Figure 1.
Multi-stage Differentiation System.
(A) 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), BMP4 (C), PI3KI (D) 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.
Figure 2.
Morphological analysis of the cells after (A) DE induction showing heterogeneous populations under all conditions (Scale bar: 12.5 µM), and (B) Cell death after 24 h of DE treatment. Death was comparable in all groups, except for PI3KI which resulted in considerably higher death (C) Increase in cell number was observed after DE and PP stage. Beyond the PP stage there is a slight decrease in cell number for all conditions except PI3KI. (D–F) Cell cycle analysis of the differentiating cellular population under different conditions, as analyzed and quantified by flow cytometry. Shown is the fraction of the population in the G1 (D), S (E), and G2/M (F) phases of the cell cycle. Data are represented as mean +/− STDEV.
Figure 3.
Stage Specific Marker Expression.
Relative expression of (A) DE specific markers after DE induction under all differentiation conditions. Upregulation was obtained for all groups with PI3KI consistently yielding highest expression. (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. (C) Pancreatic hormone expression after maturation for all groups with WNT3A and FGF2 groups achieving highest upregulation of INS (p>0.05), while BMP4 obtained lowest INS upregulation but highest GLUC expression. Data are represented as mean +/− SEM.
Figure 4.
A representative sample (based on INS expression) for each group was analyzed and compared to in-vivo (A) pancreatic development [24] 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) FGF2, (C) BMP4, (D) WNT3A and (E) 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).
Figure 5.
Transcription factor dynamics.
(A) 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) 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 in vivo 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.
Figure 6.
Clusters obtained for each induction condition. (A) WNT3A (B) PI3KI (C) FGF2 and (D) 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 SOX17, FOXA2, HLXB9 are closely regulated under all the induction conditions.
Figure 7.
Partial least squares regression performed on the mean expression. Most PP markers show high degree of correlation to INS 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 INS expression. R2 values were above 0.995.