Il 11

Sorted single Tom⁺ cells were cultured in 96-well plates containing serum-free medium StemSpan SFEM II (STEMCELL Technologies) supplemented with 50 ng/ml recombinant murine SCF and 50 ng/ml TPO (Peprotech). Cultures were examined on days 3–4, and wells with living cells were ascertained by brightfield and (where appropriate) fluorescent microscopy for Tom expression. Wells were scored throughout the culture period based on morphology as either U colonies, differentiating into Mk’s, or a mixture of undifferentiated cells and Mk’s (U+Mk). After 7 d in culture, an equal number of randomly selected U and U+Mk colonies was replated whereby half of the well was transferred into a 96-well plate containing a fresh batch of the medium described above and the other half was cultured in semisolid MethoCult GF M3434 (STEMCELL Technologies) supplemented with TPO to assay for CFU potential.
To assess megakaryocytic differentiation, bulk sorted Tom⁺ cells were transferred to 24-well plates at a density of 1,000–2,000 cells/well and cultured in the same liquid medium as above with the addition of 20 ng/ml IL-6 and 20 ng/ml IL-11 (Peprotech). Brightfield and fluorescent microphotographs were acquired using BZ-X800 microscope (Keyence) with manufacturer’s software.

Mouse Lin⁻ BM cells were isolated and kept at −80°C overnight. Cells were thawed the following day and seeded in a 24-well plate at 1 × 10⁵ cells/well in StemSpan medium supplemented with complete growth medium supplement (CGMS; 1% penicillin/streptomycin, 50 ng/mL mouse SCF, 100 ng/mL FLT-3L, 100 ng/mL IL-11, and 20 ng/mL mouse IL-3; all from PeproTech) and incubated for 2 days. Plates coated with Retronectin (Takara Bio) were used to preload viral constructs, and the first round of transduction was completed with prestimulated Lin⁻ cells. A new Retronectin-coated plate was used for preloading viral vectors on the following day (day 0), and a second round of transduction was conducted by transferring transduced cells to the new plate. After overnight incubation, cells were transferred to 12-well plates and cultured in Iscove's modified Dulbecco's medium (IMDM; Biochrom) supplemented with CGMS. After day 15, cells were seeded at 100 cells/well in 96-well plates and cultured for an additional 2 weeks. Trypan blue exclusion counting was performed on days 1, 4, 6, 8, 11, and 15.