Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro$ Yanhong Yu a,1, Layla Al-Mansoori a,b,1, Michal Opas a,n a b

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Chemistry & Earth Sciences, College of Arts and Science, University of Qatar, P.O. Box 2713, Doha, Qatar

art ic l e i nf o

a b s t r a c t

Article history: Received 10 June 2014 Received in revised form 11 November 2014 Accepted 17 December 2014

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. One such lineage is osteoblasts, which are responsible for forming bone tissue in the body. There are many osteogenic differentiation protocols in the literature utilizing different soluble factors. The goal of the present study was to increase the efficacy of our osteogenic differentiation protocol from R1 cells. We have studied the effects of the addition of the following factors: dexamethasone, retinoic acid, and peroxisome-proliferator-activated receptor-gamma inhibitor, which have been reported to enhance osteogenesis. We found that among the 6 different protocols that were tested, the addition of retinoic acid with later addition of dexamethasone gives the most enrichment of osteogenic lineage cells. Thus, our findings provide valuable guidelines for culture condition to differentiate mouse R1 ESCs to osteoblastic cells in vitro. & 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Keywords: Embryonic stem cells Osteogenic differentiation In vitro culture

1. Introduction Embryonic stem cells (ESCs) are primitive cells with the potential to give rise to all types of cells in the body. Thus, they have tremendous potential in clinical applications and they have been extensively studied in the past few decades. Embryoid bodies (EBs) are aggregates of ESCs formed in vitro. They represent a unique tool to investigate developmental processes and the generation of the three germ layers (endoderm, ectoderm and mesoderm), since their growth is similar to embryonic development (Weitzer, 2006). Several studies generated osteoblasts via the formation of EBs within 3–4 weeks in the presence of osteogenic factors, while others have also successfully differentiated osteoblasts directly from ESCs in 10 days,

Abbreviations: AA, ascorbic acid; Alp, alkaline phosphatase; Bmp-2, bone morphogenetic protein-2; Bsp, bone sialoprotein; B-Gly, β-Glycerolphosphate; Dex, Dexamethasone; EB, embryoid body; ESC, Embryonic stem cell; Ppar, peroxisome proliferator-activated receptor; Ocn, osteocalcin; Osx, osterix; RA, retinoic acid; Runx2, runt-related transcription factor 2 ☆ This work was supported by Grants from CIHR MOP-102549 and MOP-106461 to M.O. n Correspondence to: Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, room 6326, Toronto, Ontario, M5S 1A8 Canada. Tel.: þ1 416 978 8947; fax: þ1 416 978 5959. E-mail address: [email protected] (M. Opas). 1 These authors had equal contribution.

without the formation of EBs (Zur Nieden et al., 2003; Kawaguchi et al., 2005; Hwang et al., 2006; Duplomb et al., 2007). The mammalian skeleton is formed by two distinct processes. In endochondral ossification, a cartilage template is created first, which is subsequently replaced by mineralized bone tissues deposited by osteoblasts. In contrast, intramembranous ossification involves the direct differentiation of osteoblasts without chondrogenesis. Furthermore, progenitors in endochondral ossification are mesodermal, while in intramembranous ossification, progenitors possess neural crest characteristics (Sakurai et al., 2006; Aihara et al., 2010). The in vitro formation of osteoblasts and bone tissue has been characterized by the capacity of the differentiated cells to mineralize and to express elevated mRNA levels of osteoblastic marker genes, such as osteocalcin (Ocn), the runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osterix (Osx) and bone sialoprotein (Bsp). Runx2 is indispensable for osteoblast differentiation where Runx2-null mice were found to have an abnormal and cartilaginous skeleton. In these mice, osteoblast differentiation is arrested at early stages (Komori et al., 1997; Schroeder et al., 2005). In the process of endochondral ossification, higher Runx2 expression induces chondrocyte hypertrophy and osteoblast differentiation. Furthermore, Runx2 expression is also upregulated at early embryonic development in the osteo-chondroprogenitor cell during mesenchymal condensation (Ducy et al., 1997; Otto et al., 1997). Osx is another transcription factor required for the regulation of osteoblast differentiation and bone formation. Osx-deficient mice displayed abnormal bone

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Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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Table 1 A summary of the studies investigated the effects of different factors promoting osteogenesis from ESCs. References

Cell line used

Factors studied

Phillips et al. (2001) Kuske et al. (2011) Kawaguchi et al. (2005) Buttery et al. (2001)

CGR8 (mouse) D3 (mouse) CGR8, E14Tg2a (both mouse) CEE (mouse)

Β-Gly, AA, RA, BMP-2, compactin. Β-Gly, AA, BMP-2, vitamin D3. Β-Gly, RA, AA, RA, Dex.

Bourne et al. (2004) Woll et al. (2006) Yamashita et al. (2006) Rose et al. (2012) Yamashita et al. (2005)

CEE (mouse) HM1 (mouse) SV6 (mouse) D3 (mouse) CMS-A-2 (monkey)

Β-Gly, AA, RA, Dex (both early and late addition). Β-Gly, AA, Dex. Β-Gly, AA. PPARγ siRNA. Basic fibroblast growth factor. Β-Gly, AA, BMP-2, Dex.

These studies have either used mouse or monkey ESCs. Abbreviations: B-Gly: beta-glycerophosphate; AA: ascorbic acid; RA: retinoic acid; Dex: dexamethasone, PPARγ: peroxisome proliferator-activated receptorgamma; BMP-2: bone morphogenetic protein-2.

formation, suggesting a major role of Osx in osteoblast differentiation. Interestingly, Osx-null osteoblast precursors still express Runx2, suggesting that Osx acts downstream of Runx2 (Nakashima et al., 2002). Finally, Ocn, an extracellular matrix protein, is a marker for late osteoblastic differentiation and it is required for proper bone mineralization (Koga et al., 2005). Mouse ESCs have been used as an in vitro model to study the molecular pathways underlying osteogenesis. There are many osteogenic differentiation protocols in the literature, each utilizing different soluble factors on different cells lines (Buttery et al., 2001; Phillips et al., 2003; Chaudhry et al., 2004; Yamashita et al., 2006). There have been reports for the presence of non-osteogenic lineage cells in a variety of osteogenic-inducing cultures (Zur Nieden et al., 2003). Therefore several studies, including the present one, attempted to enhance osteogenesis in vitro by the use of different combinations of various factors in order to increase the efficacy of ESC commitment to the osteoblastic lineage (Table 1). The majority of these protocols incorporate β-glycerophosphate (B-Gly) and ascorbic acid (AA), which was a protocol first demonstrated by Buttery et al. (2001) to induce mineralization in culture. Among the pro-osteoblastic factors, B-Gly figures out prominently as it is required as a source of organic phosphate for proper matrix mineralization. B-Gly induces differentiation and decreases proliferation in the osteoblastic cell population (Coelho and Fernandes, 2000). Prior to differentiation, osteoblasts secrete a collagenous matrix. AA was found to be essential for osteoblast differentiation in vitro and collagen synthesis (Coelho and Fernandes, 2000). Hydroxyapatite-containing mineral is then deposited on this collagenous matrix if a source of organic phosphate such as B-Gly is provided (Coelho and Fernandes, 2000). Due to their critical roles in the proper osteogenic differentiation from ES cells, BGly and AA were both included in all the tested osteogenic protocols in the present study. Dexamethasone (Dex) is a glucocorticoid drug known for its antiinflammatory properties and thus it is often included in the treatment regimens of inflammatory diseases (Lian et al., 1997). Several studies have reported its stimulatory effects on osteogenic differentiation (Leboy et al., 1991; Ogston et al., 2002). During ESCs differentiation, retinoic acid (RA) is commonly added to the cultures during the growth phase in which EBs are in suspension; it has been shown to be critical in adipogenesis and the generation of neurectoderm cells (Duester, 2008). It has also been suggested that RA promotes osteogenesis by increasing the expression of Runx2 (Dingwall et al., 2011) and inhibiting Pparγ (Shao and Lazar, 1997). Some studies have indicated the inhibitory role of peroxisomeproliferator-activated receptor-gamma (Pparγ) in osteogenic differentiation (Lecka-Czernik et al., 2002; Jeon et al., 2003; Takada et al., 2007; Kawai and Rosen, 2010), reviewed by Lecka-Czernik and Suva

(2006). Hence, inhibiting this transcription factor has been reported to enhance osteogenic differentiation from human mesenchymal stem cells (Krause et al., 2010) using either the potent inhibitor GW9662 or siRNA-mediated inhibition in ESCs (Yamashita et al., 2006). In application of pro-osteogenic supplements in the culture, factors such as concentration and timing of addition are extremely important and can lead to different yields of osteogenic lineage cells. The aim of the current study was to further increase the efficacy of our osteogenic differentiation protocol (which utilizes B-Gly and AA) from mouse R1 cells, using some of the soluble factors that are reported in the literature to enhance osteogenesis. We examined the effect of several factors reported to enhance osteogenic differentiation: RA, Dex, and Pparγ inhibitor GW9662. We studied their effects alone, as well as in combination. We also looked at the effect of differential timing of addition of Dex, since this has been shown by Buttery et al. (2001) to have different effects on osteogenic induction.

2. Materials and methods 2.1. Cell culture Osteogenic lineage differentiation was performed from R1 mouse ESCs (derived from J1 129/Sv mice, purchased from Dr. Janet Rossant, Mount Sinai Hospital, Toronto, Canada) (Nagy et al., 1993) by the hanging drop method (Doetschman et al., 1985). The CGR8 cell line (Nichols et al., 1990) was initially used in parallel but as preliminary data indicated no substantial differences between CGR8 and R1 cells we focused on the latter as its use is far more widespread (Elliott et al., 2004; Gundry et al., 2010). The ESCs were maintained on mitomycin C-treated mouse embryonic fibroblast feeder cells on gelatin-coated plates. At passage 2, cells were trypsinized and collected for hanging drops. 250 ESCs per 25 μL drops were placed on the lids of tissue culture dishes for 3 days. The medium consisted of high glucose Dulbecco modified Eagle's medium with sodium pyruvate and L-glutamine (Multicell, Wisent), 20% Fetal Bovine Serum (Multicell, Wisent), nonessential amino acids, and β-mercaptoethanol. After 3 days in hanging drop, these ESCs aggregated and formed EBs and then were floated in differentiation medium for 2 days. On day 5, the EBs were then plated in tissue culture dishes coated with 0.1% gelatin (in phosphate-buffered saline [PBS]) in differentiation medium with 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate for commitment to the osteogenic lineage. The medium was changed every 2 days for the entire 21 days of differentiation. The effects of Dex, RA, and Pparγ inhibition on osteogenic differentiation from ESCs were examined in six different protocols: A) Control: only AA and B-Gly addition; B) Dex E: 0.1 μM Dex

Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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Fig. 1. Osteogenic differentation timeline and protocols. The hanging drop method was used to study the effects of different factors on the osteogenesis from mouse ESCs. The different combinations of osteogenic factors tested are as follows: (A) control with AA and B-Gly addition; (B) 0.1 μM Dex Early, added at day 6 and kept throughout differentiation; (C) 0.1 μM Dex Late, added at day 10. (D) 1 μM PPARγ inhibitor GW9662 is added at day 3 and kept throughout differentiation; 0.1 μM Dex is added at day 10 to further enhance mineralization. (E) 0.1 μM RA added from day 3 and kept until day 5; (F) 0.1 μM RA added from day 3 and kept until day 5þDex Early (added at day 6 until day 21. (G) 1 μM RA added from day 3 until day 5þ Dex Late (added at day 10 until day 21).

(Sigma-Aldrich) early addition, added at day 6 and kept throughout differentiation; C) Dex L: 0.1 μM Dex late addition, added at day 10 and kept throughout differentiation; D) GW9662: Pparγ inhibitor: 1 μM GW9662 (Sigma-Aldrich) is added at day 3 and kept throughout differentiation; 0.1 μM Dex is added at day 10 to further enhance mineralization; E) RA: 0.1 μM RA (Sigma-Aldrich) added during the floating stage (from day 3 and kept until day 5), and media was changed every day; F) RA Dex E: RA added from day 3 and kept until day 5þDex early addition (added at day 6 until day 21); G) RA Dex L: RA added from day 3 until day 5þlate addition of Dex (added at day 10 until day 21). A schematic diagram representing the above protocols and the timeline of differentiation can be found in Fig. 1. 2.2. Mineralization assays Von Kossa staining is a routine method for detecting calcium deposits in culture. Osteoblasts are known to produce a matrix of osteoid, which is mainly composed of type I collagen. Calcium and phosphate deposition takes place later by osteoblasts, resulting in the formation of carbonated hydroxyapatite crystals (Field et al., 1974). Von Kossa staining is based on the visualization of reduced silver ions that replaced calcium ions which were bound to phosphate in tissue deposits (Rungby et al., 1993). Von Kossa staining was performed on differentiated day 21 nodules for the presence of mineral deposits. Before staining, cells were washed three times with PBS, fixed in 10% neutral formalin buffer for 2 h, and then washed 3 times with distilled water. Nodules were then stained with 2.5% silver nitrate solution for 30 min. Finally, they were washed 3 times with distilled water and were examined for the presence of mineralization. For a quantitative measure of the amount of calcium in the mineralized nodules, the Arsenazo III method was used (Zayzafoon et al., 2004). Briefly, the differentiated bone nodules at day 21 were collected and incubated with 3 M HCl at 80 C for 1–2 h. The resulting suspension was neutralized to pH 5 with 3 M Trizma base, and mixed with Arsenazo

III (100 μM); after 5 min incubation the intensity of resulting color is measured at 595 nm. The total calcium amount was calculated by dividing it by the total protein determined from a duplicate well. The resulting values were expressed as mmol of calcium/μg of total protein. 2.3. Real-time Polymerase Chain Reaction (PCR) Total RNA was extracted from day 21 nodules using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. 4 μg of RNA was reverse transcribed to cDNA using Superscript II (Invitrogen) in a total reaction volume of 48 μL. To examine the mRNA expression of osteogenic markers, cDNA was amplified using Real-time PCR. Real-time PCR analysis was performed in a Bio-Rad's CFX384 TouchTM detection system. The accumulation of PCR products was monitored by measuring the fluorescence caused by the binding of SYBR Green (Applied Biosystems) to double-stranded DNA. The cDNA levels were normalized using L32 (a housekeeping gene). The primer sequences that were used are as follows: for L32, forward primer 50 -CATGGCTGCCCTTCGGCCTC-30 and reverse primer 50 -CATTCTCTTCGCTGCGTAGCC-30 ; for Runx2, forward primer 50 -CCTCTGACTTCTGCCTCTGG-30 and reverse primer 50 -TAAAGGTGGCTGGGTAGTGC-30 ; for Ocn forward primer 50 -AAGTCCCACACAGCAGCTG-30 and reverse primer 50 -AGCCGAGCTGCCAGA GTTTG-30 ; for Osx, forward primer forward primer 50 -GCAACTGGCTAGGTGGTGGTC-30 and reverse primer 50 -GCAAAGTCAGATGGGTAAGTAGGC-30 ; for sonic hedgehog (Shh), forward primer 50 -CAATTA CAACCCCGACATCA-30 and reverse primer 50 -AGTCACTCGAAGCTTCACTCC-30 ; for Indian hedgehog (Ihh), forward primer 50 -ATCACCACCTCA-GACCGTGA-30 and reverse primer 50 -ACCCGGTCTCCTG GCTTTAC-30 ; for Gli1, forward primer 50 -TTCAACTCGATGACCCCACC30 and reverse primer 50 -GGCACTAGAGTTGAGGAATT-30 ; for Smad1, forward primer 50 -CTCACATGCCACTGAACGCC-30 and reverse primer 50 -TCCGCATACACCTCTCCTCC-30 ; for Smad5, forward primer 50 -TTT GCAGAGCCATCACGAGC-30 and reverse primer 50 -TTTCCAATATGCC

Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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GCCTAGT-30 ; for FoxD3, forward primer 50 -GCGGCATCTGCGAGTTCATC-30 and reverse primer 50 -GGCTCCGAAGCTCTGCATCA-30 ; for sex determining region Y box 10 (Sox10), forward primer 50 -CTAGGCAAGCTCTGGAGGTT-30 and reverse primer 50 -AGGTATT GGTCCAGCTCAGT-30 ; for brachyury, forward primer 50 -GTGACTGCC TACCAGAATGA-30 and reverse primer 50 -ATTGTCCGCATAGGTTGGAG30 ; and for Snail, forward primers 50 -CTGCTTCGAGCCATAGAACTAAAG30 and reverse primer 50 -GAGGGGAACTATTGCATAGTCTGT-30 . 2.4. Immunofluorescence staining Day 21 differentiated nodules grown on coverslips were rinsed 3 times with PBS. They were fixed in 3.7% formaldehyde for 15 min. After washing (three times for 5 min each time) in PBS, the cells were permeabilized with 0.1% Triton X-100 in buffered containing 100 mM 1,4-piperazinediethanesulfonic acid, 1 mM EGTA, and 4% (w/v) polyethylene glycol 8000 (pH 6.9) for 2 min. Nodules were then rinsed 3 times in PBS for 5 min and they were incubated with primary antibodies (dilution of 1:50) for 30 min at room temperature. The primary antibodies used include anti-Runx2 (Abcam) and anti-Osx (Abcam). After washing in PBS (3 times for 5 min), the cells were stained with the secondary antibody (fluorescein isothiocyanateconjugated donkey anti-mouse, or anti-rabbit; diluted 1:50) for 30 min at room temperature. After, nodules were washed 3 times in PBS for 5 min. Nodules were incubated with 100 μg/mL DNasefree RNase in PBS for 30 min at 37 1C, followed by rinsing in PBS 3 times, 1 min each. For nuclear staining, 500 nM Propidium Iodide solution was added to cover the nodules for 5 min. After final rinsing in PBS (3 times for 1 min each), the slides were mounted in fluorescent mounting medium (Dako). A confocal fluorescence microscope (MRC 600; Bio-Rad Laboratories) equipped with a 60/ 1.40 planapochromatic oil immersion objective (Nikon) and krypton– argon laser was used for fluorescence imaging. To avoid bleedthrough of fluorescence between channels, we imaged FITC and PI using separate, single excitation filters and single fluorochrome filter/ mirror blocks: 10 nm 488 nm bandpass excitation filter and BHS block with 510 nm longpass dichroic mirror and 515 nm long pass OG filter versus 10 nm 514 nm bandpass excitation filter and YHS

block with 540 nm longpass dichroic mirror and 550 nm long pass OG filter, which prevented any spillover of fluorescent signal. 2.5. Transmission electron microscopy Samples for hydroxyapatite imaging by transmission electron microscopy (TEM) were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer followed by rinsing in the same buffer. They were then fixed in 1% osmium teroxide in buffer, dehydrated in a graded ethanol series followed by propylene oxide, and embedded in Quetol-Spurr resin. Sections 100 nm thick were cut on an RMC MT6000 ultramicrotome, stained with uranyl acetate and lead citrate and viewed in an FEI Tecnai 20 TEM. 2.6. Statistical analysis All statistical analyses were performed using the IBM SPSS (version 21) data analysis program for two-way analysis of variance (ANOVA) with post-hoc Tukey's test. Experiments were independently repeated at least three times, each repeated in triplicate unless otherwise stated. Values are expressed as the mean 7SD. Differences between mean values for different treatments were considered to be significant at P o0.05 and P o0.01. 3. Results 3.1. The effect of osteogenic protocols on mineral deposition The amount of minerals present in day 21 differentiated nodules from different osteogenic treatments was evaluated using two different methods: Von Kossa stain and Arsenazo III methods. Von Kossa staining is a method based on the visualization of reduced silver ions that have replaced calcium ions present in the extracellular matrix of nodules. The quantification of the amount of calcium ions present is done by the Arsenazo III method. The treatment regimens of Dex L, the Pparγ inhibitor GW9662, RA, RA Dex E and RA Dex L all enhanced mineralization compared to control according to Von Kossa staining (Fig. 2A) and

Fig. 2. Mineralization assays revealed that the RA and later addition of Dex treatment resulted in the highest level of mineral deposition. (A) Von Kossa staining was used to detect mineralization in day 21 nodules treated with different combinations of osteogenic factors. Nodules were fixed in formaldehyde and then stained with 2.5% silver nitrate solution. The combination of RA and late addition of Dex gives the most mineralization compared to other groups upon Von Kossa staining. (B) Quantification of extracellular calcium content by Arsenazo III kit confirmed the Von Kossa results. Conditions were performed in triplicates; data are presented as mean7 SD. Abbreviations: RA: retinoic acid; Dex: dexamethasone, GW9662: PPARγ inhibitor; E: early; L: late.

Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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Fig. 4. Immunofluorescence staining of the osteogenic markers, Runx2 and Osterix (OSX). At day 21 of differentiation, nodules were immunolabelled for the transcription factors Runx2 or OSX. Propidium iodide (PI) was used to identify the nuclei. The table shows the degree of nuclear localization denoted as: (Cyto)¼ predominately cytosolic, (Mixed)¼ both cytosolic and nuclear, (Nuc) ¼predominately nuclear, for different treatment groups as indicated in the “Protocol” column. Control: only AA and B-Gly addition. The lower panel shows representative immunofluorescence pictures depicting predominately cytosolic, mixed (nuclear and cytosolic), and predominately nuclear localization. In all the protocols studied, RA and Dex (late) induced the highest level of nuclear translocation of both Runx2 and OSX. Scale bar division represents 0.1 μm. Abbreviations: RA: retinoic acid; Dex: dexamethasone, GW9662: PPARγ inhibitor; E: early; L: late.

Fig. 3. Expression of osteogenic markers in day 21 nodules treated with different culture conditions. Total mRNA of nodules from each condition was isolated and reverse transcribed. The mRNA expressions of Runx2, OSX and OCN were detected using Real-time PCR. All treatments increased the expression of Runx2, OSX and OCN. However, RA and late addition of Dex yielded best results. Conditions were performed in triplicates; data are presented as mean7 SD. Abbreviations: RA: retinoic acid; Dex: dexamethasone, GW9662: PPARγ inhibitor; E: early; L: late.

quantification of extracellular calcium (Fig. 2B). However, the combination of RA and Dex (late addition at day 10) gave the most mineralization, according to Von Kossa staining (Fig. 2A). 3.2. Expression of osteogenic markers The mRNA expression of osteogenic markers, Runx2, Ocn, and Osx, were evaluated using real-time PCR. The effect of late addition

of Dex at day 10 leads to more profound results compared to early addition at day 6 (Fig. 3). Early addition of Dex resulted in only a 2-fold increase in osteogenic marker Ocn mRNA, while there was 4-fold increase in late addition (Fig. 3). Similarly, early addition of Dex enhanced the mRNA expression of Runx2 by 12-fold compared to the control, while in the cells treated with Dex later in differentiation, there was a 22-fold increase in Runx2 mRNA expression. Fig. 3 shows that both early and late addition of Dex increased the expression of Osx compared to their control. RA treatment also leads to higher abundance of osteogenic lineage cells. Real-time PCR revealed that Runx2 and Ocn had a 33-fold and 5.5-fold increase in their transcription, respectively (Fig. 3). The expression of Osx was increased by 22.3-fold compared to the non-treated control (Fig. 3). Under the treatment of GW9662, the mRNA expression of Runx2 and Ocn increased as well. There was a 26.9-fold increase in Runx2

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Fig. 5. Transmission electron microscopy images of differentiated day 21 nodules in RA and Dex (late) protocol. The presence of hydroxylapatite depositions is detected in the extracellular matrix of the nodules. Classic modes such as bright field were used to image the texture of the deposit (A, B), while C shows a selected area diffraction pattern identifying the crystalline material as biological hydroxyapatite.

mRNA transcript; a 5.6-fold increase in the Ocn mRNA expression; and a 5.8-fold increase in Osx mRNA expression (Fig. 3). The increase in the osteogenic induction by GW9662-treated nodules was not as significant compared to the addition of RA and (late) Dex. RA and Dex both enhanced osteogenic differentiation from ESCs. When they were both added together into the media, this resulted in the highest increase in osteogenic markers expression compared to all other protocols (Fig. 3). The addition of both RA and Dex (early addition at day 6) leads to a 46.7-fold increase in Runx2 mRNA expression and a 7.3-fold increase in Ocn mRNA expression (Fig. 3). The mRNA expression of Osx was increased 43.3-fold in the treatment regime of RA and late Dex addition (Fig. 3).

confirm that the improved protocol (RA and late Dex treatment) does not induce nonphysiological mineral deposition using more sensitive methods such as TEM for hydroxyapatite imaging. Fig. 5 shows the transmission electron micrographs illustrating the presence of hydroxyapatite crystals in the extracellular matrix which has been further confirmed with the crystalline microdiffraction pattern obtained with 1 nm electron probe (Fig. 5C). Thus, the high levels of mineral deposition in the RA and Dex (late) protocol observed by Von Kossa staining were hydroxyapatite and not any other nonphysiological forms.

3.3. RA and late Dex treatment induced the highest level of Runx2 and Osx nuclear localization

Although the aim of the present study is not to elucidate mechanisms in detail further than what is already known, nevertheless, we have verified that some of the most well-known pathways induced by RA and Dex in osteogenesis are progressing as reported in our system. Bone formation proceeds via two distinct pathways, endochondral and intramembranous ossification. Progenitor cells which give rise to osteoblasts in the endochondral pathway are of mesodermal origin, while in intramembranous ossification, progenitor cells possess neural crest characteristics. To determine whether RA or Dex affects these two pathways, we examined the expressions of mesodermal (brachyury and Snail) and neural crest (FoxD3 and Sox10) markers. The addition of Dex enriched the population of mesodermal cells early in differentiation. The mRNA abundance of both mesodermal markers brachyury and Snail was markedly increased by Dex Treatment (Fig. 6). RA enhanced neural crest osteogenesis in our culture. The mRNA expressions of neural crest markers, FoxD3 and Sox10, were increased with RA treatment (Fig. 6). We also examined the effects of RA and Dex on crucial osteogenic signaling pathways, Shh, Ihh and BMP2. Dex increased the expression of the signaling molecule, Shh (Fig. 7). The abundance of signaling molecules required for osteogenesis such as Ihh, BMP2, Smad1, and Smad 5 was induced by RA treatment (Fig. 7).

Since Runx2 and Osx are both crucial osteogenic transcription factors, their nuclear localization is indispensable for their regulatory functions. Using confocal microscopy, the nuclear localization of Runx2 and Osx was examined. The treatments of Dex L, GW9662, RA, and RA Dex L enhanced the degree of nuclear translocation of Osx (Fig. 4). The treatments of RA and RA Dex E enhanced the nuclear translocation of Runx2 (Fig. 4). Compared to all other protocols that were studied, RA Dex L treatment gave rise to the highest extent of nuclear translocation of both Runx2 and Osx (Fig. 4). 3.4. Confirmation of hydroxyapatite deposition in RA and late Dex treatment Thus far, we found that the differentiation protocol of RA and late Dex resulted in the highest enrichment of osteogenic lineage cells compared to other treatments examined in this study. Although Von Kossa staining is routinely used to detect the level of mineralization in osteogenic cultures, it is not specific for mineralization since it measures the level of calcium which is known to be induced due to other biological processes such as apoptosis. Thus, it is imperative to

3.5. Mechanisms of RA and Dex in osteogenic differentiation

Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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Fig. 6. Mesodermal and neural crest markers expressions in osteogenic differentiation. The mRNA expressions of brachyury, Snail, FoxD3 and Sox10 were detected using Real-time PCR. Conditions were performed in triplicates; data are presented as mean 7 SD. Abbreviations: RA: retinoic acid; Dex: dexamethasone, GW9662: PPARγ inhibitor; E: early; L: late.

4. Discussion In an attempt to further increase the efficacy of our differentiation protocol, some candidate molecules were excluded since they were hormones or soluble factors which had controversial effects on osteogenesis. One such molecule is compactin, which has been indicated to inhibit cholesterol synthesis by reducing the enzymatic activity of HMG-CoA reductase. Although it has been reported that compactin enhances osteogenesis, its use can induce

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pleiotropic effects. We examined the effect of several factors reported to enhance osteogenic differentiation: RA, Dex, and Pparγ inhibitor GW9662. We discovered that the combined treatment of RA and Dex, in particular later Dex addition, was the most efficacious osteogenic differentiation protocol. This treatment regimen leads to a significant increase in mineralization, Runx2 and Osx transcription and nuclear translocation. Mechanistically, RA and later Dex treatment enhanced BMP2, Ihh and Shh signaling, a result that is consistent with the increased mRNA expressions of osteogenic markers. RA signaling is vital for organogenesis in utero and the maintenance of adult organs (Duester, 2008; Niederreither and Dolle, 2008). RA is a steroid hormone and it exerts its effects on cell differentiation and metabolism by binding to its nuclear receptors, the retinoic X and the RA receptors (Chawla et al., 2001; Mark et al., 2006). It remains controversial regarding the effects of RA on skeletogenesis (Weston et al., 2000; Wan et al., 2006; Wang et al., 2008). It has also been shown that RA promotes osteogenesis by increasing the expression of Runx2 (Dingwall et al., 2011). This is accomplished by RA interacting with Smad1 and thus enhancing BMP2 signaling (Drissi et al., 2003). In our study, the addition of RA during the floating stage of differentiation did in fact enhance mineralization as well as the expressions of Runx2 and Ocn. Furthermore, we found that RA facilitates the function of Runx2 by promoting its nuclear translocation. RA increases the expressions of BMP2, Smad1, Smad5, Ihh. The BMP2 signaling pathway is crucial for osteogenesis and bone repair. It has been reported to induce Runx2-activated transcription of Ocn, Osx, osteopontin, bone sialoprotein, and alkaline phosphatase (Wang et al., 2011). The Ihh pathway has important regulatory roles in skeletal development and tissue patterning. It has been shown to induce Runx2 expression (Shimoyama et al., 2007). The differentiation of neural crest cells from ESCs was enhanced with RA addition; thus, RA seems to enhance the pathway of intramembranous ossification. Although several studies have reported Dex's stimulatory effects in osteogenic differentiation (Leboy et al., 1991; Ogston et al., 2002), others have also pointed to its negative effects in osteogenesis (Shi et al., 2000; Pereira et al., 2002). Critical factors that may influence such effects of Dex are the maturity and density of the differentiating cells in culture (Mikami et al., 2011). It is also interesting to note that Dex is required to be added in the media continuously to induce maximal osteogenesis; removal of Dex from the culture may cause osteogenic lineage cells to dedifferentiate (Porter et al., 2003). In our study, Dex enhanced the osteogenic differentiation as demonstrated by higher levels of mineralization, expression of osteogenic markers, as well as higher extent of nuclear translocation of Osx. In accordance with another recent study (Ma et al., 2013), we found that Dex stimulates the Shh signaling pathway by increasing the expression of Shh. The hedgehog signaling pathways have key roles in skeletogenesis, and mutations in these pathways lead to defective craniofacial bone formation. Shh signaling has been shown to promote mesenchymal stem cell differentiation to the osteogenic lineage (Spinella-Jaegle et al., 2001). Thus, in our culture, the mechanism of action of Dex is at least in part due to enhancement of the Shh pathway. Furthermore, Dex enhanced the yield of osteogenic lineage cells by enriching the culture with mesodermal cells, which are known to subsequently undergo endochondral ossification. In addition, we also examined the effect of differential time treatments of Dex on osteogenesis. The specific time points (early versus late) chosen were based on studies in the literature that pointed to the importance of the temporal treatment for Dex in its effect on osteogenesis, and in particular the study by Buttery et al. (2001). This study illustrated that in mESCs, later addition of Dex leads to more potent osteogenic stimulation than earlier treatment. Due to slight differences in the timeline of our protocol, we decided the comparable times to add Dex would be around day 6 and day 10. In our hands, late addition led to better

Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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Fig. 7. Expression of Ihh, Shh and BMP2 signaling pathway molecules in osteogenic differentiation. The mRNA expressions were detected using Real-time PCR. Conditions were performed in triplicates; data are presented as mean7 SD. Abbreviations: RA: retinoic acid; Dex: dexamethasone, GW9662: PPARγ inhibitor; E: early; L: late.

osteogenic differentiation than early addition of Dex, a result that is in agreement with a study by Buttery et al. (2001). We also observed an enhancement in osteogenic differentiation from ESCs with the addition of 1 μM of GW9662, although this was not as prominent as osteogenic enhancement achieved with the addition of RA and (late) Dex, based on the expressions of osteogenic markers and the extent of nuclear translocation of transcription factors. Furthermore, Pparγ is a transcription factor that is controlling the transcription of several other genes in addition to adipogenic markers. Due to this pleiotropic effect, its inhibition may have other nonspecific downstream effects. Our study revealed that different optimizing protocols led to varying degrees of Runx2 and Osx nuclear localization. Other studies have also observed cytosolic localizations of these two transcription factors and gave insights on how this may impact osteoblast differentiation and maturation. STAT1, a member of the Signal Transducers and Activators of Transcription family, was found to be a negative regulator of osteoblastogenesis. Differentiation of osteoblasts and subsequent bone formation can be prevented by cytosolic retention of Runx2 by STAT1 (Long, 2012). Huang et al. (2013) have described that frameshift

mutations, which caused at least partially impaired nuclear translocation of Runx2, lead to the phenotype of cleidocranial dysplasia in patients. Another study by Deepak et al. (2011) showed that Tbox3, a previously reported negative regulator of osteoblastogenesis, interferes with Runx2 transcriptional activity. In addition, overexpression of Tbox3 leads to the mis-localization of Runx2 in the cytosol. Although it is less known about what may affect the subcellular localization of Osx, Tai et al. (2005) showed that Osx nuclear localization and activation are required for early osteoblast development (mesenchymal stem cell and pre-osteoblast stages) and remain nuclear in differentiated osteoblasts, while in non-osteogenic cells such as fibroblasts, Osx remains inactive in the cytosol. In summary, in all of the 6 tested osteogenic protocols (Fig. 1), consisting of osteogenic factors alone and in combination, with varying timings of addition, RA and late addition of Dex yielded the most pronounced enhancement of osteogenic differentiation based on the results of mineralization assays, the expression of osteogenic markers, and the extent of nuclear localization of Runx2 and Osx. The treatment of RA and late Dex also increased the yield of mesodermal and neural crest cells, as well as induced the

Please cite this article as: Yu, Y., et al., Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro. Differentiation (2015), http://dx.doi.org/10.1016/j.diff.2014.12.003i

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expression of several crucial osteogenic signaling molecules (Ihh, Shh, BMP2, Smad1, and Smad5). Thus, our findings provide valuable guidelines into fine-tuning of culture condition to differentiate R1 mouse ESCs to osteoblastic cells in vitro.

Disclaimer Authors declare no conflict of interest and no financial conflict.

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