A Novel, Efficient Method to Isolate Chicken Primordial Germ Cells from Embryonic Blood Using Cell Culture Inserts

Abstract

Primordial germ cells (PGCs) play a crucial role in preserving poultry genetic resources and conducting transgenic research. A system for the rapid isolation of PGCs from single chicken embryonic blood was established in this paper. We found that PGCs can migrate to the lower layer of chicken embryonic fibroblasts (CEFs) through pores smaller than their diameter, while blood cells cannot, when co-cultured with CEFs of passages two to three. Based on the characteristics of PGCs, we developed a new PGC isolation method (cell culture insert/CEF adhesion method) that utilizes a 3 μm cell culture insert and CEFs of passages two to three. Using this method, approximately 700 PGCs can be isolated from the blood of a single chicken embryo at Hamburger and Hamilton (H&H) stage 17 of development. The separation rate achieved was 87.5%, with a separation purity of 95%. The separation rate of this method was 41.4% higher than the common Percoll density gradient centrifugation method and 33.6% higher than lysis with ACK buffer. PGCs isolated from embryonic blood could proliferate 37-fold within 2 weeks when cultured in a feeder-free culture system. They also continued to express the SSEA-1 and DAZL proteins and retained the ability to migrate in vivo. Overall, PGCs separated using cell culture inserts/CEF adhesion method retain their stem cell characteristics and migration ability. PGCs also exhibit good proliferation efficiency, making them suitable for subsequent transgenic experiments or genetic resource preservation.

Keywords: cell culture insert; feeder-free culture system; primordial germ cells.

Figure 1

Chicken PGCs pass through 1 µm cell culture inserts. Chicken embryonic blood was placed in a 1 µm cell culture insert and co-cultured with CEFs for 3 days. (A) Some of the PGCs migrated to the lower layer of CEFs through the cell culture insert; (B) PGCs, blood cells and other impurities were retained in the insert. The orange arrow indicates PGCs. (C) Chicken PGCs labeled with the fluorescent dye PKH67 (green) were placed in a 1 µm cell culture insert and co-cultured with CEFs for 3 days.

Figure 2

Effect of isolating PGCs using cell culture inserts with different pore sizes. (A) Place PGCs into three different pore size cell culture inserts and co-culture with CEFs. PGCs adhered to the lower layer of CEFs. The scale bar in the large image is 100 µm, while the scale bar in the small inset image is 20 µm. (B) Separation rate of PGCs using cell culture inserts. (C) Purity of PGCs isolated using cell culture inserts. The orange arrow shows PGCs.

Figure 3

Characteristics of PGCs isolated using the cell culture insert/CEF adhesion method. (A) PGCs isolated using the cell culture insert/CEF adhesion method and DF1 cells were collected, and the expression of cDAZL, Nanog, cPou5fl and Sox-2 was detected using RT-PCR. (B) The expression of cDAZL and SSEA-1 in PGCs was labeled using immunofluorescence. Scale bar: 10 µm. (C) PGCs labeled with eGFP were injected into the egg at HH stage 13, and gonadal tissue was separated on the 6th day of incubation and compressed for observation under a fluorescence microscope. Scale bar: 100 µm.

Figure 4

Separation of PGCs using three different separation methods. The distribution of PGCs (orange arrows) and blood cells (black arrows) in chicken embryonic blood (A). PGCs were separated using Percoll density gradient centrifugation (B), ACK lysis (C) and the cell culture insert/CEF adhesion method (D). PGCs isolated using the cell culture insert/CEF adhesion method had pseudopodia-like cytoplasmic protrusions on the surface. Scale bar: 100 µm (E). Magnification of the pseudopod-like cells within (E). Scale bar: 10 µm (F).

Figure 5

The proliferation of PGCs isolated using different methods. (A) The proliferation efficiency of PGCs obtained using three different separation methods. Transfer the PGCs obtained through different separation methods to a 24 well plate containing 500 µL of culture medium, continue to culture for 15 days, and then observe the condition of PGCs. Percoll density gradient centrifugation (B), ACK lysis (C), and cell culture insert/CEF adhesion method (D). PGCs obtained using the cell culture insertion/CEF adhesion method were cultured until day 30 (E). Scale bar: 100 µm.

Figure 6

Schematic diagram of the cell culture insert/CEF adhesion method.