Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions

We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional ‘slow freezing’ methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS + 0.5 M sucrose at 37 °C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions

Role of the Mitochondria in Immune-Mediated Apoptotic Death of the Human Pancreatic β Cell Line βLox5

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ0 cells. βLox5 ρ0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways

Role of Proinsulin Self-Association in Mutant INS Gene–Induced Diabetes of Youth

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene-induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production

Inflammatory cytokines rewire the proinsulin interaction in human islets

Corrected by: Correction to “Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets”, The Journal of Clinical Endocrinology & Metabolism, Volume 108, Issue 4, 1 April 2023, Page e61, https://doi.org/10.1210/clinem/dgac713, an error occurred in the Correspondence section: The Correspondence section listed one corresponding author: “Pamela Itkin-Ansari, PhD, Adjunct, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA. Email: [email protected].” The article should state that author Randal J. Kaufman is a co-corresponding author. The corrected Correspondence section is: “Randal J. Kaufman, PhD, Sandford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Email: [email protected] or Pamela Itkin-Ansari, PhD, Adjunct, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA. Email: pitkin@ sbpdiscovery.org.” The article has been corrected online. doi.org/10.1210/clinem/dgac493CONTEXT: Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both Type I and Type II diabetes (T1D, T2D). Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes but the mechanisms remain unclear. OBJECTIVE: We sought to determine the effect of the diabetes associated cytokines on proinsulin folding, trafficking, secretion, and b-cell function. DESIGN: Human islets were treated with interleukin-1β and interferon-γ for forty-eight hours, followed by analysis of IL6, nitrite, proinsulin and insulin release, RNAseq, and unbiased profiling of the proinsulin interactome by Affinity Purification-Mass Spectrometry (AP-MS). RESULTS: Cytokine treatment induced secretion of IL6, nitrites, and insulin, as well as aberrant release of proinsulin. RNAseq showed that cytokines upregulated genes involved in ER stress and consistent with this, AP-MS revealed cytokine induced proinsulin binding to multiple ER chaperones and oxidoreductases. Moreover, increased binding to the chaperone BiP was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and T1D and T2D GWAS candidate proteins not previously known to interact with proinsulin (e.g., Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. CONCLUSION: Together, the data shed new light on mechanisms by which diabetes associated cytokines dysregulate β-cell function. For the first time we show that even short term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.Duc T. Tran, Anita Pottekat, Kouta Lee, Megha Raghunathan, Salvatore Loguercio, Saiful A. Mir, Adrienne W. Paton, James C. Paton, Peter Arvan, Randal J. Kaufman, and Pamela Itkin-Ansar