Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage

Abstract

Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro disease modeling, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age‐related macular degeneration. Here we show development of an efficient method for deriving homogeneous RPE populations in a period of 45 days using an adherent, monolayer system and defined xeno‐free media and matrices. The method utilizes sequential inhibition and activation of the Activin and bone morphogenetic protein signaling pathways and can be applied to both human embryonic stem cells and induced pluripotent stem cells as the starting population. In addition, we use whole genome transcript analysis to characterize cells at different stages of differentiation that provides further understanding of the developmental dynamics and fate specification of RPE. We show that with the described method, RPE develop through stages consistent with their formation during embryonic development. This characterization— together with the absence of steps involving embryoid bodies, three‐dimensional culture, or manual dissections, which are common features of other protocols—makes this process very attractive for use in research as well as for clinical applications. Stem Cells Translational Medicine 2017;6:490–501

Dynamic expression of SMAD3 is critical in�osteoblast differentiation of PDMCs

“…Human pluripotent stem cells have the potential to assist in the identification of genes associated with �a��alian develo�� genes associated with mammalian develop-s associated with mammalian develop-mammalian development (1,2), and the plasticity of stem cells suggests that they may have a wide range of clinical applications (3,4). Bone tissue engineering has also been explored for clinical purposes (5,6).…”

Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells

“…Previous studies have evaluated purity by combined image analysis of pigmentation together with staining for several intracellular markers 28 . However, global transcriptional analysis has revealed that contaminants of cells with alternative fate can be found, such as lense-like cells expressing genes encoding crystallins in <10% of otherwise apparently homogeneous cultures 29 . Such contaminants may be difficult to identify by image analysis for pigmentation and intracellular staining of RPE markers.…”

Induced pluripotent stem cell-based therapy for age-related macular degeneration

“…This hurdle was overcome in 2012 by using human fibroblast feeder cells [103] as opposed to non-human feeder cells, and later by methods obviating the need for feeder cells [104]. Differentiation of desired cells is a time-consuming process, and from 2013-2016, numerous investigators have published successively more efficient methods of RPE differentiation [105][106][107][108][109][110][111].…”