J Ge1, Y Guo, Z Wang, H Liu, Z Yang, Y Li, S Li. 1. Zhongshan Ophthalmic Center, Sun Yat-sen University of Medical Sciences, Guangzhou 510060, China.
Abstract
PURPOSE: To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy. MATERIALS AND METHODS: Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium, BRL-CM) was used for culturing embryonic stem cells (ES-D3 cell line). Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy. Based on the methods used by Bain et al, we modified the methods and used retinoic acid (RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside (Ara-C) as inhibitor of proliferative cells. The growth of the cells was observed under phase contrast microscope. RESULTS: ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated. Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces. In the first day after the adding of retinoic acid, some neuron-like cells with one, two or more processes were present. In the second day after adding RA and the first day after the plus of 10 microns Ara-C, a large amount of neuron-like cells appeared, with the formation of neuron-like networks. CONCLUSIONS: Combined use of RA and Ara-C can induce ES cells to differentiate into neuron-like cells. Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.
PURPOSE: To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy. MATERIALS AND METHODS: Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium, BRL-CM) was used for culturing embryonic stem cells (ES-D3 cell line). Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy. Based on the methods used by Bain et al, we modified the methods and used retinoic acid (RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside (Ara-C) as inhibitor of proliferative cells. The growth of the cells was observed under phase contrast microscope. RESULTS: ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated. Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces. In the first day after the adding of retinoic acid, some neuron-like cells with one, two or more processes were present. In the second day after adding RA and the first day after the plus of 10 microns Ara-C, a large amount of neuron-like cells appeared, with the formation of neuron-like networks. CONCLUSIONS: Combined use of RA and Ara-C can induce ES cells to differentiate into neuron-like cells. Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.