Real-time quantitative PCR analysis of chondrogenic markers in iMPC with different treatments
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE140330 | GEO | 2019/11/14
REPOSITORIES: GEO
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Project description:Multipotent progenitor cells (iMPCs) created from induced pluripotent stem cells (iPSCs) is a promising cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) were shown to significantly enhance transforming growth factor-b (TGFb)-induced iMPC chondrogenesis. In contrast, TGFb alone is sufficient to induce robust chondrogenesis of human primary mesenchymal stromal cells (MSCs). Currently, the mechanism underlying this difference between iMPCs and MSCs has not been fully understood. In this study, we first generated iMPCs from human iPSCs and examined their differentiation capacity at different passages. We then optimized the conditions for chondrogenesis and determined that medium supplemented with TGFb and BMP6 led to robust cartilage formation from iMPCs with minimal hypertrophy. Moreover, the cartilage generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and led to a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFb alone induced phosphorylation of Smad2/3 in iMPCs but not Smad1/5, which led to poor chondrogenesis. In contrast, TGFb resulted in the phosphorylation of both Smad2/3 and Smad1/5 and robust chondrogenesis in human MSCs. We further discovered that the remarkably low level of Activin receptor-like kinase 1 (ACVRL1/ALK1) in iMPCs, when compared to MSCs, partially accounts for this difference. For instance, overexpression of ALK1 in iMPCs activated both Smad1/5 and Smad2/3 upon TGFb only treatment and resulted in a high level of chondrogenesis, which was not observed in control iMPCs. In summary, this study describes a robust method to generate chondrocytes with low hypertrophy for hyaline cartilage repair and elucidates the difference between MSCs and iMPCs in response to TGFb.
2022-12-20 | GSE197170 | GEO
Project description:Multipotent progenitor cells (iMPCs) created from induced pluripotent stem cells (iPSCs) is a promising cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) were shown to significantly enhance transforming growth factor-b (TGFb)-induced iMPC chondrogenesis. In contrast, TGFb alone is sufficient to induce robust chondrogenesis of human primary mesenchymal stromal cells (MSCs). Currently, the mechanism underlying this difference between iMPCs and MSCs has not been fully understood. In this study, we first generated iMPCs from human iPSCs and examined their differentiation capacity at different passages. We then optimized the conditions for chondrogenesis and determined that medium supplemented with TGFb and BMP6 led to robust cartilage formation from iMPCs with minimal hypertrophy. Moreover, the cartilage generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and led to a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFb alone induced phosphorylation of Smad2/3 in iMPCs but not Smad1/5, which led to poor chondrogenesis. In contrast, TGFb resulted in the phosphorylation of both Smad2/3 and Smad1/5 and robust chondrogenesis in human MSCs. We further discovered that the remarkably low level of Activin receptor-like kinase 1 (ACVRL1/ALK1) in iMPCs, when compared to MSCs, partially accounts for this difference. For instance, overexpression of ALK1 in iMPCs activated both Smad1/5 and Smad2/3 upon TGFb only treatment and resulted in a high level of chondrogenesis, which was not observed in control iMPCs. In summary, this study describes a robust method to generate chondrocytes with low hypertrophy for hyaline cartilage repair and elucidates the difference between MSCs and iMPCs in response to TGFb.
2022-12-20 | GSE197169 | GEO
Project description:A combination therapy of electromagnetic fields (EMF) and simulated microgravity (SMG) has not been examined in regenerative medicine of cartilage. In the present study, a bioreactor system using extremely low-frequency EMF and SMG was applied during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). It was hypothesized that a beneficial effect of EMF regarding chondrogenesis (COL2A) could be combined with an avoiding effect of SMG regarding hypertrophy (COLXA1) of cartilage. Pellet cultures of hMSCs formed cartilaginous tissue under the addition of growth factors (FGF; TGF-β3). Pure SMG reduced COLXA1 expression but also COL2A expression of hMSCs. Pure EMF showed no gene expression changes of hMSCs during chondrogenic differentiation. Combining EMF/SMG resulted in a re-increase of COL2A but did not reach control levels. The COL2A to COLXA1 ratio of combined EMF/SMG was not significantly different from control levels. The combination therapy of EMF/SMG did not significantly improve the chondrogenic potential of hMSCs.
2015-12-31 | GSE57298 | GEO
Project description:Osteoarthritis (OA) is the most prevalent chronic joint disease that affects a majority of the elderly. Chondrogenic progenitor cells (CPCs) reside in late stage OA cartilage tissue, producing a fibrocartilagenous extra-cellular matrix and can be manipulated in-vitro, to deposit proteins of healthy articular cartilage. CPCs are under control of SOX9 and RUNX2 and in order to enhance their chondrogenic potential, we found that RUNX2 plays a pivotal role in chondrogenesis. In another approach, CPCs carrying a knockout of RAB5C, a protein involved in endosomal trafficking, demonstrated elevated expression of various chondrogenic markers including the SOX trio and displayed an increased COL2 deposition, whereas no changes of COL1 deposition was observed. We report RAB5C as an attractive target for future therapeutic approaches to increase the COL2 content in the diseased joint.
2021-09-09 | PXD021785 | Pride
Project description:We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants Comparison of gene expression patterns in 4 BM-MSC donors, before and after chondrogenic differentiation in alginate (BM-MSCs and DIF)
2012-08-17 | E-GEOD-40175 | biostudies-arrayexpress
Project description:We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants
2012-08-17 | GSE40175 | GEO
Project description:A combination therapy of electromagnetic fields (EMF) and simulated microgravity (SMG) has not been examined in regenerative medicine of cartilage. In the present study, a bioreactor system using extremely low-frequency EMF and SMG was applied during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). It was hypothesized that a beneficial effect of EMF regarding chondrogenesis (COL2A) could be combined with an avoiding effect of SMG regarding hypertrophy (COLXA1) of cartilage. Pellet cultures of hMSCs formed cartilaginous tissue under the addition of growth factors (FGF; TGF-β3). Pure SMG reduced COLXA1 expression but also COL2A expression of hMSCs. Pure EMF showed no gene expression changes of hMSCs during chondrogenic differentiation. Combining EMF/SMG resulted in a re-increase of COL2A but did not reach control levels. The COL2A to COLXA1 ratio of combined EMF/SMG was not significantly different from control levels. The combination therapy of EMF/SMG did not significantly improve the chondrogenic potential of hMSCs. chondrogenic differentiation, electromagnetic stimulation-control, 1 timepoint with/without stimulation
2015-12-31 | E-GEOD-57298 | biostudies-arrayexpress
Project description:Osteoarthritis (OA) is a degenerative joint disease that involves destruction of articular cartilage and eventually leads to disability. Mesenchymal stem cells (MSCs) reside in healthy and diseased cartilage, and through their chondrogenic potential may provide a strategy for cartilage repair. To this end, we performed an image-based, high throughput screen and identified the small molecule, kartogenin, that promotes selective MSC differentiation into chondrocytes (EC50=100nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A and induces chondrogenesis by regulating the CBFbeta-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to an effective stem-cell based therapy for osteoarthritis. one compound, three doses, two time points, a vehicle control
2012-05-01 | E-GEOD-35546 | biostudies-arrayexpress
Project description:Long non-coding RNAs profiling of human mesenchymal stem cells comparing undifferentiated HMSCs with differentiated HMSCs during chondrogenesis. The chondrogenic differentiation of HMSCs was induced by chondrogenic medium. The chondrogenic marker genes (Col2a1, Sox9 and ACAN) has been detected upregulating during this process by Q-PCR. The aim of this study is to determine key lncRNAs regulating the chondrogenic differentiation process.
2020-03-18 | GSE128533 | GEO
Project description:A miRNA microarray was used to identify candidate miRNAs regulated by PTHrP treatment in chondrogenic hMSC differentiation. miRNA-expression patterns after PTHrP treatment for 1 week (CM-T-Pt-L1) or 3 weeks (CM-T-Pt-3) in chondrogenic culture were compared with reference levels obtained from cell pellets treated with TGF-β only (CM-T). Under identical chondrogenesis conditions (except for PTHrP treatment), miRNA-expression patterns were relatively unchanged when compared with the TGF-β-only control, which simplified the selection of candidate miRNAs. Microarray analysis revealed that only 4 miRNAs in CM-T-Pt-L1 cells and 7 miRNAs in CM-T-Pt-3 cells were differentially expressed following PTHrP treatment. Hsa-miR-590-5p and hsa-miR-892b were commonly down-regulated or up-regulated in both PTHrP-treated groups. Interestingly, hsa-miR-892b expression was not detected in the TGF-β-only control, while hsa-miR-877*, hsa-miR-1288, and hsa-miR-1305 were up-regulated. Therefore, PTHrP treatment induced hsa-miR-892b expression during TGF-β-mediated hMSC chondrogenesis. hsa-miR-892b expression in CM-T-Pt-L1 cells was 2.43-fold higher than that in CM-T-Pt-3 cells.
2016-12-01 | GSE89188 | GEO