1. Preparation of culture media and coating solutions
2. Thawing, maintenance, and passaging of iPSCs
3. Generation of iPSC-derived endothelial progenitors (Figure 1).
4. FACS of endothelial progenitors
5. Encapsulation and long-term culture of iPSC-EP-laden collagen hydrogels
6. Fixing, immunostaining, and visualization of EP-based vascular networks
7. Using the computational pipeline to analyze and compare vascular network topologies
After differentiation (Figure 1), FACS and encapsulation of iPSC-EPs in collagen hydrogels, the cells will typically remain rounded for 24 h before beginning to migrate and form initial lumens. After about 6 days of culture, a primitive capillary plexus will be visible in the hydrogel when viewed with brightfield microscopy (Figure 2). After imaging the fixed, stained cell-laden hydrogels on a confocal microscope (Movie 1, Supplemental Movie 1), the pre-processed images are converted to a skeleton which enables an analysis of the overall length and connectivity of the network. These quantitative measures can then be used to determine which set of conditions are optimal for producing robust vascular networks.
This protocol allows for the development of a robust, three-dimensional capillary plexus that is responsive to local physical and chemical cues. In previous work, this network formation has been shown to be sensitive to matrix density, matrix stiffness, matrix metalloprotease inhibition, and the type and concentration of various angiogenic mitogens20,23.
Figure 1: Generation of iPSC-EPs from pluripotent stem cells. (A) WiCell 19-9-11 iPSCs, which stained positive for Oct4, were cultured in E8 medium supplemented with 10 μM Y-27632 ROCK inhibitor for 48 h. (B) The iPSCs were then induced with 6 μM of CHIR99021 in LaSR Basal medium for 48 h, at which point the cells were positive for Brachyury, a mesoderm marker. (C) The cells were further cultured in LaSR Basal media until they expressed CD34, a marker for endothelial progenitors. (D) Roughly 15%–25% of the differentiated cells expressed CD34. All scale bars represent lengths of 200 μm. Please click here to view a larger version of this figure.
Figure 2: Generation and analysis of iPSC-EP vascular networks in collagen hydrogels. (A) A cross-section of the 3D microenvironment used in this assay to promote vascular network formation from iPSC-EPs. A floating collagen hydrogel is seeded with iPSC-EPs and exposed to EGM-2 supplemented with 50 ng/mL VEGF and a temporal dose of Y-27632. (B) The resulting capillary plexus is highly branched and interconnected, as visualized via staining with F-actin (cyan). The binarized image, shown on the left, is generated by pre-processing with ImageJ. This z-stack is then analyzed via a previously developed algorithm, which skeletonizes the network (shown in a collection of thin red lines) and then analyzes the network topology for branches (yellow), end points (blue), isolated vessels (black), and connected vessels (red). The scale bar represents a length of 100 m. (C) Morphological changes of iPSC-EPs-laden collagen hydrogels: 24 h after seeding, the iPSC-EPs remain spherical and within 96 h gradually take on a more elongated phenotype. Further culture results in assembly of lumen-containing VE-Cadherin network, as shown in the inset at the 144-h time point. The scale bars represent lengths of 400 μm; green = VE-Cadherin, red = F-actin, and blue = DAPI. Please click here to view a larger version of this figure.
Movie 1: Z-stack of VASCULATURE GENERATED FRom iPSC-EPs. Vascular networks were fixed, stained with F-actin, and visualized by acquiring z-stacks on a confocal microscope. Slices were acquired at 17 μm intervals. Please click here to view this video. (Right-click to download.)
Supplemental Movie 1: 3D rendering of vessels. Vascular networks were fixed, stained with F-actin (red) and VE-cadherin (green), and visualized by acquiring z-stacks on a confocal microscope. Please click here to download this file.
µ-Slide Angiogenesis | Ibidi | N/A | A flat, glass bottom tissue-culture plate with side walls enables facile confocal imaging |
96 well, round bottom, ultra low attachment microplate, sterile | Corning | 7007 | Prevents the binding of cell-laden collagen hydrogels to the cell culture dish |
Accutase | STEMCELL Technologies | 7920 | Gentle cell detachment solution; does not degrade extracellular epitopes vital for FACS |
Advanced DMEM/F12 | Thermo Scientific | 12634010 | The base media for iPSC-EP differentiation. |
Barnstead GenPure xCAD Plus | Thermo Fisher Scientific | 50136165 | Water purification system; others can be readily substituted |
Bovine Serum Albumin solution,7.5% in DPBS, sterile-filtered, BioXtra, suitable for cell culture | Fisher Scientific | A8412 | To preserve cell viability when FACs sorting |
CD34-PE, human (clone: AC136) | Miltenyi Biotec | 130-098-140 | Antibody used for FACs isolation of iPSC-EPs |
CHIR99021 | LC Laboratories | C-6556 | Induces the formation of mesoderm from pluripotent stem cells |
Collagen I Rat Tail High Protein 100 mg | VWR | 354249 | Main component of the 3D microenvironment |
Conical centrifuge tubes (15/50 mL) | Fisher Scientific | 14-959-49D/A | Used to store and mix relatively large volumes of reagents and cell culture media |
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | To counterstain and visualize cell nuclei |
DMEM/F12 | Thermo Fisher Scientific | 11320-082 | For dilution of Matrigel and thawing of pluripotent stem cells |
Dulbecco's phosphate-buffered saline (DPBS) | ThermoFisher | 14190-250 | To wash monolayer cultures |
EDTA | Sigma-Aldrich | E8008 | For passaging of pluripotent stem cell colonies and to prevent cell aggregation when FACs sorting |
Endothelial Cell Growth Medium 2 | PromoCell | C-22011 | Promotes endothelial cell viability and proliferation |
Essential 8 Medium | Thermo Fisher Scientific | A1517001 | For maintenance of pluripotent stem cells |
Glycine,BioUltra, for molecular biology, >=99.0% (NT) | Sigma-Aldrich | 50046 | Neutralizes remaining detergent |
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate,>=95% | Sigma-Aldrich | A8960 | Component of iPSC-EP differentiation medium |
MATLAB | MathWorks | 1.8.0_152 | Multi-paradigm numerical computing environment (free available at most academic institutions) |
Matrigel Matrix GFR PhenolRF Mouse 10 mL (gelatinous protein mixture) | Fisher Scientific | 356231 | Diluted in DMEM/F12 to coat plates for iPSC-EP differentiation |
Medium-199 10X | Thermo Fisher Scientific | 1825015 | Used to balance final hydrogel osmolarity and pH |
Microcentrifuge tubes (1.7 mL) | VWR | 87003-294 | Stores small volumes of reagents |
Phosphate-buffered saline (PBS) | Sigma-Aldrich | P3813 | The main ingredient of the immunostaining solutions |
Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | Antibiotic used after sorting to remove possible contamination from FACS instrument |
Recombinant Human VEGF 165 Protein | R&D Systems | 293-VE | Mitogen that stimulates endothelial cell proliferation and tubulogenesis |
Rhodamine phalloidin | Themo Fisher Scientific | R415 | To identify F-actin deposition and therfore outline the borders of the vascular networks |
Triton X-100 (nonionic surfactant) | Sigma-Aldrich | X-100 | Detergent used to gently permeabilize cells |
Tween-20 (emulsifying reagent) | Fisher Scientific | BP337 | Increases the binding specificity of the added antibodies |
VE-Cadherin (F-8) | Santa Cruz Biotechnology | sc-9989 | To identify 3D endothelial lumen in collagen hydrogels |
Vitronectin | ThermoFisher | A14700 | For maintenance of pluripotent stem cells |
Y-27632 | Selleck Chemicals | S1049 | Preserves pluripotent stem cell and iPSC-EP viability when dissociated and re-seeded |
Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.
Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.
Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.