In collaboration with the New York Stem Cell Foundation (NYSCF, see letter of collaboration), we will attempt de-differentiation of cryopreserved splenocytes to iPSC: this has not been attempted before to our knowledge. If successful, these iPSC may be differentiated into sc-islets and serve as an isogenic sources of sc-islets for interactions with the islet-derived T cell lines the Kent lab has generated and banked. If successful, we will submit a full addendum to this project for the study of the interaction of isogenic iPSC-derived sc-islets and autologous islet-derived T cells. This technology may be used by other investigators in their examination of the isogenic interaction of sc-islets and immune cells.
Addendum: In collaboration with investigators at the City of Hope, we will attempt de–differentiation of cryopreserved, islet–derived T cell lines from nPOD donors to iPSC: this has not been attempted before to our knowledge. In addition, we would transfer autologous, cryopreserved spleen cells to the investigators at COH. Primary splenocytes from the donor, previously supplied by nPOD to Dr. Kent, will be used in whole genome sequencing (WGS) to establishing the donor’s true germline reference genome in order to monitor Identify any donor–specific or disease–related germline variants and confirm genetic stability and absence of reprogramming–associated mutations in the iPSCs. These data will be made available to nPOD. If successful, these iPSC will be differentiated into sc–islets and serve as an isogenic sources of islets for interactions with the islet–derived T cell lines the Kent lab has generated and banked. These sc–islets may be used by other investigators in their examination of the interaction of sc–islets and immune cells.
Addendum: As previously approved,we will attempt de–differentiation of cryopreserved, islet–derived T cell lines or in vitro polyclonally expanded splenocytes (from cryopreserved primary donor splenocytes, generated in the Kent laboratory) from nPOD donors 6578 and 6579 to iPSC: this has not been attempted before to our knowledge. We will also include samples from2 control nPOD donors (6643 and 6645) and 2 recent onset T1D nPOD donors (6550 and 6563).If successful, these iPSC will be differentiated into sc–islets and serve as an isogenic sources of islets for interactions with the islet–derived T cell lines the Kent lab has generated and banked from nPOD donors and TCR–transductants generated from islet–infiltrating CD8+T cells from nPOD donors 6578 and 6579 (demise at T1D onset and 1 years duration of T1D, respectively: rare samples close to T1D onset) as proof–of–principle of islet–infiltrating T cells recognizing isogenic sc–islets. Any other iPSC generated by other investigators may be used to examine of the interaction of sc–islets or other differentiated cells and immune cells.
