Induction of differentiation of embryonic stem cells into insulin secreting cells by fetal soluble factors. Export

@article{citeulike:300842, abstract = {Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that developing pancreas releases soluble factors responsible for "in vitro" endocrine pancreatic differentiation from embryonic stem (ES) cells. A mouse D3 ES cell line was transfected with a human insulin promoter-beta-geo/pGK-hygro(r) construct. To direct differentiation cells were cultured for 7 days to form embryoid bodies (EB) and then plated for an additional 7 days. During this 14-day period, besides eliminating LIF, cells were cultured in low serum concentration with the addition of conditioned media from e16.5 pancreatic buds (PB). Islet cell differentiation was studied by: i) X-gal staining after neomycin selection; ii) BrdU studies; iii) simple and double immunohistochemistry for insulin, C-peptide and Glut-2; iv) RT-PCR for insulin and PDx-1; v) insulin and C-peptide content and secretion assays; vi) intraperitoneal glucose tolerance test; vii) electrophysiology (patch-clamp studies in inside-out configuration) and viii) transplantation of differentiated cells under the kidney capsule of streptozotocin (STZ)-diabetic mice. The differentiated ES cells showed: changes in the mRNA levels of insulin and PDX-1; co-expression of insulin, c-peptide and glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study we establish a battery of techniques that could be used together to properly characterise islet-cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental to engineer beta cells from stem cells.}, address = {University Miguel Hern\'{a}ndez, E-03550, San Juan de Alicante, Spain.}, author = {Vaca, Pilar and Mart\'{\i}n, Franz and Vegara-Meseguer, Josefina and Rovira, Juan and Bern\'{a}, Genoveva and Soria, Bernat}, citeulike-article-id = {300842}, citeulike-linkout-0 = {http://dx.doi.org/10.1634/stemcells.2005-0058}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/16109755}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=16109755}, day = {18}, doi = {10.1634/stemcells.2005-0058}, issn = {1066-5099}, journal = {Stem Cells}, keywords = {beta-cell, pancreas, stem-cell}, month = {August}, posted-at = {2005-08-23 15:15:14}, priority = {2}, title = {Induction of differentiation of embryonic stem cells into insulin secreting cells by fetal soluble factors.}, url = {http://dx.doi.org/10.1634/stemcells.2005-0058}, year = {2005} }

TY - JOUR ID - citeulike:300842 L3 - citeulike-article-id:300842 N2 - Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that developing pancreas releases soluble factors responsible for "in vitro" endocrine pancreatic differentiation from embryonic stem (ES) cells. A mouse D3 ES cell line was transfected with a human insulin promoter-beta-geo/pGK-hygro(r) construct. To direct differentiation cells were cultured for 7 days to form embryoid bodies (EB) and then plated for an additional 7 days. During this 14-day period, besides eliminating LIF, cells were cultured in low serum concentration with the addition of conditioned media from e16.5 pancreatic buds (PB). Islet cell differentiation was studied by: i) X-gal staining after neomycin selection; ii) BrdU studies; iii) simple and double immunohistochemistry for insulin, C-peptide and Glut-2; iv) RT-PCR for insulin and PDx-1; v) insulin and C-peptide content and secretion assays; vi) intraperitoneal glucose tolerance test; vii) electrophysiology (patch-clamp studies in inside-out configuration) and viii) transplantation of differentiated cells under the kidney capsule of streptozotocin (STZ)-diabetic mice. The differentiated ES cells showed: changes in the mRNA levels of insulin and PDX-1; co-expression of insulin, c-peptide and glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study we establish a battery of techniques that could be used together to properly characterise islet-cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental to engineer beta cells from stem cells. JF - Stem Cells SN - 1066-5099 AD - University Miguel Hernández, E-03550, San Juan de Alicante, Spain. TI - Induction of differentiation of embryonic stem cells into insulin secreting cells by fetal soluble factors. KW - beta-cell KW - pancreas KW - stem-cell AU - Vaca, Pilar AU - Martín, Franz AU - Vegara-Meseguer, Josefina AU - Rovira, Juan AU - Berná, Genoveva AU - Soria, Bernat PY - 2005/08/18/ UR - http://dx.doi.org/10.1634/stemcells.2005-0058 ER -