Effects of Culture Conditions and Bone Morphogenetic Protein 2 on Extent of Chon - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:56 |只看该作者 |倒序浏览 |打印

作者：Wei Seong Toha, Zheng Yanga,b, Hua Liua, Boon Chin Henga, Eng Hin Leeb, Tong Caoa # `* l7 e/ e2 I$ f% }1 H

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      【摘要】
   The study of human embryonic stem cells (hESCs) can provide invaluable insights into the development of numerous human cell and tissue types in vitro. In this study, we addressed the potential of hESCs to undergo chondrogenesis and demonstrated the potential of hESC-derived embryoid bodies (EBs) to undergo a well-defined full-span chondrogenesis from chondrogenic induction to hypertrophic maturation. We compared chondrogenic differentiation of hESCs through EB direct-plating outgrowth system and EB-derived high-density micromass systems under defined serumfree chondrogenic conditions and demonstrated that cell-tocell contact and bone morphogenetic protein 2 (BMP2) treatment enhanced chondrocyte differentiation, resulting in the formation of cartilaginous matrix rich in collagens and proteoglycans. Provision of a high-density three-dimensional (3D) microenvironment at the beginning of differentiation is critical in driving chondrogenesis because increasing EB seeding numbers in the EB-outgrowth system was unable to enhance chondrogenesis. Temporal order of chondrogenic differentiation and hypertrophic maturation indicated by the gene expression profiles of Col 1, Col 2, and Col 10, and the deposition of extracellular matrix (ECM) proteins, proteoglycans, and collagen II and X demonstrated that the in vivo progression of chondrocyte maturation is recapitulated in the hESC-derived EB model system established in this study. Furthermore, we also showed that BMP2 can influence EB differentiation to multiple cell fates, including that of extraembryonic endodermal and mesenchymal lineages in the EB-outgrowth system, but was more committed to driving the chondrogenic cell fate in the EB micromass system. Overall, our findings provide a potential 3D model system using hESCs to delineate gene function in lineage commitment and restriction of chondrogenesis during embryonic cartilage development.
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Disclosure of potential conflicts of interest is found at the end of this article. 7 |7 Q; h( M5 C9 \
      【关键词】 Bone morphogenetic protein Chondrogenic Differentiation Embryonic stem cells Human+ S* G# e# Y2 D
   INTRODUCTION

Human embryonic stem cells (hESCs), derived from the inner cell mass of the blastocyst stage embryos, represent a promising cell source for transplantation because of their unlimited self-renewal and ability to differentiate into various somatic cell lineages .' }8 Z# c. w7 [* h/ s

Previously, murine ESCs have been induced through embryoid body (EB) formation and then plated directly to differentiate into prechondrogenic cells that aggregated to form cartilage nodules . No study to date has yet achieved growth factor-induced direct chondrogenesis of hESCs in vitro. Furthermore, the temporal order of chondrogenic induction and hypertrophic maturation of human EB-derived cells in different culture systems has yet to be explored. Establishing a good model system for studying hESC chondrogenesis would undoubtedly provide a powerful tool for analyzing the contribution of specific genes to the process of chondrogenesis and, in particular, the study of genes in which alteration or deletion results in embryonic lethality in genetically altered mice.. P9 A$ w- d3 W
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Hence, this study attempted to achieve and characterize the full-span chondrogenesis of hESC-derived EBs in both EB direct-plating/outgrowth and micromass systems, while also investigating the effects of bone morphogenetic protein 2 (BMP2) on EB chondrogenesis in both systems.
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MATERIALS AND METHODS

Culture of Human Embryonic Stem Cells
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The NIH-registered H1 and H9 cell lines, isolated and established at the University of Wisconsin, were used in this study . Briefly, murine embryonic fibroblasts (MEFs) were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY, http://www.invitrogen.com), supplemented with 10% (vol/vol) fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com) and 1% (vol/vol) nonessential amino acids (Gibco BRL, Gaithersburg, MD, http://www.invitrogen.com). Feeder layers of MEFs were prepared by chemically inactivating subconfluent cultures with 10 µg/ml mitomycin C (Kyowa, Tokyo, http://www.kyowa.co.jp) for 2 hours at 37¡ãC and reseeding at 2 x 105 cells per well of a six-well 0.1% (wt/vol) gelatin-coated plate. hESCs were grown on the inactivated MEF feeder layer with hESC medium composed of DMEM/F12 supplemented with 20% Knockout Serum Replacement (KSR; Gibco BRL), 1% (vol/vol) nonessential amino acids, 1 mM L-glutamine (Gibco BRL), 4 ng/ml fibroblast growth factor 2 (FGF-2; Invitrogen), and 0.1 mM ß-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) with media change every other day. Under these conditions, most of the cells were kept in an undifferentiated state. Confluent hESC cultures on day 7 were passaged by treatment with 1 mg/ml collagenase type IV (Gibco BRL) for 5 minutes, followed by trituration to small clumps. hESCs were replated at 2 x 105 cells per well of a six-well MEF-seeded plate, and proliferated to approximately 1.2 x 106 hESCs after 5¨C6 days.
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Embryoid Body Formation- e3 U! w% W4 J( Q
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hESC colonies were dissociated into small clumps through 5-minute treatment with 1 mg/ml collagenase type IV and then transferred to low-attachment six-well culture plates (Corning, Lowell, MA, http://www.corning.com/lifesciences) in EB formation media consisting of 80% DMEM/F12 and 20% (vol/vol) KSR supplemented with 1% (vol/vol) nonessential amino acids, 1 mM L-glutamine and 0.1 mM ß-mercaptoethanol. The seeding density was approximately 8.0 x 105 cells per well of a nonadherent six-well culture dish. In the presence of a nonadherent surface, the suspended hESC clumps would form three-dimensional (3D) free-floating aggregates or EBs. There were approximately 300 EBs per well. The culture medium was changed every 2 days for a period of 5 days.

Chondrogenic Differentiation via EB Outgrowth and Micromass Systems
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For EB direct-plating culture, 5-day-old (5d) EBs were suspended in high-glucose DMEM supplemented with 10% FBS and 10% KSR before being plated directly onto a 12-well plate precoated with 0.1% gelatin. From the 6-well plate of EBs, 1 well was split to 10 wells of the 12-well plate, maintaining approximately 30 EBs per well (1:10 splitting ratio). The human EB cultures were incubated at 37¡ãC for 24 hours to enable cell attachment before induction of differentiation. EBs attached rapidly upon plating and heterogeneous outgrowth of cells formed a monolayer. This culture system will be abbreviated as "EB outgrowth" for the rest of the article for clarity. For some experiments, the number of EBs seeded was increased accordingly from the initial seeding numbers of EBs.4 m) _3 I# Z) D* c7 D& v2 k

For micromass culture, 5d EBs were dissociated into single cells by means of trypsinization using 0.25% trypsin/EDTA (Sigma-Aldrich) for 5¨C10 minutes, followed by passing the cell suspension through a 22-gauge needle and 40-µm cell strainer (BD Biosciences Inc., Franklin Lakes, NJ, http://www.bd.com) to obtain a single-cell suspension. The dissociated single cells were then resuspended in high-glucose DMEM supplemented with 10% FBS and 10% KSR, and washed twice with the same medium before being cultured at a high density of 3 x 105 cells per 15-µl spot in a 12-well plate precoated with 0.1% gelatin. After incubation for at least 1 hour, 1 ml of the same medium was carefully added to each well. These EB-derived micromass cultures were then incubated at 37¡ãC for 24 hours to enable cell attachment before induction of differentiation.

Chondrogenic differentiation of human EB-derived cells was induced under serum-free conditions modified from the protocol previously described . Because BMP2 provided statistically similar effects on the development of the chondrogenic lineage from both H1 and H9 hESCs, all results from both cell lines were combined for this study.

Alcian Blue Staining$ a2 L/ E4 M4 D; @

Cell cultures were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 30 minutes, washed once with PBS, and rinsed with distilled water (dH2O), then processed for Alcian Blue staining. Specimens were incubated with 0.05% (wt/vol) Alcian Blue solution overnight. Excess stain was removed by washing in PBS, rinsing with 5% acetic acid to remove nonspecific staining, and washing with PBS again.! O/ q$ N3 r# y2 v

Immunofluorescence and Immunohistochemistry$ G, O- b" Q! }$ d$ J
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EBs cultivated on chamber slides were rinsed twice in PBS, fixed for 5 minutes with methanol:acetone (7:3) at ¨C20¡ãC, rinsed three times in PBS again, and incubated for 15 minutes in 10% (vol/vol) goat serum at room temperature (RT). For collagen II detection, specimens were incubated with the monoclonal antibody II-II6B3 (Chemicon, Temecula, CA, http://www.chemicon.com) at a dilution factor of 1:40 for 1 hour at 37¡ãC in a humidified chamber. After incubation, specimens were rinsed three times with PBS, and incubated for 2 hours at RT with Qdot 655 goat anti-mouse IgG antibody (Quantum Dot, Hayward, CA, http://probes.invitrogen.com) diluted 1:200. Slides were then washed three times in PBS and mounted with Vetashield mounting medium with 4',6-diamidino-2-phenylindole for nuclear counterstaining (Vector, Burlingame, CA, http://www.vectorlabs.com). For negative control, primary antibodies were omitted. Analysis was done using the Olympus inverted microscope and its Microimage software (Olympus, Tokyo, http://www.olympus-global.com). To study the effects of growth factors, the area of the immunostained regions in individual EBs was measured and expressed as the percentage of the size of the EB.. S' R' `" x$ s/ p0 ]

For immunohistochemical analysis, cultures were rinsed with PBS and fixed for 30 minutes in 10% (vol/vol) neutral buffered formalin. To facilitate antibody access, cultures were predigested for 20 minutes at 37¡ãC in pepsin (Labvison Inc., Fremont, CA, http://www.labvision.com). Endogenous peroxidase activity was quenched by incubation with hydrogen peroxide block (Labvision) for 15 minutes at RT. The cultures were blocked using Ultra V Block (Labvision) for 5 minutes at RT and then incubated with the respective primary antibodies diluted in PBS for 1 hour at RT. Monoclonal antibodies to collagen II (II-II6B3; Chemicon) and collagen X (clone X-53; Quartett Immunodiagnostika, Berlin, http://www.quartett.com) were used at 1:500 and 1:25 dilution respectively. The control mouse IgG isotype was from Zymed Laboratories Inc. (San Francisco, http://www.zymed.com). After washing with PBS, cultures were treated for 30 minutes with prediluted biotin-conjugated goat-derived anti-mouse secondary antibody and then visualized using streptavidin-conjugated horseradish peroxidase (HRP) and diaminobenzidine chromogen provided in the UltraVision HRP Detection System (Labvision).
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Reverse Transcription Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction
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Total RNA was extracted from each well from a 12-well plate for EB outgrowth and micromass cultures using the RNeasy Mini Kit (Qiagen, Chatsworth, CA, http://www1.qiagen.com), and passing through the Qiashredder following the manufacturer's instructions. Total RNA was also extracted from human articular chondrocytes, human neuroblastoma cell line SH-SY5Y (ATCC CRL-2266, American Type Culture Collection, Rockville, MD, http://www.atcc.org), human vein umbilical endothelial cells (Cambrex, East Rutherford, NJ, http://www.cambrex.com) for use as controls.

Each sample was treated with RNase-Free DNase (Qiagen) to avoid genomic DNA contamination. For each batch of samples, a test polymerase chain reaction (PCR) with housekeeping gene ß-actin was performed to verify the absence of genomic contamination. Reverse transcription reaction was performed using a PCR thermocycler, Mycycler (Bio-Rad, Hercules, CA, http://www.bio-rad.com). cDNA synthesis was generated from 500 ng of total RNA per 20 µl of reaction volume using the iScript cDNA synthesis kit (Bio-Rad).  X  X; D* P1 c
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PCR of the cDNA samples were performed at 95¡ãC for 5 minutes, followed by amplification cycles of 30-second denaturation at 95¡ãC, 45-second annealing at 55¡ãC¨C65¡ãC, 60-second elongation at 72¡ãC, and a final extension at 72¡ãC for 5 minutes. The number of cycles varied between 26 and 40, depending on the abundance of a particular mRNA. In addition, all RNA samples were adjusted to yield equal amplification of ß-actin as an internal control to normalize PCR reactions. The amplified products were subjected to electrophoresis on 2% agarose gels and subsequently stained with ethidium bromide and photographed using the Light Imaging System (Bio-Rad). For semiquantitative analysis, mean pixel intensity of each band was measured using the NIH public domain imaging software, and normalized to mean pixel intensity of the corresponding ß-actin. Each sample was repeated at least three times for each gene of interest. PCR primers, annealing temperature, and their expected product sizes are described in supplemental online Table 1.+ r( x% K9 u/ ~8 x1 _# B: ?+ ^

For quantitative analysis, Oct4, Col 1, and Col 2 gene expressions were analyzed by real-time reverse transcription PCR reactions using the SYBR Green PCR Master Mix System (Qiagen) on a PCR thermocycler, Stratagene MX3000P (Stratagene, La Jolla, CA, http://www.stratagene.com). cDNA samples (1 µl for a total volume of 25 µl per reaction) were analyzed for the gene of interest normalized to reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The level of expression of each target gene was then calculated as 2¨CCt, as previously described . Each sample was repeated at least three times for each gene of interest. Real-time reverse transcription PCR was performed at 95¡ãC for 15 minutes followed by 40 cycles of 15-second denaturation at 94¡ãC, 30-second annealing at 55¡ãC, and 30-second elongation at 72¡ãC. PCR primers, annealing temperature, and their expected product sizes are described in supplemental online Table 2.; C# i, z9 F" E9 I& A1 {
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Col 10 was analyzed by customized probe-based real-time reverse transcription PCR reaction obtained for Taqman gene expression system (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems). cDNA samples (2 µl for a total volume of 20 µl per reaction) were analyzed in duplicates and normalized to the internal reference gene GAPDH. Data analysis was carried out using the Sequence Detector V program (Applied Biosystems 7500 Real-Time PCR System). The level of expression of Col 10 gene was then calculated as 2¨CCt, as previously described . Real-time reverse transcription PCR was performed at 95¡ãC for 10 minutes, followed by 45 cycles of 15-second denaturation at 95¡ãC and 1-minute extension at 60¡ãC.

Sulfated Glycosaminoglycan and DNA Quantitation
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To measure total glycosaminoglycan accumulation in chondrogenic cells with time in different culture conditions, cultures were digested with 200 µl of papain digestion buffer (125 µg/ml in sterile phosphate buffered saline, pH 6.0, with 5 mM cysteine hydrochloride and 5 mM Na2EDTA) for 18 hours at 60¡ãC. Sulfated glycosaminoglycan (s-GAG) content was measured spectrophotometrically at 630 nm using Biocolor Blyscan Glycosaminoglycan Assay kit (Biocolor Ltd, Newtownabbey, U.K., http://www.biocolor.co.uk), and normalized to the DNA content measured spectrophotometrically using the Hoechst 33258 method . The fluorescence measurement of Hoechst 33258 dye was performed using a fluorescence plate reader (Tecan Safire, Männedorf, Switzerland, http://www.tecan.com). The standard curve of s-GAG was constructed using bovine trachea chondroitin sulfate, according to the manufacturer's instructions. Calf thymus DNA was used for construction of the standard curve for DNA quantitation.

Alkaline Phosphatase Activity" x  [, b$ W. v# O6 d6 M
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The total alkaline phosphatase (ALP) activities of cells and matrix were measured according to the method described previously ). Cell suspensions were sonicated before enzyme assays to dissociate the ECM and liberate membranous ALP. Specific ALP activity was assayed as the release of p-nitrophenol from p-nitrophenylphosphate and measured spectrophotometrically at 405 nm, assuming that 1 A405 = 64 nmol of product. Protein concentration was determined using the Pierce BCA protein assay kit (Pierce Chemicals, Rockford, IL, http://www.piercenet.com).
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Statistical Analysis

All quantitative data reported here were analyzed using Student's t test. Data are presented as the mean ¡À SD, with the level of significance set at p
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RESULTS6 I- |$ @* F  `2 j6 h

Effects of BMP2 on Chondrogenesis of Human EB-Derived Cells' O) d+ _5 X, m% p% @$ _. P, Z

Recent studies  showed that BMP2 is able to induce chondrogenic differentiation of murine ESCs using the EB direct-plating method. Initial experiments, therefore, examined the effects of BMP2 on the intact EBs. EBs attached rapidly on plating, and heterogeneous outgrowth of cells formed a monolayer, that, on confluence, developed highly condensed areas in some parts of the EB outgrowth.

Under our control chondrogenic condition, only little collagen II deposition was detected in highly condensed areas of the EB outgrowths in control cultures (Fig. 1A¨C1F). In contrast, BMP2-treated cultures resulted in collagen II deposition in the EB outgrowths (Fig. 1G¨C1L) found predominately in monolayer cells (Fig. 1I, 1J) and nodules in the periphery of EBs (Fig. 1K, 1L). Subsequent image analysis yielded a significant enhancement (p
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Figure 1. Effects of BMP2 on human EB-derived cells. Interference contrast (A, C, E, G, I, K) and indirect immunofluorescence micrographs (B, D, F, H, J, L) of control (A¨CF) and BMP2-treated (G¨CL) EB outgrowth 21 days after treatment. Expression of cartilage-specific collagen II protein was analyzed by immunofluorescence staining. Representative areas of the EB and its outgrowth are shown. BMP2 induced EB differentiation with intense staining of collagen II-producing cells at the boundary of the EB (H) and outgrowth of round-shaped chondrogenic cells that could exist as single cells in monolayer (J) or in three-dimensional nodular aggregates (L). Scale bar = 100 µm. Graph showing the percentage of collagen II immunostained areas in the EB and its outgrowth per size of EB after BMP2 treatment (M). Mean values ¡À standard derivation derived from duplicate analysis of two independent experiments are shown, with statistical significance at p ( g0 r- f, g4 m! I/ H5 b  W
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This finding was further validated by reverse transcription PCR analysis (Fig. 1N) carried out on day 21 of differentiation, showing a significant degree of chondrogenic differentiation under control chondrogenic conditions, marked by an increase in transcript level of chondrogenic gene markers Sox9, Col 2a1, and Link protein compared to their day-0 expression level. Under continuous BMP2 treatment, there was further upregulation of these chondrogenic genes, as well as Col 1, when compared to the control. Furthermore, there was also an increase in the level of endogenous BMP2 transcripts in the control culture, which was further enhanced in the BMP2-treated culture.

Modulation of Chondrogenesis in EB Outgrowth and Micromass Cultures

To further investigate the full span process of chondrogenesis, EBs were either plated directly at approximately 30 EBs per well or trypsinized into single cells and plated at a high density of 3 x 105 cells per micromass to facilitate cell-to-cell contact at the beginning of differentiation. Cells expressed Col 2 in both EB outgrowth and micromass cultures in chondrogenic conditions, but the temporal expression profiles between the two systems differed dramatically. For control EB outgrowth, the expression of Col 2 mRNA level increased from day 3 and reached a plateau by day 11, without significant changes thereafter. The Col 2 mRNA level in the BMP2-treated EB outgrowth was consistently higher than in the control, with a slow and steady increase in the Col 2 mRNA level from day 3 onward before it peaked on day 14 with a more than twofold increase compared to the corresponding control (p , K5 E1 }& {0 w/ K5 D

Figure 2. Chondrogenic induction in EB outgrowth and EB-derived micromass. 5-day-old EBs were either plated directly or dissociated into single cells and plated as a high-density micromass. Expression of Col 2 (A, B) and Col 1 (C, D) were analyzed along the course of chondrogenesis by quantitative real-time reverse transcription polymerase chain reaction in both EB outgrowth (A, C) and micromass cultures (B, D). Mean magnitude of mRNA levels was normalized to GAPDH and expressed relative to the day-0 expression level of 5-day-old EBs. Values are means ¡À standard deviation (SD) from duplicate analysis of at least two experiments, expressed as mean ¡À SD, and statistical significance was set at p % }7 j) W- f6 O: [' h

We also analyzed the Col 1 gene expression profile in both systems along the course of chondrogenesis. The expression of Col 1 mRNA level increased in a time-dependent manner in control EB outgrowth, and was consistently higher in the BMP2-treated EB outgrowth throughout the entire course of differentiation (Fig. 2C). In contrast, the profile of Col 1 gene expression differs dramatically in the micromass system (Fig. 2D). In the control micromass culture, there was increase in Col 1 mRNA level up to day 7 before it reached a plateau. In the BMP2-treated micromass culture, Col 1 mRNA level initially peaked at days 7 and 11, with significantly higher levels than in the corresponding control, before it declined to levels similar to the those in the control by day 14.
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Collagen II immunohistochemistry carried out at the end of the 21-day cultures showed weak deposition of collagen II in EB aggregates/nodules of the EB outgrowth. Comparatively, collagen II deposition was intense in the micromass cultures. BMP2 supplementation was able to enhance collagen II deposition in the EB-outgrowth culture, but to a much higher degree in the micromass culture (Fig. 2E).; }) r5 Y$ `- {- H
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The accumulation of s-GAG in control and BMP2-supplemented culture in both systems was compared to the 5d EBs at day 0. Under the EB-outgrowth system, there was accumulation of s-GAG in both control and BMP2 treated groups along the course of differentiation, with no significant additive effect from BMP2 (Fig. 3C). EB-derived cells plated in monolayer exhibit extensive proliferation, as shown by a fourfold increase in DNA levels by day 7 of differentiation (Fig. 3E). As a result, the s-GAG/DNA analysis showed continuous low levels in both control and BMP2-treated cultures when analyzing the s-GAG synthesis on a single-cell level (Fig. 3G). Alcian Blue staining at the end of 21 days showed no apparent difference between the control and BMP2-treated EB outgrowth (Fig. 3A), which is reflective of the quantitative data. By dissociating the EB cells and plating under micromass culture condition, the cells exhibit minimal proliferation, with less than a twofold increase in DNA levels by day 7 of differentiation (Fig. 3F). There was no significant difference in the DNA levels between the control and the BMP2-treated samples. BMP2 treatment upregulates s-GAG deposition from day 7 to day 14 (Fig. 3D), which, after normalizing to the DNA content, resulted in a significant increase in s-GAG/DNA levels compared to the controls (p ; R5 Z$ k% }% J

Figure 3. Effects of BMP2 on s-GAG synthesis in EB outgrowth and EB-derived micromass. 5-day-old EB cells were either plated directly or dissociated and cultured as high-density micromass under control and BMP2 supplemented serum-free culture conditions for 21 days before staining with alcian blue. No significant enhancement in staining was observed in the cultures of EB outgrowth (A). Cartilage nodules with dark blue appearance were observed in BMP2-treated micromass culture, compared to the control (B). For quantitative analysis, s-GAG content (C, D) as well as changes in DNA content (E, F) per sample were determined. The s-GAG content was normalized against the DNA content to determine the s-GAG synthesis per cell (G, H). Measurements reported were averaged from duplicate analysis of at least two independent experiments, and data are expressed as mean ¡À standard deviation, with statistical significance set at p

Hypertrophic Development in EB Outgrowth and Micromass Cultures; `! I! l  U* L6 A5 o, w

To investigate the hypertrophic maturation of human EB-derived chondrogenic cells in the two systems, Col 10 mRNA expression was analyzed under both control and BMP2-treated conditions along the course of differentiation (Fig. 4A, 4B). When compared to day-0 expression levels, no significant increase in Col 10 mRNA level was observed in control EB outgrowth throughout the entire course of differentiation. Conversely, in the BMP2-treated EB outgrowth, a significant increase in Col 10 mRNA level was observed from day 14 onward, with an approximately twofold increase compared to the control on day 21 (Fig. 4A).
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Figure 4. Hypertrophic development in EB outgrowth and EB-derived micromass. 5-day-old EBs were either plated directly or dissociated into single cells and plated as a high-density micromass. Expression of hypertrophic marker gene Col 10 was analyzed along the course of chondrogenesis by quantitative real-time reverse transcription polymerase chain reaction in EB outgrowth (A) and micromass cultures (B). Mean magnitude of mRNA levels was normalized to GAPDH, and expressed relative to the day-0 expression level of 5-day-old EBs. Values are displayed as means ¡À standard deviation (SD) from duplicate analysis of at least two independent experiments, expressed as mean ¡À SD, and statistical significance was set at p

When compared to the day-0 expression level, the high-density micromass system promoted a rapid increase in Col 10 mRNA level as early as day 7, and the upregulation progressively increased until day 21 with a significant 700- and 1,000-fold increase in the Col 10 mRNA levels observed in the control and BMP2-treated micromass cultures, respectively (Fig. 4B). This phenomenon appeared to be independent of the BMP2 stimulation at the early stages of differentiation (day 7 to day 18), and a further increase in Col 10 mRNA expression level was induced only in BMP2-treated micromass cultures from day 18 onward (Fig. 4B). Col 10 expression levels in the high-density micromass system were consistently higher than in the EB outgrowth counterparts throughout the course of differentiation, with a remarkable 1,000-fold upregulation observed on day 21.5 V. M5 G% o1 Z4 f: B
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Collagen X immunohistochemistry carried out at the end of 21 days showed some collagen X staining in EB aggregates/nodules of the EB outgrowth, with no significant difference between the control and the BMP2-treated cultures. In contrast, the micromass cultures promoted a significant increase in collagen X deposition, which was further enhanced in the presence of BMP2 (Fig. 4C).

The chondrocyte hypertrophic phenotype was further confirmed with analysis of alkaline phosphatase activity. The ALP activity profile differed dramatically between the two culture systems (Fig. 5). ALP activity of 5d EB at day 0 was high, and on plating, there was an acute increase in ALP activity by day 7, which then decreased in a time-dependent manner up to day 21. Conversely, ALP activity decreased in the micromass system to almost basal levels on day 7. Under continuous chondrogenic culture conditions, there was an increase in ALP activity on day 14, when chondrogenic cells entered hypertrophic maturation. ALP activity was significantly enhanced by BMP2-treatment on day 21 when compared to the control (p
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Figure 5. Effects of BMP2 on ALP activity in EB outgrowth and EB-derived micromass. Both systems displayed strikingly different temporal patterns of ALP activities. In the EB-outgrowth culture, there was an increase in ALP activity on d7, followed by a time-dependent decrease up to d21 and no significant induction of activity by BMP2 was observed. In contrast, there was a decrease in ALP activity at d7 in the micromass culture, followed by a time-dependent increase up to d21, and significant increase in ALP activity by BMP2 was observed on d21. Measurements reported were averaged from duplicate analysis of at least two independent experiments, and data are expressed as mean ¡À standard deviation, with statistical significance set at p # C! L& f2 k8 N5 t& t

Modulation of Chondrogenesis in Different EB Seeding Densities
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Next, we examined the effects of increasing the seeding numbers of EBs on chondrogenic induction at day 7 of differentiation to investigate whether provision of a denser outgrowth of cells could facilitate cell condensation (henceforth, "chondrogenic differentiation"). However, we observed by real-time PCR analysis (Fig. 6) that Col 2 was markedly decreased when a higher number of EBs were seeded, and supplementation of BMP2 further reduced the Col 2 mRNA expression levels in a density-dependent manner. On the other hand, BMP2 induced significant increase in Col 1 and FP mRNA levels in all densities tested. In fact, FP expression was increased in a density-dependent manner even in the controls, and further augmented by BMP2, suggesting that the default differentiation pathway of EBs in the outgrowth conditions may be more inclined toward extraembryonic endoderm lineages. Analysis of the Oct4 expression indicated a loss of pluripotency, which was enhanced by BMP2 in all densities tested. Similar results were observed at day 14 of differentiation (data not shown).% `7 E8 R$ a, ^# @# ?+ n

Figure 6. Effects of different EB seeding numbers on chondrogenic induction in an EB-outgrowth system. 5¡®d' EBs were plated directly at seeding numbers 30, 60, and 120 EBs. Expression of Oct4 (A), Col2 (B), Col1 (C) were analyzed at day 7 of chondrogenic differentiation by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Mean magnitude of mRNA levels were normalized to GAPDH, and expressed relative to the day-0 expression level of 5¡®d' EBs. -FP expression, representative of the extraembryonic endoderm, was analyzed by RT-PCR (inset) and quantified by image analysis. Mean pixel intensities of -FP expression were normalized against ß-actin and expressed relative to the day-0 expression level of 5¡®d' EBs (D). Abbreviations: 5¡®d', 5-day-old; B, BMP2; BMP2, bone morphogenetic protein 2; C, control; EB, embryoid body; -FP, -fetoprotein./ `0 o7 ]( u9 ]% a
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Effects of BMP2 and Culture Systems in Lineage Selection During Chondrogenesis
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We also investigated how the two systems may modulate the differentiation of other lineages under our chondrogenic conditions (Fig. 7). In the EB-outgrowth system, differentiation to neuroectodermal lineage, as marked by the expression of neuroD1, was highly upregulated, whereas the high-density micromass system was able to inhibit neuronal differentiation even in the absence of BMP2. BMP2 supplementation was required to completely inhibit the neuroectodermal lineage in the EB-outgrowth system by day 21. The high-density micromass system was able to partially inhibit differentiation of extraembryonic endoderm lineage (indicated by FP) and hematopoietic and endothelial lineages (indicated by VEGFR-2). Adipogenesis (indicated by PPAR) was highly upregulated in the outgrowth system, but was almost completely inhibited in the micromass system.; Y# j# ~0 w( H/ F6 G5 S' g6 f& c

Figure 7. Lineage restriction analysis of EB-derived cells cultured as EB outgrowth and EB-derived micromass. 5¡®d' EBs were either plated directly at 30 EBs per well or dissociated into single cells and plated as a high-density micromass. RNA was extracted on days 7, 14, and 21, and reverse transcription polymerase chain reaction analysis was performed to detect the expression of genes denoted. Expressions of pluripotency and differentiation gene markers of neuroectodermal-, endodermal-, and mesodermal-derived hematopoietic, endothelial, and adipogenic lineages were analyzed. Appropriate positive and negative controls were included. Abbreviations: 5¡®d', 5-day-old; B, BMP2; BMP2, bone morphogenetic protein 2; C, control; Cho, chondrocytes; EB, embryoid body; -FP, -fetoprotein; Huv, human umbilical vein endothelial cells; Neu, neuroblastoma cell line; NTC, no transcript control.

DISCUSSION

BMPs, as members of the transforming growth factor (TGF)-ß superfamily of growth and differentiation factors, were originally identified as potent inducers of bone and cartilage formation at ectopic sites .

In our study, BMP2 was able to enhance initiation of mesenchymal condensation in regions of EB outgrowth, forming chondrogenic nodules as observed by collagen II immunostaining. Single chondrogenic cells were also observed at the periphery of the chondrogenic nodules and EBs. Presumably, these chondrogenic cells migrate out of the EB to the periphery, where they proliferate to form centers of mesenchymal condensation, in which cells synthesize ECMs that are abundant in collagen II. Interestingly, there was also significant induction of Col 1, Col 2a1, Link protein, and Sox9 genes under control chondrogenic condition, which was further enhanced by BMP2 treatment. Because the BMP2 gene was upregulated under control chondrogenic conditions, we speculate that endogenous BMP2 signaling could be triggering chondrogenic differentiation in the control cultures.
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Our findings on hESCs correlate with the murine ESC studies, which showed that BMP2 is capable of inducing chondrogenic differentiation in vitro . Therefore, one would envisage that a serum-free, growth factor-supplemented system is able to better mimic the cartilage microenvironment for chondrogenic differentiation of hESCs compared to a serum-supplemented system.5 ?# p! L) }4 m7 M4 w" X! D$ W
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In an attempt to enhance chondrogenic differentiation, we further explored the possibility of disrupting the 5d EBs and culture the single cells obtained in a 3D high-density micromass configuration. In micromass cultures, under both control and BMP2 supplemented conditions, morphological mesenchymal condensations were apparent by day 7 of differentiation, and distinct cartilaginous nodules could be observed by day 14, which further increased in number and size up to day 21 (data not shown). Gene expression analysis further showed that chondrogenic differentiation occurred much earlier and with enhancement in the micromass system when compared to the EB-outgrowth system. Comparatively, there was a slower and weaker induction of chondrogenesis in the EB-outgrowth system that became efficient only when the cells reached postconfluence, between days 11 and 14 of culture.
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The differences in temporal order and magnitude of early chondrogenesis in the two culture systems were further demonstrated in subsequent hypertrophic maturation. Results from Hwang et al.  suggested a possible inhibitory role of BMP2 in hypertrophic differentiation. However, our observations suggested that hypertrophic maturation can be induced by cell-to-cell contact and BMP2 treatment, whereas in combination, these conditions further resulted in much increased collagen X deposition and ALP activity. It is evidenced that cell-to-cell contact promoted not only earlier chondrogenic activation but also an earlier and more robust activation of hypertrophic development, marked by a striking 1,000-fold increase in Col 10 expression level compared to the EB outgrowth counterparts, which exhibited only a fivefold increase. This phenomenon appeared to be independent of BMP2 stimulation in the micromass cultures with increase in Col 10 expression level and ALP activity by BMP2 only being observed at the later time points (after 18 days of differentiation) of the differentiation. Whether endogenous BMP2 or other growth factors triggered by cell-cell contact are responsible for the dramatic Col 10 expression in the 3D micromass culture is currently under investigation.
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The gene expression profile of the chondrogenic development within the micromass system was further substantiated by determinations of the s-GAG synthesis and ALP activity. BMP2 induced significant mesenchymal condensation to form chondrogenic nodules and increased the ratio of s-GAG/DNA by approximately twofold on days 7 and 14, before decreasing to a level similar to that in the control. Decrease in ratio of s-GAG/DNA in the presence of BMP2 was observed only from day 14 onward, the same time point at which an increase in ALP activity and in Col 10 expression level occurred.% ~7 ?& q9 c% G' M0 G
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Although there was marked accumulation of s-GAGs in the EB outgrowths along the course of differentiation, there was no significant difference in the s-GAG levels, and an acute decrease in ratio of s-GAG/DNA levels in both control and BMP2-supplemented conditions was observed. This results from the uncontrolled outgrowth of EB-derived cells, where there is only a small proportion of committed chondrogenic cells responsive to exogenous BMP2 stimuli. Other cell types, presumably the extraembryonic endodermal cells and the mesenchymal intermediate cells, fibroblastic or osteoblastic, might also be induced by BMP2 to proliferate in the outgrowth, as evidenced by continuous expression of high levels of FP and Col 1 in the outgrowth system, respectively .' f7 {, P4 C7 K( z; y+ t7 N
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The conventional EB direct-plating system, strictly speaking, represents a mixture of 3D environment within the EBs as well as monolayer in the EB outgrowth. Previous studies demonstrated differentiation of mouse EBs into the chondrogenic lineage, which further progressed to hypertrophic maturation in the EB direct-plating system  instead of solely chondrogenic lineage in an EB-outgrowth monolayer system.6 N( ^2 Y' S: E/ ?

In contrast, with the derivation of single cells from the EBs, controlled cell seeding and provision of 3D high-density micromass system at the beginning of differentiation allows immediate cell condensation to form chondrogenic nodules, and, in combination with BMP2, results in formation of cartilaginous tissue that is rich in both s-GAGs and collagen II deposition. This system, in a way, omits the need for cells to migrate out of the EB before condensation can occur at cell confluence, and provides a better selection of chondrogenic cells for differentiation in the high-density microenvironment. We observed that, in the micromass culture system, BMP2 upregulated the expression of Col 1 at the early stage of chondrogenesis before declining to lower levels. This has been reported in the case of mesenchymal progenitor cells, chondrogenic induction by BMP2 depends greatly on cell-to-cell interactions . In contrast, in the micromass system, the EBs were first dissociated enzymatically into a single-cell suspension, which, in turn, breaks down the intercellular contact and gap junctional coupling involved in endodermal differentiation. There was also an overall reduction in expression of mesodermal-derived hematopoietic marker VEGFR2 and adipogenic gene marker (PPAR), suggesting that the capability of dissociated EB-derived cells to differentiate into multiple lineages may be limited after chondrogenic differentiation in a high-density micromass microenvironment.
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We hypothesized that, under the micromass culture conditions, differentiation toward the chondrogenic lineage is enhanced, and other lineages could be inhibited. The culture conditions may be selecting for the growth of a subpopulation of EB-derived progenitor or stem cells with the capability of mesenchymal differentiation. The expression of Oct4 is compatible with the kinetics of chondrogenesis in the EB-outgrowth system in that directly plated EBs still retain a certain degree of the pluripotency that may hinder the differentiation process. This is also substantiated by the high ALP activity of EB outgrowths detected on day 7. In the micromass system, where cells were dissociated and cultured in a high-density 3D microenvironment, earlier onset of chondrogenic differentiation, accompanied by a rapid loss of pluripotency, indicated by disappearance Oct4 and a decline in ALP activity, was observed.
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Taken together, our findings suggest that the high-density environment created by increasing the seeding numbers of EBs is not exactly the same as the high-density 3D microenvironment created by the dissociated EBs, and is insufficient to drive the cells to a chondrogenic cell fate. The micromass culture system that creates the high-density 3D microenvironment at the beginning of differentiation facilitates overall cell-to-cell contact and mimics in vivo limb development, in which there is condensing mesenchyme prior to induction of chondrogenesis . In combination with BMP2, the micromass culture system resulted in both enhancement and enrichment of chondrogenic differentiation.
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CONCLUSION* c) ^; O3 x( V
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In this study, we found that the kinetics of chondrogenesis are significantly affected by the dissociation of the EB cells and plating of the cells in high-density 3D microenvironment at the time of differentiation. By dissociating the EBs and plating them as a high-density micromass, we showed that chondrogenesis is greatly hastened, with further progression to hypertrophic maturation. This process is further augmented with BMP2 supplementation. In addition, we also showed that differentiation to other lineages maybe limited after chondrogenic differentiation in a high-density 3D microenvironment. Collectively, these findings provide a potentially efficient and reproducible 3D experimental system to delineate unknown genes or signaling molecules involved in different stages of chondrogenesis by means of molecular and genetic manipulation, as well as in combination with appropriate gene targeting and selection strategies to generate functional chondroprogenitors and chondrocytes for repair of cartilage defects .' O4 G1 Q  _3 f- ~( l$ z9 t3 d% ^
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST9 I0 ?9 l2 X/ r( v6 t3 y' B3 }

The authors indicate no potential conflicts of interest.  V; J# o: k5 v6 |- ^% `  X

ACKNOWLEDGMENTS

We thank Prof. Lawrence W. Stanton and Dr. Paul Robson from Genomic Institute of Singapore for their advice on human ESC culture work; Dr. James Hui, H.P., for the cartilage tissue biopsies; and Dr. Mitchell Lai, K.P., for the human neuroblastoma cells.- E. M7 ~) f! `) V8 k0 [' Z, q
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