Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization

Abstract

BackgroundDysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation.MethodsWe have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions.ResultsUsing highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function.ConclusionsThis report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0652-9) contains supplementary material, which is available to authorized users.

Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interactions

“…The TER in AMD patients (187 ± 6, 194 ± 7, 189 ± 4, and 198 ± 4 Ω × cm 2 ) vs the TER in normal controls (202 ± 5, 191 ± 7, and 197 ± 3 Ω × cm 2 ) revealed no significant difference ( P > .05) (Figure M). These TER results are consistent with TER in other reported embryonic stem cell (ESC)‐derived and iPSC‐derived RPE cell lines . Given the TER, high expression of claudin‐3 and claudin‐19 (Supplemental Table S3) and circumferential bands of ZO‐1, these iPSC‐derived RPE cells from patient samples can establish barrier function in vitro.…”

“…An important function of RPE cells is the phagocytosis of ROS. Since iPSC‐derived RPE cells expressed tyrosine‐protein kinase Mer (MERTK), which plays an important role in engulfing ROS, the ability of these cells to phagocytize ROS was explored. Phagocytized ROS particles localized within the cytoplasm of iPSC‐derived RPE cells are shown in Figure H.…”

“…These TER results are consistent with TER in other reported embryonic stem cell (ESC)-derived and iPSC-derived RPE cell lines. 29,40,41 Given the TER, high expression of claudin-3 and claudin- Table S3) 42,43 and circumferential bands of ZO-1, these iPSC-derived RPE cells from patient samples can establish barrier function in vitro. In addition, RPE marker genes were expressed and there was little difference in all six lines of iPSC-derived RPE cells ( Supplemental Table S3).…”

Self-Formation of RPE Spheroids Facilitates Enrichment and Expansion of hiPSC-Derived RPE Generated on Retinal Organoid Induction Platform

“…RPE spheroids or aggregates were also observed in previous studies, which were often reattached to the adherent surface to allow RPE cells to migrate out and expand for 3 to 5 weeks. 23,37,40,41 However, in our study, RPE sheets were directly dissected from the spheroids and digested into single cells for quick expansion in our optimized culture conditions with serum medium, low seeding density (2.0 to 5.0 3 10 4 /cm 2 ) and 7-day passage (SL7). Under these conditions, singularized RPE cells from RPE spheroids at varying timepoints after differentiation (day 53 to day 139) were all successfully expanded and routinely passaged at least 5 times.…”

Gene editing prospects for treating inherited retinal diseases

“…Disease-specific iPS cells represent a platform for human disease modelling to investigate the molecular and cellular mechanisms underlying the disease, in particular for those IRDs still missing an animal model fully recapitulating the human disorder, as well as for regenerative medicine. iPS cells, indeed, offer a valid alternative to stem cell therapy, exploiting the optimisation of differentiation protocols for the generation of RPE cells or photoreceptor-like cells, for instance 62–65…”