Elsevier

Experimental Eye Research

Volume 123, June 2014, Pages 16-26
Experimental Eye Research

Immediate differentiation of neuronal cells from stem/progenitor-like cells in the avian iris tissues

https://doi.org/10.1016/j.exer.2014.04.007Get rights and content

Highlights

  • Neuronal stem/progenitor cells were found in chick iris stroma.

  • By applying a new method immediate neuronal differentiation occurs.

  • Neither serum nor growth factors are required for neuronal differentiation.

  • EGF enhances neuronal differentiation and neurite growth.

Abstract

A simple culture method that was recently developed in our laboratory was applied to the chick iris tissues to characterize neural stem/progenitor-like cells. Iris tissue is a non-neuronal tissue and does not contain any neuronal cells. In the present study we isolated iris tissues from chick embryos just prior to hatching. The isolated iris pigmented epithelium (IPE) or the stroma was embedded in Matrigel and cultured in Dulbecco's MEM supplemented with either fetal bovine serum or the synthetic serum replacement solution B27. Within 24 h of culture, elongated cells with long processes extended out from the explants of both tissues and were positively stained for various neuronal markers such as transitin, Tuj-1 and acetylated tubulin. After a longer culture period, cells positive for photoreceptor markers like rhodopsin, iodopsin and visinin were found, suggesting that the iris tissues contain retinal stem/progenitor-like cells. Several growth factors were examined to determine their effects on neuronal differentiation. EGF was shown to dramatically enhance neuronal cell differentiation, particularly the elongation of neuronal fibers. The addition of exogenous FGF2, however, did not show any positive effects on neuronal differentiation, although FGF signaling inhibitor, SU5402, suppressed neuronal differentiation. The results show that neuronal stem/progenitor-like cells can differentiate into neuronal cells immediately after they are transferred into an appropriate environment. This process did not require any exogenous factors, suggesting that neural stem/progenitor-like cells are simply suppressed from neuronal differentiation within the tissue, and isolation from the tissue releases the cells from the suppression mechanism.

Introduction

Neural stem/progenitor cells have been found in several different regions of the ocular tissues, including the ciliary marginal zone, ciliary body, neural retina, retinal pigmented epithelium (RPE) and the iris (Ohta et al., 2008, Wohl et al., 2012). In lower vertebrates, such as fish and amphibians, these stem cells are recruited during in vivo retinal regeneration after the retina has been damaged. In amphibians, for example, the ciliary marginal zone (CMZ) cells, as well as the retinal pigmented epithelial (RPE) cells play a crucial role in the regeneration of the lost part of the retina in mature animals (Hitchcock et al., 2004, Yoshii et al., 2007, Karl and Reh, 2010, Miyake and Araki, 2014). The mode of regeneration, however, considerably differs from species to species (Raymond and Hitchcock, 1997, Reh and Fisher, 2001, DelRio-Tsonis and Tsonis, 2003, Araki, 2014). In the chick, the intraretinal stem-like cells seem to play the most important role in regeneration (Fisher and Reh, 2001).

The ciliary body and the iris are not in direct contact with the retina. Therefore, these structures are not thought to play any role in in vivo retinal regeneration and repair. Neural stem/progenitor cells in these tissues, however, have been shown to differentiate into neuronal and photoreceptor cells under certain culture conditions, providing possible sources of stem cells for neural regeneration, especially in mammals (Ahmad et al., 2000, Tropepe et al., 2000, Haruta et al., 2001, Sun et al., 2006, Asami et al., 2007, Wohl et al., 2012). The regenerative capacity of the retina is very limited in mammals and birds, particularly in mature animals, compared to lower vertebrates, such as amphibians (Osakada et al., 2007). Therefore, the use of potential tissues outside the retina has practical importance. The iris is particularly interesting because this tissue is one of the few tissues that can be isolated from human eyes; therefore, the iris has significant clinical importance as a possible donor tissue.

Little information is available on the neurogenic potential of the iris tissue of mammals and birds (Davis-Silberman and Ashery-Padan, 2008). Chick and mouse iris pigmented epithelium (IPE) at the postnatal stage have been shown to exhibit neural stem/progenitor properties, and these cells can give rise to neurons and glial cells under certain culture conditions (Sun et al., 2006, Asami et al., 2007). In those studies the standard protocol for neuronal stem cells, which is generally used in stem cell biology, was applied; the IPE was first dissociated into single cells, which were then subjected to rotation culture to form neurospheres. The spheres were then cultured under stationary conditions on a substratum that enhances neuronal differentiation in the presence of FGF2 and/or analogous neural growth factors.

Recently it has been shown that a much wider repertoire of tissues contain potential sources of neuronal stem cells. For example, tissues of mesenchymal origin have been shown to be a source of neuronal cell types; a multipotent precursor cell population has been isolated from adult mammalian dermis and these cells were shown to differentiate into cells with neuronal phenotypes (Toma et al., 2001, Toma et al., 2005, Fernandes et al., 2008). In the ocular tissues, multipotent stem cells were isolated from the cornea stroma tissue of the adult mouse, and these cells were shown to differentiate into chondrocytes and neuronal cells. A similar result was obtained with the adult mouse iris stroma (Kikuchi et al., 2011), although the potential for neuronal differentiation was not well examined in these studies. From these results, the concept of a neural crest-derived stem/progenitor cell population was proposed, and these tissues are thought to maintain multipotency even into adulthood (Motohashi et al., 2014).

We have been studying amphibian retinal regeneration, and we recently developed a novel gel-embedding culture method that enables RPE cells to regenerate a 3-D structure of the retina under tissue culture conditions (Araki, 2007, Kuriyama et al., 2009). The method consists of several steps, including treating target tissues with dispase and then embedding the tissue in Matrigel (Nabeshima et al., 2013). In the present study, we cultured IPEs and iris stroma using this gel-embedding method. This new procedure is very simple compared to the standard protocol for neural differentiation from stem cells, and the results we obtained were very unique and significant in several aspects. First, neuronal differentiation occurred immediately after the tissue was transferred to the culture conditions, normally within 24 h. This pace is tremendously fast compared to previously reported cases. Second, neuronal differentiation did not require growth factors, such as FGF2, or fetal bovine serum to be added to the medium. Third, the properties of mesenchyme-derived ocular stem/progenitor-like cells were fully described. Thus, the present culture system provides a novel approach to characterize tissue stem/progenitor-like cells, and our results suggest that tissue stem/progenitor-like cells may not have to be induced into neurons using artificial means. Instead, these cells only need to be released from restraints within the tissue to differentiate into neuronal cells.

Section snippets

Tissue culture

Chick embryos at 17–19 days of incubation were used. Eyes were enucleated from the embryos, and the anterior parts were cut along the equatorial plane. The lens and ciliary body were removed. The iris was dissected as a donut shape and was treated with dispase (50 unit/ml, Godo Shusei Ltd.) at 37 °C for 10–24 h. The iris pigmented epithelium (IPE) and the stroma were separated from each other using forceps (Fig. 1). Each tissue was further cut into smaller fragments, typically 8–10 pieces. Each

Differentiation of neuronal cells from cultured iris tissues

When tissue fragments of the iris pigmented epithelium (IPE) were embedded in Matrigel and cultured, cells were observed migrating out from the explant on Day 1 (1 day in culture) (Fig. 2). These migrating cells were elongated and often contained melanin granules. On Day 2, after 48 h in culture, more cells were found outside of the explant, and these cells were more elongated and had longer processes than the cells on Day 1. On Day 4, a dense fiber-like process network had formed around the

Discussion

In the present study, we demonstrate that neuronal cells differentiate immediately (within 24 h after culturing starts) from iris tissue when it is transferred to culture conditions. We found that such neuronal differentiation did not require any additives, such as fetal bovine serum or FGF2. These results were not expected and are surprising considering previous reports on stem/progenitor-like cells. The present results suggest a new mechanism for the regulation of tissue stem cells, as

Acknowledgments

We would like to thank Dr. Molday, Dr. Kuo and Dr. Shichida for providing anti-rhodopsin, anti-visinin and anti-iodopsin antibodies, respectively. We would also like to thank Dr. T Weimbs (UCSB) for his careful reading of the manuscript. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT KAKENHI Grant Number 23124506) and by Nara Women's University Intramural Grant for Project Research.

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