Influence of self-assembling peptide nanofibre scaffolds on retinal differentiation potential of stem/progenitor cells derived from ciliary pigment epithelial cells.

Aim of the study was to investigate the influence of the self-assembling peptide nanofibre scaffolds (SAPNs) on the growth, proliferation and retinal neuronal differentiation of the stem/progenitor cells (SCs) derived from the ciliary pigment epithelium (CPE) of human cadaveric eye. Here SAPNs (RADA16-I, PM), which is well described in previous studies, commercially available and xeno-free. The CPE cells isolated were cultured in DMEM/F12 supplemented with N2 and growth factors such as basic fibroblast growth factor and epidermal growth factor, encapsulated in the scaffolds. The entrapped SCs actively expanded and formed clone-like clusters in the scaffolds. Many cells in the cluster were proliferating, as revealed by 5-bromo-2-deoxyuridine uptake and could be maintained for up to 6 days and expressed neural progenitor markers such as ß-III tubulin, Nestin, Pax6 and Musashi1. Upon differentiation of these cells in conditioned medium, the cells exhibited retinal neuronal markers such as s-Opsin, rhodopsin and Recoverin. The RT2 profiler polymerase chain reaction array experiments showed selective gene expression, possibly involved in neural stem/progenitor cell adhesion and differentiation. These findings suggest the suitability of the three-dimensional culture system for the proliferation and maintenance of CPE stem/progenitor cells (CPE-NS) and for possible use in ex vivo studies of small molecules, drug deliveries for retinal diseases and for use in combination with directed stem/progenitor cell differentiation. and ultimately for tissue replacement therapies. Copyright © 2014 John Wiley & Sons, Ltd.

Profiling of DNA and histone methylation reveals epigenetic-based regulation of gene expression during retinal differentiation of stem/progenitor cells isolated from the ciliary pigment epithelium of human cadaveric eyes.

Millions of people around the world suffer from retinal degenerative diseases at varying degrees of vision loss including, complete blindness that are caused by the damage to cells of the retina. The cell replacement therapy could be a promising tool in treating these conditions, since the stem/progenitor cells could be isolated form adult ciliary pigment epithelial cells and could be differentiated into retinal phenotypes in vitro and could be of great importance. The present study aims to identify the role of epigenetic regulators during cellular differentiation, which involves loss of pluripotency and gain of lineage and cell type-specific characteristics. We analyzed DNA methylation and Histone methylation-H3K4me3 and H3K27me3 in ciliary body derived lineage committed progenitor to terminally differentiated cells. Our results demonstrate that several promoters including pluripotency and lineage specific genes become methylated in the differentiated population, suggesting that methylation may repress the pluripotency in this population. On the other hand, we detect bivalent modifications that are involved in the process of differentiation of stem/progenitor cells. Therefore, this data suggest a model for studying the epigenetic regulation involved in self renewal, pluripotency and differentiation potential of ciliary stem/progenitor cells. This work presents the first outline of epigenetic modifications in ciliary derived stem/progenitor cells and the progeny that underwent differentiation into retinal neurons/glial cells and shows that specific DNA methylation and histone methylations are extensively involved in gene expression reprogramming during differentiation.

Gene expression profiles and retinal potential of stem/progenitor cells derived from human iris and ciliary pigment epithelium.

The aim of our study was to isolate and characterize the properties of neurospheres and differentiated cellular progeny derived from iris and ciliary pigment epithelial (IPE and CPE) cells of human cadaveric eyes. In this study we investigated the gene expression profiles of the stem/progenitor cells and the differentiated progeny derived from IPE and CPE cells, as the changes underlying differentiation of the stem/progenitor derived from the IPE and CPE cells from human cadaveric eye are essentially unknown. The IPE and CPE cells from human cadaver eyes were cultured in the presence of mitogens to generate neurospheres (NS) and the growth characteristics were evaluated. The Neurospheres (NS) were plated under conditions inducing differentiation and their potential was analyzed by RT-PCR, immunocytochemistry, calcium imaging studies and microarray studies to measure the changes involved in the process of differentiation. The IPE and CPE cells can generate NS containing progenitor cells in the presence of mitogens and were capable of producing different retinal cell types as demonstrated by RT-PCR and immunocytochemistry. The cluster analyses of the differentially expressed genes show the dynamic changes that occur during the course of IPE and CPE neurospheres differentiating into retinal progeny. The study gives clues towards the changes that occur during differentiation from NS into retinal progeny. In the present study we have demonstrated the expansion and maintenance of SCs from IPE and CPE of cadaveric eyes. These cells maintain their self-renewal properties and the ability to differentiate along retinal cell lineages and hence could be a practical source of donor cells for ex-vivo stem cell therapy during retinal degeneration.

Signal transduction pathway involved in the ex vivo expansion of limbal epithelial cells cultured on various substrates.

BACKGROUND & OBJECTIVE: Ex vivo expansion of the limbal epithelial cells activates the nerve growth factor (NGF) mediated downstream signal transduction pathway. It is not clear as to which factors control the stemness of the corneal limbal stem cells, i.e., the maintenance of stem cell properties. It is likely that various signaling pathways are involved, including Notch, Wnt and NGF signaling, etc. In the present study, we investigated the activation of phosphoinositide-3-kinase (PI3K) pathway on cells cultured over the chitosan matrix, chitosan silver matrix, chitosan gold matrix, intact and denuded human amniotic membrane (HAM). METHODS: Human limbal biopsies obtained from the cadaveric donor eyes were used in this study. The cells cultured over different substrate and observed for the activation of the downstream signaling molecules of PI3K/Akt/FKHRL1 pathway. Western blotting was done to prove the results. RESULTS: The cells cultured over the intact HAM showed the activation of the downstream signaling molecules of PI3K/Akt/JNK pathway compared to the cells grown over other substrates. On inhibition of the PI3K activity there was absence of phosphorylation of downstream effectors in the limbal epithelial cells from the explant culture over the intact HAM. INTERPRETATION & CONCLUSION: The ex vivo expansion of human limbal epithelial progenitor cells on intact HAM was mediated by PI3K/Akt/FKHRL1 pathway, which is known to govern cell survival, and the mitogen-activated protein kinase (MAPK) pathway, known to control the cell mitosis.

Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells.

BACKGROUND & OBJECTIVE: The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. METHODS: Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12). RESULTS: The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions. INTERPRETATION & CONCLUSION: The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.