Comparative analysis of chondrogenic and epigenetic differences in fore or hind limb bud-derived chicken micromass cell cultures

Abstract

Purpose: Chicken embryos are often-used experimental models in developmental biology researches. Micromass cell culture system derived from mesenchymal cells of the embryo limb buds can serve as a reliable in vitro model for cartilage differentiation. In order to harvest the required high number of cells, these cell cultures are established from a mixture of fore and hind limb buds. However, the epigenetic regulation of the two limb bud types may vary. Our objective was to investigate the different chondrogenic and epigenetic regulation of fore or hind limb bud-derived high density cultures. Methods: Chicken embryos of Hamburger-Hamilton 22-24 stages were used to establish primary micromass cell cultures of chondrifying mesenchymal cells. Fore or hind limb buds were collected separately. Cell cultures established from a mixture of fore and hind limb buds served as control groups. Cultures were harvested on the 3rd and 6th day of culturing (according to the major steps of in vitro chondrogenesis). Changes in the quantity of cartilage-specific extracellular matrix were examined by dimethyl-methylene blue and toluidine blue metachromatic staining methods. Real-time quantitative PCR reactions were carried out to study the alterations of cartilage-specific (Sox9: SRY-box 9, Col2a1: collagen type II alpha 1 chain, Col1a1: collagen type I alpha 1 chain, Acan: aggrecan, Has2: hyaluronan synthase 2) and epigenetic marker gene expression levels (Dnmt3a: DNA methyltransferase 3 alpha, Tet1: tet methylcytosine dioxygenase 1, Ogt: O-linked N-acetylglucosamine (GlcNAc) transferase, Hdac3: histone deacetylase 3, Hdac4: histone deacetylase 4). Protein expressions of specific chondrogenic and epigenetic markers were examined by Western Blot technique. Measurements of mitochondrial activity with MTT-assay were also carried out. Results: Fore limb bud-derived micromass cell cultures showed a significantly pronounced and more advanced cartilage formation in comparison with hind limb bud- as well as mixed limb bud-derived micromass cell cultures. This phenomenon was verified not only with specific histological staining methods, but also with modern molecular biological techniques on mRNA and protein levels. Interestingly, our results indicated that the epigenetic regulatory pathway was altered in a negative way in the two examined cell culture types compared to the control cell culture. Results of the MTT-assay showed that the mitochondrial activity of hind limb bud-derived high density cultures was significantly lower compared to the other two experimental groups. Conclusions: Chicken limb bud-derived micromass cell culture system is a popular experimental model for chondrogenic researches. However, no modern scientific results can be found on specific molecular characteristics of high density cultures established from fore or hind limb buds separately. Our research aims to optimise this cell culture system for later scientific investigations, with a special regard to epigenetic analysis. Our experiments were supported by the EFOP-3.6.1-16-2016-00022, Debrecen Venture Catapult program.