Novel Pancreatic Endocrine Maturation Pathways Identified by Genomic Profiling and Causal Reasoning

<div><p>We have used a previously unavailable model of pancreatic development, derived <em>in vitro</em> from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.</p> </div

IL6, the top CRE prediction, has effects on expression of endocrine markers.

<p>(A) Treatment of pancreatic aggregates with IL-6 induces de novo gene expression of the pro-endocrine transcription factors NEUROG3 and NKX2.2, indicating commitment of pancreatic progenitor cells into the endocrine lineage. Noggin induction of these genes resulted in 8-fold increases (data not shown) (B) Gene expression in response to IL-6 was compared between whole aggregates (mixture of pancreatic progenitors and endocrine cells) and cultures of enriched endocrine cells (depleted of pancreatic progenitors). Induction of NKX2.2 expression was only seen in whole aggregates, consistent with the role of IL-6 in converting pancreatic progenitors into new endocrine cells. Enhanced expression of NEUROD1, IAPP, and SOMATOSTATIN seen in response to IL-6 in purified endocrine cells, suggesting IL-6 has additional roles in committed endocrine cells. No significant differences seen in INSULIN or GCG gene expression. Statistical testing using a standard t-test was performed.</p

Expression of cholesterol metabolizing genes shows a strong time dependence.

<p>(A) The cholesterol biosynthesis pathway from squalene-PP (top left) to cholesterol (top right) showing all enzymes and some intermediates. Enzymes whose gene expression patterns fall into the largest cluster seen in the heatmap of the expression levels for these enzymes (B) are colored purple, other enzymes are light blue. LIPA and SOAT1/2 interconvert cholesterol and cholesterol ester. The cholesterol sensitive miRNA-33a shows a similar expression pattern (C) to the main cluster.</p

Induction of insulin expression is not accompanied with epigenetic changes.

<p>(A) Gene expression (blue) and H3K4me³ level (red) profiles for SOX17. The horizontal dashed line indicates the background H3K4me³ level. (B) H3K4me³ reads piled up over the SOX17 gene body at days 0, 2 and 11. The start and end points of SOX17 are indicated by dashed lines. (C&D) As for (A&B) but for Insulin.</p

Top 20 protein causal drivers of early pancreatic endoderm formation between day 8 and day 11.

<p>The number of correctly, incorrectly and ambiguously explained gene expression observations are given for each gene as well as the predicted direction of regulation (up meaning activation/down meaning inhibition). The notes for each gene indicate that in cases where the gene is already associated with beta cell function whether it is generally considered a positive or negative regulator of beta cell differentiation, proliferation (growth) or apoptosis. All hypotheses pass correctness and enrichment p-value thresholds of 10⁻⁵.</p

Epigenetic changes can be used to identify novel regulatory miRNAs.

<p>(A–C) Gene expression (blue), miRNA expression (green) and H3K4me³ levels for CD47, ITGB1 and ANP32B and the miRNAs associated with them. In all 3 cases the miRNA is predicted to regulate the relevant gene and is also anti-correlated in level. In (A) H3K4me³ levels correlate closely with the gene expression, whilst in (B & C) there is no correlation, suggesting a stronger role for the miRNA regulation.</p

Transcriptome analysis shows the reproducibility of the differentiation protocol.

<p>(A) Genome wide gene expression correlation heatmap between samples. Samples are clustered by the Euclidean distance between rows/columns and single linkage clustering. The colored bar along the top of the heatmap indicates the timepoint at which the sample was taken (pink: day 0, maroon: day 11). (B) Heatmap of expression of selected markers. The developmental stage is indicated by the labels to the left of the heatmap.</p

Adipocytes derived from C2C12 cells by BMP6 action exhibit bioenergetics phenotype characteristic of brown adipocytes.

<p>Oxygen consumption rate (OCR) measurements for (A) basal respiration, (B) ATP turn over, (C) proton leak and (D) fatty acid oxidation using a Seahorse XF24 extracellular flux analyzer following sequential administration of oligomycin, FCCP and rotenone. Results in (A) are plotted relative to unstimulated condition set to 100. Data in (B) and (C) are expressed as percent of basal respiration. Results represent cumulative analyses of multiple independent biological replicates from three independent experiments (n≥6 for each condition per experiment). (D) Fatty acid (palmitate) oxidation capacity is represented as increased OCR in response to palmitate-BSA complex compared to BSA only as control. Results represent cumulative analyses of multiple independent biological replicates from three independent experiments (n≥9 for palmitate-BSA and ≥9 for BSA for each condition, mean ± SD). <i>*p<0.05</i> versus no BMP.</p

Two days of BMP6 stimulation in C2C12 cells induces genes characteristic of preadipocytes.

<p>Bar graph representation of genes characteristic of (<b>A</b>) fibroblast derived preadipocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092608#pone.0092608-Gupta1" target="_blank">[39]</a> and (<b>B</b>) murine interscapular brown preadipocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092608#pone.0092608-Yin1" target="_blank">[14]</a>, derived from Affymetrix array dataset generated from C2C12 cells stimulated with 250 ng/mL BMP6 and harvested at 0, 6, 12, 24 and 48 hours post BMP6 stimulation. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092608#pone-0092608-g007" target="_blank">Figure 7B</a>.</p

BMP6 induces lipid accumulation and <i>Ucp-1</i> expression in primary human skeletal muscle precursor cells from multiple donors.

<p>Primary human skeletal muscle precursor cells from type II diabetic or non diabetic donors isolated from distinct muscle sites were stimulated with or without BMP6 or BMP7 (250 ng/mL) for two days followed by adipogenic differentiation (as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092608#pone-0092608-g001" target="_blank">Figure 1A</a>). Age/gender/diseased state for each donor and the site of myoblast isolation is specified. <i>Left panels</i>: Lipid accumulation in different donor cells was assessed by Oil-Red-O staining and a representative image is shown (magnification: 40X). <i>Right panels</i>: Q-PCR analyses of <i>Ucp-1</i> in BMP6 or BMP7 (250 ng/mL) pretreated cells followed by adipogenic differentiation and stimulation with or without 10 μM forskolin for 4 hours. The expression in untreated cells (no BMP pretreatment and no forskolin stimulation: no BMP-no F) was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), <i>*p<0.05</i> versus no BMP-F, Student's <i>t</i> test.</p