Chondrogenesis

SCP-1 (47^th passage, PDL 57), SCP-11 (68^th passage, PDL 82) and hTERT-hMSCs (52^nd passage, PDL 57) were differentiated towards adipogenic, osteogenic and chondrogenic lineages [18 (link)]. Untransduced cells were used as a positive control. As a negative control both untransduced and transduced cells were cultured under identical conditions in standard medium (D-MEM, high glucose, glutamin, pyruvate) supplemented with 10% FBS and 1% Pen-Strep solution without differentiation supplements.
In vitro osteogenic differentiation of hMSCs was performed as previously published [18 (link)] using 100 nM dexamethasone, 10 mM β-glyc-erophosphate, 50 μM L-ascorbic acid-2-phosphate (Sigma). A total of 5 × 10³ cells/well were seeded in a 6-well plate. After 16 days, cells were assayed by von Kossa staining using a standard protocol.
Adipogenic differentiation was accomplished as previously published [18 (link)] by 1 μM dexamethasone, 0.2 mM indomethacin, 0.1 mg/ml insulin, 1 mM 3-isobutyl-1-methylxanthin (IBMX) (Sigma). The maintenance medium consists of 0.1 mg/ml insulin in standard medium. A total of 4 × 10³ cells/well were seeded in a 12-well plate. Stimulation was started when cells reached full confluency. Cells were grown for 5 days in induction medium, thereafter for 2 days in maintenance medium and then switched to induction medium again. After 16 days of stimulation, the cells were assayed by oil red O staining using a standard protocol.
Chondrogenic differentiation was achieved in aggregate cultures as previously published [18 (link)] with 100 nM dexamethasone, 1 mM pyruvate, 195 μm L-ascorbic acid-2-phosphate, 350 μM L-proline, 1.25% (v/v) insulin-transferrin-selenious acid mix (ITS, 100×), 5.35 μg/ml linolic acid, 1.25 mg/ml bovine serum albumine (BSA) (Sigma) and TGF-β₃ (10 ng/ml; R&D Systems, Minneapolis, MN, USA). A total of 2.5 × 10⁵ cells were used per pellet. Sections of the size of 12 μm were cut with a cryostat vacutome HM 200 OM (Microm, Walldorf, Germany). Anionic sulphated proteoglycans were detected by toluidine blue metachromasia. Slices were stained in 1% toluidine blue solution (Sigma, Munich, Germany), 1% sodium tetraborate (Sigma, Munich, Germany).

Fresh umbilical cords were obtained from informed, consenting mothers at the First People’s Hospital of Nanjing (China) and rapidly processed. The cords were rinsed twice with phosphate-buffered saline (PBS) supplemented with penicillin and streptomycin (pen/strep; Gibco, Carlsbad, CA), and the cord vessels were removed. The washed cords were subsequently cut into small fragments that were individually attached to the substrate of culture plates. This was followed by the addition of stem cell culture medium (Cyagen, Guangzhou, China) and incubation at 37 ℃ with 5% CO₂. The medium was replaced every 3 days after the initial culture, and well-developed colonies of fibroblast-like cells appeared ~10 days later. The colonies were then trypsinized (Gibco) and passaged into new plastic plates for further expansion. The human umbilical cord MSCs (hUCMSCs) from passages 3–7 were used for all the experiments. The experimental protocol was approved by the Nanjing Medical University Ethics Committee.
The osteogenic, adipogenic, and chondrogenic differentiation identified by Alizarin Red staining, Oil Red O staining, and Alcian Blue staining, respectively. To accomplish the differentiation, passages 3–7 MSCs were cultured in OriCell™ hUCMSCs osteogenic, adipogenic, or chondrogenic differentiation media (all from Cyagen) as described by manufacturer.
After the third passage, the hUCMSCs were trypsinized and washed twice with PBS; they were then stained with human anti-CD105, anti-CD73, anti-CD90, anti-CD45, anti-CD34, anti-CD14, anti-CD19, or anti-HLA-DR. Identical concentrations of FITC- or PE-conjugated mouse IgG isotype antibodies were used as negative controls (all from BD Biosciences Pharmingen, San Jose, CA). At least 10,000 events were acquired on a FACSVerse instrument (BD Bioscience), and the results were analyzed using FlowJo software (Tree Star, Ashland, OR).