Journal article
Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells
Stem cell research & therapy, Vol.11(1), pp.1-409
09/23/2020
DOI: 10.1186/s13287-020-01903-4
PMCID: PMC7510078
PMID: 32967716
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.
Details
- Title: Subtitle
- Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells
- Creators
- Kelly Mulfaul - Iowa City, IA 52242 USAJoseph C Giacalone - Iowa City, IA 52242 USAAndrew P Voigt - Iowa City, IA 52242 USAMegan J Riker - Iowa City, IA 52242 USADalyz Ochoa - Iowa City, IA 52242 USAIan C Han - Iowa City, IA 52242 USAEdwin M Stone - Iowa City, IA 52242 USARobert F Mullins - Iowa City, IA 52242 USABudd A Tucker - Iowa City, IA 52242 USA
- Resource Type
- Journal article
- Publication Details
- Stem cell research & therapy, Vol.11(1), pp.1-409
- DOI
- 10.1186/s13287-020-01903-4
- PMID
- 32967716
- PMCID
- PMC7510078
- NLM abbreviation
- Stem Cell Res Ther
- ISSN
- 1757-6512
- eISSN
- 1757-6512
- Publisher
- BioMed Central; London
- Grant note
- ; EY-025580 / ; EY-024605 / ;
- Language
- English
- Date published
- 09/23/2020
- Academic Unit
- The University of Iowa Institute for Vision Research; Iowa Neuroscience Institute; John and Marcia Carver Nonprofit Genetic Testing Laboratory; Ophthalmology and Visual Sciences
- Record Identifier
- 9984070358802771
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