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 hormone-specific 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 cell-derived β cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

Keywords: L‐type voltage‐gated calcium channel; RNA‐Seq; graft recovery; insulin; β cell.

Figure 1

Overview. Human islet cells were transplanted into mice, recovered, and assessed as shown

Figure 2

Islet graft transplant and functional assays. A, Human islets prior to transplant; stained with dithizone (inset). B, Islet graft following transplant under the kidney capsule (left); higher magnification shows vascularization of the graft (right). C, Human plasma INS recovered from mice challenged with glucose (2 g/kg body weight). D, Islet grafts (n = 2) were excised from the kidney, and GSIS was performed by perifusion. Insulin levels are shown in response to 20 mM glucose (gray bar) and KCl (yellow bar) as a percentage of total INS; the levels from each of two grafts are shown as a point with the average as a dashed line. E, INS (green) and GCG (red) staining of the kidney capsule and associated islet graft pre‐ (left) and post‐ (right) graft recovery, demonstrating successful removal of the graft from the kidney capsule

Figure 3

Hormone‐specific islet cell recovery from human islet engrafted mice. A, Dissociated live cells from pretransplant islets and recovered human islet grafts were fixed, stained, and sorted based on their hormone expression; shown is a representative FACS sort of cells recovered from islet grafts from a single donor. B, The percentages of hormone‐specific cells were similar for each individual donor pretransplant and posttransplant. Abbreviations: GCG, glucagon; INS, insulin; Neg, triple negative for INS, GCG, and SST; pre, pretransplant; post, posttransplant; SST, somatostatin

Figure 4

Transcript abundance pretransplant and posttransplant. The expected counts output from RNA‐Seq by Expectation‐Maximization (RSEM) for the 200 most abundant pretransplant transcripts (average of three human donors) are plotted on the x‐axis and the corresponding values for posttransplant transcripts are plotted on the y‐axis for β and α cells. Perfect correlation is indicated by the dotted line

Figure 5

Single‐cell transcriptomes from posttransplant human islets. Engrafted human islets were recovered for scRNA‐Seq analysis; UMAP shows distinct subpopulations, including four hormone‐specific cell subsets

Figure 6

Assessment of SC‐β cells recovered from grafts. A, Engrafted SC‐β cells were assessed for viability with 7‐AAD. B, Viable cells were measured for intrinsic autofluorescence (FITC) found in β cells. C, Live autofluorescent cells were confirmed as INS positive by immunostaining. D, Patch‐clamp recordings of SC‐β cells show robust calcium currents (a feature of calcium‐dependent insulin exocytosis) and sensitivity to the L‐type calcium channel agonist FPL 64176