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Differentiation and Molecular Profiling of Human Embryonic Stem Cell-Derived Corneal Epithelial Cells

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Differentiation and Molecular Profiling of Human Embryonic Stem Cell-Derived Corneal Epithelial Cells INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 33: 1597-1606, 2014 Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells J. BRZESZCZYNSKA1, K. SAMUEL2, S. GREENHOUGH3, K. RAMAESH4, B. DHILLON5, D.C. HAY3 and J.A. ROSS1 1Tissue Injury and Repair Group, MRC Centre for Regenerative Medicine, 2SNBTS Cellular Therapy Group, 3Pluripotent Stem Cell Hepatocyte Development Group, MRC Centre for Regenerative Medicine, Chancellor's Building, University of Edinburgh, Edinburgh EH16 4SB; 4Tennent Institute of Ophthalmology, Gartnavel General Hospital, Glasgow G12 0YN; 5Department of Clinical and Surgical Sciences, Ophthalmology Section, Princess Alexandra Eye Pavilion, Royal Infirmary of Edinburgh, Edinburgh EH3 9HA, Scotland, UK Received January 16, 2014; Accepted February 14, 2014 DOI: 10.3892/ijmm.2014.1714 Abstract. It has been suggested that the isolation of scalable patients with such problems require extensive surgical populations of limbal stem cells may lead to radical changes procedures, which include stem cell transplantation and immu- in ocular therapy. In particular, the derivation and transplanta- nosuppressive therapy. As a result, this approach is limited and tion of corneal stem cells from these populations may result a number of other concerns exist, which include the potential in therapies providing clinical normality of the diseased or for infection, the limited supply of donor tissue and the absence damaged cornea. Although feasible in theory, the lack of donor of a well-defined specific limbal stem cell marker. In addition, material in sufficient quantity and quality currently limits such the long-term preservation of the corneal rims decreases the a strategy. A potential scalable source of corneal cells could viability of limbal stem cells, thereby reducing the quality of be derived from pluripotent stem cells (PSCs). We developed the available biological material for transplantation. an in vitro and serum-free corneal differentiation model which An alternative source of corneal cells are pluripotent stem displays significant promise. Our stepwise differentiation model cells (PSCs) of embryonic origin [embryonic stem cells (ESCs)] was designed with reference to development and gave rise to or induced PSCs (iPSCs). PSCs can expand into large numbers, cells which displayed similarities to epithelial progenitor cells whilst maintaining their differentiation potential, promising which can be specified to cells displaying a corneal epithelial an almost unlimited supply of human somatic cell types. phenotype. We believe our approach is novel, provides a robust Moreover, iPSCs can serve as an immunologically-matched model of human development and in the future, may facilitate stem cell resource which has obvious clinical application. the generation of corneal epithelial cells that are suitable for We developed a robust in vitro corneal derivation proce- clinical use. Additionally, we demonstrate that following dure from human ESCs (hESCs) and examined the molecular continued cell culture, stem cell-derived corneal epithelial cells and cellular changes in ocular biology as differentiation undergo transdifferentiation and exhibit squamous metaplasia ensued. hESCs, grown on collagen IV in medium containing and therefore, also offer an in vitro model of disease. corneal limbal fibroblast-conditioned medium and fetal calf serum, have previously been shown to express limbal epithe- Introduction lial markers, such as CK3 and CK12, but also CK10, a skin epithelial cell marker (1). In order to address this anomaly, Chronic ocular surface problems result in debilitating visual the central aim of this study was to differentiate true corneal impairment and corneal stem cell dysfunction. Invariably, epithelial progenitor cells from human ESCs under defined conditions. In this study, we evaluated the gene expression profiles of hESCs (H9 and RCM1) following a corneal differentiation protocol which relied on conditioned medium from human Correspondence to: Professor James A. Ross, Tissue Injury and limbal fibroblasts. Crucially, both hESC lines were maintained Repair Group, MRC Centre for Regenerative Medicine, Chancellor's on Matrigel without a mouse embryonic fibroblast feeder layer in Building, University of Edinburgh, 49 Little France Crescent, Edinburgh serum-free growth medium (mTeSR-1; StemCell Technologies, EH16 4SB, UK E-mail: [email protected] Vancouver, BC, Canada) which creates a safer environment (lower risk of contamination or disease transmission during Key words: corneal epithelium, squamous metaplasia, human culture) for the differentiation of hESC-derived corneal cells embryonic stem cells, transdifferentiation, cytokeratins, leucine-rich with features and functions similar to limbal stem cells (LSCs) repeat containing G protein-coupled receptor 5 which could potentially facilitate ocular surface regeneration. In order to verify the veracity of our approach, we characterised 1598 BRZESZCZYNSKA et al: DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELL-DERIVED CORNEAL CELLS the molecular profile of hESC-derived corneal cells by investi- secondary biotinylated antibodies (Dako-Agilent Technologies, gating 12 different molecular markers of corneal epithelial and Santa Clara, CA, USA) were added at a dilution of 1:500 for limbal stem cells, but also included CK10, CK19, vimentin and 1 h at room temperature. The cells were finally counterstained α-actin, which are markers characteristic of pathological ocular with haematoxylin and then visualized and captured using a conditions. We wished to determine whether the differentiation Leica DM IRB microscope. system generates healthy corneal-like epithelial cells with no pathological features, such as abnormal transdifferentiation Flow cytometry. Flow cytometry was performed as previ- (CK10, CK19 or vimentin expression). We also examined the ously described (6) to determine cell differentiation within expression of genes which are global markers of adult stem the culture and to asses any apoptotic response of the hESCs cells, including p63α (a marker of epithelial stem cells, which to the culture conditions. The samples were analysed using a supports stem cell proliferation and regulation in epithelial FACSCalibur flow cytometer and the results were analysed cells) and ATP-binding cassette, sub-family G (White), using CellQuest Pro software (both from BD Biosciences, member 2 (ABCG2; a marker of basal limbal epithelial stem San Jose, CA, USA). cells) but also leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), which has been identified as a novel stem Total RNA isolation. RNA was isolated using TRIzol reagent cell marker (2) and limbal stem cell marker (3). Furthermore, (following the manufacturer's instructions) and 1 µg of RNA the expression of paired box 6 (Pax6) (4), the master control was reverse transcribed into cDNA using random hexamers gene for eye morphogenesis, was investigated. according to the manufacturer's instructions (all reagents were obtained from Promega, Madison, WI, USA). Prior Materials and methods to reverse transcription (RT), total RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Labtech Isolation and culture of human limbal epithelial cells. Surplus International, Ltd., Uckfield, UK). corneal buttons were obtained from the Manchester Eye Bank, Manchester Royal Eye Hospital, Manchester, UK. All corneas Reverse transcription-polymerase chain reaction (RT-PCR) were received from donors with specific consent for research and quantitative PCR (qPCR). In order to analyse the gene purposes, as previously described (3). The corneal rings were expression profiles of the cells, several genes were selected washed with CnT-20 medium and the cells were prepared and for RT-PCR analysis, using total RNA derived from the H9 cultured as previously described (3). Corneal epithelial cells and RCM1 cells before and during differentiation toward a were cultured up to passage 2 (P2) and were then assessed for corneal phenotype. In addition, the quantitative validation of molecular profiles. the expression of selected genes was performed by qPCR on Custom TaqMan array plates (Applied Biosystems, Foster City, Culture and differentiation of hESCs into epithelial-like CA, USA) (n=3 for H9 and RCM1 differentiated samples at cells. Both hESC (H9 and RCM1) lines were maintained on each time point). Matrigel without a mouse embryonic fibroblast feeder layer. RT-PCR reactions were performed for the differenti- The day after the plating of the colonies, the serum-free growth ated H9 and RCM1 cells at each selected time point using medium (mTeSR-1; StemCell Technologies) was replaced by master mix [Taq buffer, 25 mM MgCl2, 10 mM dNTPs, human limbal fibroblast conditioned medium and this was Taq polymerase (1:5), 10 µM primers, dH2O, cDNA (20 ng/ changed every second day according to a previously described µl)] (all reagents were from Promega). RT-PCR reactions were method (1). Differentiation was identified based on changes performed using the Veriti 96 well Thermal Cycler (Applied in the morphology of the cells, the downregulation of the Biosystems), at 94˚C for 5 min, followed by 35 cycles at 94˚C expression of stem cell-specific markers [Nanog homeobox for 1 min, 56˚C for 1 min, 72˚C for 1 min. The PCR products (Nanog) and octamer-binding transcription factor 4 (Oct4)], were resolved and visually analysed on a 2% agarose gel in and concomitant upregulation of markers for differentiated cell 1X TAE (triacetate
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