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. 2021 Apr 10;67(1):17-26.
doi: 10.5387/fms.2020-25. Epub 2021 Feb 17.

Comprehensive analysis of gene expression of isolated pancreatic islets during pretransplant culture

Junichiro Haga  1 , Naoya Sato  1 , Takayuki Anazawa  2 , Takashi Kimura  1 , Akira Kenjo  1 , Mitsukazu Gotoh  1 , Shigeru Marubashi  1
Affiliations

Comprehensive analysis of gene expression of isolated pancreatic islets during pretransplant culture

Junichiro Haga et al. Fukushima J Med Sci. .

Abstract

Background: The aim of this study was to investigate the effect of pretransplant culture on the survival of pancreatic islet grafts, and to determine the biological characteristics of isolated islets during pretransplant culture.

Methods: The survival of islets from Wistar rats, transplanted to diabetic C57BL/B6 mice, was compared between fresh islets and cultured islets. A comprehensive gene expression analysis was employed to investigate biological processes during pretransplant culture, and in vitro validation studies were performed.

Results: Survival of cultured xenografts was significantly prolonged as compared to that of fresh islets (fresh:12.5 ± 1.9 days, 1-day cultured:16.0 ± 1.3 days (p= 0.017), 3-day cultured:17.0 ± 2.6 days (p= 0.014)). Comprehensive gene expression analysis identified significant upregulation of annotated functions associated with inflammation in cultured groups. Six proinflammatory genes, including heme oxygenase 1 (HO-1) and IL-6, were significantly upregulated during culture. Validation studies revealed significantly higher levels of IL-6 in the supernatant of cultured islets and HO-1 in the cultured islets when compared with fresh islets.

Conclusion: Transplantation of cultured islets induced significant but minimal prolongation of graft survival in xenogeneic combinations. Comprehensive analysis of gene expression in cultured islets showed biological processes associated with proinflammation during culture.

Keywords: IL-6; culture; gene expression; heme oxygenase 1; islet transplantation.

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Conflict of interest statement

The authors declare no conflicts of interest associated with this manuscript.

Figures

Figure 1.
Figure 1.
Islets recovery and stimulation index after culture (A) Percentage of islet equivalents (IEQ) recovery of cultured islets compared to that of fresh islets (fresh-islets). Recovery rate of IEQ was measured in each group (n = 6, d1- and d3-islets). (B) Stimulation index of cultured islets. The supernatants were collected and insulin levels were determined using a commercially available ELISA kit. The stimulation index was not significantly different between the three groups (each group, n = 6).
Figure 2.
Figure 2.
Blood glucose levels of diabetic mice with transplantation of islet rat xenografts (A) Rat islets (n = 300) were transplanted under the kidney capsule of diabetic mice. The levels of blood glucose were measured in each group of mice. (B) The engraftment periods of fresh-, d1-, and d3-islets compared by Kaplan-Meier method. The engraftment period of the cultured islets was significantly increased compared to that of the fresh islets.
Figure 3.
Figure 3.
Heme oxygenase 1 (HO-1) protein expression in cultured islets. Western blotting for HO-1 and β-actin expression in cultured islets. The expression of HO-1 protein was significantly higher in d1- and d3-islets than in fresh-islets.
Figure 4.
Figure 4.
Levels of IL-6 in islet culture medium IL-6 levels in islet culture supernatants were measured using an ELISA kit. IL-6 release from d1- and d3-islets was confirmed.
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