Enhanced differentiation of human embryonic stem cells towards definitive endoderm on ultrahigh aspect ratio nanopillars

Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have highlighted that the properties of the physical environment, such as substrate stiffness, affect cellular behavior. Here, mass-produced, injection molded polycarbonate nanopillars are presented, where the surface mechanical properties, i.e., stiffness, can be controlled by the geometric design of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.0003). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of definitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure population compared to planar control. By contrast, further differentiation toward the pancreatic ­endoderm is less successful on “soft” pillars when compared to “stiff” pillars or control, indicating differential cues during the different stages of differentiation. To accompany the mechanical properties of the nanopillars, the concept of surface shear modulus is introduced to describe the characteristics of engineered elastic surfaces through micro or nanopatterning. This provides a framework whereby comparisons can be drawn between such materials and bulk elastomeric materials

Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation

The dynamic differentiation of hES cells to DE is influenced by ECMPs.

<p>(<b>a)</b> Schematic overview of the semi-optimal protocol for DE differentiation. hES cells were seeded and kept undifferentiated for 4 days to allow expansion. Subsequently the cells were treated with Wnt3a to direct the cells from the pluripotent stage into the mesendoderm stage, followed by 3 days with Activin A to direct the cells from the mesendoderm stage to the DE stage. <b>(b)</b> Representative immunofluorescence images from three independent experiments of cells cultured on the five different ECMP combinations at different time points during the DE differentiation protocol. The tested ECMP combinations were fibronectin (Fn), collagen II+fibronectin (Col2+Fn), collagen I (Col1), netrin 1+fibronectin (Ne+Fn) and vitronectin (Vn) (scale bar = 200μm). <b>(c)</b> Magnification of cultures on Col1 at day 5 (scale bar = 200μm).</p

(a-d) DE differentiation of hES cells in microtitre plates on selected ECMP combinations.

<p>(<b>a-d</b>) Quantification of Sox17 positive cells and total cell number based on immunofluorescence image analysis of cells differentiated on different ECMP combinations in microtitre plates. n = 3–6, mean ± S.E.M.). * and ** indicate statistical significant differences to Fn, P<0.05 and P<0.005 respectively. Note that not all statistical significant differences are displayed in the graphs. Full data set is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.s007" target="_blank">S3 Table</a>. Abbreviations are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.s005" target="_blank">S1 Table</a>. <b>(a)</b> Scatter plot of percentage of Sox17 positive cells and the total number of cells for each respective ECMP combination tested (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.s007" target="_blank">S3 Table</a> for descriptions), <b>(b)</b> Scatterplot of percentage Sox17 positive cells and the total yield of Sox17 positive cells (calculated as percentage of Sox17 positive cells * total cell number /100). Linear regression of data excluding six outliers (out of 65) not considered following the linear correlation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#sec009" target="_blank">results</a>). r² = >0.95. <b>(c)</b> Percentage of Sox17 positive cells of a selected ECMP combinations. <b>(d)</b> The total cell number of a selected ECMP combinations.</p

Quantitative immunofluorescence of cell cultures at different time points during the DE differentiation protocol on the different ECMP substrates.

<p>(<b>a</b>) Quantification of number of cells, which were attached 24h after seeding on the respective ECMP substrates (day 1). (<b>b</b>) Quantification of the number of cells at the different days during DE differentiation. (<b>c</b>) Quantification of the fraction Sox17 positive cells different days during DE differentiation. (<b>d</b>) Quantification of the fraction Oct3/4 positive cells at different days during DE differentiation. (n = 3, mean ± S.E.M. and * indicate statistical significant differences, P<0.05). ECMP abbreviations are explained in legend in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.g003" target="_blank">Fig 3</a>.</p