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Evaluation of transplanted islet beta-cell proliferative response in an ectopic site during pregnancy

Antonello Pileggi^1,2,3,4, R. Damaris Molano¹, Elsie Zahr-Akrawi¹, Carmen Fotino¹, Lucas Ramcharran¹, Silvia Alvarez¹, Juan Dominguez-Bendala^1,2, Sergio San Jose¹, Valia Bravo-Engana¹, Susana Villate¹, Camillo Ricordi^1,2,3,4,5, Luca Inverardi^1,5

¹Diabetes Research Institute, University of Miami; ²Surgery; ³Microbiology & Immunology; ⁴Biomedical Engineering; ⁵Medicine, University of Miami Miller School of Medicine, Miami, FL, United States

Beta-cell proliferation is rare in adulthood, while during pregnancy lactogenic hormones and serotonin mediate selective beta-cell proliferation peaking mid-pregnancy (day 12.5-14.5) in mice. Islets represent ~1-2% of pancreatic tissue. Enrichment of endocrine cells allows performing molecular studies aimed at identifying the transcriptome pattern of proliferating beta-cells, but requires isolation of islet at different stages of pregnancy. Islet isolation induces cellular stress responses that may alter basal expression profile, thus interfering with the detection of most relevant molecular pathways involved in beta-cell replication. We developed a model of syngeneic islet transplantation under the kidney capsule of chemically-induced diabetic female mice. Following completion of engraftment (≥4 wks), to allow recovery from islet isolation and implantation-related stress, animals are mated. Using this approach, the large mass of endocrine pancreatic tissue (that is, purified islets) can be easily retrieved without harsh manipulation for analysis.

Immunohistopathology assessment of bromodeoxyuridine incorporation in transplanted beta-cells showed proliferation rates comparable to those of the native pancreas. The proportion of proliferating beta-cells in the pancreas was 7.4±2.9% in control mice (n=5) and 22.8±7.2% in pregnant mice (n=5) on day 12.5 (p<0.01). A similar pattern was observed in transplanted islets, with 4.5±2.1% in control (n=6) and 30.8±6.8% in pregnant mice (n=6) on day 12.5 (p<0.001). Microarray analysis of mRNA expression performed on islet grafts obtained from control and pregnant mice (day 12.5) showed that 296 of the ~25,000 genes detected were significantly differentially expressed in islet grafts of pregnant vs. control mice (n=3 independent microarrays with a p<0.08). 68 genes resulted significantly differentially expressed both in our analysis and a previous report on isolated islets (Rieck and coll. 2009). Differential expression of multiple genes was confirmed by qRT-PCR.

Transplanted islets proliferate under physiological conditions (e.g., pregnancy) in an ectopic site. This may open new avenues for in depth molecular studies.