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Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

Chan, AA and Hertsenberg, AJ and Funderburgh, ML and Mann, MM and Du, Y and Davoli, KA and Mich-Basso, JD and Yang, L and Funderburgh, JL (2013) Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype. PLoS ONE, 8 (2).

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Abstract

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities. © 2013 Chan et al.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Chan, AA
Hertsenberg, AJanh77@pitt.eduANH77
Funderburgh, MLmlfunder@pitt.eduMLFUNDER
Mann, MMmmm28@pitt.eduMMM28
Du, Yyiqindu@pitt.eduYIQINDU
Davoli, KAkatherine.davoli@pitt.eduKAD6
Mich-Basso, JDjdm114@pitt.eduJDM114
Yang, Llyang@pitt.eduLYANG
Funderburgh, JLjlfunder@pitt.eduJLFUNDER
Date: 21 February 2013
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 8
Number: 2
DOI or Unique Handle: 10.1371/journal.pone.0056831
Schools and Programs: School of Medicine > Developmental Biology
School of Medicine > Ophthalmology
Refereed: Yes
Other ID: NLM PMC3578879
PubMed Central ID: PMC3578879
PubMed ID: 23437251
Date Deposited: 28 Mar 2013 16:46
Last Modified: 05 Feb 2019 03:55
URI: http://d-scholarship.pitt.edu/id/eprint/17871

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