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The source of insulin released from insulin‐releasing cell clusters (IRCCs) differentiated from embryonic stem cells remains unclear. Rajagopal et al. have suggested that IRCCs do not synthesize but secrete insulin that had been absorbed from media during the multistep protocol. We report here further data relevant to this controversy. No radioisotopic labeling of insulin was observed when IRCCs were incubated in a medium containing  35 S‐cysteine. Less than 1% of the extra‐cellular stoichiometric C‐peptide equivalent to insulin was secreted during glucose stimulation. However, intracellular immunostaining and immunogold labeling were both positive for C‐peptide. Finally, a mass balance calculation showed that simple equilibration of IRCCs by Fickian diffusion from media accounted for at most 4% of secreted insulin. These findings and further analysis of the results of others suggest that the mechanism of insulin secretion by IRCCs is a combination of sequestration and de novo synthesis.

Sequestration and Synthesis: The Source of Insulin in Cell Clusters Differentiated from Murine Embryonic Stem Cells