Studies on avian germline competent stem cells and practical application for bioreactor system

Abstract

Avian species have become valuable models for biotechnological purposes including production of functional proteins, disease resistance model, and industrial traits as well as developmental studies of vertebrates. To produce genome modified aves, germline-competent stem cells will be required to achieve robust, expedite, and precise genetic modification. Therefore, here, we demonstrated the derivation and manipulation of germline-competent stem cells for various purposes.

Firstly, we attempted the derivation of induced pluripotent stem cells-like cells (iPSLCs) from avian feather follicle cells (FFCs). The induced pluripotent stem cell (iPSC) as a novel class of pluripotent stem cells contribute to all three germ layers including germ line. Therefore, we attempted the derivation of induced pluripotent stem cells-like cells (iPSLCs) from avian feather follicle cells (FFCs) as a novel approach to stem cell production. The FFC-iPSLCs can proliferate with the pluripotent property and differentiate into all three germ layers in vitro. This experimental strategy should be useful for conservation and restoration of endangered or high-value avian species without sacrificing embryos.

The primordial germ cell (PGC) is the most well established germline-competent stem cells in avian species especially in chicken. In second part, we generated the production of anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B lymphoma and B lymphoblast cells. This experiment well represent as an efficient system for producing anti-cancer therapeutic antibodies from transgenic chicken with consistent expression and highly enhanced Fc effector functions. Meanwhile, to produce efficient genome modification in quail PGCs, we optimize the transfection and drug-selection system into quail genome of primary PGCs. Applying the CRISPR/Cas9 system on quail genome of primary PGCs using electroporation and puromycin drug-selection system, we confirmed that the efficient genome editing ratio compared to non-selected cells.

Apart from the PGCs, the spermatogonial stem cells (SSCs) also regarded a reliable way to produce genome-edited animals. In terms of adopting genome modification techniques on quail genome, we produced the germline chimeric quail using testicular cells (TCs) and SSCs transplanted into busulfan-treated recipient testis. The transplantation of male germ cells including spermatogonia and SSCs is an efficient method to study spermatogenesis, control male fertility and transgenesis. Compared with the embryo-mediated method, this strategy is simple and leads to rapid generation of quail germline chimeras. This will lead to production of transgenic models using adult germ cells and, through the production of germline chimeras, also help in efforts to conserve avian species.

In conclusion, these experimental strategies for derivation of avian stem cells, manipulation of them and its application using genome editing tools can be used to efficient way for produce birds of economic traits or non-sacrificible avian conservation system.