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 Turbinate-derived Mesenchymal Stem Cells Differentiated into Keratocyte Progenitor Cells

“…The similar tendency was observed in human embryonic cells and corneal stromal stem [11,28]. The expressions of embryonic ocular precursor markers, PAX6 (Figure Figure 1D), which are not detected in adult stem cell or fibroblast, decreased but were still detectable after 14 d in KDM.…”

“…The only effective therapy for corneal opacity is transplantation of corneal allografts. The transplantation of allograft has a high success rate, but still has problems, including shortage of donor corneas, and immunological rejection [11]. And CSSC derived from neural crest spontaneously differentiate into keratocyte in vivo or in vitro but stromal population of CSSC is less than 1% [12].…”

“…Mesenchymal stem cells (MSCs) are adult mesenchymal progenitor cells that can differentiate into cells of various connective tissue lineages, including bone [19], cartilage [20], adipocyte [21] and keratocyte [11]. MSCs are good materials for cell therapy because of their accessibility, easy isolation, capability of preservation, and failure to induce immunological rejection [22].…”

Generating minicorneal organoids from human induced pluripotent stem cells

“…Cell replacement therapy using autologous or allogeneic adult limbal grafts has been the standard treatment for patients with severe limbal stem cell deficiency (LSCD) (Rama et al, 2010;Sangwan et al, 2011;Basu et al, 2016). However, in the case of bilateral epithelial defects and for the treatment of conditions affecting the stromal and endothelial cell layers, alternative stem cell sources such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been explored with a view to generating the various corneal cell types (Ahmad et al, 2007;Shalom-Feuerstein et al, 2012;Hayashi et al, 2012;Sareen et al, 2014;Mikhailova et al, 2014;Chan et al, 2013;Zhang et al, 2014;Chen et al, 2015;McCabe et al, 2015).…”

Role of aquaporins in cell proliferation: What else beyond water permeability?

“…58,[137][138][139] Upregulation of AQP expression has been observed under stimuli such as dehydration in the kidney leading to overexpression of AQP2 and AQP3, [140][141][142] or hypoxia in the lung producing overexpression of AQP1 108,111,142,143 and in none of these cases have the proposals to explain AQPs overexpression been at all related to tumor proliferation. More recent studies have demonstrated involvement of AQP1 in the differentiation of stem-like cells in rat bile duct formation, 144 development of human corneal keratocytes 145 and renewal of limbal basal epithelium from a corneal epithelial stem cell niche, 146 all important roles associated with cell proliferation and differentiation. Moreover, impaired proliferation, migration and neuronal differentiation has been demonstrated in adult neural stem cells derived from AQP4 knockout mice 87 indicating a role for AQP4 in the activation and differentiation of the mice SVZ neurogenic niche.…”