Derivation, characterization and retinal differentiation of induced pluripotent stem cells

Abstract

Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

“…Work from Ali et al clearly demonstrates that the post-mitotic photoreceptor precursor cell is the optimal cell type for efficient rod photoreceptor cell replacement (Lakowski et al, 2011; Pearson et al, 2012). A variety of different protocols, utilizing both two- and three-dimensional culture systems, have succeeded in deriving photoreceptor precursor cells from less differentiated precursors (Osakada et al, 2008; Hirami et al, 2009; Meyer et al, 2009; Osakada et al, 2009; Lamba et al, 2010; Meyer et al, 2011; Tucker et al, 2011; Nakano et al, 2012; Phillips et al, 2012; Sasai et al, 2012; Homma et al, 2013; Mekala et al, 2013; Tucker et al, 2013). Although cultured three-dimensional eyecups will undoubtedly have many applications in developmental biology (Nakano et al, 2012; Sasai et al, 2012), two-dimensional systems have the advantage of easier identification and isolation of specific cell types for post-differentiation subculture.…”

In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)

“…Different protocols for the differentiation of human pluripotent stem cells (hPSCs) into RPE have been described. Acquired cells were reported to display characteristic phenotypes as well as gene expression and exhibit key physiological functions (Ukrohne et al 2012; Buchholz et al 2013; Zhu et al 2013; Rowland et al 2013; Mekala et al 2013; Maruotti et al 2013; Singh et al 2013a). RPE derived from hESCs has even reached the necessary standards for use in human clinical trials of cell-based transplantation therapy (Schwartz et al 2012).…”

Pluripotent stem cells: A therapeutic source for age-related macular degeneration

“…[737475767778] With respect to the protocols for deriving RPE from the iPSC lines, several studies have successfully employed the protocols already established in hESCs on most iPSC lines. [6779808182838485] Almost all of these studies provide evidence that iPSCs have potential similar to hESCs in terms of RPE differentiation.…”

“…[91] With respect to the use of iPSCs in ophthalmology, there has been a single study utilizing mouse iPSCs to generate retinal progenitors and RPE. [82] However, there are no reports available on the differentiation of human iPSC lines to retinal cells from India.…”