Differentiation of Tonsil-Tissue-Derived Mesenchymal Stem Cells

May 7, 2014 - Labeled cells were then viewed under an Olympus IX71 fluorescence microscope, and the images were captured using the Olympus DP2-DSW sof...
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Differentiation of Tonsil-Tissue-Derived Mesenchymal Stem Cells Controlled by Surface-Functionalized Microspheres in PEGPolypeptide Thermogels Eun Jeong Kye,† Seung-Jin Kim,† Min Hee Park,† Hyo Jung Moon,† Kyung Ha Ryu,‡ and Byeongmoon Jeong*,† †

Department of Chemistry and Nano Science, Ewha Global Top 5 Research Program, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Korea ‡ Departments of Molecular Medicine, OtorhinolaryngologyHead and Neck Surgery and Pediatrics, School of Medicine Ewha Womans University, Ewha Global Top 5 Research Program, Seoul, Korea ABSTRACT: Poly(ethylene glycol)-poly(L-alanine) diblock copolymer (PEG-L-PA; molecular weight of each block of 1000−1080 Da) aqueous solutions undergo sol-to-gel transition in a 3.0−8.0 wt % concentration range as the temperature increases. By incorporating the polystyrene microspheres with different functional groups with a size of 100−800 μm in in situ formed PEG-L-PA thermogels, the differentiation of tonsiltissue-derived mesenchymal stem cells (TMSCs) was investigated. The mRNA expression and immunohistochemical assays suggested that the TMSCs preferentially undergo adipogenesis in the ammonium (−NH3+)or thiol (−SH)-functionalized microsphere incorporated thermogels; chondrogenesis in the thiol-, phosphate (PO32−)-, or carboxylate (−COO−)-functionalized microsphere incorporated thermogels; and osteogenesis in the phosphate-, carboxylate-functionalized, or neat polystyrene microsphere incorporated thermogels. This paper provides a new TMSC 3D culture system of a sol−gel reversible matrix and suggests that the surface-functional groups of microspheres in the thermogel can control the preferential differentiation of stem cells into specific cell types during the 3D culture.

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INTRODUCTION The isolation of bone marrow derived mesenchymal stem cells (BMSCs) requires a surgical procedure accompanying pain of the donor as well as exposure to bacterial infection. In addition, a significant loss of differentiation and proliferation ability of MSCs with donor ages was also reported.1 Unlike BMSCs, tonsil-tissue-derived mesenchymal stem cells (TMSCs) can be obtained from otherwise waste tissues of tonsils after tonsillectomy, which is a common pediatric operation.2 Therefore, age-related problems of MSCs can also be avoided. The nucleated cell numbers per unit volume from tissues are about 10−100 times greater for tonsil tissues than bone marrow. Proliferation rate of TMSCs is 2−3 times faster than that of BMSCs.2,3 The isolated TMSCs not only undergo mesodermal differentiations of osteogenesis, chondrogenesis, and adipogenesis, but also express endodermal genes such as fork head box A2, SIX home box, and chemokine ligand 21 in appropriate induction media.2,4 Recently, chimerism was reported; therefore, TMSCs from several donors can be mixed without a significant loss of their stem cell properties.5 Differentiation of stem cells into a specific cell type is a critical issue in succeeding the stem cell therapy. Many biochemical supplements such as dexamethasone, ascorbic acid, and growth factors have been reported in this regard.6,7 In © 2014 American Chemical Society

addition, physicochemical parameters of polymeric scaffolds such as topography, mechanical properties, roughness of substrates, nanofiber diameter and orientation, electrical stimulation, and incorporation of RGD have been reported to be effective in controlling stem cell differentiation.8−17 In particular, effect of functional end groups on stem cell differentiation have been investigated in 3D hydrogels as well as on 2D substrates of silane modified glass, self-assembled monolayer on gold surfaces, and surface modified polymer films.18−24 On the 2D culture systems, amino groups (−NH2), phosphate groups (−PO43−), and thiol groups (−SH) promoted osteogenesis, whereas carboxylate groups (−COO−), hydroxyl groups (−OH), and RGDS enhanced chondrogenesis of the MSCs.18−24 They suggested that extracellular matrix proteins adsorbed onto the functional group modified hydrogel might be responsible for the preferential differentiation of hMSC.19 Varying the monolayer surface with methyl groups (−CH3) and hydroxyl groups (−OH) in a different composition, the binding of fibronectin and vitronectin could be controlled, which in turn affected the Received: March 5, 2014 Revised: May 4, 2014 Published: May 7, 2014 2180

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used as purchased from Acros (U.S.A.). PS-N, PS-S, PS-P, and PS-C microspheres were used as purchased from Sigma (U.S.A.). Synthesis of PEG-L-PA. The PEG-L-PA was synthesized by the ring-opening polymerization of the N-carboxy anhydrides of L-alanine in the presence of α-amno-ω-methoxy PEG. The α-amino-ω-methoxy poly(ethylene glycol) (2.5 g, 2.5 mmol; MW 1000 Da) was dissolved in anhydrous toluene (50 mL), and the residual water was removed by azeotropic distillation to a final volume of about 5 mL. Anhydrous chloroform/N,N-dimethyl formide (30 mL; 2/1 v/v), N-carboxy anhydrides of L-alanine (5.6 g, 48.7 mmol) were added to the reaction mixtures. They were stirred at 40 °C for 24 h under anhydrous nitrogen conditions. The polymer was purified by repeated dissolution in chloroform, filtration of an undissolved fraction, followed by precipitation into diethyl ether, and then evaporation of the residual solvent under vacuum. The polymer was dialyzed in water using a membrane with a molecular weight cutoff of 1000 Da and freeze-dried. The yield was about 70%. 1 H NMR Spectroscopy. 1H NMR spectra in CF3COOD (500 MHz NMR spectrometer; Varian, U.S.A.) were used to determine the composition and average molecular weight (Mn) of the polymer. Gel Permeation Chromatography (GPC). The gel permeation chromatography system (Waters 515) with a refractive index detector (Waters 410) was used to obtain the molecular weights and molecular weight distribution of the polymers. N,N-Dimethylformamide was used as an eluting solvent. Poly(ethylene glycol)s with a molecular weight range of 400−20000 Da were used as the molecular weight standards. An OHPAK SB-803QH column (Shodex) was used. Phase Diagram. PEG-L-PA aqueous solutions in a concentration range of 1.0−12.0 wt % were prepared at 4 °C. The sol-to-gel transition temperature was determined by the test tube inverting method. A flow (sol)-no flow (gel) criterion was used for defining the transition temperature at the temperature increment of 1 °C/step. The transition temperature is an average of three measurements. Dynamic Mechanical Analysis. The modulus of the polymer aqueous solution (8.0 wt %) was investigated by dynamic rheometry (Rheometer RS 1; Thermo Haake, Germany) at 4 and 37 °C. The aqueous polymer solution was placed between parallel plates with 25 mm in diameter and a gap of 0.5 mm. During the dynamic mechanical analysis, the samples were placed inside a chamber with water-soaked cotton to minimize water evaporation. The data were collected under a controlled stress (4.0 dyn/cm2) and frequency of 1.0 rad/s. Scanning Electron Microscopy (SEM). The SEM image of polystyrene and surface-functionalized polystyrene was obtained by the field emission scanning electron microscopy (FE-SEM) instrument (JSM-6700F, JEOL, Japan). Zeta Potential Measurement. To identify the electrochemical nature of the functional groups on the microsphere surfaces, the microspheres were suspended in water (0.08 wt % in neutral water) and their zeta potentials were measured at 37 °C by using Zetasizer (ALV 5000−60 × 0, U.S.A.). TMSC 3D Culture. Human TMSCs were received from Ewha Womans University Medical School, and cultured in high glucose Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone, U.S.A.), 1% antibiotic-antimitotic (Gibco, U.S.A.), and 1% penicillin/streptomycin (Hyclone, U.S.A.) under a 5% CO2 atmosphere at 37 °C. The stem cells were subcultured in passage 5. Harvested TMSCs (passage 6, 0.25 × 106 cells) were mixed in a microsphere-suspended PEG-L-PA aqueous solution (8 wt %, 0.25 mL), and were incubated in 24-well culture plates at 37 °C, during which TMSCs were encapsulated in the PEGL-PA gel by the sol-to-gel transition of the polymer aqueous solution. DMEM (2.0 mL) at 37 °C containing 10% FBS, 1% antibioticantimitotic and 1% penicillin/streptomycin was added on the top of the cell-encapsulated hydrogel and the cells were cultured under a 5% CO2 atmosphere at 37 °C. The medium was replaced every 3 days. After 21 days of cell culture, the total RNA content was extracted from the cell-encapsulated hydrogels using the TRIZOL reagent (Invitrogen, U.S.A.), according to the manufacturer’s protocol. The extracted RNA pellet was dissolved in nuclease-free water, and the RNA quality and concentration were determined using the Experion

differentiation of MSCs through the difference in focal adhesion of the MSCs.20 In addition, preferential differentiation of MSCs were reported during the 3D culture of MSCs by using the growth medium in the chemical functional group modified PEG hydrogels in the absence of specific induction supplements.21 This study proved that the chemical functional groups really affected the differentiation of the MSCs into specific cell types. The MSCs preferentially underwent osteogenesis, chondrogenesis, and adipogenesis in the phosphate group (−PO43−), carboxylate group (−COO−), and t-butyl group modified PEG hydrogel (3D), respectively, while maintaining its spherical cellular morphology.21 Natural biological systems including the human body provide three-dimensional (3D) environments for cells. Compared with traditional cell cultures by using polystyrene plates on a 2D system, the cells undergo changes in phenotypes and exhibit different biomarker expressions in 3D environments. For example, human breast epithelial cells cultured in 2D environments became tumor-like cells and then reverted into normal cells in 3D culture conditions.25 Chondrocytes expressed collagen type I more than type II on the 2D culture, whereas the opposite trend was observed in a 3D culture.26 Therefore, understanding the cell behavior in a 3D environment is a very important subject. Hydrogels have been actively investigated as a 3D matrix.27 However, chemical, photochemical, or enzymatic reactions are required to prepare a cellincorporated 3D matrix.28 Thermogelling polymer aqueous solutions undergo sol-to-gel transition as the temperature increases. Pharmaceutical agents can be incorporated in an in situ formed gel by injecting aqueous formulation of the pharmaceutical agents in a sol state into a warm environment, typically 37 °C. Due to the simple procedure for hydrogel implantation, thermogelling polymers have been applied for drug delivery, injectable tissue engineering, embolization around tumor tissues, wound dressing, and post-surgical adhesion prevention.29−33 A 3D cell culture system, where the cells are embedded in a 3D matrix of a hydrogel, can be simply prepared by increasing the temperature of a cell suspended aqueous polymer solution to 37 °C.28−35 In this paper, we prepared a 3D matrix of TMSCs by increasing the temperature of the cell suspended poly(ethylene glycol)-poly(L-alanine) aqueous solution to 37 °C. In contrast with the chemically cross-linked PEG hydrogel system, we used a thermoreversible PEG-L-PA hydrogel that undergoes reversible sol-to-gel transition, and no chemical reaction is needed during the stem cell encapsulation. Neat polystyrene microspheres (PS), ammonium- (PS-N), thiol- (PS-S), phosphate- (PS-P), or carboxylate-functionalized polystyrene microspheres (PS-C) were incorporated in thermogelling PEGL-PA systems. The characteristics of stem differentiation into adipogenesis, chondrogenesis, and osteogenesis were investigated by focusing on mRNA and protein expression for peroxisome proliferator-activated receptor gamma (PPARγ), collagen type II (COL II), and osteocalcin (OCN) in the 3D hydrogel matrix.

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EXPERIMENTAL SECTION

Materials. α-Amino-ω-methoxy poly(ethylene glycol) (PEG; M.W. = 1000 Da, IDB Chem, Korea) and N-carboxy anhydrides of L-alanine (KPX life, Korea) were used as received. Chloroform was dried over magnesium sulfate before use. Anhydrous N,N-dimethyl formide was used as received from Sigma-Aldrich (U.S.A.). Toluene (Daejung, Korea) was distilled over sodium before use. PS microspheres were 2181

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system (Bio-Rad, U.S.A.). After synthesizing the cDNA from the isolated RNA, real-time reverse transcription polymerase chain reactions (RT-PCR) were performed with the CFX96 system using the IQ SYBR Green Supermix. The sequence of primers of PPARγ, COL II, OCN, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) are listed in Table 1. The relative expression level of

glycol peak of PEG), and 4.50−4.80 ppm (methine peak of PA; Figure 1). In addition, two small peaks were also shown at

Table 1. Primer Sequences and PCR Conditions for RealTime RT-PCRa gene

primer sequences

PPARγ

F: 5′-AAGACCACTCCCACTCCTTTG3′ R: 5′-GTCAGCGGACTCTGGATTCA-3′ F: 5′-AGGAGGCTGGCAGCTGTGTGC3′ R: 5′-CACTGGCAGTGGCGAGGTCAG3′ F: 5′-TGAGAGCCCTCACACTCCTC-3′ R: 5′-ACCTTTGCTGGACTCTGCAC-3′ F: 5′-ATGGGGAAGGTGAAGGTCG-3′ R: 5′-TAAAAGCAGCCCTGGTGACC-3′

COL II

OCN GAPDH

annealing temp (°C) 54

64

63 57

a PPARγ, COL II, OCN, and GAPDH indicate peroxisome proliferator-activated receptor gamma, type II collagen, osteocalcin, and glyceraldehyde-3-phosphate-dehydrogenase, respectively. F and R indicate forward and reverse primers, respectively.

Figure 1. 1H NMR spectra (CF3COOD) and gel permeation chromatogram of PEG-L-PA: a and a′ come from the methyl groups of internal alanine and terminal alanine of the PEG-L-PA, respectively.

target genes was calculated as 2−ΔΔCt, where target gene expression was normalized as ΔΔCt = (Gene A − GAPDH)t − (Gene A − GAPDH)t0. t0 (zero day) indicates on the day when the experiment started. After 21 days of 3D cell-culture, the cell-encapsulated hydrogels were fixed in natural buffered formalin (NBF). The fixed gels were embedded in an optical cutting temperature compound (OCT, Sakura, Netherlands) for 12 h, and then they were frozen. The frozen gel samples were then sliced into 10 μm-thick sections at −20 °C and attached to the slide glass. The cryo-sections were stored at −80 °C before staining. For the immunofluorescence study on PPARγ, COL II, and OCN, the sections embedded in OCT were incubated at 15 °C with aqueous solutions (500 μL/cryo-section) of anti-PPARγ antibody, anti-COL II antibody, or anti-OCHN antibody (Abcam, U.K.). After washing the section, antibodies were detected using aqueous solutions (500 μL) of corresponding secondary antibodies (Abcam, U.K.) according to the manufacturer’s protocol. The section was then incubated with 4′,6′diamidino-2-phenyl indole (DAPI) (Molecular Probes, U.S.A.) for nucleus staining and phalloidin (Molecular Probes, U.S.A.) for actin staining. Labeled cells were then viewed under an Olympus IX71 fluorescence microscope, and the images were captured using the Olympus DP2-DSW software. For alcian blue staining, the sections embedded in OCT were fixed in cold acetone (4 °C) for 20 min and formaldehyde solution (ethyl alcohol/acetic acid/formaldehyde = 85:5:10) for 20 min and stained by the alcian blue staining method at 15 °C. Samples were treated with a mounting solution, and then viewed under an Olympus TH4-200 halogen microscope, and the images were captured with Olympus DP2-DSW software. Statistical Analysis. Data are expressed as means ± standard deviation from the triplicate experiments (n = 3). The significance of differences in the mean values was evaluated using the Student t-test. Differences were considered significant when the p value was less than 0.01 and 0.05, which were marked as * and **, respectively, on the appropriate data.

1.69−1.76 ppm and 3.56−3.61 ppm coming from the end groups of alanine (methyl) and methoxy of the PEG-L-PA, respectively. The molecular weight (Mn) of the PEG-L-PA was determined by the integration area of the peaks at 3.80−4.10 ppm (ethylene glycol peak of PEG) and 1.40−1.76 ppm (methyl peak of PA). Assuming that the molecular weight of PEG (as received) was 1000 Da, the molecular weight of PEGL-PA was calculated to be 2080 Da, where the molecular weights of each block of PEG-L-PA were 1000−1080 Da. The molecular weight (Mw) and the molecular weight distribution determined by gel permeation chromatography relative to PEG standards were 1810 Da and 1.25, respectively. PEG-L-PA aqueous solutions in a concentration range of 3.0−8.0 wt % underwent sol-to-gel transition as the temperature increased (Figure 2a). The sol-to-gel transition temperature decreased from 55 to 9 °C as the concentration of the polymer increased from 3.0 to 8.0 wt %. The gel maintained their transparency without significant syneresis or gel-to-sol transition (gel melting) behavior up to 100 °C. At polymer concentrations less than 3.0 wt %, viscosity of the polymer aqueous solution slightly increased as the temperature increased. However, the solution remained as a sol state by the flow (sol) and no flow (gel) criterion over the investigated temperature range of 0−70 °C. At polymer concentrations greater than 8.0 wt %, the system remained as a gel state over the investigated temperature range. The polymer aqueous solution at 8.0 wt % was selected to provide a rather stiff gel at 37 °C because the gel modulus decreased as the concentration decreased. The moduli of the polymer aqueous systems in sol (4 °C) and gel (37 °C) states were compared by the dynamic mechanical analysis (Figure 2b). The storage modulus (G′) and loss modulus (G″) are measures of the elastic component and the viscous component of a complex modulus (G*), respectively.36,37 In the sol state (4 °C), the G″ was greater

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RESULTS AND DISCUSSION H NMR spectra of PEG-L-PA showed large three peaks at 1.40−1.76 ppm (methyl peak of PA), 3.80−4.10 ppm (ethylene

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were chosen in this study. The SEM images of the microspheres exhibited the size of 100−750 μm (Figure 3). The size and chemical functional groups of specific microspheres were summarized in Table 2. We used the commercially available microspheres as received from the vendors (Acros and Sigma) as a proof of concept to control the differentiation of stem cells by the microsphere in the thermoreversible hydrogel. The microspheres and TMSCs were to be incorporated in the gel through sol-to-gel transition of the polymer aqueous solution. The microspheres can be recovered by temperature sensitive gel-to-sol transition, which can be distinguished from previous 3D culture systems using chemically cross-linked gels.21 To identify the nature of the functional groups on the microspheres, zeta potentials of the microspheres were measured in neutral water (Figure 4). Zeta potentials of PS and PS-S microspheres were 0.3 (±0.5) mV and −0.3 (±0.2) mV, indicating practically uncharged species in neutral water. Zeta potential of PS-N microsphere was 33.1 (±4.1) mV, whereas those of PS-P and PS-C microspheres were −86.9 (±2.9) and −40.5 (±6.6) mV in neutral water, respectively. Therefore, the major chemical species of functional groups of PS-N, PS-S, PS-P, and PS-C on the microsphere surfaces were ammonium (−NH3+), thiol (-SH), phosphate (-PO32−), and carboxylate (−COO−), respectively. That is, both PS and PS-S microspheres were uncharged, and PS-N microspheres were positively charged, whereas both PS-P and PS-C microspheres were negatively charged in neutral water. TMSCs (passage 6) and microspheres were suspended in the PEG-L-PA aqueous solution in a sol state, and the mixture was injected into a preheated culture system at 37 °C. The sol-togel transition was immediately observed and a 3D culture system of the TMSCs was constructed. The effectiveness of functional groups was significant when the functional monomer content was 5−50 mM in 10 wt % of polymerizable PEG dimethacrylate aqueous solution, indicating that the functional monomer content should be 1−10 wt % of polymer.21 In this study, the amount of the functionalized microsphere was selected to be 0.08 wt % in a 8.0 wt % of PEG-L-PA aqueous

Figure 2. (a) Phase diagram of PEG-L-PA aqueous solution determined by the test tube inverting method. (b) Modulus of the PEG-L-PA aqueous polymer solution (8.0 wt %) at 4 °C and its thermogels at 37 °C.

than G′. However, G′ was greater than G″ in the gel state (37 °C). G″ is same with the viscosity of the system at the frequency of 1 rad./s in the dynamic mechanical analysis. G″ was less than 0.1 Pas at 4 °C, and increased to more than 20 Pas at 37 °C. G′ is a parameter related to Young’s modulus by E = G/(2(1 + ν), where ν is the passion ratio, a fraction of strain in a longitudinal direction (x) when stress is applied in a transverse direction (z).38 G′ increased from 380 Pa (37 °C), which was good enough to hold the TMSCs during the 3D culture over 21 days. Neat polystyrene, ammonium-, thiol-, phosphate-, and carboxylate-surface-functionalized polystyrene microspheres

Figure 3. SEM image of the microspheres used in the study. Neat polystyrene microspheres (PS), ammonium-functionalized polystyrene microspheres (PS-N), thiol-functionalized polystyrene microspheres (PS-S), phosphate-functionalized polystyrene microspheres (PS-P), and carboxylate-functionalized polystyrene microspheres (PS-C). The scale bar is 500 μm. 2183

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Table 2. Sizes and Surface Functional Groups (X) of Polystyrene Microspheres Used in the Study microsphere size (μm) X

PS 125−250

PS-N 100−400 -CH2CH2-NH2

PS-S 100−400 -C6H5-SH

PS-P 350−750 -PO3H2

PS-C 350−750 -CH2-COOH

Differentiation of TMSCs into specific cell types was analyzed by the mRNA expression using real-time RT-PCR. The differentiation of the TMSCs into adipose, cartilage, and bone were investigated through specific biomarker expressions of PPARγ, COL II, and OCN, corresponding to adipogenesis, chondrogenesis, and osteogenesis, respectively. All biomarker expressions were normalized by the GAPDH and 0 day level. As for the adipogenesis, a significant (p < 0.05) biomarker expression was observed in the PS-N or PS-S microsphere incorporated PEG-L-PA thermogels at 21 days of incubation (Figure 6a). In case of PEG gels, PPARγ expression was significant in the t-butyl- and fluoro-functionalized systems, indicating that adipogenic differentiation might be preferred in hydrophobic environments.21 Significant (p < 0.01 or p < 0.05) expression of COL II was observed in the PS-S, PS-P, or PS-C microsphere incorporated PEG-L-PA thermogels at 7−21 days of incubation (Figure 6b).

Figure 4. Zeta potential of the microspheres in neutral water at 37 °C.

solution, corresponding to 1.0 wt % of PEG-L-PA. The polymer concentration at 8.0 wt % was chosen to allow sol-to-gel transition of the system and to maintain a durable 3D culture system which could hold the cells and microspheres in the gel during the 3D culture. DMEM containing 10% FBS, 1% antibiotic-antimitotic, and 1% penicillin/streptomycin was added to the cell-encapsulated hydrogel, and the medium was replaced every 3 days. Live/Dead assay using fluorescence microscopy showed that the cells were healthy in the hydrogel system (Figure 5). Live

Figure 5. Cell images of zero day (0d) and 21 days (21d) after the 3D culture started. The scale bar is 50 μm.

cell images of green color were developed through their enzymatic activity, converting the virtually nonfluorescent calcein AM to the intensely fluorescent calcein.39 The polyanionic dye (calcein) was well retained within live cells, producing an intense uniform green fluorescence in live cells. The TMSCs maintained their original spherical phenotypes in the all of the 3D culture systems. Some protruded fibers were observed from the cells in 21 days of culture.

Figure 6. Biomarker expression of TMSCs in 3D-cultured samples analyzed by real-time RT-PCR. The gene expressions for adipogenesis (PPARγ; a), chondrogenesis (COL II; b), and osteogenesis (OCN; c) are shown. The data were normalized by the GAPDH and 0 day data. The data are presented as the mean ± SD of three independent experiments. * and ** indicate p < 0.01 and p < 0.05 (Student t- test), respectively, relative to 0 day. 0 day indicates 4 h after the 3D culture started. 2184

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Figure 7. Immunofluorescence stained images for adipogenesis (PPARγ; a), chondrogenesis (COL II; b), and osteogenesis (OCN; c). The scale bar is 20 μm. Nucleus and actin were stained by DAPI and phalloidin, respectively. PPARγ, COL II, and OCN were stained by corresponding antibodies. (d) Histological staining of the cell encapsulated gels by the alcian blue staining method at 21 days after 3D culture. The scale bar is 30 μm.

microspheres. In particular, OCN expression was the most pronounced in PS-P microsphere incorporated thermogel. It is also noteworthy that both PS-P and PS-C microspheres are similar in size, and both microspheres are negatively charged. The OCN expression was much more effective in the PS-P microsphere incorporated thermogel than the PS-C microsphere incorporated thermogel, suggesting the specificity of phosphate functional groups in inducing osteogenic differentiation during the 3D culture of stem cells. OCN expression was also significant on thiol-functionalized 2D culture on the glass surface and amino-functionalized 2D culture on the gold surface.18,22 The matrix proteins adhered to the surface on the 2D system accompanying focal adhesion of the cell was suggested to be responsible for directing the stem cell differentiation.19 However, adhesion in the 3D environment is quite different from 2D, and thus, a simple generalization of the functional groups effect on the MSC differentiation in both 3D and 2D is not possible at this stage due to the difference focal adhesion as well as corresponding differences in mechanisms involved in each case. For instance, amino groups did not significantly induce osteogenesis in amino group functionalized PEG hydrogels as well as our PS-N microsphere containing thermogels.21 In the PEG hydrogel, osteogenesis

The extent of mRNA expression was much more significant than PPARγ. The * and ** indicate the significance of the biomarker expression compared with that of 0 day by the Student t-test with p < 0.01 and p < 0.05, respectively. In particular, the COL II expression was much higher in the PS-S microsphere incorporated thermogel than the PS-N microsphere incorporated thermogel, even though the PS-S and the PS-N microspheres were similar in size, indicating that the surface functional groups certainly played a role in determining the stem cell differentiation. In 2D culture, COL II expression was suppressed for all glass functionalized with amine, thiol, or carboxylate groups, compared with clean glass.18 Only in the chondrogenic medium, the slightly improved expression of the COL II was reported for 7 days culture of MSC on the 2D glass with hydroxyl groups. However, chondrogenesis, as measured by aggrecan expression, was increased in carboxylate-functionalized PEG gel than unmodified PEG gel.21 The biomarker expression of OCN was also significant for all microspheres, compared with that of zeroth day. However, the biomarker expression was particularly high for PS, PS-P, or PS-C microsphere incorporated PEG-L-PA thermogel at 7−21 days of incubation (Figure 6c). In other words, the OCN expression was suppressed in the thermogel containing PS-N or PS-S microspheres, compared with the thermogel containing neat PS 2185

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stem cell differentiation.19 A specific functional group might recruit its appropriate molecules to interact with the cell surface to direct the differentiation. Currently, our study suggests that similar principles can be applied to 3D culture systems where the incorporated surface-functionalized microspheres play the role.

was significantly enhanced in the phosphate-functionalized system.21 Immunofluorescence staining for biomarker expressions in a protein level was studied by using antibodies for PPARγ, COL II, and OCN to confirm again the biomarker expressions in the mRNA level. The nucleus and actin of a cell were stained by DAPI and phalloidin as blue and green, respectively. The specific red color of PPARγ expression was not developed in all the 3D culture systems (Figure 7a). On the other hand, the COL II expression was clearly shown as a red color in the PS-S, PS-P, or PS-C microsphere incorporated PEG-L-PA thermogels (Figure 7b). The red color image was not shown in the PS or PS-N microsphere incorporated PEG-L-PA thermogels. This result coincided with the mRNA expression data, where the COL II gene expression was significantly (P < 0.01) enhanced in the PS-S, PS-P, or PS-C microspheres incorporated PEG-LPA thermogels. The specific red color of OCN expression was also observed in PEG-L-PA thermogels containing PS, PS-P, or PS-C microspheres (Figure 7c). The images also coincided with the mRNA expression data. Spreading of the cell is quite apparent for osteogenic differentiation of the stem cell in the 2D culture.19,21,40 However, the spreading of the cell is not evident in 3D environments as in the case of phosphate functionalized PEG hydrogel and poly(ethylene glycol)-silica thixotropic gel.12,21 The encapsulated MSC underwent osteogenic differentiation while maintaining their spherical cell morphology during the 3D culture in the above systems. Similarly, the cells underwent osteogenesis while maintaining their spherical morphology in the phosphate-functionalized microsphere incorporated PEG-L-PA thermogels. The red color image indicating OCN expression was not observed for the PS-N or PS-S microsphere incorporated thermogels, suggesting that OCN expression was not significant in these microsphere incorporated thermogels. Alcian blue staining of the cell encapsulated gel, where the cells were 3D cultured over 21 days, significantly exhibited that glycosaminoglycan (blue color) production around the cells (red) for the PS-S, PS-P, and PS-C microsphere encapsulated thermogels, compared with the PS and PS-N microsphere encapsulated thermogels (Figure 7d). Along with the significant Col II expression in the PS-S, PS-P, and PS-C microsphere encapsulated thermogels, the biomarker expressions indicated the preferential chondrogenic differentiation of the TMSCs in the PS-S, PS-P, and PS-C microsphere encapsulated thermogels. To conclude, biomarker expressions confirm that the surface functional groups of the incorporated microspheres play a critical role in determining the TMSC differentiation in the PEG-L-PA thermogel. Based on the gene and protein expression, the effect of surface functional groups of the microsphere incorporated in PEG-L-PA thermogel can be summarized as follows. Differentiation into adipogenesis was increased in the thermogels containing PS-N or PS-S microspheres. In particular, differentiation into chondrogenesis was significantly increased in thermogels containing PS-S, PS-P, or PS-C microspheres, whereas differentiation into osteogenesis was also significantly increased in thermogels containing PS-P or PS-C microspheres. The detailed mechanism on the specificity of the functional groups in inducing the differentiation of the stem cells into a specific cell type is open for further study. As suggested by Anseth et al., anchored chemical functional groups direct cell−matrix interactions to induce tissue specific matrix molecules, which might in turn control the

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CONCLUSIONS

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AUTHOR INFORMATION

Differentiation of stem cells into a specific cell type is a very important issue for stem cell research as well as stem cell therapy. In particular, TMSCs as a new stem cell resource were reported recently.2,41,42 Tonsil tissues provide a plentiful source of stem cells, with a higher population density than bone marrow. Basic studies of TMSCs are very important from the standpoint of recycling of human tissue, otherwise wasted, as well as a fundamental understanding on stem cell behavior. Various physicochemical parameters of hydrogels such as topography, mechanical properties, nanofiber incorporation, and chemical-functionalization of the hydrogel have been investigated to control the stem cell differentiation. Starting from the breakthrough papers that reported the control of the stem cell differentiation by chemical modification,18,20,21 we investigated the control of differentiation of TMSCs by incorporating the surface-functionalized microspheres in a 3D environment provided by sol-to-gel transition of the PEG-L-PA aqueous solution. The commercially available neat polystyrene microspheres (PS), ammonium- (PS-N), thiol- (PS-S), phosphate- (PS-P), or carboxylate-functionalized polystyrene microspheres (PS-C) were simply incorporated into the hydrogel by temperature-sensitive reversible sol−gel transition of the polymer aqueous solution. The in situ formed gel matrix showed a sustainable modulus enough to hold the cells and microspheres at 37 °C. In addition, the reversible sol−gel transition provides the system with improved the extraction of the RNA from the 3D matrix, and the incorporated microspheres can be recovered by lowering the temperature of the gel. PPARγ, COL II, and OCN were selected as a biomarker of adipogenesis, chondrogenesis, and osteogenesis, respectively. mRNA expression and immunofluorescence studies consistently proved that the preferential differentiation into a specific cell type could be controlled by the microspheres functionalized with a specific functional group. This paper suggests a new method for stem cell differentiation by incorporating recoverable microspheres with a specific surface functional group in a thermoreversible 3D hydrogel matrix.

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MSIP; 2009-0080447 and 2012M3A9C6049835). E.J.K. and S.J.K. equally contributed to the paper. S.J.K. was supported by RPGrant 2013 of Ewha Womans University. 2186

dx.doi.org/10.1021/bm500342r | Biomacromolecules 2014, 15, 2180−2187

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