Derivation and Comparative Assessment of Retinal Pigment Epithelium from Human Embryonic Stem Cells Using Transcriptomics

Abstract

Human stem-cell derivatives are likely to play an important role in the future of regenerative medicine. Evaluation and comparison to their in vivo counterparts is critical for assessment of their therapeutic potential. Transcriptomics was used to compare a new differentiation derivative of human embryonic stem (hES) cells--retinal pigment epithelium (RPE)--to human fetal RPE. Several hES cell lines were differentiated into putative RPE, which expressed RPEspecific molecular markers and was capable of phagocytosis, an important RPE function. Isolated hES cell-derived RPE was able to transdifferentiate into cells of neuronal lineage and redifferentiate into RPE-like cells through multiple passages (>30 Population doublings). Gene expression profiling demonstrated their higher similarity to primary RPE tissue than of existing human RPE cell lines D407 and ARPE-19, which has been shown to attenuate loss of visual function in animals. This is the first report of the isolation and characterization of putative RPE cells from hES cells, as well as the first application of transcriptomics to assess embryonic stem-cell derivatives and their in vivo counterparts--a "differentiomics" outlook. We describe for the first time, a differentiation system that does not require coculture with animal cells or factors, thus allowing the production of zoonoses-free RPE cells suitable for subretinal transplantation in patients with retinal degenerative diseases. With the further development of therapeutic cloning, or the creation of the banks of homozygous human leucocyte antigen (HLA) hES cells using parthenogenesis, RPE lines could be generated to overcome the problem of immune rejection and could be one of the nearest term applications of stem-cell technology.

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

“…Mouse and human ESC in serum-free medium could differentiate into RPE after culturing on bare (for mouse) or laminin-coated (for human) amniotic membrane (Ueno et al, 2006). Putative RPE could be isolated from spontaneously differentiating hESC grown on gelatin-coated matrix (Klimanskaya et al, 2004). In all of these studies only a small percentage of the stem cells expressed RPE markers, thus necessitating harvesting of RPE-like clumps prior to expansion of the population.…”

Stem Cells for Retinal Replacement Therapy

“…It is not surprising, in this context, that ESCs readily differentiate into RPE [33,34]. ESCs expand extensively to produce large quantities of cells that can differentiate into all progeny types, and thus there is great interest in developing ESCs to treat diverse diseases.…”

Enhanced Functional Integration of Human Photoreceptor Precursors into Human and Rodent Retina in anEx VivoRetinal Explant Model System

“…35 The ARPE-19 line has compromised barrier and polarization defects 21 and does not express bestrophin, Rpe65, and transducin, all key RPEspecific functional proteins. 23 Our results suggest that RPE-J and ARPE-19 cell lines likely fail to secrete the necessary Percentage of cells expressing protein after 1-week exposure to A-culture media only; B-rodent retina and ARPE19 cells; Crodent retina and human-RPE; D-human retina and human-RPE. Results were compared with and without exposure to thrombospondin-1.…”

“…Human RPE derived from hESCs (hESC-RPE) was generated using published protocols 23,24 Briefly, the hESC line WA09 (WiCell Research Institute) 25 was allowed to spontaneously mature in standard hESC media (7-10 days). The medium was then changed to differentiation media, which consisted of knockout DMEM (koDMEM), 15% knockout serum replacer (KOSR), 1% l-glutamine, 1% penicillin/ streptomycin solution, 1% NEAA, and 0.1 mM b-mercaptoethanol (b-ME; Sigma).…”