Serum-free, Chemical Defined Conditions For Chondrocytes Differentiation From Human Embryonic Stem Cells

Articular cartilage is vitally important in the joints, which relieve joint surfacefriction and reduce the pressure of skeletal motion. Because its lacks the intrinsiccapacity to repair itself through regeneration, articular cartilage is highly susceptibleto damage in injury or degenerative conditions, and it is difficult to be cure.One promising treatment for repairing damaged cartilage is the transplantation ofstem cells. Human embryonic stem cells (hESCs) have considerable advantages overadult stem/progenitor cells as seed cells, due to their capacities of unlimitedproliferation and multi-lineage differentiation. They have so small individualdifferences that they can quickly build differentiated platform, while laying thefoundation for studies of iPSCs. The differentiation from hESCs in vitro proposesnew ways different from traditional, there is access to appropriate tissue and sufficientnumbers of chondrocytes.In this study, the process of hESCs differentiating into cartilage consisted of twostages, including mesoderm differentiation and Chondrogenesis from mesoderm cells.The differentiation of hESCs took a variety of growth factors and small molecules inabsolutely serum-free medium. hESC-derived chondrogenic mesoderm emerged in achemically defined medium containing CHIR (GSK-3Inhibitor，WNT signalingactivator), and Noggin (BMP-inhibitor), the late Chondrogenesis of the mesodermcells with chondrogenic factors PDGF, TGFβ, BMP showed robust chondrogenicactivity in3-dimensional (3D) micropellet culture, generated a hyaline-like cartilageparticle. The morphology，FACS, histological staining as well as the expression ofchondrogenic specific genes were detected in each stage,and further invivo experiments verified the formation of chondrocytes. They both provided sometheory and technology for the application of hESCs in clinical therapy of cartilagedisease.The experimental results showed that the effective chondrogenic specification ofparaxial mesoderm from hESCs expressed the specific transcripts for MEOX1, andthe emergence of the KDR-PDGFRα+progeny. The late chondrogenesis from mesodermal cells was monitored by expression of the chondroprogenitor genes SOX9etc. and the chondrocyte/cartilage matrix genes COL2, COMP etc.Immunohistochemistry demonstrated we got COL2+COL1-particles. Theexpression of proteoglycans was confirmed by Alcian Blue and Toluidine Blue.These results showed that hESCs, in vitro under the serum-free conditions, formedhyaline-like cartilage particles. Adding continuously to chondrogenic mesoderm cellswith BMP4,which resulted in staining lighter with Alcian blue indicating lower levelsof proteoglycans. According to the analysis of cytokine combination differentiationconditions, continuous adding BMP4to chondrogenic mesoderm cells caused Alcianblue staining lighter, which indicated lower levels of proteoglycans. We transplantedhESCs-derived chondrocyte, or tissue into immunodeficient recipients and observedthe maintain of cartilage tissue phenotype and cells became further differentiationand maturationIn conclusion, our data demonstrated that hESCs are able to generatecartilage-like tissue in serum-free chemical defined medium. By this method, wecan achieve the ideal differentiation results and high differentiation efficiency. Theseresults not only provided new experimental evidence for the inducedchondrogenic differentiation of embryonic stem cells, but also lay the foundationfor future application of stem cell therapy to cure osteoarthritis and articular cartilageinjury of cartilage disorders.