Recovery of viable endocrine‐specific cells and transcriptomes from human pancreatic islet‐engrafted mice

Abstract

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormonespecific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted β, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate β, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cellderived β cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of 1902 |

Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

“…For electrophysiological experiments, β-cells were initially identified using the high autofluorescence signature of β-cells to 488-nm excitation as these cells have high concentrations of unbound flavin adenine dinucleotide (72,73). Dithizone (Sigma-Aldrich) staining was then used to further confirm β-cell identity at the end of each recording (74). Donor information for specific experiments is provided in Table 1.…”

Single Nucleus RNA Sequencing of Human Pancreatic Islets In Vitro and In Vivo Identifies New Gene Sets and Three β-Cell Subpopulations with Different Transcriptional Profile

“…Further, scRNA-seq analysis of human islet biopsies or human islet grafts in mice requires mechanical and enzymatic cell dissociation, which causes cellular stress and eliminates specific cell subpopulations sensitive to these stresses. Finally, in this in vivo context, scRNA-seq data sets yield very low cell numbers that may not reflect the complete universe of the normal islet cell populations (18)(19)(20)(21)(22).…”

Cell Heterogeneity and Paracrine Interactions in Human Islet Function: A Perspective Focused in β-Cell Regeneration Strategies

“…Moreover, long-term analysis or the whole range of metabolic stresses including pregnancy, obesity, pathogenic infection, or extreme fasting [where β-cells undergo major modifications which must be quickly reversed after refeeding ( 88 )] have not been explored. Besides, the impact of cell heterogeneity in SC-β strategies remains elusive, with only one report indicating that β-cell subpopulations were not detected after SC-β transplantation in mice ( 89 ). Although there are not such studies in human, heterogeneity is crucial in mice for β-cell adaptation to pathological stressors like obesogenic diets ( 90 ).…”