Cartilage Repair Using Human Embryonic Stem Cell-Derived Chondroprogenitors

Abstract

In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions. STEM CELLS

LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

“…To further assess whether the SIRT1 repressive effects on LEF1 are conserved throughout chondrogenesis, we used a previously described 3-step protocol in which human embryonic stem cells are efficiently directed toward chondrogenesis (32,33). At d 13 of chondrogenesis, SOX9, COL2A1, and ACAN gene expression was significantly increased compared to d 0 (Fig.…”

“…MAN7 human embryonic stem cells (hESCs) were cultured as previously described by Oldershaw et al (32) and Cheng et al (33) with slight modifications. hESCs were cultured in a feeder-free system on vitronectin-coated (Thermo Fisher Scientific) tissue culture plates with mTESR1 medium (StemCell Technology, Vancouver, BC, Canada) and subcultured using EDTA passage.…”

Augmentation of Musculoskeletal Regeneration: Role for Pluripotent Stem Cells

“…A substantial expansion of the cell population was reported during the differentiation protocol (8.5-fold), and between 75 and 97% SOX9-positive cells were obtained with four different hESC lines [91]. The protocol was also applied to two iPSC lines with similar success [91]. Following the 14 day protocol, there was no evidence of pluripotent cells remaining; this was particularly encouraging as residual pluripotent cells following transplantation could result in teratoma formation.…”

“…Growth factors are used to direct differentiation of the hESCs toward chondrocytes over the course of 14 days. A substantial expansion of the cell population was reported during the differentiation protocol (8.5-fold), and between 75 and 97% SOX9-positive cells were obtained with four different hESC lines [91]. The protocol was also applied to two iPSC lines with similar success [91].…”

“…This high efficiency and purity makes this a potentially useful protocol for both clinical and laboratory applications. To test the potential for clinical application of this technique, hESC-derived chondroprogenitors embedded in a fibrin gel were transplanted into an osteochondral defect in athymic RNU rats [91]. The transplanted cells formed hyaline cartilage, identifiable from 4 weeks, and fully developed by 13 weeks postimplantation.…”