Isolation of Adult Gonadal Stem Cells from Coturnix japonica and Characterization of Embryonic Gonadal Stem Cells in Gallus gallus for Xenotransfer.

Abstract

Interspecific (xeno) transfer of embryonic primordial germ cells (PGCs) from quail (Coturnix japonica) to chicken (Gallus gallus) has produced germ cell chimeras that generate viable donor-derived germ cells. The practical application of this technique for the preservation of endangered species requires the use of gonadal stem cells (GSCs) recovered from adult birds at necropsy. Using the quail to chicken model, we examined the feasibility and efficacy of isolating adult GSCs for xenotransfer into host embryos. In this study, GSCs from adult quail testes were quantified by flow cytometry using antibodies against GSC-specific antigens: stage-specific embryonic antigen-1 (SSEA1; 88%), stage-specific embryonic antigen-4 (SSEA4; <1%), CD9; <1%, epithelial membrane antigen-1 (EMA1; <1%), integrin alpha-6 (alpha6; <1%), and integrin beta-1 (beta1; 3%). The high percentage of SSEA1 positive cells indicates that this antigen is too widely expressed to be a differential stem cell marker for adult testicular cells. The percentage of beta1 positive cells suggests that this marker is more suitable for stem cell identification in this model. GSC populations were then enriched by magnetic activated cell sorting (MACS). Following MACS, the proportion of testicular cells expressing stem cell antigens increased: SSEA1, 1.1 fold; SSEA4, 40.3; EMA1, 70.1; CD9, 209.7: alpha6, 114.6; beta1, 23.4. Finally, we quantified the expression of stem cell markers in the embryonic gonad of 11-day incubated chicken embryos to determine the characteristics of control, non-chimeric host tissue for comparison to post-xenotransfer chimeric gonads. In the male embryonic gonad, we found a greater number of SSEA1 and β1 antigen expressing cells and low expression levels of the other antigens tested (SSEA4, CD9, EMA1, alpha6). We found a similar pattern in the female embryonic gonad, with a SSEA1 and β1 expressed at higher levels than the other antigens tested. Ideally, we aim to transplant a GSC enriched population coincident with endogenous PGC migration that will best integrate into the host gonad and outnumber, thus supplant, the endogenous PGCs. The quail to chicken germ cell chimera using adult gonadal tissue is a model for the development of xenotransfer of endangered avian GSCs. With this technique, GSC rescued from genetically important or endangered species may survive to produce functional gametes in the gonads of non-endangered hosts. (poster)