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ISSN : 1976-7447(Print)
ISSN : 2287-7363(Online)
Journal of Biomedical Research Vol.13 No.4 pp.310-319
DOI : https://doi.org/10.12729/jbr.2012.13.4.310

Chondrogenesis of human umbilical cord-derived mesenchymal stem cells in vitro by TGFβ3 and BMP6

Jaejin Cho1,2 *, Eu Gene Park1,2, TaeJun Cho1,2, Soon-Keun Kwon1,2, Dong-Sup Lee3
1Lab of Dental Regenerative Biotechnology Major, School of Dentistry, Seoul National University, 2Dental Research Institute, Seoul National University
3Department of Biomedical Science, School of Medicine, Seoul National University
(Received 9 Nov. 2012, Accepted 14 Dec. 2012)

Abstract

Human umbilical cord is easy to obtain because it is discarded after birth, so that ethical issues can be avoided. Chondrogenesis studies using MSCs from bone marrow, cord blood, and adipose have indicated that TGFβ3 and BMP6 stimulate chondrogenesis. Therefore, we investigated chondrogenesis of hUC-MSCs on TGFβ3, BMP6, and combination of the two growth factors. We initiated chondrogenesis of cells by application of physical forces to form 3D cell clusters. After initiation, we designated four experimental groups for differentiation of cells, as follows: control, 10 ng/mL TGFβ3, 100 ng/mL BMP6, and the combination of 5 ng/mL TGFβ3 and 50 ng/mL BMP6. For analysis of chondrogenesis, GAG contents, mRNA expression, histological analysis and immunohistochemistry (IHC) were performed. For analysis of GAG contents, GAG assay was performed and RT-PCR was performed for determination of chondrogenic markers. Histological analysis was performed through safranin O, alcian blue, and IHC was performed using collagen type I and II. GAG contents were increased 184% by TGFβ3, 147% by BMP6, and 189% by the combination of TGFβ3 and BMP6, compared to control. The growth factors improved collagen II and aggrecan expression; in particular, TGFβ3 and BMP6 showed a synergistic effect, compared to only TGFβ3 or BMP6 treated. The results of histological and IHC analysis indicated that chondrogenic differentiation in TGFβ3 and the combination of TGFβ3 and BMP6 showed more cartilage deposition. In conclusion, TGFβ3 and BMP6 differentiated hUC-MSCs into chondrogenic clusters of the combination treatment of the two growth factors showed more efficient chondrogenic ability.

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Introduction

Mesenchymal stem cells (MSCs) are generally defined as multi-potent cells with self-renewal ability; they are undifferentiated [1] and exist in the many mesenchymal tissues, including fat [2], umbilical cord blood [3], amniotic fluid [4], placenta [5], dental pulp [6], the salivary glands [7], the synovial membrane [8], the vocal fold [9], and the umbilical cord [10]. Despite the great interest in MSCs, there is no standard methodology for cell preparation at present [11]. MSCs are characterized by their ability to adhere to plastic cell culture dishes [12] and their specific surface markers as well as their multi-lineage differentiation capacity under adequate conditions [11]. Because of this ability, MSCs are considered an attractive material for tissue engineering and cell-based therapies [13, 14]. 

 Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are isolated from discarded human umbilical cords after birth. The human umbilical cord is composed of 2 arteries and 1 vein surrounded by Wharton’s jelly. These cells are characterized by the expressions of cell surface markers, including CD14, CD29, CD44, CD73, CD90, and CD105, and the absence of the expressions of CD14, CD34, and CD45. In addition, they have fibroblastoid morphology [15, 16]. Human bone marrow MSCs (hBM-MSCs) are currently the main source of MSCs. However, the number of hBM-MSCs decreases significantly with donor age [17]. Therefore, the umbilical cord is considered an alternative source of MSCs. Human umbilical cord-derived MSCs (hUC-MSCs) have greater expansion potential than do adult tissue-derived MSCs [18]. Furthermore, hUC-MSCs can avoid ethical dilemmas because the umbilical cord is discarded after birth. Recently, Wang et al. showed that hUC-MSCs possess the ability to differentiate into insulin-secreting cells both in vitro and in vivo [19]. In another study, Weiss et al. provided encouraging results for the utility of hUC-MSCs in a rodent model of Parkinson’s disease [20]. Furthermore, hUC-MSCs have been used for chondrogenesis research [21] and studied for their effects on engineered tissue structure [22].

 Articular cartilage is a viscoelastic connective tissue that covers the bone at the joint surface [23]. Chondrogenesis is the earliest phase of skeletal development and involves mesenchymal cell recruitment and migration, the condensation of progenitors, and chondrocyte differentiation and maturation [24]. Since cartilage has limited blood vessels, nerves and lymphatic vessels, it is difficult to repair itself. In particular, damage to cartilage can cause osteoarthritis (OA) and related articular cartilage disease. OA is one of the most common causes of pain and disability in older people. Although various surgical treatments have been implemented for OA, they provide limited reduction in pain. Therefore, cell-based tissue engineering research is required to replace damaged cartilage with new tissue. Brittberg et al. showed that deep cartilage defects in the knee can be treated by autologous chondrocyte transplantation [25]. First, healthy chondrocytes are obtained from the patient and cultured in the laboratory. Second, cultured cells are injected into the affected area. However, the transplantation of autologous chondrocytes has a disadvantage because 2 surgeries are required. Furthermore, cell dedifferentiation of mature chondrocytes makes it difficult to obtain enough cells during monolayer culturing [26]. Since MSCs have the ability to differentiate into chondrocytes, they are regarded as a substitute material for chondrocytes in cartilage repair [27].

MSCs barely differentiate into chondrocytes when cultured in monolayers. Therefore, 3-dimensional (3D) culturing is performed to induce the chondrogenesis of MSCs [28]. To form the 3D cluster, appropriate centrifugal force, a defined medium [29], and further supplementation are required. Growth factors such as transforming growth factor β (TGFβ) and bone morphogenetic proteins (BMPs) are used for further supplementation since they are essential for cartilage homeostasis [30-32]. Chondrogenesis with 3D clusters has been studied using hBM-MSCs and adipose-derived MSCs (hADSCs). According to the literature, TGFβ and BMPs promote chondrogenesis [33, 34]. In particular, TGFβ3 and BMP6 enhance chondrogenesis in hBM-MSCs [35]. Therefore, the main purpose of the present experiment was to demonstrate the chondrogenesis ability of hUC-MSCs with the growth factors TGFβ3 and BMP6, which are known to stimulate chondrogenesis. 

Materials and Methods

1. Isolation and purification of hUC-MSCs

 Human umbilical cord was obtained and processed within 24 h after delivery. All experiment was approved by institutional review board (IRB No.860-20110087). Human umbilical cord was carried in HBSS (Welgene, Daegu, South Korea) containing 3× antibiotic antimycotic (Gibco, Grand Island, NY, USA) and washed DPBS (Welgene) several times. Cord was sectioned along with a vein 2~3 cm long. Then put down with a sectioned-side in a culture dish which contained with 0.1% collagenase type Ι (Gibco) and cultured for 30 min at 37°C, 5% CO2. After incubation, the umbilical cord vein was scrapped with a scrapper to isolate endothelial stem cells. Then, umbilical cord vein put down again with sectioned-side in a culture dish which contained with 0.1% collagenase type Ι (Gibco) and 0.24% dispase (Gibco) and cultured for 2 h at 37°C, 5% CO2. After 2 h, the umbilical cord vein was scrapped with a scrapper. Enzyme solution was gathered and centrifuged for 4 min at 400 g. Then cell pellet was suspended in cell culture medium which DMEM (Welgene) containing 20% FBS, 1% antibiotic antimycotic (Gibco) and plated in 35 cm2 cell culture dish (Nunc, Naperville, IL, USA). Cell culture was maintained at 37°C, 5% CO2. After 3 d, cell culture medium was replaced and cell culture medium was changed every 2 d.

2. FACS analysis

 MSCs markers such as CD10, CD29, CD44, CD73, CD90, CD105 and hematopoietic markers such as CD14, CD31, CD34, CD45 were attached and analyzed. Each antibodies were conjugated follow as: FITC-conjugated mouse anti-human CD14, CD34, CD45, CD90 and PE-conjugated mouse anti-human CD73, CD117 and PE. Cy5-conjugated mouse anti-human CD10 and PE. Cy7-conjugated mouse anti-human CD44 and APC-conjugated mouse anti-human CD29, CD105 (All from eBioscience, San Diego, CA, USA). hUC-MSCs passage 5 was used. hUC-MSCs 2.5 × 105 cells/mL were washed in FACS buffer (PBS, 2% FBS, 0.02% Sodium azide) and incubated with primary antibodies for 30 min in ice. After incubation, cells were washed and fixed with 4% paraformaldehyde at 4°C and analyzed through FACS calibur (Becton Dickinson, San Joes, CA, USA).

3. Macroscopic analysis

 After 14 d, 3D clusters were observed using a stereoscopic microscope (SMZ645, Nikon, Tokyo, Japan), also micro-ruler was observed to size analysis.

4. Chondrogenic differentiation of hUC-MSCs

 hUC-MSCs passage 8 was used to induce chondrogenic differentiation. To forming the 3 D cluster, cells were seeded in 15 ml polypropylene conical tube with 2.5 × 105 cells and centrifuged for 5 min at 500 g. Then supernatant was removed and replaced four types of cell culture medium. We divided four types of group for experiment. The first group was optimized chondrogenic medium containing high-glucose DMEM (Welgene), 100 nM dexametasone, 50 μg/mL ascorbate-2-phosphate, 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, and ITS + Premix (all from Sigma-Aldrich, St. Louis, MO, USA) and the second group the optimized chondrogenic medium was mixed with 10 ng/mL TGFβ3 (R&D system, Minneeapolis, MN, USA) and the third group the optimized chondrogenic medium was mixed with 100 ng/mL BMP6 (R&D system, Minneeapolis, MN, USA) and the fourth group the optimized chondrogenic medium was mixed with 5 ng/mL TGFβ3 and 50 ng/mL BMP6. TGFβ3 and BMP6 were known as chondrogenesis promoting growth factor. All groups were maintained at 37°C, 5% CO2 for 14 d and the medium were changed every 3 or 4 d.

5. Glycosaminoglycan (GAG) assay

 After 14 d, the GAG and DNA assay was measured. For GAG assay, we were used three 3D clusters per each group. GAG assay was performed by GAG assay kit (Blyscan Sulfate Glycoaminoglycan assay kit, Biocolor, Belfast, Ireland) and all steps were carried out according to the manufacturer’s instructions. Shortly, 3D clusters were dissolved in 500 ml papain buffer solution which 100 ml of 0.2 M sodium phosphate buffer, add 0.1 M sodium acetate, 10 nM EDTA, 5 mM L-cysteine HCl, pH 6.4 containing papain (Sigma-Aldrich) 10 μL/ml in water bath at 60°C for 24 h. After incubation, the dissolved solution was centrifuged at 6,000 rpm for 5 min and the supernatant 100 μL mixed with reagent solution 100 ml then settled in room temperature for 30 min. The pellet was dissolved 1ml alkali reagent. The absorbance measured at 656 nm in ELISA reader (S500, BIO-RAD, Camarillo, CA, USA).

 DNA assay was measured using Picogreen assay kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. GAG content was analyzed versus total cellular DNA contents, and the experiment was performed to triplicate.

6. RT-PCR

 We used ten of 3D clusters per each group. Total RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed with according to the complementary DNA synthesis kit (M-MLV RT, Invitrogen) using total RNA 1 μg with oligo-dT primers. To PCR reactions, PCR premix (Accupower PCR premix, Bioneer, Daejun, South Korea) was used with 1 μL cDNA that used as template. RT-PCR condition was that pre denaturation at 94°C for 5 min, denature at 94°C for 30 sec, annealing at each temperature for 30 sec and extension at 72°C for 20 sec followed by final extension at 72°C for 5 min. The PCR reaction carried out Mastercycler gradient (Eppendorf, Hamburg, Germany). Then gel electrophoresis was performed with 1.2% agarose gel containing 0.01 mg/ml ethidium bromide at 100 V for 40 min. RT-PCR primer sequence and annealing temperature is arranged Table 1 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as normalization of mRNA level.

7. Histological analysis

 3D clusters were fixed in 4% paraformaldehyde for overnight then embedded in paraffin and sectioned by 4 μm. We carried out three types of histology staining: H&E staining, safranin O and alcian blue which for glycosaminoglycan detection. H&E staining was performed with hematoxylin (Sigma-Aldrich) for 3 min and eosin (Sigma-Aldrich) for 2 min. Safranin O (Thermo Fisher Scientific, Bremen, Germany) staining was performed 0.1% hematoxylin (Sigma-Aldrich) for 1 min 30 sec, 0.4% fast green (Thermo Fisher Scientific) for 3 min and 0.12% safranin O (Thermo Fisher Scientific) for 15 min. Alcian blue staining was performed alcian blue for 1 h and fast red for 5 min. All experiments were carried out at room temperature.

8. Immunohistochemistry

 Immunohistochemical analysis was performed with rabbit polyclonal collagen I (Abcam, Cambridge, UK) and mouse monoclonal collagen II (Calbiochem, San-diago, CA, USA) antibody. The antibodies were diluted by 1:100. ABC detection kit (Cap-plus detection Kit, Invitrogen, Carlsbad, CA, USA) was used for detect collagen type I and collagen type II antibodies. All experiments were performed according to the manufacturer’s instructions. Shortly, the sectioned tissue was treated by 3% peroxidase for 10 min and washed 3 times by DPBS. In order to retrieval, we treated the pepsin and the sample was incubated for 30 min at 37°C. After incubation, the sample was washed 3 times by DPBS and treated by blocking solution for blocking then primary antibody and secondary antibody was treated. To confirm the color degree, streptavidin and DAB was used.

9. Statistical analysis

 GAG values were analyzed by t-test to compare parameters between four experimental groups using PASW (Version 18.0; SPSS, Inc., Chicago, IL, USA). Difference at P<0.05 was considered significant.

Results

1. Characterization of the morphology of hUC-MSCs

 Image analysis was performed to characterize the morphology of hUC-MSCs (Fig. 1). hUC-MSCs exhibited fibroblast-like morphology at passage 7.

Fig. 1. Characterization of the morphology of hUC-MSCs. The morphology of hUC-MSCs are shown in (A) and (B). The cells have a fibroblast-like morphology. hUC-MSCs passage 7 were cultured in 20% FBS/DMEM and observed. Magnification is (A) ×40 and (B) ×100.

2. Cell surface phenotype characterization of hUC-MSCs

 In the FACS analysis (Fig. 2), hUC-MSC were positive for MSC markers, including CD10 (71.01%), CD29 (100%), CD44 (84.92%), CD73 (84.92%), CD90 (63.98%), and CD105 (99.89%), and negative for hematopoietic markers, including CD14 (1.76%), CD34 (1.65%), CD45 (1.74%), and CD117 (1.84%).

Fig. 2. FACS analysis of surface-marker expression on hUC-MSCs. The surface-marker expression of hUC-MSCs was detected by FACS analysis. The hUC-MSCs were cultured in 20% FBS/DMEM. Each antibody was stained at 4°C for 30 min and then detected. Under same culture condition, hUC-MSCs were positive for CD10, CD29, CD44, CD73, CD90 and CD105 but negative for CD14, CD34, CD45 and CD117.

3. Morphology of 3D clusters

 The sizes of the 3D clusters cultured for 14 d were similar among the 4 experimental groups at approximately 1 μm (Fig. 3).

Fig. 3. Macroscopic view of hUC-MSCs chondrogenic 3D clusters after 14 d. The chondrogenic 3D clusters shown similar size for all groups. Chondrogenic 3D clusters were cultured for 14 d and observed by stereoscope. Pictures are 3 ×magnification with 1 μm scale bar.

4. Glycosaminoglycan (GAG) assay

 A GAG assay was performed to compare the glycosaminoglycan contents of the 3D clusters. Figure 4A shows that the GAG levels increased by 184% and 147% with TGFβ3 and BMP6 compared with the control, respectively; a combination of TGFβ3 and BMP6 increased GAG contents by 189%. These results indicate that a combination of TGFβ3 and BMP6 induces significantly more GAG synthesis than TGFβ3 or BMP6 treatment. Although BMP6 treatment exhibited higher GAG contents than the control, the difference was not significant (>0.05%). The results of total DNA levels indicated similar OD values for all groups (Fig. 4B). GAG contents were normalized to the total DNA values.

Fig. 4. GAG assay for detection to the glycosamioglycan contained in chondrogenic 3D clusters. (A) The values of GAG are normalized total DNA. The total DNA values shown in (B). The chondrogenic 3D clusters were treated TGFβ3 and/or BMP6 for 14 d then used. We used three 3D clusters per groups for GAG and total DNA analysis and experiments were performed to triplicate. Error bars represent standard deviations (□: Defined, ▤: TGFβ3 at 10 ng/mL, ▦: BMP6 at 100 ng/mL, ■: TGFβ3 at 5 ng/ml and BMP6 at 50 ng/mL).

5. Expression of mRNA of chondrogenesis- and smad signaling-related markers

 RT-PCR was performed to confirm the gene expression levels associated with chondrogenesis (Fig. 5). The chondrogenic markers collagen II and aggrecan showed similar patterns; in addition, these patterns were similar to the results of the GAG analysis. TGFβ3 increased collagen II and aggrecan expression by 1.66- and 2.27-fold, respectively; BMP6 increased collagen II and aggrecan expression by 1.34- and 1.6-fold, respectively. Moreover, the combination of TGFβ3 and BMP6 increased collagen II and aggrecan expression 2.18- and 3.53-fold compared with the control, respectively. Sox9 expression was increased by 1.21-, 1.5-, and 1.65-fold by TGFβ3, BMP6, and a combination of TGFβ3 and BMP6, respectively. The expression of collagen X, a chondrocyte hypertrophy marker, increased by 3.27-, 1.24-, and 1.72-fold compared with the control for TGFβ3, BMP6, and both, respectively.

Fig. 5. RT-PCR analysis. The gene expression was analyzed by RT-PCR. (A) lane 1: defined; lane 2: TGFβ3 at 10 ng/mL; lane 3: BMP6 at 100 ng/mL; lane 4: TGFβ3 at 5 ng/ml and BMP6 at 50 ng/mL. (B) RT-PCR image analysis of relative absorbance (□: Defined, ▤: TGFβ3 at 10 ng/mL, ▦: BMP6 at 100 ng/mL, ■: TGFβ3 at 5 ng/ml and BMP6 at 50 ng/mL). Sox9: Sry-type homeobox protein-9; COL I: collagen I; COL II: collagen II; TGFβ: Transforming growth factor beta; TGFβR: TGF-beta receptor; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

To investigate smad signaling, the gene expressions of smad 1, 2, 3, 5, and 7 as well as related genes such as TGFβ receptors I and II were detected using RT-PCR. TGFβ receptor I showed a similar pattern to those of collagen II and aggrecan: TGFβ3, BMP6, and a combination of both increased TGFβ receptor I levels by 3.17-, 2.84-, and 5.79-fold, respectively. However, TGFβ3 and BMP6 increased the gene expression of TGFβ receptor II by 4.12- and 5.63-fold, respectively. TGFβ3 prominently increased the expression smad 1 by 8.84-fold, but this was not apparent with BMP6 or a combination of TGFβ3 and BMP. Smad 2 and 7 had similar patterns in that the combination of TGFβ3 and BMP6 increased gene expression to a greater extent than TGFβ3 alone: 1.22-fold with TGFβ3 and 1.46-fold with TGFβ3 and BMP6 for smad 2; 1.24-fold with TGFβ3 and 1.27-fold with TGFβ3 and BMP6 for smad 7. However, BMP6 did not increase smad 2 expression. The combination of TGFβ3 and BMP6 increased the gene expression of smad 3- by 1.6-fold, whereas TGFβ3 and BMP6 alone increased by it by 1.11- and 1.09-fold, respectively. BMP6 increased the gene expression of smad 5 by 1.64-fold, and the combination of TGFβ3 and BMP6 increased it by 1.3-fold. However, TGFβ3 increased smad 5 expression by only 1.04-fold compared with the control.

6. Histological analysis

 We performed special staining with safranin O and alcian blue for GAG detection as well as H&E staining (Fig. 6). H&E staining showed that all types of 3D clusters appeared to have 2 layers: an inner layer and an outer one. H&E staining showed that the inner layer had round nuclei, whereas the outer layer had spindle-shaped nuclei. Safranin O staining revealed that TGFβ3 and the combination of TGFβ3 and BMP6 were detected more strongly than BMP6 in both layers. TGFβ3 and a combination of TGFβ3 and BMP6 in particular showed more spindle-shaped nuclei in the outer layer. Alcian blue staining indicated that TGFβ3 resulted in more compact and prominently stained cells arrangements than did the others.

Fig. 6. Histological analysis. The histological analysis of the chondrogenic 3D clusters were stained with H&E (A-D), safranin O (E-F) and alcian blue (I-L). The chondrogenic 3D clusters were fixed for overnight in 4% paraformaldehyde then embedded in paraffin. The paraffin blocks were sectioned by 4 μm and then used. Pictures are ×200 magnification and small picture is ×100. (A, E, and I: defined; B, F, and J: TGFβ3 at 10 ng/mL; C, G, and K: BMP6 at 100 ng/mL; D, H, and L: TGFβ3 at 5 ng/ml and BMP6 at 50ng/mL).

7. Immunohistochemistry

 For collagen type I, BMP6 was stained in the outer layer with round nuclei, while TGFβ3 and the combination of TGFβ3 and BMP6 were stained in both layers with round and spindle-shape nuclei. In addition, TGFβ3 showed more spindle-shaped nuclei at the edges of 3D clusters than the combination of TGFβ3 and BMP6. Meanwhile, collagen type II, a chondrogenic marker, showed that BMP6 was sparsely stained with a pale brown color, whereas TGFβ3 showed dense staining with a darker brown color. In addition, TGFβ showed more spindle-shaped nuclei; however, the combination of TGFβ3 and BMP6 resulted in almost no staining (Fig. 7).

Fig. 7. Immunohistochemistry for collagen I and collagen II. The chondrogenic 3D clusters were investigated by immunohistochemistry for collagen I (A-D) and Collagen II (E-H). The chondrogenic 3D clusters were fixed for overnight in 4% paraformaldehyde then embedded in paraffin. The paraffin blocks were sectioned by 4 μm and then used. Pictures are ×200 magnification and small picture is ×100. (A and E: defined; B and F: TGFβ3 at 10 ng/mL; C and G: BMP6 100 ng/mL; D and H: TGFβ3 at 5 ng/ml and BMP6 at 50ng/mL).

Discussion

 hUC-MSCs were isolated by enzymatic digestion from umbilical cord matrix (i.e., Wharton’s jelly) [36] or the umbilical cord vein [16]. To isolate hUC-MSCs, whole umbilical cords were chopped up and digested in enzyme; alternatively, the umbilical cord vein was digested in enzymes and gently mixed. In this study, we successfully isolated hUC-MSCs from the subendothelial layer by scraping. Their characteristics were the same as those reported in previous hUC-MSCs studies [20, 36]. As shown in Fig. 2, the cells expressed several MSC-positive markers including CD10, CD29, CD44, CD90, and CD105 but not CD14, CD34, CD45, or CD117. Also, the cells have a fibroblast-like morphology (Fig. 1).

 These results indicate that we successfully isolated MSC subendothelial layers from the human umbilical cord vein. The scraping procedure had no effect on the expressions of specific surface markers of hUC-MSCs.

 Estes et al. induced chondrogenesis using an alginate scaffold for hADSCs over 7 d [37]; however, we did not use such a scaffold in the present study. Instead, to form 3D clusters, we used a 14-d pellet culture system [28]. According to the chondrogenesis study of hUC-MSCs, larger pellets (1~2 mm in diameter) were observed in 10 ng/mL TGFβ3 than with no TGFβ3 within 21 d [38]. However, the 3D clusters of our 4 tests groups had similar sizes of approximately 1 mm (Fig. 3). We investigated the effects of the growth factors TGFβ3 and BMP6, and a combination of both on chondrogenesis by GAG analysis, RT-PCR, histological assays, and immunohistochemistry.

 TGFβ3 and BMP6 accelerated MSC chondrogenesis. BMP6 induced more chondrogenesis than TGFβ3, especially in hADSCs [37]. In addition, a combination of TGFβ3 and BMP6 is considered the most effective combination of growth factors for hADSCs [39]. Mark et al. found that TGFβ1 was not significantly different from the control in GAG synthesis for hUC-MSCs at 21 d [40]. Our GAG results indicate that TGFβ3 promotes GAG synthesis to a greater extent than the control at 14 d. The combination of TGFβ3 and BMP6 also promoted GAG synthesis (Fig. 4). Sekiya et al. demonstrated that a combination of 10 ng/mL TGFβ3 and 500 ng/mL BMP6 enhances the proteoglycan synthesis of hBM-MSCs by Safranin O staining at 21 d. However, TGFβ3 and BMP6 alone had no discernible effects [35]. Our results indicate that a combination of 10 ng/mL TGFβ3 and BMP6 or 10 ng/mL TGFβ3 alone increase the proteoglycan synthesis of hUC-MSCs at 14 d. However, 100 ng/mL BMP6 had a weaker effect than a combination of TGFβ3 and BMP6 or TGFβ3 alone.

 Indrawattana et al. also reported that a combination of 10 ng/mL TGFβ3, 500 ng/mL BMP6, and 100 nM dexamethasone induces mRNA expressions of collagen II, aggrecan, and Sox9 in hBM-MSCs for 21 d. Moreover, the expressions of these molecules, except aggrecan, were slightly higher than with a combination of 10 ng/mL TGFβ3 and 100 nM dexamethasone. A combination of TGFβ3 and BMP6 increased Sox9, aggrecan, and collagen II expression compared to the control. BMP6 alone significantly induced Sox9 and aggrecan gene expression; aggrecan showed a higher mRNA expression level with 500 ng/mL BMP6. With TGFβ3 alone, collagen I expression is reported to increase by approximately 25~30 fold [41]. However, we found that collagen II and aggrecan showed similar mRNA expression levels, while Sox9 showed greater expression levels with TGFβ, BMP6, and a combination of TGFβ and BMP6. Aggrecan and collagen I showed greatly increased gene expression with a combination of TGFβ and BMP6 compared to TGFβ or BMP6 alone. Estes et al. reported that collagen X is suppressed by 500 ng/mL BMP6 [37]. In the present study, even with 100 ng/mL BMP6, the mRNA expression of collagen X was suppressed.

 TGFβ3 is an essential growth factor for promoting chondrogenesis. Some investigators report that the TGFβ3 signal is transmitted into the nucleus via the smad pathway [42]. Furumatsu et al. report that smad 3 promotes the transcription of Sox9 and collagen II. Therefore, smad 3 overexpression strongly promotes the primary chondrogenesis of hMSCs [43]. Our results indicate that smad 3, Sox9, and collagen II are strongly expressed with a combination of TGFβ3 and BMP6. Hellingman et al. indicate that the phosphorylation of smad 2 and 3 is essential for the deposition of collagen II. In addition, the phosphorylation of smad 1, 5, and 8 is correlated with terminal differentiation and mineralization [44]. Furthermore, BMP-induced smad 1 is important for chondrocyte hypertrophy [24]. Our results indicate that smad 1 is only expressed with TGFβ3. Smad 2, 3, 5, and 7 showed similar gene expressions in all groups. However, image analysis revealed that smad 2 and 3 had the highest gene expression with a combination of TGFβ3 and BMP6. Meanwhile, smad 5 showed the highest gene expression with BMP6. The TGFβ receptor II phosphorylates TGFβ receptor I and subsequently activates smad 1, 5, and 8 [45]. In the present study, TGFβ receptor I was strongly expressed with a combination of TGFβ3 and BMP6. However, TGFβ receptor II showed the highest gene expression with BMP6. The expressions of TGFβ receptors I and II and smad 1 were not observed in the control (Fig. 5).

 Winter et al. reported that the histological detection of collagen type II and proteoglycans is imperative to successfully confirm chondrogenic differentiation. However, for the most part, collagen I and osteopontin are stained in hUC-MSCs [46]. In this study, collagen II labeling was observed with TGFβ3 and BMP6. However, no effect was observed with a combination of TGFβ3 and BMP6. In addition, chondrogenic clusters of hUC-MSCs were stained positive for collagen I, especially in TGFβ3 and a combination of TGFβ3 and BMP6 (Fig. 7). Indrawattana et al. reported that TGFβ3 and a combination of TGFβ3 and BMP6 are labeled by safranin O, but observed no effect of BMP6 alone on hBM-MSCs for 21 d [41]. We observed the same labeling pattern in our hUC-MSCs for 14 d. In Alcian blue staining, which indicates proteoglycan deposition, TGFβ3 was prominently labeled but not BMP6 or a combination of TGFβ3 and BMP6 (Fig. 6).

 In conclusion, our results demonstrate that human umbilical cords are a pioneering source of MSCs and an alternative to bone marrow. Results of a GAG assay, gene expression assay, histological analysis, and immunohistochemistry indicate that hUC-MSCs have chondrogenic ability upon treatment with growth factors such TGFβ3 and BMP6. Moreover, our results indicate that TGFβ3 and BMP6 stimulate the chondrogenic differentiation of hUC-MSCs. TGFβ3 and BMP6 have synergistic effects on the chondrogenesis of hUC-MSCs. Therefore, hUC-MSCs can be used as an alternative to bone marrow for cell therapy for osteoarthritis as well as cartilage reconstruction.

Acknowledgements

 This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (No. 860-20110087).

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