Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro

Biochimica et Biophysica Acta 1790 (2009) 110–118

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n

Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro Elerin Kärner a, Carl-Magnus Bäckesjö a, Jessica Cedervall b, Rachael V. Sugars a,⁎, Lars Ährlund-Richter b, Mikael Wendel a a b

Center for Oral Biology, Institute of Odontology, Karolinska Institutet, P. O. Box 4064, SE-141 04 Huddinge, Sweden Department of Woman and Child Health, Karolinska Institutet, Karolinska University Hospital Solna, SE-141 57 Stockholm, Sweden

a r t i c l e

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Article history: Received 13 May 2008 Received in revised form 16 September 2008 Accepted 8 October 2008 Available online 25 October 2008 Keywords: Osteogenesis Differentiation Extracellular matrix Gene expression Embryonic stem cell

a b s t r a c t Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using preosteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, β-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a prematurational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Bone generating, osteogenic culture conditions were first established using bone tumor cell lines or tissue-derived cells of nonhuman origin [1–4], and may not be appropriate for the study of signaling systems in normal human osteogenesis. Moreover, tumor cell lines often have an impaired cell cycle, and thus do not exhibit the true phenotype of bone tissue [5]. The traditional bone nodule assay, a standard culture model originating from early studies using fetal rat calvaria cells, has contributed significantly to the increased understanding of osteoblast differentiation [6]. Currently, the consensus is that two distinguishable events occur during osteogenic differentiation, the cellular compartment proliferates and differentiates, while the extracellular matrix (ECM) matures and mineralizes [7]. The signaling pathways and the transcriptional regulation processes occurring during osteogenic differentiation are not well understood. Genes needed for osteoprogenitor cell commitment and differentiation are intermittently up-regulated, thereby creating ⁎ Corresponding author. Tel.: +46 8 746 02 35; fax: +46 8 779 31 66. E-mail address: [email protected] (R.V. Sugars). 0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2008.10.004

recognizable stages of maturation from primitive to more mature cells within the osteoblastic lineage [8]. Disturbances in the osteoblastic differentiation process towards functioning bone cells are found in bone cancer [9]. Improvements of human embryonic stem cell (HESC) models are required for their further use in strengthening the understanding of early aspects in human developmental processes, as well as for developing reliable methods for industrial applications. Currently, it is not possible to obtain pure cultures of differentiated HESCs that exhibit suitable homogeneity for therapeutic applications, thus it is necessary to establish directed differentiation technologies. Osteogenic culture models, utilizing stem cells, potentially provide a unique system and an unlimited source of cells that can be used for differentiation into functional osteoblasts for cell therapies or drug development applications. Studies on directed differentiation of embryonic stem cells have provided useful information as to the osteogenic potential of HESCs [10–18], albeit, all were conducted to investigate whether the osteoblastic phenotype was achievable. Many investigations to date have relied upon differences in marker expression, rather than functional outcome, such as capacity to form bone-like nodules. However, the precise relationship between the

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proliferative phase of cells, matrix maturation and expression of markers associated with the differentiated osteoblast is unknown in the HESC model. In the current study, the HESCs were allowed to differentiate in the presence of dexamethasone to stimulate cell differentiation, ascorbic acid to stimulate ECM synthesis and β-glycerophosphate to promote mineralization [14]. Such medium is routinely used for the differentiation of bone marrow stromal cells and more mature osteogenic cells, although the concentrations vary in different studies. In addition, several other growth-inducing factors have been successfully used for the initiation of osteogenesis (for review see [19]). However, a system based on undifferentiated pluripotent stem cells might not activate the “correct” signaling pathway and thus, the addition of growth factors to undifferentiated cells might also result in an undesired outcome. For example, bone morphogenetic proteins (BMPs) up-regulate osteoblast-associated genes, and BMP2 specifically has successfully been used in osteogenic studies in several culture models including stem cells [20–22]. Interestingly, in the HESC model, activation of the Smad pathway induced cardiac differentiation [23], and regulated differentiation towards extra-embryonic endoderm [24]. Recruitment, proliferation and differentiation of osteoblasts is regulated by many factors besides BMPs, including systemic hormones and other modulators, and during embryonic development osteogenesis is also tightly connected to the vascular system [25,26]. Vascular endothelial growth factor (VEGF) is released by endothelial cells, and it has been reported that expression of VEGF receptors on osteoblastic cells depends on the state of differentiation [25]. In addition, supplementation with exogenous VEGF stimulates osteoblastic differentiation [25], in an action that is independent from BMPs [27]. To address the requirement for well-characterized bone differentiation systems, it is important to investigate the effect of these two growth factors on HESCs. Here, we report the first attempt to characterize the gene expression pattern during osteoblastic differentiation in HECSs. Furthermore, we establish a timeframe for the supplementation of additional growth factors to the culture system. This study builds upon our previous work, which showed that in vitro HESCs can produce a mineralized matrix possessing all the major bone markers, and that the mode of differentiation is highly dependent on the cell line [14]. In the current study we have focused on the HESC line, HS181 only, which was cultured on human embryonic fibroblasts, and as we have previously reported this line exhibits a greater tendency to follow the osteogenic pathway [14]. 2. Materials and methods 2.1. Osteogenic differentiation in vitro HESC line HS181 (passages 35–66) (from Karolinska Institutet Stem Cell Network, Stockholm, Sweden) with a normal karyotype was cultured as published [14]. Experiments were repeated on three separate occasions. To initiate differentiation, HESCs were removed from culture by incubation with collagenase NB5 (1 mg/ml) solution in KnockOut-Dulbecco's Modified Eagle Medium (KO-DMEM) for 8 min and mechanically scraping from the plate. The equivalent of 5 colonies consisting of approximately 400 cells each (1000 cells/cm2) were seeded onto 24-well plates coated with 0.1% gelatin. Differentiation was induced using osteogenic media ((OM) KODMEM, 20% FBS, 1% GlutaMAX, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 10 mM β-glycerophosphate (Sigma-Aldrich, St Louis, MO, USA), 50 μg/ml ascorbic acid phosphate and 1 μM dexamethasone (both Sigma-Aldrich)), as described before [14]. Control (CTR) cultures of HESCs were treated only with 20% FBS and no osteogenic supplements. The cultures were maintained for 25 days and the medium changed every second day. For treatment with growth factors, recombinant human (rh)-BMP2 (100 ng/ml) was purchased from Sigma-Aldrich, and rhVEGF (25 ng/

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ml) from Invitrogen. Both were added to the cultures along with OM from day 10. 2.2. Cell counting assay HESCs were plated onto 24-well plates at a density equivalent to 5– 6 colonies per well (each colony ∼400 cells) and cultured in OM. Cells were washed with phosphate buffered saline (PBS) (Invitrogen) followed by treatment with TrypLE Express for 15 min, and resuspended in 1 ml medium. Cell number was assessed after 3, 7, 10, 17, 21 and 25 days in 4 wells of a 24-well plate, and this was repeated in three separate experimental setups. 2.3. Cellular metabolic activity Cellular metabolic activity was determined by measuring with MTT colorimetric assay at days 0, 7, 13, 19 and 25, according to the manufacturer's instructions (Roche Molecular Biochemical's, Bromma, Sweden). Briefly, 30 μl of MTT reagent was added to the cells in 24well plates and incubated for 4 h at 37 °C. 300 μl of solubilization solution was added to each well and the plates incubated overnight at 37 °C. Colored formazan products were quantified by measuring absorbance at 540 and 690 nm. The cellular activities of cells treated with OM, and OM with the addition of BMP2, VEGF or in combination were normalized to undifferentiated HS181 cells, which were considered as 100% active. 2.4. Semi-quantitative and quantitative real-time RT-PCR Total RNA was extracted from undifferentiated (day 0) and differentiating OM-treated HS181 cells (after 36 h, 72 h, and every other day from day 5 to 25 in culture) and from irradiated human fibroblasts, using the RNeasy Kit (Qiagen, VWR International AB, Stockholm, Sweden) as described by the manufacturer. First-strand cDNA was reverse transcribed from Dnase-treated 2 μg total RNA using 100 U SuperScript™ III reverse transcriptase, 200 ng random primers and RNase inhibitor (all from Invitrogen). For the detection of Oct-4 semi-quantitative RT-PCR was performed as described [14]. The Oct-4 sequences were as previously published [28] and 18S (Accession number M10098), forward CGT TGA TTA AGT CCC TGC CCT T, reverse TCA CCT ACG GAA ACC TTG TTA CG. Quantitative real-time RT-PCR (Q-PCR) was performed using 80 ng cDNA, TaqMan Universal PCR Mastermix, and human TaqMan gene expression assays for hydroxymethylbilane synthase (HMBS, Hs0 0609297_m1), Nanog (Hs0238740 0_g1), osterix (OSX, Hs00541729_m1), bone sialoprotein (BSP, Hs00173720_m1), osteocalcin (OCN, Hs01587813_g1), collagen type I A1 (Col IA1, Hs00164004_m1), osteoadherin (OSAD, Hs00192325_m1), parathyroid hormone receptor 1 (PTHR1, Hs00174895_m1), osteonectin (ON, Hs00234160_m1) and osteopontin (OPN, Hs00959010_m1) (all from Applied Biosystems, Foster City, CA, USA). The reactions were run at 50 °C for 2 min, 10 min at 95 °C, followed by 50 cycles of 95 °C 15 s and 60 °C 40 s on Applied Biosystems 7500 Fast Real-Time PCR system. The comparative cycle threshold method was used to analyze data, HMBS was used to standardize the Ct values, and undifferentiated HS181 (day 0) was used to calibrate the values of the osteogenically differentiating HESCs. 2.5. SDS-PAGE and Western blotting Cells were lysed using TRIzol reagent (Invitrogen) and protein extracts were quantified using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). 7 μg of each sample in Laemmli buffer containing β-mercaptoethanol was electrophoresed on a SDS-PAGE 4– 15% mini-gel (Bio-Rad). Proteins were electroblotted onto nitrocellulose membranes (Hybond-ECL, GE Healthcare, Buckinghamshire, UK) and blocked in 3% non-fat milk solution in Tris-buffered saline (TBS) and 0.1%

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Tween-20. The membranes were probed with human anti-BSP (1:2000, Chemicon, Temecula, CA), anti-Col I (1:200, Santa Cruz Biotechnology, Inc., SDS Biosciences, Sweden), anti-OSX (1:200, generous gift from B. Ganss, Toronto University, Canada), anti-actin (1:20 000, SigmaAldrich), and bovine anti-OSAD (1:10 000, generated in house) [29] antibodies, diluted in blocking solution, followed by a corresponding horseradish peroxidase-conjugated secondary antibody (1:1000, DAKO, Glostrup, Denmark). Proteins were detected with ECL Plus Western Blotting Detection System (GE Healthcare). 2.6. Immunocytochemical studies To investigate if pluripotent cells remained within the differentiated HESC cultures, TRA-1-81 was analyzed by immunohistochemical staining. Cultures were fixed for 10 min in 4% paraformaldehyde and rinsed with PBS, treated with 0.2 M HCl for 15 min, washed again using PBS and treated with 3% H2O2 for 5 min, all at room temperature. The antibodies were non-specifically blocked with PBS, 3% BSA (Fraction V, Sigma-Aldrich), 0.1% Tween-20, 0.1% BSA-c (Aurion) for 40 min. After blocking, the cells were incubated with primary antibody (1:20) in PBS, 0.1% Tween-20, 0.1% BSA-c for 1 h. The cells were washed with PBS, 0.01% Tween-20, 0.01% BSA-c for 15 min, and incubated for 1 h at room temperature with fluorescent labeled secondary antibody AlexaFluor594 (goat anti-mouse IgG) (Invitrogen). After washing again with PBS, the samples were mounted with Vectashield containing DAPI (Vector labs Inc., Burlingame, CA). 2.7. Statistical analysis The experimental data are shown as mean ± SD of three experiments. The significance of difference was analyzed by one-way ANOVA and p N 0.05 was considered as statistically significant.

3. Results The HESC line HS181 has previously been reported to exhibit a high osteogenic differentiation potential both in vitro and in vivo [14,30]. In this study, osteogenic cultures were maintained over 25 days without the initial induction of differentiation within embryoid bodies (EBs) [14,15]. This timepoint was chosen because it was found that prolongation of the culture time, at a similar seeding density, resulted in densely mineralized and possibly necrotic cultures. 3.1. Proliferative and self-renewal properties of HESCs in differentiation medium containing dexamethasone, ascorbic acid and β-glycerophosphate The response to osteogenic culture conditions was evaluated by measuring cell number, and the increase in cell number is presented as a mean of three independent experiments (Fig. 1A). It was evident that the cells proliferated rapidly until day 7, at which time they reached confluency as monolayers. A slower increase in cell number was observed after that, indicative of a decreased proliferation rate or increased cell death. From that timepoint onwards the cultures continued to form multi-layered structures. To examine whether the remaining proliferating cells were potential pluripotent HESCs, immunohistochemistry was performed. The timepoints were chosen based on the general outline of the study; day 4 representing conditions before reaching confluency, day 15 corresponding approximately to the ECM maturation stage, and day 25 the endpoint with mineralized matrix. Immunohistochemical staining with antibodies against human TRA-1-81, an embryonic cell surface proteoglycan linked to pluripotency, revealed at day 4 a strong immunoreactive signal dispersed throughout the colonies from both groups (Fig. 1B). By day 15, TRA-1-81 expressing cells were found distributed across the colonies in the OM, while in the

Fig. 1. HESCs were left to differentiate 25 days in OM supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid phosphate and 1 μM dexamethasone. (A) Changes in proliferation were measured after 3, 7, 10, 17, 21 and 25 days. Cells rapidly proliferated until day 7, after which a slower increase in cell number was observed. Results are presented as means ± SD of three independent experiments. ⁎ refers to p b 0.05, values which differ significantly from each other. (B) Immunohistochemical staining of HESC derived monolayer cultures with an antibody against human TRA-1-81. CTR cells were treated only with 20% FBS, and no osteogenic supplements were added. In sub-confluent cultures, at day 4, the positive signal was observed along the cell colonies in both groups (OM and CTR). In the colonies treated with OM, after 15 days in culture, TRA-1-81 positive cells were dispersed, while in the CTR group the positive cells were found along the edges of colonies. A distinct positive signal for TRA-1-81 was evident after 25 days in colonies grown with OM, however only few positive cells were detectable in the CTR group. TRA-1-81 — red, DAPI — blue, scale bar, 100 μm. (C) Semi-quantitative RT-PCR for Oct-4 expression in the OM and CTR groups. The data is presented at various timepoints, days 4, 8, 15 and 25. (D) Nanog gene expression was analyzed by Q-PCR in OM treated cells after 36 h, days 5, 7, 15, 21 and 25. ⁎ refers to p b 0.05, values differ significantly from 36 h sample.

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Fig. 2. Expression of bone-related genes and proteins in HESC cultures supplemented with OM covering the various stages of osteoblastic lineage development. (A) RNA was isolated from HESC osteogenic cultures at indicated timepoints and analyzed by Q-PCR. Undifferentiated HS181 was used to calibrate the values. For a better understanding, osteoblast differentiation and matrix formation associated genes are presented on two separate graphs. (B) Western blot analysis was performed on whole cell extracts from OM HESC cultures at indicated time points of culture. The HESC cultures were probed for production of human OSX, collagen type I, BSP, and (C) OSAD.

CTR group positive cells were found along colony edges. At day 25, a major difference in TRA-1-81 receptor expression was apparent between cells grown with and without the OM. Colonies in OM remained TRA-1-81 positive in demarcated areas, however, in the CTR group only a few positive cells were detectable. In addition, gene expression of Oct-4 and Nanog, markers for the undifferentiated state, were detected in the cultures treated with OM (Fig. 1C and D). These findings suggest that the initiation of differentiation was not synchronized in the tested medium supplemented with factors previously demonstrated to induce osteoblastic gene expression. 3.2. Expression profile of bone-related genes A panel of markers was chosen to cover the various stages of osteoblastic lineage development, from the earliest progenitors to terminally differentiated osteoblasts. The reliability of the primers used was first verified using the cell lines; human osteoblasts (Cambrex Bio Science Walkersville, Inc., MD) at day 0, and day 15 with and without OM, HEK293-EBNA (ATCC, Manassas, VA, USA) and Saos-2(HTB-85, ATCC) (data not shown). Total RNA was isolated from the OM HESC cultures at different timepoints to 25 days and the resulting cDNA was subjected to Q-PCR to illustrate the relationship between the markers during osteogenic differentiations in HESCs. The results are depicted as smooth lines representing the mean value of three independent experiments (Fig. 2A). Even though the three experimental set-ups differed to some extent in the expression levels,

similar trends in relation to each other and to the time after reaching confluency were observed. Osteoblasts synthesize and secrete collagenous and non-collagenous proteins, constituting the ECM that subsequently calcifies. For the understanding of osteoblastic differentiation towards mature functional cells it was desirable to dissect the order of the molecular events. Shortly after the cultures reached confluency, around day 7, the osteoblast-specific transcription factor OSX expression was upregulated, with peaks at days 9 and 15, followed by a down-regulation. BSP expression, normally restricted to mineralizing tissues, was upregulated day 11 and subsequently dropped to be undetectable at day 13, after that a second expression peak at day 17 occurred. OCN mRNA, characteristic only to the latest stage of mature osteoblasts, was upregulated at the end of the proliferative phase, and had a second peak of expression at day 19 and increased more towards the end of the experiment, suggesting emerging mature osteoblasts and mature matrix. OSAD, another non-collagenous protein, was expressed at the beginning of the culture period. The expression peaks of OSAD correlated with two other osteogenic markers; the first with OSX (day 15), and the second with OCN (day 19). Furthermore, OSAD followed the same expression profile as OCN at the end of the culture, suggesting that functional OSAD was needed during the commitment to the osteogenic pathway, and in osteoblast terminal differentiation. Col I A1 showed an early increase after 72 h indicating possible contamination from feeder cells or prediffentiated cells from the initial cultures. Thereafter, Col I A1 was up-regulated from day 13 until day 21, peaking at day 17, thus providing the cultures with a three-

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dimensional matrix for the deposition of mineral crystals. The Col I A1 peak also coincided with PTHR1, a hormone receptor found on osteoblasts. OPN, a potent mineralization inhibitor [31] was expressed initially but slowly decreased towards the end of the culture period, with minor peaks at days 9 and 15, correlating to the expression of OSX and OSAD. Another regulator of hydroxyapatite (HAP) crystal formation, ON [31], was expressed at low levels at the beginning of the culture time, but was up-regulated after day 11, peaking at days 17 and 23. Identification of proteins synthesized into the matrix revealed similar trends to the above described examination of mRNA levels (Fig. 2B). OSX was detected earliest at day 10, with increasing levels from day 15. At the beginning of the mineral formation phase (day 20), strong bands for BSP (70 kD) and Col I (60 kD) were observed. We also detected high levels of the OSAD core protein (60 kD) from day 10 and onwards (Fig. 2C). However, at later time points (days 20 and 25), a 120–150 kD smear was observed, suggesting the presence of the fully glycosylated OSAD, with keratan sulphate glycosaminoglycan chains. 3.3. Effect of exogenous BMP-2 and VEGF After establishing the gene expression profile in standard OM cultures, we concluded that the best time to add additional growth factors would be after confluency when the cells had reached the end of the active proliferation phase (Fig 1A). Therefore we supplemented the OM cultures with the two growth factors BMP2, VEGF or a combination of both, at day 10. Cellular metabolic activity as a measure of cell replication was assessed by MTT staining (Fig. 3). After the addition of growth factors (day 10) we observed that the cultures exhibited an increasing metabolic activity, correlating with the gene expression data, and the presence of remaining proliferating cells in the cultures. Addition of either BMP2 or VEGF induced a rapid initial active differentiation process, although with BMP2 this effect diminished by day 19 and the onset of mineralization. The results from adding both growth factors simultaneously followed the pattern of the osteogenic curve, indicating that the activity in cells was similar after these two treatments. Expression levels of bone-related gene mRNAs were standardized against a common internal control HMBS, calibrated to day 0, and normalized to osteogenic conditions (OM) of each separate experiment (Fig. 4). Significance was calculated by comparing the mean values of three experiments when treating the cells with BMP2, VEGF

Fig. 3. Effects of the different OM supplements on cellular proliferation. MTT assays were performed at day 0 and after 7, 13, 19 and 25 days to compare the proliferation rate of differentiating HESCs. Data for the curves are shown as means ± SD of three independent experiments. Cells were treated with 10 mM β-glycerophosphate, 50 μg/ ml ascorbic acid phosphate and 1 μM dexamethasone (OM) alone or with the additional supplementation of BMP2 (100 ng/ml), VEGF (25 ng/ml) or both (BMP2/VEGF). Addition of BMP2 and VEGF rapidly increased the activity, which slowed down at the onset of mineralization on day 19. A combination of both growth factors followed the OM culture curve, indicating that the proliferation activity was similar after these two treatments.

and BMP2/VEGF to the OM cultures at the given timepoint. Cultures supplemented with BMP2 displayed significantly stronger up-regulation of OSX and BSP compared to HESCs treated only with ascorbic acid, β-glycerphosphate and dexamethasone (OM). In addition to the two peaks detected for OSX expression at days 9 and 15 in osteogenic supplemented cultures (Fig. 2), the growth factors yielded a third peak day 23 (Fig. 4). Thus suggesting either that OSX expression was delayed, or that a late additional OSX expression was induced at the onset of mineralization. Hypothetically, BMP2 and VEGF could stimulate OSX expression by increasing the regulators needed for the transcription of OSX itself, or the effect could be due to asynchronous cell cultures with a delayed start of differentiation. BMP2 also increased the expression of BSP mRNA 2.7-fold at day 21, which decreased thereafter. OCN expression increased at the end of the culture period, reaching the highest levels at days 23 and 25. Exposure to ectopic BMP2 increased the expression of OSAD 1.7-fold at day 25. Supplementation with VEGF induced a late peak of OSX at day 23, being 1.5-fold higher than in cultures with only osteogenic supplements (OM), although the up-regulation was not as intense as with BMP2. In contrast, continuous treatment with VEGF significantly reduced the expression levels of OCN, which remained half the level in VEGF treated cells compared to non-supplemented conventional OM cultures. 4. Discussion Embryonic stem cells have become a widely used tool for the study of differentiation into various cell types and several studies have been published on osteoblastic differentiation using HESCs [11,12,14– 16,18,32]. Regrettably, most reports, to date, focus entirely on the final phenotype, leaving the pattern of osteogenic differentiation and the mechanisms mediating differentiation unknown. The process of osteogenesis from HESCs is often triggered by supplementing the medium with ascorbic acid, β-glycerophosphate and dexamethasone, three factors which are not specifically osteoinductive, and cells from other lineages can be generated, too [33–36]. We show here that such osteogenically treated cultures retain a potentially undifferentiated population of cells. The potential role of Oct-4 and Nanog during osteogenic HESC differentiation remains to be established. The finding of increased levels of Nanog in differentiating cells is not novel [37]. Albeit, Nanog has been shown to inhibit the switch from myogenic cells to the osteogenic lineage [38] whereas the forced expression of Nanog in human bone marrow-derived mesenchymal stem cells maintained the cells high proliferative potential and greatly facilitated osteogenic differentiation [39]. However, these studies have used nonHESCs and thus the effects maybe attributable to the different cell types. To address the requirement for well-characterized differentiation systems, the aim of the current study was to analyze the standard model system for osteogenesis of HESCs, and to establish the expression profile of bone-related genes during HESC differentiation. We have previously reported that HESC line HS181 exhibits a significant osteogenic potential, leading to the expression of a panel of known bone-matrix markers, both at the gene and protein level without the need for EB formation [14], a finding also supported by the work of Karp et al. [15]. In light of these previous results we did not repeat them in this current study and tried to move forward with other aspects. In addition, it should be noted that comparison analyses with primary osteoblasts or isolated bone marrow stromal cells were not performed due to the pre-differentiated nature of these cells, which precludes any parallel studies with the undifferentiated HESCs. During osteoblastogenesis, precursor cells are characterized by their extensive replicative capabilities and limited self-renewal, which is exceedingly dependent on cell density [40]. HESCs seeded at high density have been shown to lose the ability to form the characteristic

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Fig. 4. The expression profile of osteoblast and matrix associated genes after the addition of BMP2 (100 ng/ml), VEGF (25 ng/ml) or the combination of both. HESCs were treated continuously with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid phosphate and 1 μM dexamethasone for 25 days. Growth factors were added at day 10 and total RNA was extracted on days 13, 15, 17, 21, 23, 25. Results are presented as means ± SD of three independent experiments normalized to osteogenic conditions. a (BMP2), b (VEGF) or c (BMP2/VEGF) refers to p b 0.05, significantly different from OM at the same timepoint.

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bone-like mineralized nodules, as cell–cell interactions and secretion of factors are directly linked to the distance between individual cells. Thus, initial cell density plays an important role in differentiation, and our pilot experiments revealed that the optimal seeding density for osteogenic HESC cultures was about ∼ 1000 cells/cm2. This provides the cells with the possibility to enter the transit-amplifying phase, creating enough space to proliferate until reaching cell–cell contact at confluency, followed by the interaction with the produced ECM to switch on the optimal signaling pathways. Earlier studies on HESCs have reported that cell–cell contacts at high density can inhibit the proliferative activity and switch the cells to differentiate [41]. Osteoblastic development can be subdivided into several developmental stages: proliferation and differentiation of cells, and ECM synthesis, maturation and mineralization, each with characteristic changes in gene expression [7]. Potential osteogenic cell populations comprise cells with different morphology and activity, and changes in gene expression levels correspond to distinguishable developmental stages. Early stage osteoblasts predominantly express genes which support proliferation and ECM biosynthesis, while phenotypeassociated genes are suppressed during this period. In this study, we used an alternative approach to the HESC osteogenic model and considered separately the cellular compartment activity on one side, and matrix formation and mineralization on the other (summarized in Fig. 5). We believe that the first regulatory transition triggering the initiation of osteoblastic gene expression takes place after the active proliferation step, even though several ECM associated gene mRNAs were expressed in actively proliferating immature cells. It is important to note, that transcription factors which control proliferation of osteoblasts are modestly understood and only a few have been identified as modulators of bone formation. Committed osteoprogenitors express both Runx2 and OSX [42]. We show that at the end of active proliferation, the osteoblast-specific transcription factor OSX was up-regulated suggesting that its expression was regulated by the onset of contact-inhibition. OSX expression diminished to low levels upon entry into the early terminally differentiated osteoblast phase indicating that OSX function precedes matrix maturation. An important molecule to inhibit cellular proliferation and to regulate matrix assembly is ON, a major non-collagenous component of bone, albeit found in other tissues. In the current study we demonstrate that ON was up-regulated straight after the end of the proliferative phase. Another non-collagenous protein, which is currently believed to be mineralizing tissue-specific, is OSAD [43], and has previously been immunolocalized to the primary spongiosa in

the bovine bone growth plate [44], and a recent report showed that OSAD possessed a similar ultrastructural distribution to BSP in rat [45]. OSAD was expressed at the beginning of the culture period, supporting the possibility that it has a role in inhibiting the actively proliferating cells and is associated with the terminally differentiated osteoblastic phenotype [46] and according to our knowledge so far it is considered osteoblast-specific. The second regulatory transition mediates initiation of gene expression for ECM formation, maturation and mineralization. OPN expression appears prior to other matrix proteins including BSP and OCN [47,48], however it is also expressed by many cells other than osteoblasts. OPN gene expression was progressively down-regulated towards the end of the culture, which is in agreement with the reports that low OPN levels are required for apatite crystal growth. The initial higher levels could be attributable to contamination from cultured fibroblasts [49–51], or due to a sign of active proliferation [52–54]. Thus, considering the role of OPN in cell–cell attachment [55] and migration in vitro, we assumed that some cells at the beginning of our study were migrating cells [50,51,56]. Q-PCR analysis revealed that OCN was expressed at the end of matrix maturation, being rapidly down-regulated before mineralization, but thereafter increased again. OCN is known to appear with later stages of mineralization and inhibit this process [57,58]. Another typical property of developing osteoblasts is the expression of receptors for hormones. PTHR1, receptor for PTH [59,60] and parathyroid hormone-like hormone, was up-regulated at the second regulatory phase during matrix maturation. PTHR1 has been described as a “globally” expressed marker for osteoblastic cells, whereas OPN, BSP, and OCN can be differentially expressed at mRNA and protein levels in only a subset of osteoblasts, depending on the maturational state of the cells [60]. The direction of differentiation towards osteogenic lineage with growth factors are essential to either increase the outcome of osteoblastic cells or decrease the presence of other cell types. Due to the specificity of HESCs as an undifferentiated and pluripotent system, the timing is of utmost importance. Here, our results showed that HESCs seeded at 1000 cells/cm2, reached confluency around days 7–8, which was followed by the up-regulation of the bone-specific transcription factor OSX. This provided us with a time window to manipulate the number of osteogenic cells using BMP2 and VEGF. BMP2 is considered as a multi-purpose cytokine that stimulates migration and differentiation of many cell types, furthermore it is currently recognized as the strongest osteoinductive molecule

Fig. 5. A proposed scheme for osteogenic culture in the HESC model with recognizable stages of differentiation as detected from the experiments with HS181 line. Active proliferation of the cells occurred for approximately one week, after which the first regulatory transition phase was initiated (N 8 days). During this time the cells continued to differentiate, and started to express the osteoblast-specific transcription factor, OSX, and pre-phenotypic ECM genes. The second transition stage represents osteoblastic-driven matrix formation and maturation, and mineralization.

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inducing the expression of OSX and subsequent formation of mineralized matrix [61,62]. VEGF, conversely, the best-characterized angiogenic factor is the only mitogen that specifically acts on endothelial cells and is produced by many cell types [63]. The biological function of VEGF is well-established to be mediated through binding to specific tyrosine kinase receptors, Flk-1 (VEGFR-2 or KDR) [64], and an oncogene Flt-1 (VEGFR-1). Osteoblastic cells express both of these receptors [65], indicating a role for VEGF in osteoblastic gene expression. A direct effect of VEGF on osteoblast differentiation [66], and its distinct role in mineralized bone formation during endochondral ossification have been reported [67,68]. In our study, VEGFtreated cells demonstrated down-regulated levels of known osteoblast-associated mRNAs. However, we also show that inclusion of BMP2 rescued expression, which could be due to the fact that during osteogenic lineage progression, in addition to the BMP pathway, several signal transduction pathways mediate osteoblastic gene expression [69–71]. BMP2 and VEGF are known to act downstream of p38 MAPK and may antagonistically affect the regulation of osteoblast differentiation through OSX [72,73]. The combined addition of both growth factors demonstrated that BMP2 decreased the inhibitory effect of VEGF on most of the bone related gene mRNAs. OSX, OCN and OSAD all showed increased expression levels compared to levels in the VEGF treated cells. Addition of BMP2 induced an earlier significant up-regulation of BSP compared to “osteogenically”-treated cells, and the finding that OCN was not significantly increased by BMP2, could be because OCN is expressed at low levels in the young bone, where BSP along with other acidic phosphoproteins are expressed at high levels [74]. The overall higher expression of OSX and BSP, indicative of immature mineralized tissue formation confirms that assumption. Perhaps, continuation of the culture period would have exposed an increased expression level for OCN. Interestingly, the combination of BMP2/VEGF had an inhibitory effect on BSP expression throughout the culture time, although a similar observation was reported in another study where a cross-communication between two pathways was suggested [75]. In summary, our results extend the information concerning expression of known osteoblast-associated genes by differentiating HESCs. We have shown two distinguishable phases during osteoblastic differentiation; firstly, cellular proliferation and secretion of a distinct prematurational matrix needed for cell migration, and secondly, the appearance of committed osteoprogenitors with characteristic ECM synthesis and mineral deposition. This approach helps to identify expression patterns of the required characteristics in further studies using the HESC model, as well as facilitating comparisons with other model systems. Acknowledgements

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