Cryopreservation of stem cell-derived beta-like cells enriches for beta cells with improved function

The generation of stem cell-derived beta-like cells (sBC) holds extreme promise as not only an abundant insulin-producing cell source for replacement therapy of Type 1 diabetes (T1D), but also as an invaluable model system for investigating human beta cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose responsive sBC. Nevertheless, the process of generating sBC poses significant experimental challenges. It involves lengthy differentiation periods, experiences substantial variability in efficiency, and inconsistencies in obtaining functional sBC. Here, we described a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin expressing cells while simultaneous depleting proliferative progenitors. Thawed sBC display enhanced glucose-stimulated insulin release compared to controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBC for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries. Overall design: Singe cell RNA-seq libraries were created using the Chromium Next GEM Single Cell 5' Kit v2 from 10X Genomics (PN-1000265). Libraries were derived from 2 matched day 30 controls and F/T samples for a total of 4 samples. scRNA-seq libraries were sequenced via paired-end sequencing through the Illumina ® Novaseq platform by Novogene Corporation Inc. Sequencing reads were uploaded to 10X Genomic's cloud analysis platform and processed with the Cell Ranger Count v7.1.0 pipeline using the Human GRCh38 v3.0.0 reference genome to create unique molecular identifier (UMI) gene count matrices per sample. IThese individual sample matrices were then aggregated together and processed using the 10X Genomics Loupe Browser v7.0.1 platform to perform filtering, normalization, clustering and to create t-SNE projections. Cells that contained a UMI count lower than 260 or higher than 200,000, had fewer than 20 or greater than 12,000 genes expressed, or a proportion of mitochondrial genes greater than 20% were removed from the analysis.