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

Abstract

Background Endothelial 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. Methods We 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. Results We 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. Conclusion ECs 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.

Local factor H production by human choroidal endothelial cells mitigates complement deposition: implications for macular degeneration

“…Having determined that local FH production is important for controlling MAC deposition, we next investigated the extent to which restoring wild-type FH to CECs could protect against complement activation and MAC deposition. To do this, we generated iPSC-derived choroidal endothelial cells from a female patient with severe dry AMD using our published protocol [39]. This patient is homozygous for Y402H and also has a stop mutation in exon 22 of one of her FH alleles.…”

3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages

“…The Matrigel ® is the gold standard. It is a tissue formed of a mixture of gelatinous proteins derived from the basement membrane isolated from the mouse sarcoma Engelberth-Holm-Swarm (EHS) and commercially available under the brand name Matrigel ® (BD Biosciences) [107][108][109][110] (Table 2). This extract is liquid at 4 • C and turns into a gel at 37 • C under physiological pH and ionic strength.…”

Microfluidic processing of stem cells for autologous cell replacement

“…[60][61][62] Similarly, markers that are often used for stem cell derived vascular endothelial cell enrichment (eg, CD31) are expressed on a variety of different endothelial cell populations and monocytic cells, making them less than ideal for isolation of tissue specific vascular subtypes. [63][64][65]…”