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The Human Umbilical Cord Stem Cells Improve the Viability of OA Degenerated Chondrocytes

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The Human Umbilical Cord Stem Cells Improve the Viability of OA Degenerated Chondrocytes 4474 MOLECULAR MEDICINE REPORTS 17: 4474-4482, 2018 The human umbilical cord stem cells improve the viability of OA degenerated chondrocytes HAO WANG1*, XU YAN2*, YUXIN JIANG3, ZHENG WANG2, YUFEI LI4 and QINGDONG SHAO2 1Teaching Center of Experimental Medicine, Shanghai Medical College, Fudan University, Shanghai 200032; 2Department of Orthopedics, 455th Hospital of PLA, Shanghai 200052; 3School of Medicine, Dalian Medical University, Dalian, Liaoning 116044; 4Department of Plastic Surgery, 455th Hospital of PLA, Shanghai 200052, P.R. China Received July 11, 2017; Accepted December 5, 2017 DOI: 10.3892/mmr.2018.8413 Abstract. Osteoarthritis (OA) affects a large number of hUC-MSCs have a predominant expression of stem cell patients; however, human umbilical cord stem cells exhibit ther- markers, while the hematopoietic and endothelial markers apeutic potential for treating OA. The aim of the present study were absent. Osteogenic, chondrogenic and adipogenic was to explore the interaction between human umbilical cord differentiation was observed in certain induction conditions. stem cells and degenerated chondrocytes, and the therapeutic hUC-MSCs improved the proliferation of OA chondrocytes potential of human umbilical cord stem cells on degenerated and downregulated the expression of inflammatory cytokines, chondrocytes. Human umbilical cord-derived mesenchymal while OA chondrocytes promoted MSCs to differentiate into stem cells (hUC-MSCs) were harvested from human umbilical chondrocytes. Taken together, the co-culture of hUC-MSCs cords, and flow cytometry was used to analyze the surface and OA chondrocytes may provide a therapeutic potential in antigen markers, in addition, chondrogenic, osteogenic and OA treatment. adipogenic differentiation on the cells was investigated. OA cells at P3 were cocultured with hUC-MSCs in a separated Introduction co-culture system, and reverse transcription-polymerase chain reaction and western blot were used to evaluate the mRNA, Osteoarthritis (OA) is the most common articular disease and protein expression of collagen type II (Col2), SRY-box 9 characterized by a progressive degradation of joint carti- (sox‑9) and aggrecan. The level of inflammatory cytokines, lage, resulting in loss of joint mobility and function at last, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-10, were which was estimated to be the 4th leading cause of disability analyzed by ELISA in the supernatant. hUC‑MSCs grow in in Asia (1). OA affects the hands, feet, spine and large a fibroblastic shape with stable proliferation. hUC-MSCs weight‑bearing joints commonly, such as the hips and knees, expressed cluster of differentiation 44 (CD44), CD73, CD90, while OA patients manifest joint pain, stiffness, tenderness, CD105; while did not express CD34, CD45, CD106, CD133. limited joint movement, and joint cracking. OA is a major After multi-induction, hUC-MSCs were able to differatiate into disturbing in aging people as its incidence increases highly adipogenic, osteogenic and chondrogenic lineage. hUC-MSCs associated with age (2). inhibited the expression of matrix metalloproteinase-13, It is reported that OA occurred in 19.2% of adults partici- collagen type X α1 chain and cyclooxygenase-2 in OA chon- pants age ≥45 in the Framingham Project and 27.8% in the drocytes, and enhanced the proliferation of OA chondrocytes, Johnston County Osteoarthritis Survey, ~37% of participants while OA chondrocytes stimulated the production of Col2, age >60 years or older had radiographic knee OA in the sox-9 and aggrecan and promoted hUC-MSCs differentiate third National Health and Nutrition Examination Study (3,4). into chondrocytes. Flow cytometry analysis demonstrated According to a systematic review of Global Burden of Disease 2010 study, the global age‑standardized prevalence of knee OA was 3.8%, and hip OA was 0.85% (5). Articular carti- lage (AC) stand the mechanical distribution of loads across the joints, while cartilage degenerated and osteophyte formed Correspondence to: Professor Qingdong Shao, Department of in OA (6). Articular chondrocytes maintain proliferation and Orthopedics, 455th Hospital of PLA, 338 West Huaihai Road, terminal differentiation in healthy articular cartilage. While Shanghai 200052, P.R. China E-mail: [email protected] hypertrophy, vascularization and calcification were observed in OA cartilage (7). *Contributed equally Despite the global increase in the incidence of OA, there are no effective pharmacotherapies capable of restoring the Key words: human umbilical cord-derived mesenchymal stem cells, original structure and function of damaged articular carti- osteoarthritis, degenerated chondrocytes, differentiation, co-culture lage (8). Pharmaceutical or surgical therapies (osteotomies, microfracture) have limited efficacy in reversing or halting OA progression, while stem cell-based cartilage tissue engineering WANG et al: HUMAN UMBILICAL CORD STEM CELLS IMPROVED OA DEGENERATED CHONDROCYTES 4475 and cartilage regeneration that may be an effective strategy digestion, chondrocytes were cultured in low glucose DMEM in OA treatments (9-13). Numerous efforts have been made at 37˚C with 5% CO2 as previously described (23). to develop tissue-engineered grafts or patches to repair focal In the present study, the cells were divided into 2 groups: chondral and osteochondral defects, and to date several Experimental group (co-culture group) and control group. In researchers aim to implement clinical application of cell-based proliferation assay, the medium in experimental group was therapies for cartilage repair (14,15). changed with the supernatant from hUC-MSCs, while the Mesenchymal stem cells (MSCs) are reported to show medium in control group was changed with normal medium. promising clinical applications in articular cartilage regenera- In other experiments, OA chondrocytes and hUC-MSC were tion, and mesenchymal stem cells have a potential in treatment incubated in a noncontact co-culture system: Chondrocytes of OA (16,17). were cultured in the bottom well, and hUC-MSCs were Adipose mesenchymal stem cells have been reported cultured in a Transwell insert (Transwell; Corning Costar, to differentiate into chondrocytes in 3-dimensional culture Corning, NY, USA) in co-culture group; while chondrocytes express lubricin, and adipose tissue derived-mesenchymal were cultured alone in the bottom well in control group. stem cells cultured on collagen cell carrier scaffolds were to regenerated engineered cartilage (18,19). Cytoflow analysis. The expression of hUC-MSC surface Human umbilical stem cell populations was reported to be markers was tested by using flow cytometry as described previ- found in the umbilical cord, the cord lining, and perivascular ously (24). hUC-MSCs were isolated and harvested by using tissue, as well as Wharton's jelly, so they are attractive autolo- 0.1% trypsin-EDTA treatment, and washed with PBS. Then gous or allogenic cells to treat malignant and non-malignant the cells were incubated with the following antibodies: Rabbit solid and soft cancers, and they also can be the feeder layer for polyclonal to CD44 (ab157107; 1/500 dilution), rabbit poly- embryonic stem cells or other pluripotent stem cells (20,21). clonal to CD73 (ab175396; 1/500 dilution), rabbit monoclonal Human umbilical cord-derived MSCs (hUC-MSCs) to CD90 (ab92574; 1/1,000 dilution), mouse monoclonal to constitute an attractive alternative to bone marrow-derived CD105 (ab11414; 1/1,000 dilution), rabbit monoclonal to CD34 MSCs for potential clinical applications because of easy (ab81289; 1/1,000 dilution), rabbit polyclonal to CD45 (ab10559; preparation and lower risk of viral contamination, they can 1/1,000 dilution), rabbit polyclonal to CD106 (ab134047; 1/1,000 differentiate into the three germ layers that promote tissue dilution), rabbit polyclonal to CD133 (ab16518; 1/1,000 dilution) and organ repair and modulate immune responses and anti- (all from Abcam, Cambridge, MA, USA), in the dark for 30 min cancer properties (20). at room temperature, then conjugated with either fluorescein However, the role of hUC-MSCs and degenerated chon- PE or FITC [Goat Anti-Rabbit IgG H&L (FITC) (ab6717) or drocytes in OA progression is unclear. Therefore, we explore Goat Anti-Mouse IgG Secondary Antibody (PE) LS-C60691; the interaction between human umbilical cord stem cells and 1/1,000 dilution; LifeSpan BioSciences, Seattle, WA, USA]. OA degenerated chondrocytes, and the therapeutic potential of The labeled cells were washed and tested by flow cytometry human umbilical cord stem cells on degenerated chondrocytes. (Becton-Dickinson, Franklin Lakes, NJ, USA). Materials and methods Cell differentiation. A 6 well-plate was cultured with 5x104 cells/well. At 48 h, the medium was replaced by adipo- Patients. The study was approved by the Ethical Review genic medium (high glucose DMEM containing 10% FBS, Committee of 455th hospital of PLA (Shanghai, China). 500 µM isobutylmethylxanthine, 5 µg/ml insulin, 200 µg/ml After obtaining informed consents from the mothers and ascorpate-2-phosphate, 100 U/ml penicillin, 100 U/ml strep- family, the umbilical cords were harvested from the full-term tomycin, 1 µM dexamethasone), osteogenic medium (glucose natural delivery infants. WJ-MSC were isolated and cultured DMEM containing 10% FBS, 100 U/ml penicillin, by Shanghai Omnicells Biotechnology Co., Ltd. (Shanghai, 100 U/ml streptomycin, 100 nM dexamethasone, 10 mM China) as described (22): Umbilical cords were washed β-glycerophosphate
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