Arthritis Rheum.
2005;52:929-39. PMID: 15751078.
22 Meyer T, Gustafsson JA, Carlstedt-Duke J. Glucocorticoiddependent transcriptional repression of the osteocalcin geneby competitive binding at the TATA box. DNA Cell Biol.
1997;16:919-27. PMID: 9303434.
23 Martino MM, Mochizuki M, Rothenfluh DA, Rempel SA,
Hubbell JA, Barker TH. Controlling integrin specificity andstem cell differentiation in 2D and 3D environments throughregulation of fibronectin domain stability. Biomaterials.
2009;30:1089-97. PMID: 19027948.
24 Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity
directs stem cell lineage specification. Cell. 2006;126:677-89.
PMID: 16923388.
25 Anselme K. Osteoblast adhesion on biomaterials. Biomaterials. 2000;21:667-81. PMID: 10711964.

REFERENCES
1 Harada S, Rodan GA. Control of osteoblast function andregulation of bone mass. Nature. 2003;423:349-55. PMID:
12748654.
2 Wang X, Wang Y, Gou W, Lu Q, Peng J, Lu S. Role of mesenchymal stem cells in bone regeneration and fracture repair: areview. Int Orthop. 2013;37:2491-8. PMID: 23948983.
3 Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem. 1997;64:295-312.
PMID: 9027589.
4 Bultink IE, Baden M, Lems WF. Glucocorticoid-induced osteoporosis: an update on current pharmacotherapy and future
directions. Expert Opin Pharmacother. 2013;14:185-97. PMID:
23317448.
5 Nakanishi T, Kokubun K, Oda H, Aoki M, Soma A,
Taniguchi M, et al. Bioluminescence imaging of bone formation using hairless osteocalcin-luciferase transgenic mice.
Bone. 2012;51:369-75. PMID: 22732553.
6 Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin
and matrix Gla protein: vitamin K-dependent proteins in bone.
Physiol Rev. 1989;69:990-1047. PMID: 2664828.
7 Lian JB, Stein GS. Development of the osteoblast phenotype:molecular mechanisms mediating osteoblast growth and differentiation. Iowa Orthop J. 1995;15:118-40. PMID: 7634023.
8 Haussler MR, Whitfield GK, Kaneko I, Haussler CA, Hsieh D,
Hsieh JC, et al. Molecular mechanisms of vitamin D action.
Calcif Tissue Int. 2013;92:77-98. PMID: 22782502.
9 Oshimura M, Katoh M. Transfer of human artificial chromosome vectors into stem cells. Reprod Biomed Online.
2008;16:57-69. PMID: 18252049.
10 Takiguchi M, Kazuki Y, Hiramatsu K, Abe S, Iida Y, Takehara S, et al. A Oovel and Ttable Nouse BrtificialDhromosome
WFctor. ACS Synth Biol. &QVC20120DU. PMID: 23654256.
11 Ren X, Katoh M, Hoshiya H, Kurimasa A, Inoue T, Ayabe F,
et al. A novel human artificial chromosome vector provides
effective cell lineage-specific transgene expression in humanmesenchymal stem cells. Stem Cells. 2005;23:1608-16. PMID:
16141362.
12 Okamoto T, Aoyama T, Nakayama T, Nakamata T, Hosaka
T, Nishijo K, et al. Clonal heterogeneity in differentiation
potential of immortalized human mesenchymal stem cells.
Biochem Biophys Res Commun. 2002;295:354-61. PMID:
12150956.
13 Kugoh H, Mitsuya K, Meguro M, Shigenami K, Schulz
TC, Oshimura M. Mouse A9 cells containing single humanchromosomes for analysis of genomic imprinting. DNA Res.
1999;6:165-72. PMID: 10470847.

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