Density gradients for the isolation of germ cells from embryoid bodies.
In previous reports, the isolation of embryonic stem cell (ESC)-derived germ cells utilized fluorescent protein knock-in cell lines that could be sorted by flow cytometry. The present study aimed to isolate putative germ stem cells from embryoid bodies (EB) using Percoll and Nycodenz density gradients. The optimal ESC concentration to establish EB was identified as 15,000 cells per 30 mul drop and the optimal culture time to obtain the highest number of germ cells within EB was identified as 120 h, with over 25% of cells confirmed as germ cells for the specific cell line used. Germ cells were isolated from 120-hourold EB by Percoll and Nycodenz density gradients, while isolation of primordial germ cells from genital ridges of embryonic day 12.5 fetuses was used as a control. Putative germ cells were isolated from EB at proportions of 80.3 +/- 3.0% (SEM) and 75.8 +/- 0.9% for Percoll and Nycodenz respectively. Primordial germ cells were isolated from genital ridges at rates of 89.7 +/- 2.7% and 89.5 +/- 0.9% respectively. Although the purity of the isolated germ cells was similar between the two gradients, more germ cells with higher viability were obtained with the Percoll gradient.
Saiti
Deshira
D
Monash Immunology and Stem Cell Laboratories, Level 3, Strip 1, Building 75, Monash University, Wellington Road, Clayton, Australia 3800.
Lacham-Kaplan
Orly
O
D002469:Cell Separation; D004622:Embryo, Mammalian / Q000166:cytology*; D005854:Germ Cells / Q000166:cytology*; D006801:Humans