Studies On Differentiation Of ESCs Into Cartilage Stem Cells Through Ectoderm And Application Of Cartilage Injury Repair

Background Temporomandibular joint(TMJ)cartilage degeneration is a common pathological feature of many TMJ diseases,which can lead to subchondral bone resorption or destruction.It is very difficult to repair once the cartilage damage occurs since it is avascular tissue.Currently,it has attracted more and more attention that targeted differentiation of chondrocytes through stem cells to repair damaged chondrocytes.The most common seed cells include bone marrow stroma stem cells(BMSCs),embryonic stem cell(ESC)and so on.Although the technology of BMSCs to chondrogenic differentiation through mesoderm has been mature,its limited proliferation capacity and uncontrollable factors of multiple differentiation have astricted the application of BMSCs in repairing articular cartilage.On the other hand,the differentiation of ESC into articular chondrocytes has been recognized by more and more scholars,however these techniques are all though mesoderm without exception,which is not consistent with the development of TMJ.This is because during embryonic development,TMJ is generated in the neuroectoderm rather than the mesoderm,which differs from other joints.Objective we aimed to simulate the development process of TMJ,inducing the ESC to differentiate into chondrocytes through the neural ectoderm,and to detect the characteristics and directional differentiation potential of chondrocytes,so as to repair the damaged TMJ condylar chondrocytes.Materials and methods1.First,we want to explore the differentiation method of ESCs into chondrogenic stem cells by neuroectoderm and observe the morphological changes of the cells.PCR was used to detect the main markers of ESCs and chondrogenic stem cells,as well as the main markers of totipotent embryonic stem cells and key markers of each embryo layer in the induction process,so as to determine whether the cells were differentiated from the ectoderm.2.Then,we detected the clonal formation ability of the induced cells and the changes of different generations of induced cell markers by PCR in order to analyze the stem cell characteristics.The main markers of chondrocytes were detected by immunofluorescence,and the expression of common stem cell markers was also detected by flow cytometry.The induced cells,TMJ condylar cartilage and common chondrocyte line were sequenced and analyzed.3.In order to evaluate the differentiation potential of induced cells,we first induced the chondrogenic stem cells into osteoblasts,chondroblasts and lipids in vitro,and then observed the differentiation effect of chondrogenic stem cells under different spontaneous differentiation conditions.Finally,in the in vivo experiments,cartilage stem cells were transplanted into the subcutaneous of immunodeficient mice through to further clarify the characteristics and differentiation ability of cartilage stem cells.4.Finally,the cartilage injury model of the knee joint in rats were established.Cartilage stem cells were transplanted into the defect knee joint.The effect of cartilage stem cells in repairing cartilage injury was evaluated by in vivo experiments.Results1.In this part,we found that the morphology of the induced cells changed from the ESCs which like a colony before induction to the cells that grew like mesenchymal monolayer after induction.PCR results showed that OCT4,SOX2 and NANOG,markers of pluripotent stem cells,were gradually weakened and disappeared during induction.PCR and sequencing results showed that the induced cells mainly expressed ectodermal markers(NES,ALCAM,SIX1),but almost no mesoderm or endoderm markers were expressed.In addition,cartilage/bone related markers RUNX2,AGG,OCN and SOX9 were significantly expressed in cells after induction.2.Immunofluorescence staining showed that after induction,Nestin was cytoplasmic positive in the induced cells and CD29 was mainly membrane positive.SOX9,RUNX2,twist1 and SOX5 had a higher proportion in the cells after induction.Flow cytometry showed that the induced cells did not express CD146,CD24,CD45 or CD90,while CD105,CD29,CD71,CD73 and CD44 were expressed in most cells.Clonal formation experiments showed that the induced single cells could form clonal growth and express SOX9,Col1a1,SOX5,OCN,RUNX2 and Col2a1.The expression levels of the above RNAs were not significantly changed between the induced cells in different generations.Immunofluorescence co-staining showed that proliferating cell nuclear antigen(Ki67)and RUNX2 were double positive in the induced cells.Principal component analysis showed that compared with common chondrocyte line SW1353,the induced cells and P9 generation stem cells were more similar to human condylar cartilage.3.HE staining showed that the induced stem cells were basophilic with chondrogenic differentiation,with chondrogenic lacunas.Immunohistochemical staining showed that AGG,Col? and RUNX2 are positive in the induced cells.Ali's scarlet staining showed strong positive with large tufted areas after osteoblast differentiation.After lipogenic differentiation,a large number of lipid droplets accumulate in the cytoplasm.The results were similar to those of human dental pulp stem cells.In different culture conditions,like adherent culture,cultured on sponge scaffold and suspension culture,the induced cells can all show uniform basophilic staining,and the structure of cartilage lacunae is more obvious than that of orienting induced cartilage differentiation.Meanwhile,Toluidine blue,Alcian O,Safranin O,COL?,COL1 a and COL10 were also positive.The results of differentiation in vivo showed that the experimental group had scattered colony-like growth of basophilic chondroid structure in the scaffold structure at 4weeks,8 weeks and 10 weeks after transplantation.The induced chondrocytes were evenly distributed,cartilage lacunae were obvious,and the tissue structure was similar to normal cartilage tissue.In the sponge of the experimental group,the colony-like growth of the cartilage structure was positively stained with bright red.Within the cartilage structures of sponge,AGG and Col ? were strongly positive,while OCN was weakly positive.4.At 8 weeks after implantation,HE staining showed that there were no obvious structure of bone,cartilage in defect parts in the control side,with a large number of inflammatory cells infiltration.While in the experimental side,we can see scattered structure of cartilage and bone in the defect parts.Specific immunohistochemical staining showed that some cells were indeed integrated into chondroid tissue,indicating that the implanted stem cells realized chondrogenic differentiation and integrated into the damaged cartilage.Conclusion This study confirmed that it is feasible that inducing ESCs to differentiate into a cartilage stem cell through the ectodermal pathway.The induced cartilage stem cell has self-renewal ability,which is different from the common mesodermal stem cells in the past.The induced cartilage stem cell also has the potential of chondrogenic,osteogenic and lipogenic differentiation,and can spontaneously generate chondrogenic differentiation to repair damaged articular cartilage.