Expression of regulatory genes for pancreas development during murine embryonic stem cell differentiation

Abstract

Insulin-producing cells derived from embryonic stem cells could be surrogates for beta cells in diabetes therapy. However, their derivation remains hard to achieve with current protocols which rely on initial embryoid body formation. We assume that factors known to inhibit pancreas development contribute to this limitation in vitro. To evaluate this hypothesis, embryoid bodies were examined after different culture periods by real time RT-PCR to profile the expression of genes known to regulate embryonic pancreas development. Our data indicate that transcripts for pancreas markers (insulin, glucagon and amylase ) were expressed during differentiation, but the highest levels achieved were at least 10 5 times lower than in the adult mouse pancreas. Notch signalling was activated as suggested by Delta, Jagged, Ngn3 and NeuroD1 profiles. However, Sonic hedgehog, a known inhibitor of pancreas induction in vivo drastically increased in day 6 embryoid bodies, while Inhibin βA and βB were down-regulated and follistatin up-regulated. Members of the Fibroblast-and Transforming Growth Factor families which pattern the endoderm were expressed at low levels, while those that inhibit pancreas development were highly transcribed. The profile of pancreas regulators expressed in embryoid bodies is therefore not compatible with differentiation of pancreatic and insulin-producing cells. These findings provide an explanation for the limited derivation of such cells to date, in addition to basic information for establishing novel differentiation protocols.

Sonic Hedgehog and Other Soluble Factors from Differentiating Embryoid Bodies Inhibit Pancreas Development

“…Real-time polymerase chain reaction (PCR) was carried out with a cDNA pool corresponding to 50 ng (ES cells, EBs, cultured liver cells) or 7.5 ng (pancreas explants) RNA equivalent using SYBR green I (Sigma-Aldrich), as previously described [26]. Amplicon specificities were ensured by primer design encompassing introns of mouse mRNAs, primer sequences matching up with Basic Local Alignment Search Tool (BLASTing) against the National Center for Biotechnology Information database, and recording of melting profiles during each run.…”

“…Sections (5 m) were incubated overnight with primary antibodies against amylase (polyclonal, 1/5,000; Sigma-Aldrich), glucagon (polyclonal, 1/3,000; Dr. C. Van Schravendijk, Vrije Universiteit Brussel, Brussels), insulin (monoclonal, 1/2,000; Sigma-Aldrich), Ngn3 (polyclonal, 1/1,000; the laboratory of Michael German, University of California, San Francisco), and Pdx1 (polyclonal, 1/1,000; Beta Cell Biology Consortium, Nashville, TN, http://www.betacell.org), and stained as previously described [26].…”

“…Aspecific binding was blocked before exposure to primary antibodies by incubation in relevant nonimmune serum (goat, rat, or donkey). For both liver cells and EBs, tetramethylrhodamine isothiocyanate, fluorescein isothiocyanate, or biotinylated secondary antibodies were used and processed as previously described [26] with 4Ј,6-diamidino-2-phenylindole or hematoxylin-eosin used for counterstaining.…”

“…We recently reported that cells within EBs express numerous pancreas regulatory genes including Shh both at messenger and protein levels. When compared to their expression during em-bryonic development, the overall profile of pancreas regulators appeared incompatible with differentiation of pancreatic cells from ES cells [26]. Although very suggestive, demonstration of this hypothesis was made extremely difficult by the lack of a thorough protocol that would associate blockade of all inhibitory signals and supplementation of all defective factors in correct amounts and at needed time points.…”

Translational embryology: Using embryonic principles to generate pancreatic endocrine cells from embryonic stem cells

“…While addition of factors such as activin increased the number of insulin-producing cells to almost 3%, this is still an inefficient process. One explanation for why embryoid body formation is an inefficient method was suggested by Mfopou et al, who showed that secondary inductions resulted in the expression of sonic hedgehog, a known inhibitor of pancreas development (Mfopou et al, 2005).…”