Microarray analysis indicated that hESC-CEC and HCEC have the most similar genetic profile compared to HUVEC, HEK 293, and pancreatic Islet cells.

<p>Publicly available datasets for HUVEC, HEK293, and human pancreatic Islet cells were compared to ESC-CEC and pHCEC. A. Correlation analysis indicates that samples within groups have little variation. B. Principal component analysis indicates that ESC-CEC were mostly closely grouped to pHCEC, with HUVEC showing the next closest similarity. HEK 293 (HEK) and pancreatic Islet cells (ISL) were the least similar. C. Venn diagram of total probes. Of note, 76 genes appear to be uniquely expressed by ESC-CECs and HCECs which could be potential novel identifiers for corneal endothelial cells.</p

Microarray analysis indicates hESC-CEC were highly similar to primary cultured adult HCECs.

<p>A. Correlation analysis. Red color indicated little global variability between samples within group of human corneal endothelial cells (pHCECs) and human embryonic stem cell derived corneal endothelial cells (ESC-CEC). B. Volcano plot indicated most genes expressed by pHCECs and ESC-CEC were within 2 fold.</p

Genes expressed by corneal endothelial cells were present in hESC-CEC.

<p>A. COL8A1, a major component of the Descemet’s membrane was expressed is upregulated after 1 week of induction compared to hESC by QPCR utilizing the <b>ΔΔ</b>Ct method of analysis and the endogenous control PGK1. mRNA levels peaked around the second week which may have indicated that the cornea endothelial cells no longer need to produce as much COL8A1 to form Descemet’s membrane in vitro. B, C. hESC-CEC secreted COL8A1 and COL8A2 in vitro. The subcellular extracellular matrix secreted by hESC-CEC was analyzed by standard Western blot analysis for the presence of COL8A1 and COL8A2. D. Aquaporin 1 (AQP1) is a water pump that functions as part of the endothelial pump that keeps the cornea dehydrates. The increase in AQP1 expression may indicate increased ability of hESC-CEC to function as a pump. E. Many components of the corneal endothelial pump function were enriched in hESC-CEC. Carbonic Anhydrase 2 (CA2), Carbonic anhydrase 4 (CA4), Cystic Fibrosis Transmembrane conductance receptor (CFTR), Solute Carrier Family 16, member 3(SLC16A3)/Monocarboxylic acid transporter 4 (MCT4), Solute Carrier Family 16, member 7 (SLC16A7)/ Monocarboxylic acid transporter 2 (MCT2), and Solute carrier family 4, sodium bicarbonate cotransporter, member 4 (SLC4A4)/Sodium Bicarbonate cotransporter 1 (MBC1) were enriched in hESC-CECs at 1–4 weeks after induction with the exception of CFTR at 4 weeks, where it was expressed at levels similar to undifferentiated hESCs. F. hESC-CEC weeks 1–4 express about 5 fold less COL8A1 than 2 separate samples of human cultured CEC (HCEC). Normalized to hESC and the endogenous controls of 18S and GAPDH. G. hESC-CEC weeks 1–3 express similar levels of AQP1 as HCEC with the exception of 4 weeks. Normalized to hESC and the endogenous controls of 18S and GAPDH. Wk = week.</p

hESC-CEC expressed ZO-1 and Na⁺K⁺ATPaseα1 at the boundaries of cells, but not vascular endothelial markers vWF and CD31.

<p>A. Zona Occuldins-1, ZO-1 (red), an adherens tight junction marker, was expressed at the cell borders in hESC derived CEC indicating that the cells were tightly adhered and were hexagonal/polygonal in shape. B. Nuclear marker DAPI (blue) was on a different plane than ZO-1. C. Merge of ZO-1 and DAPI. D. Representative phase contrast picture from same experiment. E. Na⁺K⁺ATPase<b>α</b>1 (red) was localized to the cell borders in hESC-CEC indicating that cells had properly localized a component of the endothelial pump function. E. Nuclear marker DAPI (blue) was on a different plane than Na⁺K⁺ATPase<b>α</b>1. F. Merge of Na⁺K⁺ATPase<b>α</b>1 and DAPI. G. Human umbilical cord vein endothelial cells (HUVEC) expressed von Willebond factor (vWF, red) a marker of vascular endothelial cells, but not ZO-1 (green). H. hESC-CEC expressed ZO-1 (green), but not vWF (red). I. HUVEC expressed Platelet endothelial cell adhesion 1 (PECAM1 or CD31) but not ZO-1 (green). J. hESC-CEC expressed ZO-1 (green), but not CD31 (red). DAPI (blue) stained nucleus. Scale bars = 10 <b>μ</b>m.</p

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation

<div><p>Aim</p><p>To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.</p><p>Materials and Methods</p><p>Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.</p><p>Results</p><p>hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na⁺/K⁺ATPase<b>α</b>1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIII<b>α</b>1 and VIII<b>α</b>2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.</p><p>Conclusion</p><p>hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.</p></div

hESC markers was greatly reduced after 2 days of neural crest differentiation.

<p>A. Nanog, a marker for pluripotency, was expressed at 0.1–0.2x compared to hESC from Day 2–9. B. Nanog and another pluripotency marker, Oct4, levels were expressed at extremely low levels at Day 9 and Day 16 of cornea culture.</p

Generation of corneal endothelial cells (CEC) from human embryonic stem cells (hESC).

<p>A. Diagram outlining the process of inducing feeder free hESC with Noggin and SB431542 (dual Smad inhibitors) to become neural crest. Neural crest cells were then exposed to PDGFB and DKK2 to induce neural crest to differentiate into neural crest. Passaging cells on Day 3 improved the process. By Day 10, hexagonal/polygonal corneal endothelial cells were present in large numbers, with some progenitors remaining as clumps. B. Neural crest markers NGFR and Sox10 mRNA were expressed after 2 days of exposure to dual Smad inhibitors as determined by QPCR normalized to hESC mRNA levels of endogenous control, PGK1. C. Using 2 day method, low power phase contrast picture of corneal endothelial cells one week after induction. Center CEC colony was surrounded by progenitor-like cells. D. Using 2 day method, higher power phase contrast picture of corneal endothelial cells one week after induction. E. Using day3 dissociation method, low power phase contrast picture of corneal endothelial cells one week after induction. Notice that the CEC colony is a continuous sheet with some progenitor-like colonies. F. Using day3 dissociation method, higher power phase contrast picture of corneal endothelial cells one week after induction. G. COL8A1 mRNA, a marker for CEC, was expressed as early as 2 days of differentiation. NGFR and Sox10 mRNA, markers of neural crest, continue to be expressed during differentiation. Nanog mRNA, a hESC maker, is not detectable after 2 days of differentiation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145266#pone.0145266.g002" target="_blank">Fig 2A</a> for more detail. H. FoxC1, a marker of neural crest and potentially corneal endothelium, can be detected after 2 days of differentiation, with peak levels at Day9, similar to NGFR and Sox10. QPCR normalized to hESC mRNA levels of endogenous control, PGK1. Scale bars = 10 <b>μ</b>m. RQ = relative quantification, Endo = corneal endothelium, NC = neural crest.</p