A factor(s) secreted from MIN-6 β-cells stimulates differentiation of definitive endoderm enriched embryonic stem cells towards a pancreatic lineage

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 2 AbstractIn the mouse the developing pancreas is controlled by contact with, and signaling molecules secreted from, surrounding cells. These factors are best studied using explant cultures of embryonic tissue. The present study was undertaken to determine whether embryonic stem (ES) cells could be used as an alternative model in vitro system to investigate the role of cell-cell interactions in the developing pancreas.Transwell culture experiments showed that MIN-6 β-cells secreted a factor or factors that promoted differentiation of ES cell derived definitive endoderm enriched cells towards a pancreatic fate. Further studies using MIN-6 condition medium showed that the factor(s) involved was restricted to MIN-6 cells, could be concentrated with ammonium sulphate, and was sensitive to heat treatment, suggesting that it was a protein or peptide. Further analyses showed that insulin or proinsulin failed to mimic the effects of the conditioned media. Collectively, these results suggest that β-cells secrete a factor(s) capable of controlling their own differentiation and maturation. The culture system described here presents unique advantages in the identification and characterisation of these factors.

Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium

“…Its expression seems likely to be very important in the in vitro differentiation of ES cells along pancreatic cell lineages (Miyazaki et al ., ). The previous reports by other investigators suggested that ES cells cultivated with pancreatic rudiments, mesenchyme (Shiraki et al ., ) or MIN‐6 β ‐cell‐conditioned medium could express Hnf1 β , PDX‐1 and Ptf1 (Uroic et al ., ). The genes tested here ( PDX1 , INS‐1 and INS‐2 ) did not show a concentration‐dependent increase in expression levels (Figure A).…”

“…() reported that rat islet endothelium‐conditioned culture medium (ECCM) supports pancreatic β cell function; interestingly, both insulin release and total insulin content were increased in the islets cultured in ECCM. Mouse ES cells were cultured using MIN‐6 β cell conditioned medium; the data indicated that MIN‐6 β cells secreted factors that promoted differentiation of mouse ES cells towards a pancreatic fate (Uroic et al ., ). Talavera‐Adame et al .…”

“…More recently, Johansson et al (2009) reported that rat islet endotheliumconditioned culture medium (ECCM) supports pancreatic β cell function; interestingly, both insulin release and total insulin content were increased in the islets cultured in ECCM. Mouse ES cells were cultured using MIN-6 β cell conditioned medium; the data indicated that MIN-6 β cells secreted factors that promoted differentiation of mouse ES cells towards a pancreatic fate (Uroic et al, 2010). Talavera-Adame et al (2011) found that the differentiation of mouse EBs into pancreatic endocrine progenitors and insulin-producing β-like cells was enhanced by endothelial cell conditioned media; they detected upregulation of PDX-1, Ngn3, Nkx6.1, INS1, INS2, amylin, SUR1, GKS and amylase as well as down-regulation of SST in treated embryoid bodies (EBs).…”

The Importance of Proper Oxygenation in 3D Culture

“…Simply elevating the culture above the gas impermeable surface and allowing for mass transfer on both the basal and apical surface of the tissue improves the oxygen profiles. The apical surface is covered by a minimal height of culture medium reducing oxygen mass transfer distances but requiring frequent changes to prevent tissue drying due to evaporative loss (Bigas et al, 1995;Uroic et al, 2010).…”