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Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine
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Biomaterials 27 (2006) 6015–6023 www.elsevier.com/locate/biomaterials
Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine Nathaniel S. Hwang1, Shyni Varghese1, Parnduangjai Theprungsirikul, Adam Canver, Jennifer Elisseeff Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Clark Hall 106, 3400 North Charles Street, Baltimore, MD 21218, USA Received 5 April 2006; accepted 28 June 2006 Available online 26 July 2006
Abstract Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). We have previously reported that hydrogels provide EBs a supportive environment for in vitro chondrogenic differentiation and three dimensional tissue formation [Hwang NS, et al. The Effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine ES cells. Stem Cells 2006;24:284–91]. In this study, we report chondrogenic differentiation of murine ES cells encapsulated in photopolymerizing poly(ethylene-glycol)-based (PEG) hydrogels in the presence of glucosamine (GlcN), an amino monosaccharide found in chitin, glycoproteins and glycosaminoglycans such as hyaluronic acid, chondroitin sulfate and heparin sulfate. We examined the growth and differentiation of encapsulated EBs in standard chondrogenic differentiation medium containing 0-, 2-, and 10-mM GlcN. Morphometric analysis and examination of gene and protein expression indicated that treatment of hydrogel cultures with 2-mM GlcN for 21 days significantly increased EB size, levels of aggrecan mRNA, and tissue-specific extracellular matrix accumulation. GlcN can induce multiple aspects of cell behavior and optimal GlcN concentrations can be beneficial for directing the differentiation and tissue formation of ES cells. r 2006 Elsevier Ltd. All rights reserved. Keywords: Embryonic stem cells; Hydrogels; Chondrogenesis; Glucosamine; Three-dimensional (3-D) culture
1. Introduction Cartilage is avascular and is not able to repair when damaged, making treatment of cartilage lesions an intractable clinical problem [2]. Cell transplants with and without scaffolds are now being applied to create functional cartilage replacements through tissue engineering approaches [3–5]. However, limited proliferative capacity of differentiated chondrocytes and bone marrow-derived mesenchymal stem cells (MSCs) poses a major challenge in providing adequate cell numbers for viable transplantations and cartilage repair. The ex vivo expansion of chondrocytes results in a loss of their phenotype [6] and the self-renewal and proliferative capacity of MSCs Corresponding author. Tel.: +1 410 516 4015; fax: +1 410 516 8152. 1
E-mail address: [email protected] (J. Elisseeff). These authors contributed equally to this work.
0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.06.033
decreases with age [7]. Applications of embryonic stem (ES) cells to cartilage repair may circumvent these problems as they could provide unrestricted amounts of chondro-progenitor cells. ES cells are pluripotent precursor cells with indefinite self-renewing ability [8,9]. However, spontaneous differentiation of three-dimensional (3-D) ES cell aggregates, termed embryoid bodies (EBs), often leads to a heterogeneous population of differentiated and undifferentiated cells [10]. Controlled differentiation of EBs into a desired lineage is often achieved to some extent through a ‘‘cocktail’’ of growth factors and other signaling molecules that provide tissue specific progenitor cell niches. For example, incubation of EB-laden hydrogels in the presence of TGF-b1 induces chondrogenesis [1]. In addition to growth factors, the fate of stem cells is also moderated by small cell-permeable molecules such as dexamethasone, vitamin C, sodium pyruvate, and retinoic acid [11].
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Glucosamine (GlcN), which is converted in the body to GlcN 6-phosphate (G6-P), is the most fundamental building block required for the biosynthesis of compounds such as glycolipids, glycoproteins, glycosaminoglycans (GAGs), hyaluronate and proteoglycans. Directly or indirectly, GlcN plays a role in the formation and function of articular surfaces, tendons, ligaments, synovial fluid, skin, bone, nails, heart valves, blood vessels, and mucus secretions of the digestive, respiratory, and urinary tracts [12]. The ability of GlcN to enhance cartilage-specific proteogylcan biosynthesis and inhibit the activity of proinflammatory mediators has been widely exploited to treat symptoms associated with cartilage degeneration (arthritis), although results and efficacy remain controversial [13,14]. GlcN is used in the biosynthesis of proteoglycans and GAGs as a proposed substrate for the synthesis of these cartilage components and perhaps a direct stimulator of their synthesis [15,16]. Exposure of cells to GlcN resulted in up-regulation of TGF-b1 mRNA levels, which further stimulated matrix production [17,18]. We have cultured EB-laden poly(ethylene glycol)-diacrylate (PEGDA) hydrogels with varying amounts of GlcN, both in the presence and absence of TGF-b1, and investigated the effects of GlcN on chondrogenic differentiation of EBs. Results indicate that 3-D hydrogel culture and GlcN concentration influence chondrogenic differentiation and cartilaginous tissue formation. These findings point towards the importance of scaffold and biological signals in guiding the commitment of EBs toward specific 3-D tissue development in vitro.
Immediately before photo-encapsulation, EBs were re-suspended in the polymer solution. One hundred microliters of EB–PEGDA solution containing 300 EBs or 106 cells was transferred to a cylindrical mold and exposed to 365 nm light with intensity of 4.5 mW/cm2 (Glowmark Systems, Upper Saddle River, NJ) for 5 min to form a hydrogel. EB–PEG hydrogels were then removed from their molds and incubated up to 21 days in 12-well plates with chondrogenic medium containing 5% FBS (Hyclone) in addition to high-glucose DMEM supplemented with 10.7 M dexamethasone, 50 mg/ml ascrobate-2-phosphate, 40 mg/ml proline, 100 mg/ ml sodium pyruvate, and 50 mg/ml ITS-Premix (Collaborative Biomedical: 6.25 ng/ml insulin, 6.25 mg transferiin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid), as described previously [21]. To study the effects of GlcN on the chondrogenic differentiation of EBs, 2 or 10-mM GlcN (final concentration, Sigma) was added to the chondrogenic medium.
2.3. Water-soluble tetrazolium salt cell proliferation assay Culture medium was removed and water-soluble tetrazolium salt (WST-1) cell proliferation reagent (Roche Molecular Biochemicals, Hannheim, Germany) containing culture medium was added to each well containing hydrogel construct, according to manufacturer’s protocol. Hydrogel constructs (n ¼ 4) were incubated for 3 h and WST-1-derived precipitates, produced by metabolically active cells in the hydrogels, were quantified by spectrophotometer at A450.
2.4. Histology and immunostaining EB–PEG hydrogels were fixed overnight in 4% paraformaldehyde in PBS (pH 7.4) at 4 1C and transferred to 70% ethanol until processing. Hydrogels were embedded in paraffin, and cut into 5 mm sections that were stained with Safraonin-O/fast green. Immunohistochemistry was performed with a Histostain-SP kit (Zymed Laboratories, San Francisco, CA). Polyclonal rabbit antibodies against mouse types I, II, and X collagen (RDI, Flanders, NJ), were used with 1:40–1:100 dilutions.
2.5. Biochemical assay 2. Materials and methods 2.1. Mouse ES (mES) cell culture ES-D3 GL cell line (ATCC, Manassas, VA) was cultivated on a feederlayer of mitomycin C-inactivated mouse embryonic fibroblasts with cultivation medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Gaithersbur, MD) supplemented with 15% fetal bovine serum (FBS; Hyclone, Logan, Utah), 2-mM L-glutamine (GIBCO), 5 105 M b-mercaptoethanol (Sigma-Aldrich, Saint Louis, MS), nonessential amino acids (GIBCO) and leukemia inhibitory factor (LIF; Sigma-Aldrich) as described previously [19]. EBs were formed in liquid suspension culture. In brief, ES cells were dissociated using 0.05% trypsinEDTA (GIBCO) for 5 min and the cells were pipetted several times to obtain single-cell suspensions. ES cell solution (10 ml with 104 cells) was transferred to a Petri dish (Fisher Scientific, Hampton, NH) and cultured for 5 days to allow EB formation. Number of cells per EBs was quantified by dissociating single EBs with 0.25% trypsin-EDTA for 10 min with occasional pipetting. On an average a single EB contained approximately 3.3 104 cells.
2.2. Photoencapsulation of EBs in hydrogel PEGDA (Nektar, Huntsville, AL) solution (10% w/v) was prepared with phosphate-buffered saline (PBS) according to previously described protocol [20]. The photo-initiator, Igracure 2959 (Ciba Specialty Chemicals, Tarrytown, NY), was added to the PEGDA solution and mixed thoroughly to make a final concentration of 0.05% (w/v).
EB–PEG hydrogels (n ¼ 4) were collected after 21 days of culture, lyophilized, and digested using papain solution (1 ml of papain solution per lyophilized construct). DNA content and proteoglycan deposition by EBs were quantified using 33258 Hoechst dye and dimethylmethylene blue (DMMB), respectively [22,23]. Standard curve for DMMB assay was generated using aqueous chondroitin sulfate C (Sigma-Aldrich, St. Louis, MO) solution, with concentrations ranging from 10 to 100 mg/ml.
2.6. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR Total RNAs were extracted from three EB–PEG hydrogels (combined) using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’s instruction. Two micrograms of total RNA per 20 ml of reaction volume were reverse transcribed into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen). Real-time PCR reactions were performed and monitored using the SYBR Green PCR Mastermix and the ABI Prism 7700 Sequence Detection System (Perkin Elmer/Applied Biosystems, Rotkreuz, Switzerland). cDNA samples (2 ml for total volume of 25 ml per reaction) were analyzed for gene of interest and for the reference gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). The level of expression of each target gene was then calculated as 2DDCt as previously described [24]. Triplicate reading of each experimental sample was performed for each gene of interest. RT-PCR was performed at 95 1C for 2 min followed by 34 cycles of 30 s denaturation at 95 1C, 30 s annealing at the primer specific temperature, and 1 min elongation at 72 1C. PCR products were verified by electrophoresis. The PCR primers are listed in Table 1.
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Table 1 Sequences of primers and conditions used in reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR Gene
PCR primers Sequence (forward and reverse)
Annealing temp (1C)
Aggrecan
50 -TCCTCTCCGGTGGCAAAGAAGTTG-30 50 -CCAAGTTCCAGGGTCACTGTTACCG-30
60
Collagen II
50 -AGGGGTACCAGGTTCTC CATC-30 50 -CTGCTCATCGCCG CGGTCCGA-30
60
Sox-9
50 -TGGCAGACCAGTACCCGCATCT-30 50 -TCTTTCTT GTGCTGCACGCGC-30
57
Collagen X
50 -ATGCCTTGTTCTCCTCTTACTGGA-30 50 -CTTTCTGCTGCTAATGTTCTTGACC-30
61
TGF-bI
50 -AGAGGTCACCCGCGTGCTAA-30 50 -TCCCGAATGTCTGACGTATTG-30
60
GAPDH
50 -GCACAGTCAAGGCCGAGAAT-30 50 -GCCTTCTCCATGGTGGTGAA-30
60
2.7. Statistical analysis Data are expressed as mean7standard deviation (SD). Statistical significance was determined by analysis of variance (ANOVA single factor) with po0:05.
3. Results 3.1. Effects of GlcN on cell proliferation and matrix synthesis of EBs Five-day-old EBs were prepared in suspension culture and approximately 300 EBs (1 million cells) per construct were encapsulated in PEG-based hydrogels. Immediately after photo-encapsulation, the EBs were observed to be intact and evenly dispersed throughout the hydrogel constructs (data not shown). To test the hypothesis that GlcN may influence tissue development during EB differentiation in PEG-based hydrogels, we compared the development of EBs in standard chondrogenic differentiating medium containing TGF-b1 with or without GlcN (2, 10 mM). As the culture period extended, varying degree of EB sizes were observed depending on medium conditions (Fig. 1A–C). No significant changes in size were observed between the EBs cultured in TGF-b1 alone and those cultured in TGF-b1 along with 2-mM GlcN. However, at 10-mM GlcN, we observed smaller EBs, indicating less matrix accumulation and cell proliferation. Presence of GlcN (2 and 10 mM) resulted in reduced cell metabolic activity in a concentration dependent manner. DNA content was also reduced with GlcN treatment, indicating that the observed reduction in cellular metabolic activity was a result of reduced cell number in the presence of GlcN rather than a reduction in cell metabolism. However, the rate of aggrecan synthesis, measured by DMMB dye assay, showed that 2-mM GlcN plays a positive role in matrix synthesis. EBs treated with 2-mM
GlcN increased GAG synthesis by 20% compared to control EB cultures. 3.2. GlcN upregulates aggrecan expression and matrix accumulation Supplementation of 2-mM GlcN resulted in upregulation of cartilage-specific markers. After 21 days in culture, quantitative gene expression indicated that exposure to 2-mM GlcN increased aggrecan expression approximately 20% compared to controls (Fig. 2A). Since GlcN was previously demonstrated to increase levels of TGF-b1 expression, we quantified mRNA levels of TGF-b1. However, there was no upregulation in TGF-b1 expression observed in the EBs (Fig. 2B). Safranin-O staining demonstrated that supplementation of 2-mM GlcN in chondrogenic medium containing TGF-b1 increased staining for GAG compared to other groups (Fig. 3). Cells with a round morphology and significant extracellular matrix accumulation were present in the hydrogel, producing typical cartilage-like tissue structures. Negatively charged proteoglycans were not observed in EBs supplemented with 10-mM GlcN. At 10-mM GlcN, most of the cells were apoptotic as observed by tunnel live/ dead assay (data not shown). 3.3. Cartilage-relevant markers are enhanced by GlcN treatment The expression of chondrogenic genes in the EBs treated with GlcN progressed at a faster rate (Fig. 3D). Type II collagen is found specifically in articular cartilage and is synthesized as a procollagen in two forms (IIA and IIB), generated by differential splicing of the gene primary transcript. Type IIA collagen is expressed in juvenile or prechondrogenic cells, whereas type IIB collagen is expressed in adult or fully differentiated chondrocytes.
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Fig. 1. The influence of glucosamine (GlcN) supplementation on murine ES cell-derived embryoid bodies (EBs) in hydrogels. Gross image of EBs encapsulated in PEG-hydrogels after 21 days of culture. (A) EBs in chondrogenic medium with TGF-b1. (B) EBs in chondrogenic medium with TGF-b1 and supplemented with 2-mM GlcN. (C) EBs in chondrogenic medium with TGF-b1 and supplemented with 10-mM GlcN. Cell metabolic activity and DNA content was reduced in GlcN concentration dependent manner (D, E). (F) Glycosaminoglycan (GAG) content was quantified by dimethylmethylene blue (DMMB) assay and normalized to the DNA content. 2-mM GlcN supplemented EBs significantly increased amount of GAG production per cell basis. Scale bar ¼ 100 mm. *po0:05.
1.4
*
(B) 2
1.2 1 0.8 0.6 0.4 0.2
**
0
Relative fold induction
Relative fold induction
(A)
1.5 1 0.5 0
0 mM GlcN 2 mM GlcN 10 mM GlcN
0 mM GlcN 2 mM GlcN10 mM GlcN
Fig. 2. Real-time PCR of matrix protein aggrecan expression (A) and TGF-b1 (B) was quantified by using SYBR green fluorescence after 21 day of culture in TGF-b1 treated EBs with varying amount of GlcN. 2-mM supplementation of GlcN upregulated aggrecan but did not change TGF-b1 expression. *po0:05, **po0:01.
Both control cultures as well as EBs treated with 2-mM GlcN expressed the juvenile splice variant of type IIA collagen at day 1 of culture. In control cultures, type IIA collagen expression reached the highest levels at day 16. In contrast, EBs treated with 2 mM GlcN showed the greatest expression of type IIA collagen at day 11, followed by a progressive upregulation of type IIB collagen, suggesting a faster shift from juvenile to mature collagen splice variants. The upregulation of Aggrecan and Sox-9 expressions of both control EBs and EBs treated with 2-mM GlcN was
time dependent. Real-time PCR assessment of relative Sox9 expression at different days compared to day 1 of control EBs show that 2-mM GlcN treatment resulted in early upregulation of Sox-9 compared to control EBs (Fig. 3E). Type X collagen was expressed as early as day 16 in control EBs, but 2-mM GlcN exposure delayed expression until day 21, indicating that GlcN may play a role in inhibiting hypertrophic differentiation of chondrocytes (Fig. 3F). EBs treated with 10-mM GlcN had detectable levels of aggrecan, while other chondrogenic markers were absent.
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Fig. 3. Basophilic extracellular matrix (ECM) deposition in EBs after 21 days of culture in PEG-based hydrogels. Safranin-O staining of (A) EBs in chondrogenic medium with TGF-b1. (B) EBs in chondrogenic medium with TGF-b1 and supplemented with 2-mM GlcN. (C) EBs in chondrogenic medium with TGF-b1 and supplemented with 10-mM GlcN. (D) Reverse transcription-polymerase chain reaction (PCR) indicated that 2-mM GlcN treatment upregulated expression of cartilage markers earlier compared to untreated cultures. (E) Real-time PCR of Sox-9 indicates early upregulation in 2-mM GlcN-treated EBs over their untreated counterparts. Relative expression of Sox-9 at different time points were compared to Day 1 control EBs without GlcN treatment. (F) 2-mM GlcN treatment resulted in down-regulation of type X collagen expression. Scale bar ¼ 100 mm.
3.4. Tissue morphology and matrix composition of EBs in treated with 2-mM GlcN are comparable to TGF-b1 treated EBs We subsequently investigated if GlcN alone would induce chondrogenic differentiation of EBs. EBs cultured without TGF-b1 were small and stained minimally for negatively charged proteoglycans (Fig. 4). However, treatment with 2-mM GlcN increased the size of EBs and histological analysis confirmed the presence of Safranin-O positive extracellular matrix. Deposition of cartilagespecific extracellular matrix was also confirmed by RTPCR. The extracellular matrix of EBs cultured without TGF-b1 was minimal, but 2-mM GlcN treatment produced matrix with strong immunoreactivity for types I and II collagen, predominantly within the pericellular matrix of cells in the EBs (Fig. 5). Type X collagen was also detected in both cultures.
4. Discussion The aim of the present study was to examine the effects of GlcN and TGF-b1 on mouse ES cell (via EBs) growth and development, by evaluating gross morphology, histomorphology, and gene expression in a 3-D hydrogel culture system. Recent evidence for efficient chondrogenic differentiation of ES cell-derived EBs has indicated the possibility of using ES cells for cartilage tissue-engineering application. We have previously reported that PEG-based hydrogels can support 3-D structure of EBs and their efficient differentiation towards the chondrogenic lineage in the presence of TGF-b1 [1]. In this study, we demonstrated positive effects of GlcN supplementation during in vitro chondrogenic differentiation of ES cells in PEG-based hydrogels. 3-D tissue culture is a more realistic mimicry of the in vivo histoarchitecture. A biomaterial scaffold provides a
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Fig. 4. Gross image and Safranin-O staining of embryoid bodies (EBs) encapsulated in PEG-hydrogel after 21 days of culture without TGF-b1 treatment. (A, D) EBs in chondrogenic medium without TGF-b1. (B, E) EBs in chondrogenic medium without TGF-b1 and supplemented with 2-mM GlcN. (C, F) EBs in chondrogenic medium without TGF-b1 and supplemented with 10-mM GlcN. (G) Reverse transcription-polymerase chain reaction (PCR) analysis confirms 2-mM GlcN treatment resulted in up-regulation of cartilage markers. Scale bar ¼ 100 mm.
3-D structure and can regulate cell-to-cell and/or matrix interactions. Photo-polymerizable hydrogels are attractive scaffolds for tissue-engineering applications since temporal and spatial control of the polymerization and gelation process is readily achieved, making the system amenable to implantation [20]. The polymerization conditions and hydrogel properties support cell viability and maintain intact structure of EBs. The induction of chondrogenic gene expression observed in this study is consistent with previous studies reporting the role of TGF-b1 in promoting chondrogenesis in 3-D culture. In the present study, GlcN-enhanced chondrogenesis by stimulating an earlier shift to the mature type IIB collagen splice variant in addition to upregulating aggrecan and Sox-9 expressions. However, higher GlcN concentrations were toxic.
Previous studies demonstrated that presence of glucose and GlcN in culture enhanced extracellular matrix production in murine fetal bone explants [25]. Recently, Khoo et al. investigated the dose dependent effect of glucose on differentiation of human EBs, showing enhanced cartilaginous matrix formation within the EBs and efficient chondrogenic differentiation [26]. GlcN promoted the production of cartilage matrix proteins and also prevented degradation of proteoglycans in the culture medium [25,27]. GlcN’s primary biological role in improving extracellular matrix synthesis have been proposed to be its ability to act as an essential substrate for, and to stimulate the biosynthesis of, the GAGs and the hyaluronic acid backbone used in the formation of the proteoglycans found in the structural matrix of cartilage [16]. Our observation indicates that 2-mM GlcN concentration
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Fig. 5. Types I, II, and X collagen immunostaining of EBs cultured for 21 days. EBs treated with 2-mM GlcN showed strong immunoreactivity for type I and type II collagen (B, D) compared to controls (A, C). Type X collagen is weakly stained in both conditions (E, F). Scale bar ¼ 100 mm.
enhances matrix formation of differentiating EBs while greater concentrations (10-mM GlcN) are toxic to cells. At Day 21, there was evidence of marked proliferation within EBs cultured in 2-mM GlcN whereas an increase in apoptosis was observed as dark pyknotic cells in EBs cultured with 10-mM GlcN (condensed rounded dark nuclear material). The hexosamine biosynthesis pathway is the metabolic pathway that enables glucose to be utilized for glycosaminoglycan synthesis, such as hyaluronic acid [28]. The hexosamine pathway is readily up-regulated by the addition of GlcN [29]. However, the action of an up-regulated hexosamine pathway, particularly by the inclusion of GlcN, is known to cause other cellular stresses such as depletion of ATP levels [28,30] and oxidative stress [31]. Various other studies have shown that GlcN mediate perturbation of hexosamine pathway regulates various growth factors [17]. For example, studies with mesangial cells have shown that GlcN can modulate expression of TGF-b1 expression, thus promoting matrix production through autocrine signaling pathways [18,32]. Quantifica-
tion of TGF-b1 mRNA levels indicates GlcN did not induce TGF-b1 expression in differentiating EBs. Through biochemical and gene expression analysis of cartilage matrix markers, our study demonstrates that 2 mM GlcN enhanced cartilaginous matrix formation by EBs. The effects of GlcN were more apparent in the absence of TGF-b1, with EBs grown in 2-mM GlcN producing cartilage-like structure without exogenous TGF-b1 treatment. Histomorphological examination revealed no major differences between TGF-b1 supplemented EBs and EBs supplemented only with 2-mM GlcN. GlcN supplementation alone increased aggrecan expression and produced basophilic extracellular matrix without exogenous TGF-b1. This indicates that specific concentrations of GlcN may have chondro-inducing effects. We believe that an optimal concentration of exogenous GlcN can play a positive role in inducing chondrogenic differentiation of EBs independent of TGF-b1 treatment. Real-time PCR of aggrecan mRNA in EBs revealed a 20% upregulation in expression for cells treated with TGF-b1 and 2 mM GlcN as compared to those treated with TGF-b1
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alone. Further evidence from immunohistochemical data confirmed the differentiation of chondrocyte-like cells from EBs in hydrogels. EBs treated with GlcN showed strong immunoreactivity for types I and II collagen, predominantly within the pericellular matrix of chondrocytes in the cartilaginous tissue structure. Recently, Qu et al. [33] indicated that mRNA levels of aggrecan were not induced by GlcN treatment of chondrocytes culture in twodimensional (2-D) culture. However, other studies have indicated significant increases in cartilage matrix production as well as matrix gene upregulation via GlcN supplementation [12,27]. Several factors, including differences in medium and culture condition (i.e., 3-D culture of cell aggregates) and duration of culture, may account for such differences. 5. Conclusion We have shown that EBs, derived from ES cells, have the potential to differentiate into cartilage-like structures in three-dimensional culture and that supplementation with GlcN may help improve structural integrity through enhanced ECM production. By examining histology, gene expression responses, and biochemical properties, we demonstrated that specific GlcN concentrations (2-mM) enhanced chondrogenic differentiation of murine EBs derived from ES cells. Acknowledgments This study was supported by the Johns Hopkins University (JHU)-Technion Program and the Whitaker Foundation. The authors are grateful to Dr. Zijun Zhang for critical review and technical assistance. References [1] Hwang NS, Kim MS, Sampattavanichet S, Baek JH, Zhang Z, Elisseeff J. The effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells. Stem Cells 2006;24:284–91. [2] Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthr Cartilage 2002;10:432–63. [3] Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889–95. [4] Temenoff JS, Mikos AG. Review: tissue engineering for regeneration of articular cartilage. Biomaterials 2000;21:431–40. [5] Solchaga LA, Goldberg VM, Caplan AI. Cartilage regeneration using principles of tissue engineering. Clin Orthop Relat Res 2001:S161–70. [6] Homicz MR, Schumacher BL, Sah RL, Watson D. Effects of serial expansion of septal chondrocytes on tissue-engineered neocartilage composition. Otolaryngol Head Neck Surg 2002;127:398–408. [7] Fehrer C, Lepperdinger G. Mesenchymal stem cell aging. Exp Gerontol 2005;40:926–30. [8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Watnitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–7.
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[32] Singh LP, Green K, Alexander M, Bassly S, Crook ED. Hexosamines and TGF-beta1 use similar signaling pathways to mediate matrix protein synthesis in mesangial cells. Am J Physiol Renal Physiol 2004;286:409–16. [33] Qu CJ, Karjalainen HM, Helminen HJ, Lammi MJ. The lack of effect of glucosamine sulphate on aggrecan mRNA expression and (35)S-sulphate incorporation in bovine primary chondrocytes. Biochim Biophys Acta 2006;1762:453–9.
Biomaterials 27 (2006) 6015–6023 www.elsevier.com/locate/biomaterials
Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine Nathaniel S. Hwang1, Shyni Varghese1, Parnduangjai Theprungsirikul, Adam Canver, Jennifer Elisseeff Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Clark Hall 106, 3400 North Charles Street, Baltimore, MD 21218, USA Received 5 April 2006; accepted 28 June 2006 Available online 26 July 2006
Abstract Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). We have previously reported that hydrogels provide EBs a supportive environment for in vitro chondrogenic differentiation and three dimensional tissue formation [Hwang NS, et al. The Effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine ES cells. Stem Cells 2006;24:284–91]. In this study, we report chondrogenic differentiation of murine ES cells encapsulated in photopolymerizing poly(ethylene-glycol)-based (PEG) hydrogels in the presence of glucosamine (GlcN), an amino monosaccharide found in chitin, glycoproteins and glycosaminoglycans such as hyaluronic acid, chondroitin sulfate and heparin sulfate. We examined the growth and differentiation of encapsulated EBs in standard chondrogenic differentiation medium containing 0-, 2-, and 10-mM GlcN. Morphometric analysis and examination of gene and protein expression indicated that treatment of hydrogel cultures with 2-mM GlcN for 21 days significantly increased EB size, levels of aggrecan mRNA, and tissue-specific extracellular matrix accumulation. GlcN can induce multiple aspects of cell behavior and optimal GlcN concentrations can be beneficial for directing the differentiation and tissue formation of ES cells. r 2006 Elsevier Ltd. All rights reserved. Keywords: Embryonic stem cells; Hydrogels; Chondrogenesis; Glucosamine; Three-dimensional (3-D) culture
1. Introduction Cartilage is avascular and is not able to repair when damaged, making treatment of cartilage lesions an intractable clinical problem [2]. Cell transplants with and without scaffolds are now being applied to create functional cartilage replacements through tissue engineering approaches [3–5]. However, limited proliferative capacity of differentiated chondrocytes and bone marrow-derived mesenchymal stem cells (MSCs) poses a major challenge in providing adequate cell numbers for viable transplantations and cartilage repair. The ex vivo expansion of chondrocytes results in a loss of their phenotype [6] and the self-renewal and proliferative capacity of MSCs Corresponding author. Tel.: +1 410 516 4015; fax: +1 410 516 8152. 1
E-mail address: [email protected] (J. Elisseeff). These authors contributed equally to this work.
0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.06.033
decreases with age [7]. Applications of embryonic stem (ES) cells to cartilage repair may circumvent these problems as they could provide unrestricted amounts of chondro-progenitor cells. ES cells are pluripotent precursor cells with indefinite self-renewing ability [8,9]. However, spontaneous differentiation of three-dimensional (3-D) ES cell aggregates, termed embryoid bodies (EBs), often leads to a heterogeneous population of differentiated and undifferentiated cells [10]. Controlled differentiation of EBs into a desired lineage is often achieved to some extent through a ‘‘cocktail’’ of growth factors and other signaling molecules that provide tissue specific progenitor cell niches. For example, incubation of EB-laden hydrogels in the presence of TGF-b1 induces chondrogenesis [1]. In addition to growth factors, the fate of stem cells is also moderated by small cell-permeable molecules such as dexamethasone, vitamin C, sodium pyruvate, and retinoic acid [11].
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Glucosamine (GlcN), which is converted in the body to GlcN 6-phosphate (G6-P), is the most fundamental building block required for the biosynthesis of compounds such as glycolipids, glycoproteins, glycosaminoglycans (GAGs), hyaluronate and proteoglycans. Directly or indirectly, GlcN plays a role in the formation and function of articular surfaces, tendons, ligaments, synovial fluid, skin, bone, nails, heart valves, blood vessels, and mucus secretions of the digestive, respiratory, and urinary tracts [12]. The ability of GlcN to enhance cartilage-specific proteogylcan biosynthesis and inhibit the activity of proinflammatory mediators has been widely exploited to treat symptoms associated with cartilage degeneration (arthritis), although results and efficacy remain controversial [13,14]. GlcN is used in the biosynthesis of proteoglycans and GAGs as a proposed substrate for the synthesis of these cartilage components and perhaps a direct stimulator of their synthesis [15,16]. Exposure of cells to GlcN resulted in up-regulation of TGF-b1 mRNA levels, which further stimulated matrix production [17,18]. We have cultured EB-laden poly(ethylene glycol)-diacrylate (PEGDA) hydrogels with varying amounts of GlcN, both in the presence and absence of TGF-b1, and investigated the effects of GlcN on chondrogenic differentiation of EBs. Results indicate that 3-D hydrogel culture and GlcN concentration influence chondrogenic differentiation and cartilaginous tissue formation. These findings point towards the importance of scaffold and biological signals in guiding the commitment of EBs toward specific 3-D tissue development in vitro.
Immediately before photo-encapsulation, EBs were re-suspended in the polymer solution. One hundred microliters of EB–PEGDA solution containing 300 EBs or 106 cells was transferred to a cylindrical mold and exposed to 365 nm light with intensity of 4.5 mW/cm2 (Glowmark Systems, Upper Saddle River, NJ) for 5 min to form a hydrogel. EB–PEG hydrogels were then removed from their molds and incubated up to 21 days in 12-well plates with chondrogenic medium containing 5% FBS (Hyclone) in addition to high-glucose DMEM supplemented with 10.7 M dexamethasone, 50 mg/ml ascrobate-2-phosphate, 40 mg/ml proline, 100 mg/ ml sodium pyruvate, and 50 mg/ml ITS-Premix (Collaborative Biomedical: 6.25 ng/ml insulin, 6.25 mg transferiin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid), as described previously [21]. To study the effects of GlcN on the chondrogenic differentiation of EBs, 2 or 10-mM GlcN (final concentration, Sigma) was added to the chondrogenic medium.
2.3. Water-soluble tetrazolium salt cell proliferation assay Culture medium was removed and water-soluble tetrazolium salt (WST-1) cell proliferation reagent (Roche Molecular Biochemicals, Hannheim, Germany) containing culture medium was added to each well containing hydrogel construct, according to manufacturer’s protocol. Hydrogel constructs (n ¼ 4) were incubated for 3 h and WST-1-derived precipitates, produced by metabolically active cells in the hydrogels, were quantified by spectrophotometer at A450.
2.4. Histology and immunostaining EB–PEG hydrogels were fixed overnight in 4% paraformaldehyde in PBS (pH 7.4) at 4 1C and transferred to 70% ethanol until processing. Hydrogels were embedded in paraffin, and cut into 5 mm sections that were stained with Safraonin-O/fast green. Immunohistochemistry was performed with a Histostain-SP kit (Zymed Laboratories, San Francisco, CA). Polyclonal rabbit antibodies against mouse types I, II, and X collagen (RDI, Flanders, NJ), were used with 1:40–1:100 dilutions.
2.5. Biochemical assay 2. Materials and methods 2.1. Mouse ES (mES) cell culture ES-D3 GL cell line (ATCC, Manassas, VA) was cultivated on a feederlayer of mitomycin C-inactivated mouse embryonic fibroblasts with cultivation medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Gaithersbur, MD) supplemented with 15% fetal bovine serum (FBS; Hyclone, Logan, Utah), 2-mM L-glutamine (GIBCO), 5 105 M b-mercaptoethanol (Sigma-Aldrich, Saint Louis, MS), nonessential amino acids (GIBCO) and leukemia inhibitory factor (LIF; Sigma-Aldrich) as described previously [19]. EBs were formed in liquid suspension culture. In brief, ES cells were dissociated using 0.05% trypsinEDTA (GIBCO) for 5 min and the cells were pipetted several times to obtain single-cell suspensions. ES cell solution (10 ml with 104 cells) was transferred to a Petri dish (Fisher Scientific, Hampton, NH) and cultured for 5 days to allow EB formation. Number of cells per EBs was quantified by dissociating single EBs with 0.25% trypsin-EDTA for 10 min with occasional pipetting. On an average a single EB contained approximately 3.3 104 cells.
2.2. Photoencapsulation of EBs in hydrogel PEGDA (Nektar, Huntsville, AL) solution (10% w/v) was prepared with phosphate-buffered saline (PBS) according to previously described protocol [20]. The photo-initiator, Igracure 2959 (Ciba Specialty Chemicals, Tarrytown, NY), was added to the PEGDA solution and mixed thoroughly to make a final concentration of 0.05% (w/v).
EB–PEG hydrogels (n ¼ 4) were collected after 21 days of culture, lyophilized, and digested using papain solution (1 ml of papain solution per lyophilized construct). DNA content and proteoglycan deposition by EBs were quantified using 33258 Hoechst dye and dimethylmethylene blue (DMMB), respectively [22,23]. Standard curve for DMMB assay was generated using aqueous chondroitin sulfate C (Sigma-Aldrich, St. Louis, MO) solution, with concentrations ranging from 10 to 100 mg/ml.
2.6. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR Total RNAs were extracted from three EB–PEG hydrogels (combined) using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’s instruction. Two micrograms of total RNA per 20 ml of reaction volume were reverse transcribed into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen). Real-time PCR reactions were performed and monitored using the SYBR Green PCR Mastermix and the ABI Prism 7700 Sequence Detection System (Perkin Elmer/Applied Biosystems, Rotkreuz, Switzerland). cDNA samples (2 ml for total volume of 25 ml per reaction) were analyzed for gene of interest and for the reference gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). The level of expression of each target gene was then calculated as 2DDCt as previously described [24]. Triplicate reading of each experimental sample was performed for each gene of interest. RT-PCR was performed at 95 1C for 2 min followed by 34 cycles of 30 s denaturation at 95 1C, 30 s annealing at the primer specific temperature, and 1 min elongation at 72 1C. PCR products were verified by electrophoresis. The PCR primers are listed in Table 1.
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Table 1 Sequences of primers and conditions used in reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR Gene
PCR primers Sequence (forward and reverse)
Annealing temp (1C)
Aggrecan
50 -TCCTCTCCGGTGGCAAAGAAGTTG-30 50 -CCAAGTTCCAGGGTCACTGTTACCG-30
60
Collagen II
50 -AGGGGTACCAGGTTCTC CATC-30 50 -CTGCTCATCGCCG CGGTCCGA-30
60
Sox-9
50 -TGGCAGACCAGTACCCGCATCT-30 50 -TCTTTCTT GTGCTGCACGCGC-30
57
Collagen X
50 -ATGCCTTGTTCTCCTCTTACTGGA-30 50 -CTTTCTGCTGCTAATGTTCTTGACC-30
61
TGF-bI
50 -AGAGGTCACCCGCGTGCTAA-30 50 -TCCCGAATGTCTGACGTATTG-30
60
GAPDH
50 -GCACAGTCAAGGCCGAGAAT-30 50 -GCCTTCTCCATGGTGGTGAA-30
60
2.7. Statistical analysis Data are expressed as mean7standard deviation (SD). Statistical significance was determined by analysis of variance (ANOVA single factor) with po0:05.
3. Results 3.1. Effects of GlcN on cell proliferation and matrix synthesis of EBs Five-day-old EBs were prepared in suspension culture and approximately 300 EBs (1 million cells) per construct were encapsulated in PEG-based hydrogels. Immediately after photo-encapsulation, the EBs were observed to be intact and evenly dispersed throughout the hydrogel constructs (data not shown). To test the hypothesis that GlcN may influence tissue development during EB differentiation in PEG-based hydrogels, we compared the development of EBs in standard chondrogenic differentiating medium containing TGF-b1 with or without GlcN (2, 10 mM). As the culture period extended, varying degree of EB sizes were observed depending on medium conditions (Fig. 1A–C). No significant changes in size were observed between the EBs cultured in TGF-b1 alone and those cultured in TGF-b1 along with 2-mM GlcN. However, at 10-mM GlcN, we observed smaller EBs, indicating less matrix accumulation and cell proliferation. Presence of GlcN (2 and 10 mM) resulted in reduced cell metabolic activity in a concentration dependent manner. DNA content was also reduced with GlcN treatment, indicating that the observed reduction in cellular metabolic activity was a result of reduced cell number in the presence of GlcN rather than a reduction in cell metabolism. However, the rate of aggrecan synthesis, measured by DMMB dye assay, showed that 2-mM GlcN plays a positive role in matrix synthesis. EBs treated with 2-mM
GlcN increased GAG synthesis by 20% compared to control EB cultures. 3.2. GlcN upregulates aggrecan expression and matrix accumulation Supplementation of 2-mM GlcN resulted in upregulation of cartilage-specific markers. After 21 days in culture, quantitative gene expression indicated that exposure to 2-mM GlcN increased aggrecan expression approximately 20% compared to controls (Fig. 2A). Since GlcN was previously demonstrated to increase levels of TGF-b1 expression, we quantified mRNA levels of TGF-b1. However, there was no upregulation in TGF-b1 expression observed in the EBs (Fig. 2B). Safranin-O staining demonstrated that supplementation of 2-mM GlcN in chondrogenic medium containing TGF-b1 increased staining for GAG compared to other groups (Fig. 3). Cells with a round morphology and significant extracellular matrix accumulation were present in the hydrogel, producing typical cartilage-like tissue structures. Negatively charged proteoglycans were not observed in EBs supplemented with 10-mM GlcN. At 10-mM GlcN, most of the cells were apoptotic as observed by tunnel live/ dead assay (data not shown). 3.3. Cartilage-relevant markers are enhanced by GlcN treatment The expression of chondrogenic genes in the EBs treated with GlcN progressed at a faster rate (Fig. 3D). Type II collagen is found specifically in articular cartilage and is synthesized as a procollagen in two forms (IIA and IIB), generated by differential splicing of the gene primary transcript. Type IIA collagen is expressed in juvenile or prechondrogenic cells, whereas type IIB collagen is expressed in adult or fully differentiated chondrocytes.
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Fig. 1. The influence of glucosamine (GlcN) supplementation on murine ES cell-derived embryoid bodies (EBs) in hydrogels. Gross image of EBs encapsulated in PEG-hydrogels after 21 days of culture. (A) EBs in chondrogenic medium with TGF-b1. (B) EBs in chondrogenic medium with TGF-b1 and supplemented with 2-mM GlcN. (C) EBs in chondrogenic medium with TGF-b1 and supplemented with 10-mM GlcN. Cell metabolic activity and DNA content was reduced in GlcN concentration dependent manner (D, E). (F) Glycosaminoglycan (GAG) content was quantified by dimethylmethylene blue (DMMB) assay and normalized to the DNA content. 2-mM GlcN supplemented EBs significantly increased amount of GAG production per cell basis. Scale bar ¼ 100 mm. *po0:05.
1.4
*
(B) 2
1.2 1 0.8 0.6 0.4 0.2
**
0
Relative fold induction
Relative fold induction
(A)
1.5 1 0.5 0
0 mM GlcN 2 mM GlcN 10 mM GlcN
0 mM GlcN 2 mM GlcN10 mM GlcN
Fig. 2. Real-time PCR of matrix protein aggrecan expression (A) and TGF-b1 (B) was quantified by using SYBR green fluorescence after 21 day of culture in TGF-b1 treated EBs with varying amount of GlcN. 2-mM supplementation of GlcN upregulated aggrecan but did not change TGF-b1 expression. *po0:05, **po0:01.
Both control cultures as well as EBs treated with 2-mM GlcN expressed the juvenile splice variant of type IIA collagen at day 1 of culture. In control cultures, type IIA collagen expression reached the highest levels at day 16. In contrast, EBs treated with 2 mM GlcN showed the greatest expression of type IIA collagen at day 11, followed by a progressive upregulation of type IIB collagen, suggesting a faster shift from juvenile to mature collagen splice variants. The upregulation of Aggrecan and Sox-9 expressions of both control EBs and EBs treated with 2-mM GlcN was
time dependent. Real-time PCR assessment of relative Sox9 expression at different days compared to day 1 of control EBs show that 2-mM GlcN treatment resulted in early upregulation of Sox-9 compared to control EBs (Fig. 3E). Type X collagen was expressed as early as day 16 in control EBs, but 2-mM GlcN exposure delayed expression until day 21, indicating that GlcN may play a role in inhibiting hypertrophic differentiation of chondrocytes (Fig. 3F). EBs treated with 10-mM GlcN had detectable levels of aggrecan, while other chondrogenic markers were absent.
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Fig. 3. Basophilic extracellular matrix (ECM) deposition in EBs after 21 days of culture in PEG-based hydrogels. Safranin-O staining of (A) EBs in chondrogenic medium with TGF-b1. (B) EBs in chondrogenic medium with TGF-b1 and supplemented with 2-mM GlcN. (C) EBs in chondrogenic medium with TGF-b1 and supplemented with 10-mM GlcN. (D) Reverse transcription-polymerase chain reaction (PCR) indicated that 2-mM GlcN treatment upregulated expression of cartilage markers earlier compared to untreated cultures. (E) Real-time PCR of Sox-9 indicates early upregulation in 2-mM GlcN-treated EBs over their untreated counterparts. Relative expression of Sox-9 at different time points were compared to Day 1 control EBs without GlcN treatment. (F) 2-mM GlcN treatment resulted in down-regulation of type X collagen expression. Scale bar ¼ 100 mm.
3.4. Tissue morphology and matrix composition of EBs in treated with 2-mM GlcN are comparable to TGF-b1 treated EBs We subsequently investigated if GlcN alone would induce chondrogenic differentiation of EBs. EBs cultured without TGF-b1 were small and stained minimally for negatively charged proteoglycans (Fig. 4). However, treatment with 2-mM GlcN increased the size of EBs and histological analysis confirmed the presence of Safranin-O positive extracellular matrix. Deposition of cartilagespecific extracellular matrix was also confirmed by RTPCR. The extracellular matrix of EBs cultured without TGF-b1 was minimal, but 2-mM GlcN treatment produced matrix with strong immunoreactivity for types I and II collagen, predominantly within the pericellular matrix of cells in the EBs (Fig. 5). Type X collagen was also detected in both cultures.
4. Discussion The aim of the present study was to examine the effects of GlcN and TGF-b1 on mouse ES cell (via EBs) growth and development, by evaluating gross morphology, histomorphology, and gene expression in a 3-D hydrogel culture system. Recent evidence for efficient chondrogenic differentiation of ES cell-derived EBs has indicated the possibility of using ES cells for cartilage tissue-engineering application. We have previously reported that PEG-based hydrogels can support 3-D structure of EBs and their efficient differentiation towards the chondrogenic lineage in the presence of TGF-b1 [1]. In this study, we demonstrated positive effects of GlcN supplementation during in vitro chondrogenic differentiation of ES cells in PEG-based hydrogels. 3-D tissue culture is a more realistic mimicry of the in vivo histoarchitecture. A biomaterial scaffold provides a
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Fig. 4. Gross image and Safranin-O staining of embryoid bodies (EBs) encapsulated in PEG-hydrogel after 21 days of culture without TGF-b1 treatment. (A, D) EBs in chondrogenic medium without TGF-b1. (B, E) EBs in chondrogenic medium without TGF-b1 and supplemented with 2-mM GlcN. (C, F) EBs in chondrogenic medium without TGF-b1 and supplemented with 10-mM GlcN. (G) Reverse transcription-polymerase chain reaction (PCR) analysis confirms 2-mM GlcN treatment resulted in up-regulation of cartilage markers. Scale bar ¼ 100 mm.
3-D structure and can regulate cell-to-cell and/or matrix interactions. Photo-polymerizable hydrogels are attractive scaffolds for tissue-engineering applications since temporal and spatial control of the polymerization and gelation process is readily achieved, making the system amenable to implantation [20]. The polymerization conditions and hydrogel properties support cell viability and maintain intact structure of EBs. The induction of chondrogenic gene expression observed in this study is consistent with previous studies reporting the role of TGF-b1 in promoting chondrogenesis in 3-D culture. In the present study, GlcN-enhanced chondrogenesis by stimulating an earlier shift to the mature type IIB collagen splice variant in addition to upregulating aggrecan and Sox-9 expressions. However, higher GlcN concentrations were toxic.
Previous studies demonstrated that presence of glucose and GlcN in culture enhanced extracellular matrix production in murine fetal bone explants [25]. Recently, Khoo et al. investigated the dose dependent effect of glucose on differentiation of human EBs, showing enhanced cartilaginous matrix formation within the EBs and efficient chondrogenic differentiation [26]. GlcN promoted the production of cartilage matrix proteins and also prevented degradation of proteoglycans in the culture medium [25,27]. GlcN’s primary biological role in improving extracellular matrix synthesis have been proposed to be its ability to act as an essential substrate for, and to stimulate the biosynthesis of, the GAGs and the hyaluronic acid backbone used in the formation of the proteoglycans found in the structural matrix of cartilage [16]. Our observation indicates that 2-mM GlcN concentration
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Fig. 5. Types I, II, and X collagen immunostaining of EBs cultured for 21 days. EBs treated with 2-mM GlcN showed strong immunoreactivity for type I and type II collagen (B, D) compared to controls (A, C). Type X collagen is weakly stained in both conditions (E, F). Scale bar ¼ 100 mm.
enhances matrix formation of differentiating EBs while greater concentrations (10-mM GlcN) are toxic to cells. At Day 21, there was evidence of marked proliferation within EBs cultured in 2-mM GlcN whereas an increase in apoptosis was observed as dark pyknotic cells in EBs cultured with 10-mM GlcN (condensed rounded dark nuclear material). The hexosamine biosynthesis pathway is the metabolic pathway that enables glucose to be utilized for glycosaminoglycan synthesis, such as hyaluronic acid [28]. The hexosamine pathway is readily up-regulated by the addition of GlcN [29]. However, the action of an up-regulated hexosamine pathway, particularly by the inclusion of GlcN, is known to cause other cellular stresses such as depletion of ATP levels [28,30] and oxidative stress [31]. Various other studies have shown that GlcN mediate perturbation of hexosamine pathway regulates various growth factors [17]. For example, studies with mesangial cells have shown that GlcN can modulate expression of TGF-b1 expression, thus promoting matrix production through autocrine signaling pathways [18,32]. Quantifica-
tion of TGF-b1 mRNA levels indicates GlcN did not induce TGF-b1 expression in differentiating EBs. Through biochemical and gene expression analysis of cartilage matrix markers, our study demonstrates that 2 mM GlcN enhanced cartilaginous matrix formation by EBs. The effects of GlcN were more apparent in the absence of TGF-b1, with EBs grown in 2-mM GlcN producing cartilage-like structure without exogenous TGF-b1 treatment. Histomorphological examination revealed no major differences between TGF-b1 supplemented EBs and EBs supplemented only with 2-mM GlcN. GlcN supplementation alone increased aggrecan expression and produced basophilic extracellular matrix without exogenous TGF-b1. This indicates that specific concentrations of GlcN may have chondro-inducing effects. We believe that an optimal concentration of exogenous GlcN can play a positive role in inducing chondrogenic differentiation of EBs independent of TGF-b1 treatment. Real-time PCR of aggrecan mRNA in EBs revealed a 20% upregulation in expression for cells treated with TGF-b1 and 2 mM GlcN as compared to those treated with TGF-b1
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alone. Further evidence from immunohistochemical data confirmed the differentiation of chondrocyte-like cells from EBs in hydrogels. EBs treated with GlcN showed strong immunoreactivity for types I and II collagen, predominantly within the pericellular matrix of chondrocytes in the cartilaginous tissue structure. Recently, Qu et al. [33] indicated that mRNA levels of aggrecan were not induced by GlcN treatment of chondrocytes culture in twodimensional (2-D) culture. However, other studies have indicated significant increases in cartilage matrix production as well as matrix gene upregulation via GlcN supplementation [12,27]. Several factors, including differences in medium and culture condition (i.e., 3-D culture of cell aggregates) and duration of culture, may account for such differences. 5. Conclusion We have shown that EBs, derived from ES cells, have the potential to differentiate into cartilage-like structures in three-dimensional culture and that supplementation with GlcN may help improve structural integrity through enhanced ECM production. By examining histology, gene expression responses, and biochemical properties, we demonstrated that specific GlcN concentrations (2-mM) enhanced chondrogenic differentiation of murine EBs derived from ES cells. Acknowledgments This study was supported by the Johns Hopkins University (JHU)-Technion Program and the Whitaker Foundation. The authors are grateful to Dr. Zijun Zhang for critical review and technical assistance. References [1] Hwang NS, Kim MS, Sampattavanichet S, Baek JH, Zhang Z, Elisseeff J. The effects of three dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells. Stem Cells 2006;24:284–91. [2] Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthr Cartilage 2002;10:432–63. [3] Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889–95. [4] Temenoff JS, Mikos AG. Review: tissue engineering for regeneration of articular cartilage. Biomaterials 2000;21:431–40. [5] Solchaga LA, Goldberg VM, Caplan AI. Cartilage regeneration using principles of tissue engineering. Clin Orthop Relat Res 2001:S161–70. [6] Homicz MR, Schumacher BL, Sah RL, Watson D. Effects of serial expansion of septal chondrocytes on tissue-engineered neocartilage composition. Otolaryngol Head Neck Surg 2002;127:398–408. [7] Fehrer C, Lepperdinger G. Mesenchymal stem cell aging. Exp Gerontol 2005;40:926–30. [8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Watnitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–7.
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