Human tpo

According to a protocol previously established by Takayama et al. [29 (link)], hiPSC colonies were removed from MEF feeders using a dissociation buffer (0.25% trypsin, 1 mg/ml collagenase IV, 20% KSR, 1 mmol/l CaCl₂ in PBS), transferred onto irradiated C3H10T1/2 cells and differentiated with IMDM medium containing 10 mg/l insulin, 5.5 mg/l transferrin, 6.7 mg/ml selenium, 2 mmol/l L-glutamine, 15% fetal bovine serum (all Gibco), 0.45 μmol/l α-monothioglycerol (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich) and 20 ng/ml recombinant human vascular endothelial growth factor (Invitrogen). On day 15, hiPSC-Sacs were disrupted with a cell scraper, crushed with a pipette and passed through a 40 μm cell strainer (BD Falcon). The yielded cells were transferred onto irradiated C3H10T1/2 cells and cultured in the same medium without vascular endothelial growth factor containing 100 ng/ml human TPO (R&D), 50 ng/ml human SCF (R&D), and 25 U/ml heparin (Sigma-Aldrich). Medium was changed every 3 days. According to Takayama et al. [29 (link)], floating cells from days 24 to 30 were collected for platelet and MK analysis.

Sorted CD34ˉLSK cells were cultured in StemSpan SFEM medium (Stem Cell Technology) suspended with purified recombinant mouse SCF (10 ng/ml, R&D Systems, Minneapolis, MN), human TPO (10 ng/ml, R&D Systems), human Flt-3L (10 ng/ml, R&D Systems) and mouse IL-3(10 ng/ml, R&D Systems)52 (link)53 (link)54 (link). Cell proliferation was measured at 490 nm using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI).

HDFs and iMEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS, Japan Bio Serum). PBMCs were cultured in StemSpan ACF (STEMCELL) with 100 ng/mL human SCF (R&D), 100 ng/mL human TPO (R&D), 100 ng/mL human Flt3/Flk2 (R&D), 50 ng/mL human IL-6 (R&D), and 20 ng/mL human IL-3 (R&D) for 5 days before the SeV vectors infection. Primed PSCs were maintained in StemFit AK02N medium (Ajinomoto) on laminin 511-E8 fragments (iMatrix-511, Nippi)-coated plates. Naive PSCs were cultured on iMEFs and maintained in t2iLGö (Takashima et al., 2014 (link)) medium composed of N2B27 medium (NDiff227, Takara Bio) with 1 μM CHIR99021 (Merck), 1 μM PD0325901 (Merck), 10 μg/mL human LIF (PeproTech), and 2.5 μM Gö6983 (Merck). The day before naive PSCs were plated, iMEF cells were seeded in cell culture dishes at a concentration of 25,000 cells/cm² and cultured overnight. The next day, the cells were washed twice with PBS(−) before plating. Medium was changed every other day and 10 μM Y27632 (Wako) was added just before every medium change. Primed iPSCs were passaged every 6–7 days, and naive iPSCs were every 3–4 days using Accutase (Innovative Cell Technologies).