Журнал исследований и терапии стволовых клеток

Абстрактный

Culture of Pig Induced Pluripotent Stem Cells without Direct Feeder Contact in Serum Free Media

Yang JY, Liu Y, Yu P, Lu Y, Hutcheson JM, Lau VW, Li X, Dove CR, Stice SL and West FD

Background: Reprogramming pig somatic cells into induced pluripotent stem cells (iPSCs) have promising applications in basic biology, disease model development and xenotransplantation. In the mouse, embryonic stem cell (ESC) technology has revolutionized the field enabling gene targeting, complex screening strategies and the creation of animals that show unique characteristics of interest. Recent breakthroughs utilizing induced pluripotent stem cell technology in the pig have made it possible to produce pig pluripotent stem cells that resemble germline chimeric competent mouse ESCs. However, an optimal culture system for piPSC expansion has yet to be developed. Most reports have maintained piPSCs in undefined systems that use xenoproducts and feeder layers, which are potential sources of contamination. Methods: In this study, new lines of pig iPSCs (piPSC) were generated from pig fibroblast cells by overexpressing six reprogramming genes: POU5F1, SOX2, NANOG, LIN28, KLF4 and C-MYC. These new lines were tested for their ability to be maintained on a Matrigel substrate in the established mouse 2i+LIF system, the human mTeSR1 system and variations of a feeder conditioned media system. Analysis and identification of piPSCs were performed using immunocytochemistry, flow cytometry and by examining embryoid body formation and differentiation. Results: The newly generated piPSCs showed morphological features, immunoreactivity and reactivation of endogenous pluripotency networks consistent with iPSCs. Similar to cells cultured on feeders, piPSCs maintained under all 7 feeder-free conditions expressed POU5F1 and NANOG, SSEA-1, SSEA-4 and TRA1-81. However, flow cytometry demonstrated that piPSCs cultured in feeder conditioned media with KnockOut Serum Replacement and basic fibroblast growth factor (FGF2) showed significantly higher levels of SSEA1 and SSEA4 expression than cells cultured in a 2i+LIF or mTeSR1 system. Conclusion: These findings demonstrate that piPSCs can be maintained in defined systems without serum and direct feeder contact, increasing their potential use in both agricultural and biomedical fields.