Biegler, M. T.; Harter, E.; Sidhu, A. V.; Szialta, C.; Durham, G.; Tchernichovski, L.; Collier, P.; Luo, J.-D.; Wang, W.; MacIsaac, R.; Belay, K.; Carroll, T.; Keyte, A. L.; Jarvis, E. D.

Primordial germ cells (PGCs) are germline stem cells that develop into sperm or egg cells and are valuable for avian biobanking and the propagation of donor-derived offspring. However, in non-poultry birds the long-term maintenance and self-renewal of PGCs in vitro remains challenging. This limitation hinders biobanking in other avian clades, particularly in the zebra finch and other songbirds that uniquely possess a germline restricted chromosome (GRC). Here, we generated and compared short-term cultures of chicken and zebra finch PGCs from the embryonic gonads or blood, as well as established long-term cultures of chicken PGCs. Using single-cell RNA sequencing, we found that the transcriptome profile of long-term chicken gonadal cultures were exclusively PGCs, whereas the short-term chicken and zebra finch cultures represented a heterogeneous mixture of cell types. The zebra finch culture further included rapidly differentiating PGCs, as well as a germ cell type not previously identified in the embryonic songbird gonad. Although zebra finch short-term gonadal cultures did not yield robust long-term PGC cultures, short-term cultured PGCs were able to integrate into host zebra finch gonads after injection into the dorsal aorta, contribute to gametic populations in adult chimeras, and give rise to phenotypically- and genomically-validated offspring. This study provides a foundation for using short-term gonadal cultures to derive donor and transgenic offspring in songbirds and further explore the unique developmental genetics of PGCs across the avian clade.