(J-368) Advancing Hypoxia Resistance in Stem Cell-Derived Islets for Type 1 Diabetes Therapy

Type 1 diabetes (T1D) is a chronic condition of uncontrolled blood glucose levels arising from the loss or dysfunction of insulin-producing β cells found within pancreatic islets. While islet transplantation can potentially reverse this condition, efficacy is limited by hypoxia-induced transplant death and dysfunction. A mechanistic understanding of the impact of hypoxia on primary donor islets and lab-grown stem-cell-derived islets (SC-islets) is crucial for improving islet transplant longevity. Our objective is to investigate the hypoxia response in primary and SC-islets using single-cell RNA sequencing (scRNA-seq) and generate CRISPR-screening stem cell systems.

Primary human and SC-islets were cultured in 142 (standard) and 7 (low) mmHg partial pressure O₂ (pO₂) for 0, 6, 24, and 48 hrs on gas-permeable silicone rubber plates. We measured glucose-stimulated insulin secretion for one hour in both 2 mM (low) and 20 mM (high) glucose to evaluate function and performed scRNAseq to evaluate cell-type-specific transcriptome.

To generate the CRISPR-screening system, we introduced dCas9 plasmids and KRAB (for interference) or VPR (for activation) into the AAVS1 locus of HUES8 human stem cell line. We then introduced a genome-wide single-guide RNA (sgRNA) library into the cells via lentivirus transduction. Finally, we performed quality control metrics, assessing gene knockdown and activation efficiency, sgRNA library representation in differentiated cells, and fold changes of targeting vs. non-targeting control sgRNAs in sequenced samples.

scRNAseq revealed a decrease in β cells identity gene transcription and upregulation of genes involved in a metabolic switch to anaerobic glycolysis and canonical apoptosis genes under hypoxic conditions. Surprisingly, hypoxia induced increased insulin secretion in response to low-glucose-stimulation in both primary and SC-islets. Live/dead staining images revealed dying cells in the cores of hypoxic SC-islets. 

The newly generated CRISPR-screening stem cell lines exhibited efficient knockdown (52% NANOG knockdown in 24 hrs) and activation (15x MAFA increase in 24 hrs), while maintaining the differentiation efficiency and function to SC-islets. The 106,193 count sgRNA library distribution in a control and screened sample were similarly distributed with an average read count ± SD of 629 ± 298 and 625 ± 303, respectively. The average fold change ± SD of targeting and non-targeting control sgRNAs were 0.998 ± 0.156 and 1.011 ± 0.166, respectively. These results indicate that our CRISPR-screening stem cell systems function as expected and retain satisfactory coverage of the sgRNA library for future screens.

Our study provides critical insights into the multifaceted impact of hypoxia on islet function and survival, unveiling unexpected effects on insulin secretion. We report on the development of CRISPR-screening stem cell lines capable of differentiating into functional SC-islets, laying the groundwork for future exploration of hypoxia-resistance cellular engineering strategies. By highlighting the potential to improve islet transplant efficacy and longevity, our work advances cell therapy as a viable treatment for T1D.