Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546.

Concise Review: Patient-Specific Stem Cells to Interrogate Inherited Eye Disease.

Whether we are driving to work or spending time with loved ones, we depend on our sense of vision to interact with the world around us. Therefore, it is understandable why blindness for many is feared above death itself. Heritable diseases of the retina, such as glaucoma, age-related macular degeneration, and retinitis pigmentosa, are major causes of blindness worldwide. The recent success of gene augmentation trials for the treatment of RPE65-associated Leber congenital amaurosis has underscored the need for model systems that accurately recapitulate disease. With the advent of patient-specific induced pluripotent stem cells (iPSCs), researchers are now able to obtain disease-specific cell types that would otherwise be unavailable for molecular analysis. In the present review, we discuss how the iPSC technology is being used to confirm the pathogenesis of novel genetic variants, interrogate the pathophysiology of disease, and accelerate the development of patient-centered treatments. Significance: Stem cell technology has created the opportunity to advance treatments for multiple forms of blindness. Researchers are now able to use a person's cells to generate tissues found in the eye. This technology can be used to elucidate the genetic causes of disease and develop treatment strategies. In the present review, how stem cell technology is being used to interrogate the pathophysiology of eye disease and accelerate the development of patient-centered treatments is discussed.

Generating iPSC-Derived Choroidal Endothelial Cells to Study Age-Related Macular Degeneration.

PURPOSE: Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols. METHODS: Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC- and CEC-specific markers. RESULTS: Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS: Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases.

Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement.