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

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

Human limbal biopsy-derived stromal stem cells prevent corneal scarring

Background: Previous research suggested that husbands’ participation in housework is positively associated with fertility choices for both women and men. We tested this association by using data of four East Asian countries. Objective: This paper examines whether the positive association between gender-equal sharing of housework participation and fertility intention in China, Japan, South Korea, and Taiwan has strengthened between 2006 and 2012. Methods: We harmonize two datasets, the 2006 East Asian Social Survey and the 2012 International Social Survey Programme. We employ OLS and ordered logit models estimators to test the association between husband’s housework participation and the ideal number of children. Results: In both 2006 and 2012, husband’s participation in housework is associated with both own and partner’s fertility intentions in 2006 and 2012. The association between the domestic division of labour and fertility has not changed between 2006 and 2012. Conclusions: Corroborating the findings of our earlier paper the results suggest that a more gender-equal domestic division of labour in East Asia is associated with higher fertility intentions in this region. The gender revolution framework offers a plausible explanation for the East Asian fertility trends between 2006 and 2012. The findings suggest that there is a stall in the pace of the gender revolution. Contribution: This paper provides a summary of the trends highlighted by the contributors to this special issue. This is also the first paper to look at the evolution of domestic division of labour and fertility preferences in four East Asian countries over time

Expression of mouse mRNA in isolated NGFR+ Cells.

<p>Expression of mouse mRNA in isolated NGFR+ Cells.</p

Co-culture with PA6 cells induces upregulation of neural crest gene expression in hES cells.

<p>hES cells were co-cultured with PA6 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. RNA was isolated at post-induction days (PID) 2, 4, 6, and 8. Expression of characteristic neural crest (NC) marker genes was determined by qPCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a> using human-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#pone-0056831-t002" target="_blank">Table 2</a>). Expression levels are calculated relative to untreated hES cells (hES = 1). Error bars show the standard deviation (S.D.) of triplicate analyses.</p

PCR Primer Sequences.

<p>PCR Primer Sequences.</p

Culture of hES cells. (

<p>A) Shows colonies of hES cells cultured on monolayers of PA6 mouse cells to induce neural differentiation. (B) NGFR+ derived hES cells after 6 days of co-culture were cultured as monolayers in serum-free N2 medium as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. The cells remain small and exhibit polygonal morphology. (C) NGFR+ derived hES cells are cultured as a monolayer in serum-containing alpha-MEM medium as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. The cells form aligned, spindle-shaped confluent monolayers. (D) Pellet from alpha-MEM cultured cells after 2-week culture. Cells are small, tightly packed and difficult to disperse. Bars show 200 µm.</p

Expression levels of CHST6 and B3GNT7.

<p>Expression of CHST6 and B3GNT7 in the hES-derived cells was compared relative to that of human corneal fibroblasts (cells that do not secrete keratan sulfate) and to freshly isolated stromal keratocytes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. Levels of both genes are comparable in NGFR+ cells to those in keratocytes but are reduced in the presence of FBS. Cell cultures are those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. Error bars show S.D. of triplicate analyses.</p

Magnetic-activated cell sorting (MACS) enriches for a population of NGFR+ cells.

<p>Single cell suspensions of hES cells after 6 days of co-culture with PA6 cells were stained with APC-labeled NGFR antibody and analyzed by flow cytometry either before or after separation of NGFR-positive cells by MACS columns as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. The blue trace shows the cells stained with non-specific isotype-matched control antibody. (<b>A</b>) Unfractionated hES+PA6 co-culture. (<b>B</b>) Flow-through, cells not bound to the NGFR MACS column. (<b>C</b>) NGFR+ cells bound and released from MACS column. The vertical bar marks the population containing <0.1% non-specific control cells (blue trace). The calculated percentages of the population (red trace) with positive staining are listed on the graph.</p

Upregulation of keratocyte-specific gene expression in pellet cultures.

<p>Expression of six genes, previously identified as up-regulated during keratocyte differentiation, was determined after 2 weeks in pellet cultures derived from either MEM+FBS or N2 monolayer cultures as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. Gene expression is calculated relative to the NGFR+ derived hES cells. Error bars represent S.D. of triplicates. All genes were significantly (p<0.05) upregulated in pellets compared to NGFR+ cells except for CHST6. Asterisks show cases in which pellet culture induced a significant (p<0.05) increase in gene expression compared to the monolayers cultures.</p