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EMBRYONIC STEM CELLS |
aStem Cell Laboratory, Faculty of Dentistry, and
bDepartment of Orthopaedic Surgery, Yong Loo Lin School of Medicine, Tissue Engineering Program, National University of Singapore, Singapore
Key Words. Bone morphogenetic protein 2 • Chondrogenic • Differentiation • Embryonic stem cells • Human
Correspondence: Tong Cao, D.D.S., Ph.D., Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119-074. Telephone: +65 651-64630; Fax: +65 677-45701; e-mail: omscaot{at}nus.edu.sg
Received May 30, 2006;
accepted for publication December 27, 2006.
First published online in STEM CELLS EXPRESS January 11, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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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 [4], in a manner similar to the condensation of differentiating mesenchymal stem cells. EB-derived chondrocytes were shown to develop further into mature chondrocytes [5]. Although studies have shown that it is possible to obtain mature mesenchymal cell types from hESCs [6–8],the cellular and molecular mechanisms underlying the mesenchymal transition, formation of mesenchymal precursors and onset of chondrogenesis remain obscure. To date, directed chondrogenic differentiation in vitro has been reported only for murine ESCs [4, 9–13], with only one report on chondrogenic differentiation of human embryonic stem cells by coculture method [14]. 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.
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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Embryoid Body Formation
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
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°C 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.
For micromass culture, 5d EBs were dissociated into single cells by means of trypsinization using 0.25% trypsin/EDTA (Sigma-Aldrich) for 5–10 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°C 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 [16, 17]. The basal serum-free chondrogenic medium consisted of high-glucose DMEM supplemented with ITS+1 (6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ml bovine serum albumin, 5.35 µg/ml linoleic acid (BD Biosciences), 1% KSR, 40 µg/ml L-proline (Sigma), 50 µg/ml ascorbic acid 2-phosphate (Sigma-Aldrich), 1% sodium pyruvate (Sigma-Aldrich), 1% nonessential amino acids (Gibco BRL), 10–7 M dexamethasone (Sigma-Aldrich), and 100 units/100 µg penicillin/streptomycin (Gibco BRL). After incubation for 24 hours to allow cell attachment (day 1 of differentiation), the medium was replaced with serum-free chondrogenic medium in the presence or absence of 100 ng/ml recombinant human BMP2 (R&D Systems, Minneapolis, http://www.rndsystems.com) for a period of 21 days with media change every alternate day. Cultures in the basal medium alone without growth-factor supplementation served as the control for this study. BMP2 was chosen because it has been demonstrated to be a potent chondrogenic factor for mesenchymal stem cells in vitro [18, 19] and plays an important role in the chondrogenesis of condensed mesenchyme in vivo [20]. 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
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.
Immunofluorescence and Immunohistochemistry
EBs cultivated on chamber slides were rinsed twice in PBS, fixed for 5 minutes with methanol:acetone (7:3) at –20°C, 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°C 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.
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°C 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).
Reverse Transcription Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction
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).
PCR of the cDNA samples were performed at 95°C for 5 minutes, followed by amplification cycles of 30-second denaturation at 95°C, 45-second annealing at 55°C–65°C, 60-second elongation at 72°C, and a final extension at 72°C 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.
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–
Ct, as previously described [21]. Each sample was repeated at least three times for each gene of interest. Real-time reverse transcription PCR was performed at 95°C for 15 minutes followed by 40 cycles of 15-second denaturation at 94°C, 30-second annealing at 55°C, and 30-second elongation at 72°C. PCR primers, annealing temperature, and their expected product sizes are described in supplemental online Table 2.
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–Ct, as previously described [21]. Real-time reverse transcription PCR was performed at 95°C for 10 minutes, followed by 45 cycles of 15-second denaturation at 95°C and 1-minute extension at 60°C.
Sulfated Glycosaminoglycan and DNA Quantitation
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°C. 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 [22, 23]. 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
The total alkaline phosphatase (ALP) activities of cells and matrix were measured according to the method described previously [24]. In brief, cultures were incubated in an extraction buffer (1.5 M Tris buffer, pH 9, with 1 mM MgCl2 containing 1% Triton X-100 [Bio-Rad]). 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).
Statistical Analysis
All quantitative data reported here were analyzed using Student's t test. Data are presented as the mean ± SD, with the level of significance set at p < .05. Each measurement reported here was based on duplicate analysis of at least two independent experiments.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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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–1F). In contrast, BMP2-treated cultures resulted in collagen II deposition in the EB outgrowths (Fig. 1G–1L) 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 < .05) compared to the control (Fig. 1M).
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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 < .05), and declined thereafter (Fig. 2A). On the other hand, the micromass culture system resulted in a sharp increase in Col 2 mRNA level that peaked on day 7 (sixfold compared to undifferentiated cells) and decreased to almost basal levels by day 14. This profile was similar between the control and the BMP2-treated micromass cultures, with BMP2 exerting an enhancing effect with approximately a twofold increase between days 7 and 14, compared to the corresponding control (Fig. 2B; p < .05). The micromass system promoted earlier chondrogenic differentiation, marked by the Col 2 expression level that peaked on day 7, compared to the EB-outgrowth system that peaked later between days 11 and 14. BMP2 supplementation was able to enhance chondrogenic differentiation in both culture systems.
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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).
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 < .05; Fig. 3H). During the later stage of differentiation at day 21, the s-GAG production plateaus (Fig. 3D), whereas there is still continuous increase in cell number, resulting in a slight decrease in s-GAG/DNA levels (Fig. 3H). Phenotypically, addition of BMP2 resulted in intensified mesenchymal condensation and cartilage nodule formation, as visualized by alcian blue staining (Fig. 3B).
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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 < .05).
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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).
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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.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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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.
Our findings on hESCs correlate with the murine ESC studies, which showed that BMP2 is capable of inducing chondrogenic differentiation in vitro [4, 11]. Unlike serum-containing culture systems, a defined "serum-free" differentiation system supplemented with synthetic serum replacement, KSR, was applied in our studies, taking into consideration that mesodermal differentiation of ESCs may be inhibited under strictly serum-free conditions [31]. KSR has been reported to be completely devoid of any undefined growth factors or differentiation-promoting factors, and would, therefore, be applicable in this instance for achieving controlled differentiation into the chondrogenic lineage in vitro [3]. The serum-free culture system has also been suggested to recapitulate in vivo chondrogenesis better [13], because it is believed to promote better cell selection and differentiation than does a serum-supplemented system. Cartilage is avascularized, and in vivo, mesenchymal cells rely on various autocrine/paracrine growth factors and cytokines for their growth and differentiation [32]. 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.
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.
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. [9] 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.
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.
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 [12, 33]. Another possibility could be that BMP2 alone may not be sufficient to promote all aspects of chondrogenesis, and that other factors such as TGFß1 may be necessary, on the basis of reports that BMP2 and TGFß1 act by distinct mechanisms to regulate chondrogenic cell fate [34–37].
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 [4, 5]. Our findings suggested that direct-plating of EBs could not provide for controlled seeding density and is insufficient to create an effective and uniform 3D microenvironment necessary for chondrogenic induction. One would envisage that, in such a system, an array of cellular differentiation events and lineages coexist and behave in a rather chaotic way, depending on the biochemical cues and microenvironment within the culture. Moreover, direct plating of EBs could not provide for controlled seeding density, which created difficulty for consistent, controlled differentiation. Earlier observations by Hwang et al. suggested that EBs did not consistently undergo chondrogenesis [9]. In our study, heterogeneous and uneven type II collagen staining in the EB outgrowths was also observed. Our study examining the effects of increasing seeding density of EBs in the outgrowth system further demonstrated the importance of an appropriate 3D high-density cell-to-cell contact system at the beginning of differentiation for directing a chondrogenic cell fate. When we increased the EB seeding numbers, the cells lost their stem cell properties marked by decreased Oct4 expression, indicating differentiation. However, the chondrogenic potential of these cells dropped drastically, marked by decreasing levels of Col 2 expression, along with increasing numbers of EBs plated. Addition of BMP2 resulted in even lower Col 2 mRNA levels. In contrast, the levels of Col 1 and FP expression were augmented by BMP2 in all densities of EBs tested. One possible explanation could be that pleiotropic effects of BMP2 are driving other lineages' differentiation, especially that of extraembryonic endodermal [33] instead of solely chondrogenic lineage in an EB-outgrowth monolayer system.
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 [18, 19], and in monolayer culture conditions, in which BMP2 leads to mesenchymal multilineage development of these cells, ending up predominantly with osteoblasts and low levels of chondrocytes and adipocytes [38–40]. A recent study by zur Nieden et al. [11] also suggested that BMP2, depending on the cofactors, was able to induce adipogenesis, chondrogenesis, and osteogenesis in differentiating mouse EB cultures in which studies were done in a two-dimensional EB direct-plating system, allowing extensive outgrowth of other unknown cell types. Further analysis of other lineage gene markers indicated that the micromass system resulted in a much more restricted chondrogenic lineage differentiation. Upregulation of neuroectoderm, endoderm, and some of the mesoderm markers were detected in the outgrowth system. Interestingly, the 3D micromass culture system was able to inhibit neuronal differentiation from the start with little expression of neuroD1, even in the absence of BMP2. On the other hand, inhibition of neuroectoderm differentiation in the outgrowth system requires the presence of BMP2. This is consistent with recent reports that suppression of BMP signaling by noggin resulted in enrichment of neural precursors [33, 41]. In addition, we also observed a time-dependent increase in the expression of FP in our outgrowth system under both control and BMP2 conditions, which further supports the earlier findings that the default differentiation pathway in the outgrowth conditions may be more inclined toward the extraembryonic endodermal lineage. By dissociation of EB cells and plating as high-density micromass, this default pathway adopted by the EB cells was disrupted and partially inhibited, as indicated by lower expression levels of FP, rendering the differentiation more permissive and directed toward the chondrogenic lineage. We speculate that, in the EB-outgrowth cultures, intercellular contact and gap junctional coupling that play a crucial role in extraembryonic endoderm development within the EBs remain intact [42–44]. 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.
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.
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 [32]. In combination with BMP2, the micromass culture system resulted in both enhancement and enrichment of chondrogenic differentiation.
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CONCLUSION |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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