Project description:Gowth plate chondrocytes include heterogenous cell population and cellular diversity of these chondrocytes remains to be elucidated. We used single cell RNA sequencing to uncover the diverssity of growth plate chondrocytes and identify noel marker genes selectively expressed in pre-hypertrophic chondrocytes.
Project description:Growth plate chondrocytes were isolated from the distal metacarpus of young dairy cattle (all under 10 mo of age), the chondrocytes were released from the extracellular matrix by digestion with Collagenase P for 4 hours, and the various zones of the growth plate were separated by density centrifugation. The least-dense Hypertrophic Zone (HZ) cells were compared to the most-dense Reserve Zone (RZ) cells. 6 pairs of HZ vs RZ were compared by microarray.
Project description:Growth plate chondrocytes were isolated from the distal metacarpus of young dairy cattle (all under 10 mo of age), the chondrocytes were released from the extracellular matrix by digestion with Collagenase P for 4 hours, and the various zones of the growth plate were separated by density centrifugation. The least-dense Hypertrophic Zone (HZ) cells were compared to the most-dense Reserve Zone (RZ) cells. 6 pairs of HZ vs RZ were compared by microarray. Experiment Overall Design: Growth plate chondrocytes were isolated from the distal metacarpus of young dairy cattle (all under 10 mo of age), the chondrocytes were released from the extracellular matrix by digestion with Collagenase P for 4 hours, and the various zones of the growth plate were separated by density centrifugation. The least-dense Hypertrophic Zone (HZ) cells were compared to the most-dense Reserve Zone (RZ) cells. Six independent sample pairs of HZ vs RZ were compared by microarray.
Project description:Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate.
Project description:Human growth plate chondrocytes were isolated from the distal femoral growth plate from 3 different adolescents undergoing epiphysiodesis. Cell were isolated and grown in monolayers, long term, in 20% serum. Cells were replated and cultured 48 h in 0.5% serum.
Project description:We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFB3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.