Sensitivity analysis of population based model.

<p>(A) Cellular growth sensitivity to each of the parameters, perturbed by 10%. Parameter definitions listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032975#pone.0032975.s003" target="_blank">Table S1</a>. (inset) Comparison of cellular growth output histogram from nominal (red) and perturbed (blue) parameters. (B) Number of sensitive parameters determined for each mechanistic model. Proliferation induced: A, all phenotypes; E&U, endoderm and uncommitted (hESC and mesendoderm); U, uncommitted only.</p

Convergence study of simulated cell population over various initial cell populations and total stochastic runs.

<p>Output is percent of the simulated population positive for CXCR4, averaged over all stochastic runs at Day 5.</p

Total protein content analysis via BCA assay.

<p>Data represent means ± S.D. (n = 3) represents pooled of EBs from 3 experimental repeats.</p>*<p>Significant difference compared with decellularized EB scaffold, P<0.01.</p

Validation of model with experimental gene expression data.

<p>Simulated dynamics of the undifferentiated (A (Condition A), B (Condition B)) and mesendoderm (D (Condition A), E (Condition B)) phenotypes were compared to experimental data of their respective genes, measured by qPCR (markers: experimental measurements; lines: linear connections between data points): Oct4 (Undifferentiated; C) and Brachyury (Mesendoderm; F). The simulated dynamics bands represent 4000 stochastic simulations using the optimized parameters of Mechanism B. mRNA levels were measured with time using qPCR. Data was first normalized to the housekeeping gene Gapdh then to undifferentiated cells. Fold change levels, determined by the 2^−ΔΔCt method, were then normalized to the maximum level for each respective gene (data reported as percent of maximum fold change).</p

Analysis of seeding efficiency of EB scaffolds by three different seeding methods.

<p>(A) Schematic illustration to demonstrate three different methods of cell seeding. (B) Whole mount fluorescence images of seeded EB scaffolds demonstrate three different seeding strategies on decellularized EB scaffolds examined at 6 hours after initial seeding. (C) Alamar Blue assay depicts higher seeding efficiency in both the orbital shaker and hanging drop seeding method than the static method. Bar = 450 µm. All values are mean ± SD, p<0.01 (**), n = 3, represents individual seeded EBs in 3 experimental repeats.</p

Collagen content via Sircol Collagen assay.

<p>Data represent means ± S.D. (n = 3) represents pooled of EBs from 3 experimental repeats.</p>*<p>Significant difference compared with decellularized EB scaffold, P<0.01.</p

ECM characterization of native EBs resulting from different treatment - SPT vs. RA.

<p>Immunofluorescence images of both groups of EBs – (A) SPT and (B) RA composing ECM molecules. Arrow heads indicate more cavities were found within the SPT EBs than the RA EBs. (C) Metamorph image analysis showing the quantification of the mean fluorescence intensity (MFI) of ECM markers stained in both group of EBs. Bar = 100 µm. All values are mean ± SD, p<0.05 (*), n = 6, represents pooled of EBs from 6 experimental repeats.</p

EB preparation, morphology and gene analysis.

<p>(A) Preparation scheme for EB derived ECM scaffold. (B–C) Generation of EB via rotary suspension culture resulted in homogenous-sized EBs. Histology showing both groups of native EB – (D) SPT EB and (E) RA treated EB. Arrow heads indicate numerous cavities within the SPT EBs more than RA treated EB. (F–H) Quantification of gene expression via qRT-PCR shows that RA induces neural differentiation of EBs. (F) Nestin, (G) Pax6, and (H) Brachyury. Relative expression is normalized to SPT EB. H&E staining of EB sections shows presence of neural rosettes (dotted lines) in the (K) RA treated EBs confirming neuroepithelial tissue formation in contrast to (I) SPT EB. Immunohistochemical analysis of consecutive sections demonstrated positive for anti-Nestin staining in (J) RA treated EB but minimally expressed in (L) SPT EB. All values are mean ± SD, p<0.01(**), n = 3, represents pooled of EBs from 3 experimental repeats.</p

Ensemble parameter estimation and model errors.

<p>(A) Parameter values for Mechanism B ensemble yielding errors of less than 0.025. Each parameter is compared to the most sensitive parameter, ‘d’. Color bar denotes the ensemble error for that particular parameter value. (B) Minimum ensemble error generated for each mechanistic model. Proliferation induced: A, all phenotypes; E&U, endoderm and uncommitted (hESC and mesendoderm); U, uncommitted only. Blue, Condition A; Red, Condition B.</p

Proposed differentiation scheme of hESC during endoderm induction as generated by the population-based model.

<p>Shown is the presence of mesendoderm, the lack of CXCR4 in mesoderm, and selective phenotype proliferation. ME: mesendoderm, VE: visceral endoderm.</p