Using a variety of experimental approaches, we found abundant cells with β-cell features, most notably including insulin production and secretion. These questions were addressed in the current study, using the differentiation of the H9 line of hES cells as described by Thomson et al. Such information is crucial to assess the feasibility of using hES cells as a potential source for β-cell replacement therapy. However, quantitative aspects, including elaboration of insulin into the medium and percentage of insulin-producing cells, were not determined. Using reverse transcriptase-polymerase chain reaction (RT-PCR) applied to RNA extracted from differentiated hES cells, detection of a variety of differentiated cell markers, including insulin, was reported ( 16). Endodermal markers, but not insulin expression, were reported in a previous general survey of different growth conditions and differentiation markers in EG cells ( 15). Removal from the MEF feeder layer is associated with differentiation into derivatives of the three EG layers, as evident from teratomas formed after subcutaneous injection in nude mice ( 11). As previously shown, they have a normal karyotype and express telomerase and embryonic cell-surface markers. hES cells grow as homogeneous and undifferentiated colonies when they are propagated on a feeder layer of mouse embryonic fibroblasts (MEFs) ( 11). The establishment of pluripotent human embryonic stem (hES) cells ( 11, 12) and embryonic germ (EG) cells ( 13) have introduced a new potential source for cell therapy in type 1 diabetic patients, especially in light of recent successes in producing glucose-sensitive insulin-secreting cells from mouse embryonic stem (ES) cells ( 14). Another more recently described approach involves extending the β-cell phenotype to other tissues using in vivo gene transfer ( 8, 9), either by expressing the insulin gene or an insulin gene analogue under the control of a glucose sensitive promoter or by ectopic expression of insulin promoter factor-1 (IPF1)/pancreatic and duodenal homeobox gene-1 (PDX1) ( 10). In addition to the problem inherent in xenobiotic sources, such cell lines have been shown to display phenotypic instability, with loss of insulin biosynthesis and regulated secretion while proliferating. It has also been reported that β-cell lines derived from rodents might provide an unlimited source for cell replacement therapy ( 5, 6, 7). Thus, attention has focused on the use of alternative sources such as xenografts, which have other disadvantages, including the potential risk of undetermined zoonotic infections ( 4). However, the shortage of donations is a primary obstacle preventing this therapeutic modality from becoming a practical solution. The promise of this approach has recently been further strengthened by a report of the use of an improved, less hazardous glucocorticoid-free immunosuppresive regimen in islet allograft transplantation ( 3). Indeed, this approach was recently shown to reverse glomerular lesions in patients with diabetic nephropathy ( 2). Yet, pancreatic cell and islet cell replacement is currently considered the only curative therapy. Recent studies have emphasized the importance of strict glycemic control in order to reduce ophthalmologic, neurological, and renal complications of the disease ( 1). These findings validate the hES cell model system as a potential basis for enrichment of human β-cells or their precursors, as a possible future source for cell replacement therapy in diabetes.Īpproximately 5–10% of all diabetic individuals suffer from type 1 diabetes. Secretion of insulin into the medium was observed in a differentiation-dependent manner and was associated with the appearance of other β-cell markers. Immunohistochemical staining for insulin was observed in a surprisingly high percentage of cells. Using hES cells in both adherent and suspension culture conditions, we observed spontaneous in vitro differentiation that included the generation of cells with characteristics of insulin-producing β-cells. In the current study, we used pluripotent undifferentiated human embryonic stem (hES) cells as a model system for lineage-specific differentiation. The relative paucity of donations for pancreas or islet allograft transplantation has prompted the search for alternative sources for β-cell replacement therapy. Type 1 diabetes generally results from autoimmune destruction of pancreatic islet β-cells, with consequent absolute insulin deficiency and complete dependence on exogenous insulin treatment.
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