2.3.1 Endothelial differentiation of hESC
Endothelial differentiation of hESC was performed using an embyroid body (EB)- based method. On d0, hESCs (90-100% confluent, 8 days post-passage) were taken to single cell suspensions using StemPro® Accutase (Life Technologies) or TrypLE Select™ (Gibco, Paisley). Cells were counted and 10,000 cells placed into each well of the inner 60 wells of a Corning® 96-well clear round bottom ultra- low attachment microplate (Corning), or 96-well clear round bottom microplate coated with 5% pluronic F-127 (Sigma-Aldrich) (Ungrin et al., 2008), with 100 µL Stemline II medium (Sigma), supplemented with BMP4, Wnt3a (both R&D Systems), Activin A, VEGF (both Peprotech) and Y-26732 (Millipore) at concentrations shown in Table 2.2. To coat plates with 5% pluronic F-127, 50 µL solution was added to each well and plates were incubated at room temperature for 30 min, before being removed prior to the addition of cells (Table 2.1).
BMP4 VEGF Wnt3A Activin A Y-26732 0 Stemline II 10ng/mL 10ng/mL 10ng/mL 5ng/mL 10µM 2 Stemline II 40ng/mL 60ng/mL 20ng/mL 10ng/mL N/A 3-7 LONZA-EBM2 with
EGM-2 bullet kit N/A 50ng/mL N/A N/A N/A Cytokine Concentration
Day Media
Table 2.2 – Cytokine concentrations used on specific days of hESC-EC differentiation
2 days after EB formation 16.6 µL of Stemline II medium, supplemented with BMP4, VEGF, Activin a and Wnt3A, was added to each well in the 96-well plate, giving final cytokine concentrations shown in Table 2.2. On d3, EBs were collected, washed in PBS and resuspended in 3mL Endothelial Growth Medium-2 (EGM-2, Lonza) containing 50 ng/mL VEGF. The EGM-2 medium used is produced from the addition of the EGM-2 bullet kit to the Endothelial Basal Medium-2 (Lonza), without the addition of the VEGF and FBS components. EBs were transferred into 6-well plates coated with 0.1% gelatin (Sigma). Medium was then changed (2 mL LONZA EGM-2 with 50 ng/mL VEGF) every 2 days until appropriate analysis time point was reached. Some earlier differentiations were performed using 6-well ultra-low attachment dishes (Corning) and StemPro® EZPassage tools for EB formation (as described in hematopoietic differentiation, section 2.3.3).
2.3.2 MACSorting of hESC-ECs and further outgrowth
After 7 days of endothelial differentiation, CD144+ cells were sorted using magnetic activated cell sorting (MACS), in order to allow for further culture of hESC-ECs (Figure 2.1). MACS was performed as follows, using commercially available microbeads, columns, magnets and holders from Miltenyi Biotec. Media was removed and cells were washed briefly with PBS, before dissociation to single cells using TrypLE Select™. Once collected, single cells were resuspended in MACS buffer; PBS, pH 7.2, 0.5% bovine serum albumin (BSA), passed through a 40 µm nylon mesh (Fisherbrand) to ensure removal of cell clumps, and counted to determine total cell number. Cells were then centrifuged at 300 x g for 5 min, and the resulting pellet resuspended in 80 µL of MACS buffer per 107 total cells.
Figure 2.1 – Overview of MACSorting
Cells were magnetically labelled using MACS microbeads conjugated antibodies, before being passed through a column within a magnetic field. Labelled cells will bind to the column, and those which are unlabelled will pass through. Labelled cells were then be collected by removal of the column from the magnetic field (Adapted from Miltenyi Biotec).
20 µL of CD144 microbeads, per 107 total cells, was then added and the mixture incubated at 4°C for 15-30 min. Cells were then washed using 1-2 mL MACS buffer and centrifuged at 300 x g for 5 min, to remove any excess or unbound CD144 microbeads, and the resulting pellet resuspended in 500 µL MACS buffer. A cell separation column (LS column) was placed in a MidiMACS™ separator and prepared for use by rinsing with 3 mL MACS buffer. The MidiMACS™ separator contains a powerful permanent magnet which applies a high-gradient magnetic field to a MACS column, allowing for cells labelled with even small amount of magnetic microbeads to bind to the column (Figure 2.1). Once the column was prepared, the cell suspension was then applied to the column and the flow through collected. The column was washed 3 times with 3 mL MACS buffer, the flow through collected and combined with that from the cell suspension. This flow through contains CD144- cells from the mixture, as they are unlabelled and have therefore been unable to bind to the MACS column. To collect the CD144+ cells, the column was removed from the MidiMACS separator and the magnetically labelled cells were flushed out using 5 mL MACS buffer.
Flow cytometry was performed to ensure efficient purification of CD144+ cells.
Sorted cells were then plated onto gelatin coated 6-well tissue culture plates at a density of 1x105 cells per well and cultured for a further 7 days in LONZA EGM- 2 media with FBS component added and supplemented with 50ng/mL VEGF.
2.3.3 Differentiation of hESC to hemogenic endothelium
Differentiation of cells to early hemogenic endothelium was performed using the first 10 days of a defined serum- and feeder-free EB-based hematopoietic differentiation protocol (Mountford lab). On d0 EBs are formed by removing medium from the wells and washing cells in PBS. 3 mL/well Stemline II medium (Sigma-Aldrich), supplemented with BMP4, VEGF, Activin A, Wnt3A and Inhibitor VIII, was added, and cells were cut into rough squares using a StemPro® EZPassage tool. Cells were then removed from the well and transferred to a Corning® 6-well ultra-low attachment plate (Corning) at a 1:3 ratio. A further 1.5 mL media, containing d0 cytokines, was then added to each well, resulting in a final volume of 3 mL/well. At d2, cells had formed EBs and a further addition of 0.5mL Stemline II medium, supplemented with BMP4, VEGF, Inhibitor VII, Wnt3A, Activin A, FGFα, SCF and β-Estradiol, was made. On d3 cells were collected and dispersed, using Tryple Select. Single cells were then resuspended in Stemline II medium, counted, and seeded at 2x105 cells per well in a standard 6-well plate, in 3 mL/well Stemline II medium, with appropriate cytokines added. A further 0.5 mL media, supplemented with specific cytokines, was added on day 5, before a complete media change was then performed on d7. On day 9, a final addition of 0.5 mL media and cytokines was made, before cells were harvested for analysis on d10. Stemline II medium was used for all stages of differentiation, and concentrations of all cytokines added are shown in table 2.3.
BMP4 VEGF Wnt3A Activin A Inhibitor VIII FGFα SCF β-Estradiol IGF2 TPO Heparin IBMX 0 10ng/mL 10ng/mL 10ng/mL 5ng/mL 2µM - - - - 2 20ng/mL 30ng/mL 10ng/mL 5ng/mL 2µM 10ng/mL 20ng/mL 0.4ng/mL - - - - 3 20ng/mL 30ng/mL - - - 10ng/mL 30ng/mL 0.4ng/mL 10ng/mL 10ng/mL 5ng/mL 50µM 5 20ng/mL 30ng/mL - - - 10ng/mL 30ng/mL 0.4ng/mL 10ng/mL 10ng/mL 5ng/mL 50µM 7 20ng/mL 30ng/mL - - - 10ng/mL 30ng/mL 0.4ng/mL 10ng/mL 10ng/mL 2.5ng/mL 50µM 9 10ng/mL 15ng/mL - - - 5ng/mL 15ng/mL 0.2ng/mL 5ng/mL 5ng/mL 1.25ng/mL 25µM Day Media Stemline II Cytokine Concentration