Potential for pancreatic maturation of differentiating human embryonic stem cells is sensitive to the specific pathway of definitive endoderm commitment

This study provides a detailed experimental and mathematical analysis of the impact of the initial pathway of definitive endoderm (DE) induction on later stages of pancreatic maturation. Human embryonic stem cells (hESCs) were induced to insulin-producing cells following a directed-differentiation approach. DE was induced following four alternative pathway modulations. DE derivatives obtained from these alternate pathways were subjected to pancreatic progenitor (PP) induction and maturation and analyzed at each stage. Results indicate that late stage maturation is influenced by the initial pathway of DE commitment. Detailed quantitative analysis revealed WNT3A and FGF2 induced DE cells showed highest expression of insulin, are closely aligned in gene expression patterning and have a closer resemblance to pancreatic organogenesis. Conversely, BMP4 at DE induction gave most divergent differentiation dynamics with lowest insulin upregulation, but highest glucagon upregulation. Additionally, we have concluded that early analysis of PP markers is indicative of its potential for pancreatic maturation. © 2014 Jaramillo et al

Stage Specific Marker Expression.

<p>Relative expression of <b>(A)</b> DE specific markers after DE induction under all differentiation conditions. Upregulation was obtained for all groups with PI3KI consistently yielding highest expression. <b>(B)</b> PP specific markers after PP induction for all DE derivatives with upregulation obtained for most markers under all conditions, except for BMP4 which consistently resulted in lowest upregulation. <b>(C)</b> Pancreatic hormone expression after maturation for all groups with WNT3A and FGF2 groups achieving highest upregulation of <i>INS</i> (p>0.05), while BMP4 obtained lowest <i>INS</i> upregulation but highest <i>GLUC</i> expression. Data are represented as mean +/− SEM.</p

Significant K-means clusters.

<p>Clusters obtained for each induction condition. <b>(A)</b> WNT3A <b>(B)</b> PI3KI <b>(C)</b> FGF2 and <b>(D)</b> BMP4. The k-means clusters show close similarity of our induction conditions WNT3A and FGF2 with pancreatic organogenesis and PI3KI with definitive endoderm commitment. The markers <i>SOX17, FOXA2, HLXB9</i> are closely regulated under all the induction conditions.</p

Transcription factor dynamics.

<p><b>(A)</b> Heat map for the entire data set of genes and conditions illustrating marker progression throughout differentiation stages. The genes are organized according to the expression clusters found through hierarchical clustering. The treatments are denoted on the right hand side as prefixes to the gene names. BMP4 induction condition typically was found to cluster separately from the rest. Hierarchical clustering was performed on the mean centered and variance scaled data of transcription factor dynamics across all the four DE induction conditions. <b>(B)</b> Biplot of transcription factor dynamics assessed by principal component analysis on the mean data-set. The first component shows a demarcation of the undifferentiated and differentiated states. The second component divides the markers according to their expected appearance during <i>in vivo</i> differentiation. The PI3KI curve moves closer to the DE markers, BMP4 curve does not perform well and the WNT3A and FGF2 curves show successful pancreatic maturation.</p

Multi-stage Differentiation System.

<p><b>(A)</b> Schematic representation of multi-stage differentiation system. Detailed media formulation found in Supp table 1. DE was induced by modulation of nodal pathway simultaneously with one of four alternate pathways. PP was achieved by SHH inhibition along with retinol signaling. Maturation was induced by notch inhibition. Differentiation using WNT3A <b>(B)</b>, BMP4 <b>(C)</b>, PI3KI <b>(D)</b> or FGF2 (E) at DE stage. IF pictures show nuclear staining of SOX17 (green) and Flow cytometry shows yield of FOXA2 after DE induction, followed by nuclear PDX1 IF pictures (purple) after PP induction and cytoplasmic C-Peptide IF (red) expression yield as measured by flow cytometry after maturation.</p

Marker Progression.

<p>A representative sample (based on <i>INS</i> expression) for each group was analyzed and compared to in-vivo <b>(A)</b> pancreatic development <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094307#pone.0094307-OliverKrasinski1" target="_blank">[24]</a> in order to identify which DE pathway modulation(s) lead to better resemblance to pancreatic organogenesis. Similarities can be observed when DE induction is achieved by modulation of <b>(B)</b> FGF2, <b>(C)</b> BMP4, <b>(D)</b> WNT3A and <b>(E)</b> PI3KI while we observed that marker progression greatly differs under BMP4 induction. The different stages of pancreatic development were grouped to represent the 3 stages of the differentiation protocol. Primitive gut endoderm (PGE) and prospective pancreatic endoderm (PPE) represent definitive endoderm induction (light green) pancreatic progenitor (PP) and early endocrine progenitors (EEP) represent pancreatic progenitor induction (medium green) and endocrine progenitors (EP), immature β- cells, mature β- cells (MC) represent the maturation stage (dark green).</p

Predictors of <i>INS</i> expression.

<p>Partial least squares regression performed on the mean expression. Most PP markers show high degree of correlation to <i>INS</i> expression while there is no significant dependence on the DE markers. WNT3A and FGF2 conditions gave positive coefficients with most of the PP and mature markers indicating that these conditions are optimal for <i>INS</i> expression. R² values were above 0.995.</p