Methocult

To test their colony formation capacity (CFC), cells with presumptive hemogenic capacity were sorted from 168 h gastruloids based on surface marker expression. Sorted cells were added at serial dilutions to 1.5 ml of MethoCult (Stem Cell Technologies, H4434). The mixture of MethoCult and cells was vortexed and transferred with a blunt end needle syringe (Stem Cell Technologies, 28110) into meniscus-free SmartDish plates (6-well-plate, Stem Cell Technologies, 27370). To allow constant humidification, the 6-well plates were placed in a large 150 mm square dish (Corning, CLS431111) containing small 3.5 × 10 mm tissue culture dishes filled with water. The cultures were incubated at 37 °C, 5% CO2 for 14 days and the CFC content was automatedly imaged after 7 and 14 days by using the StemVision imaging system (Stem Cell Technologies, Version 2.0.1.0). Additionally, higher-resolution images of colonies were acquired with a PALM Microbeam microscope (Zeiss) equipped with an AxioCamICc1 color camera and 5 × and 10 × objectives.

Mast cells were differentiated as described by Saito et al. (15 (link)), with modifications. Briefly, CD34⁺ cells from peripheral blood were isolated by positive immunomagnetic separation and cultured in 24-well plates in 100 μL of METHOCULT™ (Stem Cell) plus 200 μL of IMDM, supplemented with stem cell factor (SCF), Interleukin (IL)-6, and IL-3 (200, 50, and 5 ng/mL, respectively) per well. After 2 weeks, 100 μL of METHOCULT™ (Stem Cell) plus 200 μL of IMDM supplemented with SCF and IL-6 (200 and 50 ng/mL, respectively) were added to each well. At week 4, 1 mL of supplemented IMDM (SCF, 200 ng/mL; IL-6, 50 ng/mL; insulin–transferrin–selenium solution, Gibco^©, catalog no. 41400-045, 100 μL/mL) was added to each well. At week 6, non-adherent cells were transferred to a 12-well plate in supplemented IMDM [SCF, 100 ng/mL; IL-6, 50 ng/mL; insulin–transferrin–selenium solution (20%); 20% of 10% BSA in phosphate-buffered saline]. Two weeks thereafter, non-adherent cells were transferred to six-well plates and cultured with I-10 supplemented with SCF (100 ng/mL) and IL-6 (50 ng/mL); 1 week later, the cells were harvested.

For whole BM analysis, approximately 4 × 10⁴ cells were plated in methylcellulose M3434 (Methocult, StemCell Technologies, Vancouver, Canada). Colonies were scored after 8–10 days. Pictures were taken on Olympus IX50 microscope with ×2, ×4, and ×10 magnification. Cells were washed, resuspended, counted with trypan blue and if applicable replated into fresh methylcellulose. For colony formation analysis of human CD34⁺ cells, 5 × 10³ cells were plated into H4434 (Methocult; StemCell Technologies, Vancouver, Canada). After scoring of M3434 plates, when indicated, dishes were incubated with a mix of two volumes of 0.3% hydrogen peroxide and five volumes of 0.2% di-hydrochloride benzidine (Sigma Aldrich, Buchs, Switzerland) in 0.5 M acetic acid/1× PBS for 5 min at 37 °C.