Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility.
Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human β-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 μm in diameter, corresponding to ~6000 β cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1·7- to 12·5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human β cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
Hong
H
Guo-Parke
Jane T
JT
McCluskey
Catriona
C
Kelly
Muhajir
M
Hamid
Neville H
NH
McClenaghan
Peter R
PR
Flatt
D000596:Amino Acids / Q000494:pharmacology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D002459:Cell Fusion / Q000379:methods*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D017629:Gap Junctions / Q000502:physiology; D005947:Glucose / Q000494:pharmacology; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D016381:Islets of Langerhans Transplantation; D007651:Keto Acids / Q000494:pharmacology; D023822:Tissue Engineering / Q000379:methods*; D059467:Transcriptome
D000596:Amino Acids; D006728:Hormones; D007328:Insulin; D007651:Keto Acids; C013082:alpha-ketoisocaproic acid; D005947:Glucose