An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs

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Josefin Weber
Marbod Weber
Heidrun Steinle
Christian Schlensak
Hans-Peter Wendel
Meltem Avci-Adali

Abstract

The generation of autologous human induced pluripotent stem cells (hiPSCs) from patient’s somatic cells and the subsequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice and teratoma formation is analyzed after 4 to 6 weeks to assess the pluripotency of these cells. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. Therefore, 0.5, 1, 2, 4 x 106 hiPSCs generated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs after the application of 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was further confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. Furthermore, this simple, fast, inexpensive, and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

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How to Cite
Weber, J., Weber, M., Steinle, H., Schlensak, C., Wendel, H.-P. and Avci-Adali, M. (2021) “An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs”, ALTEX - Alternatives to animal experimentation. doi: 10.14573/altex.2005221.
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