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Abstrato

A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

Martina Johannesson, Henrik Semb, Palle Serup and Mattias Hansson

Introduction: Embryonic stem cells (ESCs) represent a prospective cell source for treating degenerative diseases such as diabetes. Several studies have addressed the generation of definitive endoderm (DE) from this cell source by attempting to recapitulate the signaling events occurring during embryogenesis. However, the subsequent differentiation of DE has failed to generate functional insulin-producing beta cells. To assure that we have the correct starting material, we need to fully characterize ESC-derived DE, by assessing whether the cells are functionally equivalent to the in vivo counterpart. Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed.

Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation.

Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed in the putative DE prior to integration and endoderm-specific proteins were assessed 48 h post transplantation.

Conclusions: We describe the detailed methodological procedure for transplanting putative DE derived from ESCs, and the subsequent analysis of the migration and development of the grafted cells. Our result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides a valuable tool for cell characterization. This is an important initial step in the differentiation process towards fully functional beta cells.