Pluripotency Evaluation of Newly Generated iPSC Lines using Chorioallantoic Membrane Assay

Objectives: Induced pluripotent stem cells (iPSCs) represent a promising cell source to obtain desired autologous cell types, such as cardiomyocytes. Patient-specific iPSCs have the unique potential to differentiate into tissues of all three germ layers. To evaluate the pluripotency of newly generated iPSC lines in vivo, these cells are generally transplanted into immunodeficient mice. In this study, a chorioallantoic membrane (CAM)-based assay was established to analyze the trilineage differentiation capacity of iPSCs by spontaneous teratoma formation as an alternative to rodent models, which have typically been used for this purpose in the past.

Methods: On the third day of the incubation of fertilized eggs, a window was opened in the eggshell to check viability and to remove albumen. The window was closed using a tape. On day 7, different numbers of iPSCs were seeded onto the CAM and the eggs were incubated for further 9 days. Fertilized chicken eggs were incubated for a total of 16 days. The formed teratomas were harvested and analyzed by immunohistochemistry.

Results: After 16 days, histological evaluation confirmed successful trilineage differentiation of applied iPSCs by formation of meso-, endo-, and ectodermal tissue. Teratoma formation was detected in approximately 70% of the eggs. The size of the teratomas was measured and a correlation between the number of seeded iPSCs and the teratoma size was detected.

Conclusion: The CAM of fertilized chicken eggs, which is immunodeficient, highly vascularized, and non-innervated, provides a well-suited extra-embryonic test environment for the engraftment of iPSCs. Overall, the CAM assay provides a simple, cost-effective, and reproducible alternative to the conventional in vivo rodent experiments, which are time-consuming, costly and face ethical issues.

No conflict of interest has been declared by the author(s).