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bioRxiv · 10.1101/108571

Novel methods of isolation and amplification of progenitor cells applied to avian primordial germ cells

Lopez Diaz, M.
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Abstract

BackgroundThe progenitor cells in adult tissues are scarce and have a great regenerative potential. In this study novel methods were used to improve the isolation and culture of the chicken primordial germ cells (PGCs) from stage X and HH 8-9 embryos. The cellular size and external glycoprotein envelope were the two criteria studied and used.\n\nResultsPGCs were segregated with high efficiency and purity, from stage X and HH 8-9 gross cell suspensions through cell strainers with 10 m of pore size. In embryos in toto, WGA Alexa 594 (affinity for N-acetylglucosamine) and Con A Alexa 488 (binding D-mannosyl) were used to characterize external polysaccharides of the PGCs. The PGCs in stage X embryos (zone pellucida), have predominately Nacetylglucosamine and later on, in HH 8-9 embryos (cephalic zone), -D mannosyl residues, in a specific manner. In coated plates with the appropriate lectin and in alkaline conditions, isolated cells from stage X and HH 8-9 embryos formed numerous clumped PGC-LCs with spherical shape \"germspheres\". In all isolates from single embryo, immunohistochemistry confirmed that they were PGCs and revealed that the \"germspheres\" were formed by hundreds of positive cells to VASA and SSEA-1. N-acethyl D+glucosamine supplementation to the culture media greatly enhances the amplification of isolated PGC-LCs.\n\nConclusionsThese gentle and quick strategies with high yields of PGCs can be potentially useful for many progenitor cells in Regenerative Medicine.

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Lopez Diaz, M.. 2017-02-15. Novel methods of isolation and amplification of progenitor cells applied to avian primordial germ cells. https://doi.org/10.1101/108571

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