Aggrewell 400 plate

Prior to cell seeding, the wells were washed twice with modified EGM-2 medium supplemented with a final concentration of 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2% (wt/vol) glucose and 0.05 mM 2-mercaptoethanol. Cell number was determined by trypan blue exclusion on an automated cell counter (TC20, Bio-Rad Laboratories). In order to form pseudoislets, which are three-dimensional aggregates of α, β and endothelial cells, the cells were trypsinised with 0.05% trypsin-EDTA for 5 min at 37°C. Subsequently, the cells seeded either in a 24-well AggreWell 400 plate (STEMCELL Technologies) to generate 1,200 pseudoislets/well or in a 96-well round-bottom plate (Corning Elplasia) for 79 pseudoislets/well. As a basis, all pseudoislets had a total of 1,500 cells, but their composition had the varying ratios of α:β (1:14), α:β:EC (1:9:5), 1× α:β:EC (1:9:2), 2× α:β:EC (1:9:5) and 4× α:β:EC (1:9:20). When studying the different sizes of pseudoislets, the ratio of α:β:EC of 1:9:5 was used with the different total number of 750, 1,500 and 3,000 cells. All cell types were seeded simultaneously into the microwell array, which was then centrifuged at 200 × g for 4 min to distribute the seeded cells into the microwells evenly. The pseudoislets were cultured for up to 5 days in a modified EGM-2 medium, with the medium refreshed daily.

ESCs were enzymatically released from the gelatin-coated plates with trypsin/EDTA (ATCC, Manassas, VA) and resuspended in Differentiation Medium (Growth Medium without LIF). Approximately 1.2 million cells were added to each well of a 24-well Aggrewell 400 plate (Stem Cell Technologies) as directed by the manufacturer. The final EB sizes were approximately 1,000 cells/EB. Once the ESCs were allowed to settle for 30 minutes at 37°C/5%CO₂, the beads were distributed to the appropriate wells and collected into the bottom of the Aggrewells using magnets, ensuring that beads were homogenously distributed among the ESCs as they aggregated. The EBs were kept in the Aggrewell 400 plates for 3 days, and medium was partially changed daily. Day 3 EBs were transferred to suspension culture in ultra-low attachment plates and medium was changed in full every 2 days. In the BMP4-treated group, 10 ng/ml BMP4 was added from Days 1 through 7 as described previously [20] (link). On Day 7, the EBs were allowed to attach to gelatin-coated plates and maintained for up to 18 days at 37°C/5%CO₂.

On transduction day (day 0), sub-confluent (50–80%) hPSC cultures were dissociated to single cells using TrypLE (Life Technologies) Fig. 2a. Embryoid body formation was initiated with 6–12E+5 viable cells per well of an Aggrewell400 plates (Stem Cell Technologies) leading to 500–1,000 cells per embryoid body following spin aggregation. Lentiviral transduction was performed concomitantly to the aggregation step in CDM supplemented with Y-27632 (10 μM, Sigma), BMP4 (10 ng ml⁻¹, R&D) and protamine sulfate. After 24 h, transduced embryoid bodies were collected and sown in ultralow adherent cell culture plates (Corning) at 1,200 embryoid bodies per 10 cm² dish in CDM with BMP4 and FGF2 (5 ng ml⁻¹). Twenty-four hours later, embryoid bodies were washed and sown in ultralow adherent plates at 600 embryoid bodies per 10 cm² in CellGroSCGM medium (CellGenix) supplemented with TPO (100 ng ml⁻¹, Cellgenix) and SCF (25 ng ml⁻¹, Gibco). At day 10, embryoid bodies were dissociated to single cells using Collagenase-IV and Dispase-II (1 mg ml⁻¹, Gibco) followed by enzyme free cell dissociation buffer (Gibco) treatment. Single cells were cultivated at 2E+5 per ml on tissue culture plates (Corning) for an additional 10 days in CellgroSCGM with TPO (100 ng ml⁻¹) and IL1-β (10 ng ml⁻¹, Miltenyi Biotec). Half of culture media was renewed every 3 days.