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

Abstract

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

The Effect of Corticosteroids on Human Choroidal Endothelial Cells: A Model to Study Central Serous Chorioretinopathy

“…24 Corticosteroid responsive genes FKBP5, PER1, GILZ, and SGK1 were selected based on available literature. [25][26][27][28][29] Markers for ECs, including vWF, CDH5, CD31, CD34, and PLVAP, the latter as a marker for fenestrated capillaries, 30 were used. The expression of VIM was measured as a marker for fibroblasts to quantify possible contamination of the EC culture.…”

Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells

“…We adopted previous publications, which use BMP-4, activin A, FGF, and VEGF to enrich for CD31-positive endothelial cells [ 20 ], and modified by using a stepwise transition from mTESR™1 to endothelial cell media with the addition of CTGF to specify choroidal endothelial cells [ 23 ]. HiPSCs were maintained on Matrigel-coated plates in mTESR™1 (Fig.…”

“…On day 5, EBs were collected and allowed to settle onto a MG-coated 6-well plate at a density of 30 EBs per well supplemented with VEGF and CTGF. VEGF was included to promote endothelial cell differentiation, and CTGF was included to promote specification of CA4-positive choroidal endothelial cells [ 23 ]. On day 6, EBs could be observed adhering to the 6-well culture dish (Fig.…”

“…Although a number of published protocols have described differentiation of ECs from pluripotent embryonic stem cells [ 18 – 21 ], differences between EC subtypes have revealed the need for refined procedures suitable for directing tissue-specific EC commitment [ 3 , 22 ]. We have previously shown that connective tissue growth factor (CTGF), when implemented during spontaneous differentiation, plays a key role in driving induced pluripotent stem cells (iPSCs) toward a choroidal endothelial cell (CEC) fate [ 23 ]. In the current report, we adopt a directed stepwise differentiation approach using bone morphogenetic protein 4 (BMP-4), fibroblast growth factor 2 (FGF2), activin A, and vascular endothelial growth factor (VEGF) [ 20 ], to enrich for mesoderm precursor cells prior to CEC specification using CTGF.…”

Restoring Vision by Regenerative Medicine Using Stem Cell: A Review

“…These patients fail to have keratoplasties due to injury or autoimmune disease and have limb stem operative by stimulating external retina addition [70]. Choroid ECs must also currently been fruitfully produced from iPSCs [71,72]. The postmitotic rod precursor secluded from the initial post-delivery rats distinguished hooked on developed poles then developed a synapsis connection with the wild-type rats.…”