Abstract
BackgroundEndothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.MethodsWe transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.ResultsWe report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.ConclusionECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.
Highlights
Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds
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 toward a choroidal endothelial cell (CEC) fate [23]
Differentiation of induced pluripotent stem cells (iPSCs)-derived choroidal endothelial cells We adopted previous publications, which use bone morphogenetic protein 4 (BMP-4), activin A, FGF, and vascular endothelial cell growth factor (VEGF) to enrich for CD31positive endothelial cells [20], and modified by using a stepwise transition from mTESRTM1 to endothelial cell media with the addition of CTGF to specify choroidal endothelial cells [23]
Summary
Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. Endothelial cells (ECs) line the walls of blood vessels throughout the body, forming the innermost monolayers of vascular tubes. ECs can aid in tissue repair by triggering blood vessel growth via angiogenesis. Regardless of their location in the body, ECs in any organ system share these basic functions. Essentially all ECs express a common set of marker genes including CD34, CD31, and von Willebrand’s factor (vWF) [1] Depending on their anatomical location, there is huge diversity in EC structure and function [2], including the expression of tissue-specific genes. Given the variability in endothelial cell subtypes, studying endothelial cells in vitro that are phenotypically similar to those from the relevant tissue is of high interest
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