In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

Abstract

Aims/hypothesis. We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. Methods. Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice. Results. The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols. Conclusions/interpretation. These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.Keywords Diabetes · Differentiation · Embryonic stem cells · Exogenous agents · Insulin-producing cells · Islet progenitors Abbreviations: Anti-Shh, antibody against sonic hedgehog · D3, undifferentiated D3 stem cell line · EB, embryoid bodies · ES, embryonic stem · FBS, fetal bovine serum · LIF, leukaemia inhibitory factor · mES, mouse embryonic stem · Ngn3, neurogenin 3 · P, gelatine-coated plates · Pdx-1, pancreatic duodenum homeobox 1

Measurement of T1, T2, and magnetization transfer properties during embryonic development at 7 Tesla using the chicken model

“…Embryonic stem cells can produce derivatives of all three primary germ cell layers: ectoderm, mesoderm, and endoderm. Numerous differentiation protocols have already been established permitting the generation of almost any somatic cell type such as cardiomyocytes for cardiac repair (5), neural precursor cells with the potential to repair or limit the damage associated with infarct or neurodegenerative diseases (6), or ␤-islet progenitor cells producing insulin for the treatment of diabetes (7,8).…”

Generation of pancreatic insulin-producing cells from embryonic stem cells — ‘Proof of principle’, but questions still unanswered

“…Functional pancreatic cells, however, were successfully generated using lineage selection strategies based on pancreas-specific promoters [5,6], by modified protocols in combination with transgene expression [7][8][9], or by addition of a phosphoinositol-3 kinase inhibitor [10]. The differentiated cells showed properties of (neonatal) beta cells, such as insulin transcripts and C-peptide/insulin co-expression, insulin-secretory granules, ion channel activity of embryonal beta cells, and normalisation of blood glucose level after transplantation into diabetic mice [5][6][7][8][9][10]. Most of the differentiation protocols required a long cultivation period, including 4-5 days of embryoid body (EB) formation, followed by 3-4 weeks of differentiation, and some protocols required genetic manipulation.…”

Gene Therapy Progress and Prospects: Gene therapy for diabetes mellitus

“…ES cells have been touted to be able to differentiate into multiple cell lineages including the pancreatic endocrine cell lineages. 54,70,71 Notwithstanding the ethical and current legal controversies regarding the use of ES cells, there are significant potential advantages and some disadvantages of using stem cells for cell therapy for diabetes. ES cells by nature are totipotent and in theory could reconstitute the entire islet and thus be the ideal replacement therapy for type I diabetes.…”