ABSTRACT
Background The 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.
Results PGCs were segregated with high efficiency and purity, from stage X and HH 8-9 gross cell suspensions through cell strainers with 10 μm 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), α-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.
Conclusions These gentle and quick strategies with high yields of PGCs can be potentially useful for many progenitor cells in Regenerative Medicine.
List of Abbreviations
- PGCs
- Primordial germ cells
- ESCs
- Embryonic stem cells
- iPSC
- induced pluripotent stem cells
- IMACS
- Magnetic-Activated Cell Sorting
- FACS
- Fluorescent-Activated Cell Sorting
- SSEA1
- Stage-specific embryonic antigen-1
- EMA
- Primordial germ cell marker (mouse)
- WGA
- Wheat germ agglutinin
- Con A
- Concanavalin A
- PGC-LC
- Primordial germ cell like cell
- PFA
- Paraformaldehyde
- PBS
- Phosphate buffer saline
- DPBS
- Dulbecco phosphate buffer saline
- BRL
- Buffalo Rat Liver
- FBS
- Fetal bovine serum
- DMEM-KO
- Dulbecco modified eagle medium knockout
- rhFGFb
- recombinant human FGF basic
- rmSCF
- recombinant murine SCF
- NPC
- Neural progenitor cell
- GlcNAc
- N-acethyl D+glucosamine
- GnTV
- N-acetylglucosaminyltransferase