Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets

Abstract

Rodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first-phase glucose-stimulated insulin secretion (GSIS) compared with cultured-intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first-phase GSIS. Lastly, the lentiviral-mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.

Keywords: Beta cell; diabetes; extracellular matrix; glucokinase; inflammation.

Figure 1

Pseudoislets exhibit a narrower size distribution compared with cultured‐intact islets. (A) Pseudoislet formation process from fresh islets using spheroid microwell plates. (B) Representative images of pseudo or cultured‐intact islets from donor 1 stained with nuclear dye Hoechst 33342 analyzed by ImageJ macro. Analysis marks shown in yellow. Scale bar = 250 μm. (C) Quantified cross‐sectional area from cultured‐intact C and pseudoislets (P) using max‐projected fluorescent images analyzed by ImageJ macro. (D) Estimation of ellipsoidal volume of culture‐intact or pseudoislets based on major and minor axis measurements extracted from fluorescent images by ImageJ macro. (E) Diameter of cultured‐intact or pseudoislets obtained as in methods. Mean ± SEM, n = 12 (donor 1, culture‐intact), 7 (donor 2, culture‐intact), 6 (donor 3, culture‐intact), 4 (all donors, pseudoislets).

Figure 2

Insulin secretion of fresh, cultured‐intact, and pseudoislets in response to glucose, glibenclamide, and KCl in perifusion. (A) Perifusion profiles of fresh (FRES), cultured‐intact (CULT), and pseudo (PSEU) islets from nondiabetic donors in response to 16.7 mmol/L glucose (Glu) expressed taking basal insulin secretion as 1. Glucose ramp is indicated in a bar on the top. (B) Area under the curve (AUC) during glucose ramp in (A). (C) Ratio of stimulation index (SI) for the first phase over the second phase of insulin secretion in (A) determined as in methods. (D) Perifusion profiles of cultured intact and pseudoislets from donor 11. Mean ± SEM, n = 9 donors for all groups. (E) Perifusion profiles of FRES and PSEU in response to 10 μmol/L glibenclamide (Gli) and 30 mmol/L KCl. Ramps with secretagogues are indicated in bars on the top.(F) Ratio of SI for the first phase over the second phase of insulin secretion in (E). AUC during ramps determined for (G) glibenclamide and (H) KCl. Mean ± SEM, n = 3 for fresh and 4 for pseudoislets. *P < 0.05.

Figure 3

Expression of markers of islet cells in fresh, cultured‐intact, and pseudoislets. qPCR probed fresh (FRES), cultured‐intact (CULT), and pseudoislets (PSEU) for expression levels of cell type markers including (A) INS, (B) GCG, (C) SST, (D) PPY, (E) GHRL, (F) ACTA2 (G) AMY2A and (H) PTPRC. Expression of PPIB was used as internal control to correct expression values as in methods. Mean ± SEM. (A), (B), (D), and (H); n = 7 donors, (C), (E), (F), and (G); n = 6 donors. *P < 0.05.

Figure 4

Expression of beta cell maturation, beta cell function, extracellular matrix, and stress markers in fresh, cultured‐intact, and pseudoislets. Expression of (A) markers associated with beta cell maturation, (B) beta cell function (C) extracellular matrix genes, and (D) stress associated genes determined by qPCR in cultured‐intact (C) and pseudo (P) islets taking an average for fresh islets as 1. Mean ± SEM. n = 5–11 donors (see Table 1 for assay performed for each donor). *P < 0.05 versus fresh islets. #; P < 0.05 versus cultured‐intact islets.

Figure 5

Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of GFP fluorescence of AV‐CMV‐GFP (AV‐GFP) transduced cultured‐intact islets (CULT) and nontransduced control (Cont) as well as LV‐CMV‐GFP (LV‐GFP) transduced pseudoislets (PSEU) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μm. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV‐GFP (AV‐C) and pseudoislets treated with LV‐GFP/LV‐ShScramble (LV‐P) prepared from four donors. (D–G) Comparison of LV‐shScramble (Scr) and LV‐shGCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM. n = 5 donors. (E) Representative insulin secretion in response to 16.7 mmol/L glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. *P < 0.05