Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

Abstract

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-moleculeonly approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxiainducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over onehalf of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.retinal pigment epithelium | pluripotent stem cells | high-throughput screening | differentiation | age-related macular degeneration A ge-related macular degeneration (AMD) is the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries. Dysfunction of the retinal pigment epithelium (RPE) is an early event associated with AMD. The RPE, a monolayer of pigmented cells directly abutting the photoreceptor cell layer, plays many important roles in vision and in maintaining the health and integrity of the retina (1). As the RPE deteriorates, there is progressive degeneration of photoreceptor cells.Successful antiangiogenesis treatments have been developed for the neovascular, or "wet," form of AMD. However, there are no Food and Drug Administration-approved treatment options available for the majority of AMD patients, who suffer from the more common nonneovascular, or "dry," form of the disease. In the past few years, however, transplantation of human pluripotent stem cellderived RPE (hPSC-RPE) has emerged as a promising new therapy for dry AMD. A Phase I clinical trial of human embryonic stem (hES)-derived RPE cells recently reported some preliminary encouraging results (2). Additionally, a Phase I trial that will use RPE cells generated from human induced pluripotent stem cells (hiPSCs) reprogrammed from the patients' own skin cells recently injected their first patient (3).If the promise of hiPSC-based approaches for AMD is to be translated into the clinic, each patient would require individualized generation of RPE cells from his or her stem cells, thereby necessitating the development of simple, efficient, safe, and affordable protocols for RPE generation. Although highly efficient protocols have been established, they rely upon mixtures of growth factors (4-6) with the use of complex biologics derived from animal cells or bacteria, presenting potential clinical challenges. As an alternative approach, use...

Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells

“…Combined application of the retinal inducing factors IGF1, noggin, the Wnt inhibitor Dkk1 and FGF2, and the RPE inducers activin A and nicotinamide, was reported to facilitate rapid RPE generation, within 14 days after the onset of differentiation and with ∼80% efficiency . Small molecule approaches to stimulate hPSC-RPE generation have also been described (Maruotti et al, 2015). Despite these advances, even the most efficient protocols can result in residual iPSCs, but manual 'picking' under the microscope or enzyme-based selection of emerging RPE colonies followed by re-plating can lead to highly pure RPE cultures (Maruotti et al, 2013).…”

“…(Maruotti et al, 2015) Additional protocols to generate RPE using 3D culture techniques are listed in Table 3. EB, embryoid bodies; SR, serum replacement; GFP, green fluorescent protein; d, day(s); w, week(s); m, month(s); fb, followed by; +, plus.…”

Strategies for retinal cell generation from human pluripotent stem cells

“…Maruotti et al described an alternative approach to hiPSC-RPE differentiation utilizing small molecules which are favored for potential clinical applications over growth factors derived from animal or bacterial cells, which are liable to vary from lot-to-lot and escalate the potential for patient infection or immune rejection. Small-molecule chetomin (CTM) was identified in a high-throughput qPCR screen to consistently upregulate RPE markers MITF, OTX2, and PMEL17 (48). CTM is a metabolite of the fungus Chaetomium species that inhibits the transcriptional activation of the hypoxia-inducible factor (HIF) pathway (49).…”

Pluripotent Stem Cell-Based Therapies in Combination with Substrate for the Treatment of Age-Related Macular Degeneration

“…[56][57][58] Therefore, efforts to optimize the clinical derivation of RPE from stem cells employ synthetic substrates such as vitronectin peptide Synthemax Ò plates as xeno-free alternatives to Matrigel and human feeder fibroblasts. [59][60][61] Also, synthetic small molecules could replace full-length recombinant proteins to avoid batch-to-batch variation in directed differentiation protocols. The small molecule chetomin, in combination with nicotinamide, yielded 67% of cells expressing a tyrosinase-GFP reporter after 35 days of iPSC differentiation.…”

“…The small molecule chetomin, in combination with nicotinamide, yielded 67% of cells expressing a tyrosinase-GFP reporter after 35 days of iPSC differentiation. 59 As the field of regenerative medicine progresses, so will the demand for stem cell-derived RPE production to be completed under defined conditions adhering to good manufacturing practices.…”