Embryoid Body Formation is Required for Differentiation of Insulin- Producing Cell Clusters from Mouse Embryonic Stem Cells

Christa Caneda

Abstract


In Type 1 diabetes, insulin-producing pancreatic cells, or beta cells, are destroyed by an autoimmune response. Current clinical treatments are indefinite insulin replacement therapy or transplantation of the pancreas or beta islets. The latter two treatments are limited in available donors; a potential alternative is the use of insulin-producing cell clusters (IPCCs) differentiated from embryonic stem cells (ESCs). We hypothesize that IPCCs will reproduce the insulin-producing capacity of healthy beta cells of an adult mouse, and we are testing the efficiency of distinct IPCC culture methods to achieve this goal. Among several existing ESC-IPCC differentiation protocols, Blyszczuk et al. developed the most successful method to date in producing IPCCs that showed similarities to pancreatic beta cells. However, this method is time-intensive, requiring approximately 41 days. We attempted to streamline the protocol by bypassing the formation of embryoid bodies (EBs), reducing the differentiation timeline to 27 days. At several time points during this protocol, IPCC cultures were analyzed by RT-PCR and immunofluorescence for genes and proteins expressed in pancreatic beta islet cells. The mRNA and protein expression of insulin was not observed. Furthermore, ELISA analysis detected low intracellular insulin response after challenging IPCCs with different glucose concentrations. These results reject the hypothesis that EB formation is not required for ESC-IPCC differentiation in vitro. However, it is possible that alpha cells can be differentiated, as glucagon was detected.


Keywords


insulin; differentiation; embryonic stem cells

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