Mechanistic Study Comparing Cartilage Formation From Human Mesenchymal Stromal Cells And IPSC-derived Multipotent Progenitor Cells

Objective:Due to the pluripotent differentiation potential of induced stem cells(iPSCs),differentiating iPSCs to mesenchymal stromal cells(MSCs)-like multipotent progenitor cells.Then,optimizing the chondrogenic differentiation protocol of induced pluripotent stem cell-derived pluripotent progenitor cells(iPSC-MPCs,iMPCs)by three-dimensional cell culture.To identify a better cell source for hyaline cartilage regeneration,in-vitro culture and in-vivo implantation experiments were utilized to compare the cartilage-like tissue formed by iMPCs and Human bone marrow-derived mesenchymal stromal cells(h BMSCs).Finally,to investigate the underlying mechanism of the different roles played by transforming growth factorβ3(TGFβ3)in the chondrogenic differentiation of iMPCs and h BMSCs.Methods:iPSCs were expanded on Vitronectin-coated,non-tissue culture,6-well plates with m Te SR-1 medium.ACF Mesenchymal Induction Medium was utilized to directly differentiate iPSCs into iMPCs.Then,the iMPCs were expanded in generic tissue culture flasks in growth medium(GM)[DMEM/F-12 supplemented with 10%FBS and 1%antibiotic-antimycotic].The cells were then subcultured to higher passages in cell culture flasks.Cells at passage 3,5 and 7 were used for the following experiments.h BMSCs were isolated from femoral heads of patients undergoing total hip arthroplasty and used as the positive control to characterize the features of mesenchymal stromal cells.Morphological observation,colony-forming assay,flow cytometry,and histological staining for tri-lineage differentiation ability(Alizarin red staining,Alcian blue staining,and oil red staining)were carried out to verify the iMPCs’MSC-like features.Then,cells with different passage numbers in two-dimensional cultures were collected,and 0.3×10~6iMPCs in 200 m L basic chondrogenic differentiation medium(BM)(without BMPs or TGFβ)were used to make pellets.The pellets were cultured for 7,14,and21 days for various analysis.The genes and proteins related to hyaline and hypertrophic cartilage formation were analyzed by real-time quantitative polymerase chain reaction(RT-q PCR),histological staining and immunohistochemistry(IHC).Three-dimensional cell pellets of the two kinds of cells were cultured in different chondrogenic differentiation media.After 21 days,RNA sequencing,RT-q PCR,determination of sulfated glycosaminoglycan content(GAG assay),histology,immunohistochemical staining,and in-vivo experiments,including subcutaneous implantation in mice(2 weeks and 3 weeks)and cartilage repair experiment in a rat osteochondral defect model(8 weeks),were utilized to figure out the optimal cell source and differentiation protocol to form hyaline cartilage-like cartilage tissue.Lastly,Samples of 3D cultured cell pellets were harvested at different time points during the chondrogenic induction process for Western blot(WB)experiments to analyze the activation of Smad pathway signaling molecules.The expression of downstream genes in the Smad pathway was further verified by RT-q PCR.The small molecule inhibitor LDN193189 was used to clarify the decisive role of Smad1/5 in the Smad pathway in promoting chondrogenic differentiation by WB,RT-q PCR and histological staining.Two different types of cells were extracted,and the distribution and expression of TGFβ-related receptors were analyzed by Single-cell Sequencing and WB to further explore the potential mechanism underlying the differential effects of TGFβon the two cell sources.The expression of differential receptors was regulated by lenti-viral system to understand their roles in connecting the Smad pathway and cartilage differentiation.Results:Generation and characterization of induced pluripotent stem cell(iPSC)-derived multipotent progenitor cells(iMPCs)In terms of cell morphology,the round iPSCs changed to polygonal iMPCs(P0),and further evolved to spindle-like iMPCs(P3-7).The iMPCs possessed a similar surface marker profile to MSCs(including CD73+,CD90+,CD105+,CD45-,CD34-,CD31-).At the same time,the results of alizarin red,alcian blue and oil red staining showed that iMPCs had the ability of three-lineage differentiation,and the staining results of different passages of iMPCs and the results of cell colony experiments showed that iMPCs had passage senescence phenotype.Optimizing the chondrogenic differentiation protocol of human induced pluripotent stem cell-derived pluripotent progenitor cells(iMPCs)to generate hyaline cartilage-like cartilage tissueIn the results of optimizing the three-dimensional chondrogenic induction protocol for iMPCs,chondrogenic differentiation medium containing a combination of bone morphogenetic protein-6(BMP)-6 and TGFβ3 outperformed other culture medium formulations led to highest cartilage-related expression of genes(SOX9,ACAN,COL2)and the lowest hypertrophy-related genes(RUNX2,ALP,COL10,MMP13).Histological staining(safranin O/fast green staining)showed that BMP4group,BMP4+TGFβ3 group and BMP6+TGFβ3 group all had better results of cartilage matrix formation.The results of immunohistochemistry(COL2 staining)showed that the BMP6+TGFβ3group had the highest COL2 deposition and the lowest expression of hypertrophy-related proteins(COL10,IHH).Comparison of cartilage tissues derived from BMSCs and iMPCsIn the experiment comparing the chondrogenic differentiation of iMPCs and BMSCs,the results showed that after differentiation induction by the traditional chondrogenic protocol(TGF group),BMSCs showed positive safranin O/fast green staining results,while iMPCs showed negative results.And by comparing the gene expression of SOX9,ACAN,COL2 and the IHC results of COL2,MSCs in the TGF group have higher gene expression and COL2 deposition.Under the optimized chondrogenic induction protocol(T+B),iMPCs exhibited the highest expression of cartilage-related genes,more homogeneous safranin O/fast green staining,and the highest COL2 deposition.The results of subcutaneous implantation in mice showed that the cartilage-like tissue formed by iMPCs had better safranin O/fast green staining results and COL2 deposition than the cartilage-like tissue formed by BMSCs after 14days and 21 days of implantation in vivo,and in the micro CT results.Also,no calcification was found,and Alizarin red staining and COL10staining were negative in iMPCs-derived cartilage tissue.In the rat knee articular cartilage defect repair experiment,the cartilage-like tissue formed by iMPCs had higher COL2 deposition and lower COL1 and COL10 expression than the cartilage-like tissue formed by BMSCs after 8weeks in vivo.Mechanistic study on the roles of TGFβin chondrogenic differentiation of h BMSCs and iMPCsWestern Blot(WB)experiments showed TGFβ3 cannot activate the expression of Smad1/5 in iMPCs on the 7th and 14th day of chondrogenic differentiation,but it could induce a higher level of Smad2/3 protein.The expression of Smad1/5 and Smad2/3 in BMSCs can be simultaneously activated by TGFβ3.In addition,BMP6 could successfully activate Smad1/5 proteins in iMPCs and BMSCs.WB results showed that LDN193189(LDN)effectively inhibited the expression of Smad1/5,and safranin O/fast green staining showed that iMPCs in the LDN group did not produce cartilage matrix under the optimized induction protocol.Finally,WB results showed that iMPCs had higher ALK5 expression and lower ALK1 expression than BMSCs,and single-cell sequencing results showed most of iMPCs showed high expression of ALK5,not ALK1.WB results showed that the expression of ALK1 was significantly increased in the ALK1 overexpression group(Ov-ALK1),the phosphorylation level of Smad1/5 was increased,and the expression of SOX9 was increased.RT-q PCR results showed that the expressions of COL2 and ACAN were also significantly increased in the Ov-ALK1group.Conclusions:iPSCs can be successfully differentiated into multipotent progenitor cells with MSCcharacteristics by direct differentiation.iMPCs showed replicative senescence and requires different chondrogenic and adipogenic schemes than BMSCs.In the experiment of optimizing iMPCs chondrogenic induction medium,it was concluded that the chondrogenic induction medium containing TGFβ3+BMP6 combination had the best chondrogenic effect.Then,by comparing the cartilage phenotypes of iMPCs and BMSCs under different chondrogenic protocols in vitro and in vivo,it was concluded that iMPCs could form better hyaline cartilage-like tissue than BMSCs.Finally,by comparing the activation of downstream Smad molecules by TGFβin cells of different origins,it was concluded that the activation failure of Smad1/5 was the decisive factor that caused iMPCs to fail to differentiate successfully and acquire cartilage phenotypes.In addition,the low expression of ALK1 may be one of the reasons for the failure of Smad1/5 activation.