Unknown,Transcriptomics,Genomics,Proteomics

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Efficient generation of stable chondrocytes from human iPSC through early induction of a pre-chondrogenic population

ABSTRACT: Regeneration of human cartilage is inherently inefficient; an abundant autologous source like human induced pluripotent stem cells (hiPSC) is therefore attractive for engineering cartilage. Here, we report a defined growth factor based protocol for differentiating hiPSC into articular-like chondrocytes within two weeks with a high efficiency. The hiPSC-derived chondrocytes (hiChondrocytes) are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production and in their ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early Sox9lowCD44lowCD140low pre-chondrogenic mesodermal population during hiPSC differentiation that eventually generates a homogenous population of Sox9highCD44highCD140high hiChondrocytes. Additionally, global gene expression analyses revealed two distinct Sox9-regulated gene networks in the Sox9low and Sox9high populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generation of hiPSC-derived articular chondrocytes in terms of safety and efficiency. 10 samples were analysed (duplicate sets of 5 time points) to assess changing gene expression over the course of differentiation from iPSC to hiChondrocyte. All samples were compared relative to the undifferentiated iPSC. Adult chondrocytes (2 samples) were also included for comparison. We analyzed the changes in gene expression with differentiation; genes with a fold-change â?¥ or â?¤1.5, with a difference in intensity of >100 and within the lower 90% confidence bound were selected.

ORGANISM(S): Homo sapiens

SUBMITTER: Sarah Taylor

PROVIDER: E-GEOD-62914 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Previous studies have demonstrated relative deficiencies of repair tissue within articular lesions when compared to articular cartilage. While cells occupying the lesions might be of mesenchymal origin, they do not recapitulate differentiation to the chondrogenic phenotype of normal articular chondrocytes. Nevertheless, attributes of repair tissue appear similar to chondrocytes and their precursors at various other different states. We hypothesized that analyses of gene expression profiles for these other cell phenotypes would elucidate the identity of repair tissue cells. Total RNA was isolated from repair tissue samples, neonatal articular cartilage, primary articular chondrocytes maintained in monolayer culture and passaged weekly over 28 days, and bone marrow stromal cells expanded to 80% confluence in monolayer culture. Total RNA was linearly amplified and applied to a 9413-probe set equine-specific cDNA microarray. Four repair tissue samples were compared with a dye-swap experimental design to four samples from each of the three other cell populations for a total of twelve comparisons, or twenty-four slides. Differential expression of genes of interest was validated by real-time quantitative polymerase chain reaction. Statistical analysis revealed that a total of 934 (9.9%), 1839 (19.5%), and 940 (10.0%) probe sets were differentially expressed for the bone marrow stromal cells versus repair tissue, de-differentiated chondrocytes versus repair tissue, and neonatal articular chondrocytes versus repair tissue comparisons, respectively. Transcriptional and translational machinery gene ontological categories were over-represented in transcripts demonstrating stable expression amongst the three comparisons. Biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue comprised much of the genes with the greatest levels of differential expression amongst the three comparisons. Overall, the profiles indicated that repair cells were more chondrogenic than bone marrow stromal cells and de-differentiated chondrocytes. However, transcript levels of key biomarkers and growth factors for repair tissue cells four months post-operatively fell far short of those of neonatal articular chondrocytes destined to undergo normal cartilage maturation.

Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, AhlbÃ¤ck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).

Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, Ahlbäck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Keywords: time course, cell type comparison, tissue engineered cartilage; osteoarthritis; Hyaff-11 scaffold; human chondrocytes; gene expression profiling; regenerative medicine; differentiation potential

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Efficient generation of stable chondrocytes from human iPSC through early induction of a pre-chondrogenic population

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