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Hiroshi Tanaka, Yoshinori Nakai, Makoto Fukuta, Morio Ueno, Makoto Ikeya, Junya Toguchida, Shigeru Kinoshita; Efficient induction of neural crest cells from human pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6040.
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© ARVO (1962-2015); The Authors (2016-present)
Neural crest cells possess multi-potency to differentiate many kinds of somatic cells, such as mesenchymal stem cells, peripheral neurons, and muscle cells. In the case of eye development, corneal endothelium, corneal keratocytes, and trabecular meshwork cells which construct the anterior segment are derived from neural crest cells. The efficient induction system of neural crest cells from human pluripotent cells is a useful tool to regenerate the anterior segment of the eye. The purpose of this present study was to develop a novel method for neural crest induction from human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells.
hES/hiPS cells were isolated from feeder cells and were then cultured on Matrigel™ (BD Biosciences, Franklin Lakes, NJ)-coated dishes in serum-free medium with chemical compounds. The proportion of CD271 (p75NTR; one of the markers of neural crest cells) highly-positive cells were detected by use of flow cytometry. The time-period and concentration of the treatment of chemical compounds for the induction of CD271 highly-positive cells was optimized. The expression of neural crest markers was examined in CD271 highly-positive cells by immunofluorescence analysis.
The induction rate of CD271 highly-positive cells peaked by 7-days treatment. The proportion of CD271 highly-positive cells was up to 30% with a 7-day treatment of a transforming growth factor beta (TGF-β) inhibitor (10µM). The efficiency of CD271 highly-positive cells increased by the 7-day treatment of a mixture of the TGF-β inhibitor and a glycogen synthase kinase 3 (GSK-3) inhibitor. The optimized concentration of GSK-3 inhibitor (1.0µM) increased CD271 highly-positive cells up to 80%. CD271 highly-positive cells co-expressed the other markers for neural crest cells, such as HNK-1 and integrin alpha4. AP2α and PAX6 were expressed in CD271 highly-positive cells.
We developed a fundamental system to regenerate corneal endothelium, corneal keratocytes, and trabecular meshwork cells from human ES/iPS cells through neural crest cells.
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