Effects of extracellular matrix and neighboring cells on induction of human embryonic stem cells into retinal or retinal pigment epithelial progenitors

Abstract

To determine the effects of extracellular matrix and neighboring cells on the differentiation of human embryonic stem cells (hESC) into progenitors of retinal cells and/or retinal pigment epithelium (RPE). HESC were cultured on mouse PA6 stromal cells for approximately 2 weeks to obtain neural progenitors. To induce photoreceptor marker expression, the neural progenitors were cultured on a confluent monolayer of ARPE19 or on laminin-coated dishes. To induce RPE markers, the neural progenitors were seeded onto human Bruch's membrane or Matrigel. Cells were examined morphologically and stained with different RPE or neural progenitor markers. Microarray techniques were used to compare the gene expression profiles of hESC cultured on mouse fibroblasts or neural progenitors on PA6 cells to the transcriptome of the adult neural retina and RPE. HESC cultured on PA6 cells expressed neural progenitor markers β-tubulin III, PAX6, neural filament, GFAP and vimentin. Culturing these neural progenitors on confluent ARPE19 monolayer induced expression of the photoreceptor progenitor cell marker CRX; culturing neural progenitors on laminin substrates induced a neuronal phenotype with neurite formation. Neural progenitors expressed the RPE marker ZO-1 after culturing on Matrigel-coated dishes and the RPE marker Bestrophin after culturing on human Bruch's membrane explants. Hierarchical clustering analysis of samples suggested that when cultured on PA6 stromal cells hESC exhibited genetic characteristics towards differentiating into neural retina. Microarray analysis showed that after culturing on PA6 cells, stem cells expressed 117 new genes; among these there were 22 genes present in neural retina or RPE cells. The functions of these genes were highly related to cell proliferation, nervous system development and cell adhesion. HESC can be induced to differentiate into neural progenitors after culturing on PA6 cells. These neural progenitors can express RPE markers when cultured on Bruch's membrane or Matrigel, or photoreceptor markers when cultured on confluent ARPE19 or laminin. Additional studies are required to assess the function of hESC induced to express retinal or RPE markers prior to successful intraocular transplantation into animal models of retinal degeneration.

In vitro and in vivo differentiation of human embryonic stem cells into retina‐like organs and comparison with that from mouse pluripotent epiblast stem cells

“…In some cases, very complex culture strategies have been used, including the sequential addition and withdrawal of factors and purification of specific cell populations during the culture period (Fang et al, 2006;Wang et al, 2005). As for the induction of ocular tissues, lens and several retinal cell types were reported to have been differentiated from ES cells (Gong et al, 2008;Lamba et al, 2006;Osakada et al, 2008). Factors regulating the differentiation of the retinal tissues, such as Wnt2b, Wnt3a, Notch, Hes1, and RA (Jadhav et al, 2006;Kubo et al, 2003;Luo et al, 2006;Osakada et al, 2007;Tomita et al, 1996), were sequentially added to induce specific retinal cell lineages such as photoreceptor cells (Osakada et al, 2008).…”

Chondroinduction from Naturally Derived Cartilage Matrix: A Comparison Between Devitalized and Decellularized Cartilage Encapsulated in Hydrogel Pastes

“…It has been established that cells exposed to a target ECM will more easily differentiate toward the target tissue, 31,32 where one of the reasons for this ECM-specific differentiation may be because native ECM may have the potential to retain the growth factors that will steer the differentiation toward the specific target tissue. 4 Decellularizing cartilage ECM may not only alter the matrix architecture but it may also furthermore remove some of these important growth factors, affecting the bioactivity of the cartilage ECM.…”

Three-dimensional early retinal progenitor 3D tissue constructs derived from human embryonic stem cells

“…In other studies, co-culture of human retinal neurospheres with RPE prolonged and enhanced the growth of human prenatal retinal neurospheres, which went on to express numerous neurodevelopmental and eye specification genes and markers characteristic of neural and retinal progenitor cells (Gamm et al, 2008). Gong et al cultured hESC-derived neural progenitors on the ARPE19 RPE cell line and also observed expression of the photoreceptor precursor markers Crx and Nrl (Gong et al, 2008). However, they never attempted to obtain 3D tissue constructs.…”