Recombinant human bmp4

TGFβR inhibitor (SB431542), NOTCH inhibitor (DAPT), BMPR inhibitor (DMH1), methylcellulose and O-glycosylation inhibitor (Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside) were purchased from Sigma-Aldrich (St Louis, MO). For PI3K inhibition, LY294002 was obtained from Calbiochem (San Diego, CA). For FGFR inhibition, BGJ398 and PD173074 were purchased from Selleckchem (Houston, TX). Recombinant human BMP4 and CXCL1 were purchased from Peprotech (Rocky Hill, NJ).

Intestinal organoids were obtained as previously described^{63 (link)}: small intestines of mice were dissected and dissociated in 8 mM EDTA/PBS for 5 min at RT followed by 20 min incubation in ice-cold 2 mM EDTA/PBS at 4 °C. The epithelia were fractionated by shaking in ice-cold PBS. 250 crypts were seeded in 20 µl of growth factor-reduced Matrigel (BD Matrigel #356231) and cultured in basic crypt medium (60/40 Advanced DMEM/F12 supplemented with N2 and B27, GlutaMax, N-Acetylcysteine and Penicillin/Streptomycin (Invitrogen)) containing 50 ng/ml mEGF (Gibco), 100 ng/ml mNoggin (Peprotech) and 500 ng/ml hR-spondin1 (Peprotech). Organoids were split every 4–6 days by mechanical disruption. Cre-mediated recombination was induced by the addition of 800 nM 4-hydroxy-tamoxifen for 2 days. β-cat^GOF organoids were selected by R-spondin1 withdrawal. All analyses were performed at 7–10 days after mutagenesis. Recombinant human BMP4 (Peprotech) was added for 24 h in Noggin-free crypt medium supplemented with EGF and R-spondin1. For serial re-plating, β-cat^GOF; Mll1^−/− organoids were dissociated into single cells with TrypLE Express (ThermoFisher Scientific) for 5 min at 37 °C, and 2000 cells were seeded in 20 µl of growth factor-reduced Matrigel and cultured in crypt medium supplemented with mEGF, mNoggin, and hR-spondin1.

Cells were starved overnight in serum-free RPMI or DMEM medium before treatment with recombinant human TGFβ1 (5 ng/ml, PeproTech EC Ltd, London, UK), recombinant human BMP4 (30 ng/mL, PeproTech EC Ltd, London, UK), recombinant human BMP7 (30 ng/mL, a gift from Kuber Sampath, Sanofi-Genzyme Research Center, Framingham, USA) or activin-A (10 ng/mL, PeproTech EC Ltd, London, UK) for the time periods indicated in the figures. The N2B27 media in glioblastoma cells were changed prior to TGFβ1 treatment. The LY2157299 TβRI kinase inhibitor (Sigma-Aldrich AB, Stockholm, Sweden) was administered to cells one hour before TGFβ1 addition at a final concentration of 2.5 nM. The MAP kinase kinase inhibitor (MEKi; PD184352, Sigma-Aldrich AB, Stockholm, Sweden) was added at a concentration of 0.5 µM, the Jun N-terminal kinase inhibitor (JNKi; SP600125, Calbiochem-Merck, Stockholm, Sweden) and the p38 MAP-kinase inhibitor (P38i; SB203580, Calbiochem-Merck, Stockholm, Sweden) were added at a concentration of 10 µM. Protein synthesis was blocked by cycloheximide (CHX; C1988, Sigma-Aldrich AB, Stockholm, Sweden), administered to the cells 1 h before TGFβ treatment at a final concentration of 40 µg/ml. Dimethyl-sulfoxide (DMSO) served as vehicle for all chemicals.

RPMI medium, DPBS without Ca²⁺ and Mg²⁺, penicillin-streptomycin, Glutamax, microplate BCA protein assay, trypsin-EDTA, and TEAB were from Thermo Scientific, Waltham, MA, USA; trypsin protease-MS grade, sodium dodecyl sulphate, acetonitrile, HPLC-grade ethanol, and water were from Sigma, Dorset, UK; human Fc block and Alexa Fluor^® anti-human CD300A Cat# 566342 were from BD Bioscience, Berkshire, UK; PE anti-human CD68 Cat# 130-118-486 was from Miltenyi Biotec, Surrey, UK; PE anti-human CD109 Cat# 323305, APC anti-human LILRB2 Cat# 338707, APC anti-human Siglec-10 Cat# 347606, APC anti-human CD206 Cat# 321109, APC anti-human CD80 Cat# 305219, APC anti-human CD86 Cat# 374208, PE/Cy7 anti-human CD14 Cat# 367112, and recombinant human M-CSF were from Biolegend, San Diego, CA, USA. X-VIVO15 Serum free medium Cat# BE02-060Q was from Lonza, Basel, Switzerland. Recombinant human BMP4 (Cat# 120-05), SCF (Cat# 300-07), VEGF (Cat# 100-20) and IL-3 (Cat# 200-03) were from Peprotech, London, UK. EmbryoMax^® 0.1% Gelatin solution was from Millipore, London, UK. Y-27632 (Cat# 1254/10) was from Bio-Techne, Abingdon, UK.

hiPSC colonies were passaged and seeded as aggregates on a Matrigel-coated plate at a density of 10–15% confluency per well in a 12-well plate. Subsequently, E8 medium supplemented with ROCKi was added before differentiation into hematopoietic cells (Day 0). After 24 h, the medium was replaced with STEMdiff™ APEL2 (STEMCELL Technologies) media supplemented with 1% P/S and stepwise cytokines for hematopoietic induction with or without ASPP 049 (the processes of isolation and purification were previously described) [27 (link), 28 (link)]. Briefly, the cells were induced by the addition of recombinant human BMP4 (50 ng/mL, PeproTech) for two days, followed by the addition of recombinant human VEGF (50 ng/mL, PeproTech) and recombinant human bFGF (50 ng/mL, PeproTech) from day 3 to 5. On day 5, in addition to VEGF and bFGF, SB431542 (20 μM, Sigma) was added. On day 7, the cytokines in the medium were substituted with recombinant human SCF (50 ng/mL, PeproTech), recombinant human IL-3 (50 ng/mL, PeproTech), and recombinant human TPO (50 ng/mL, PeproTech). During the following five days, the cells were treated with ASPP 049 and DMSO vehicle control in APEL2 media supplemented with the day 7 cytokine cocktail, and half-volume media was changed every other day. The differentiation culture was maintained at 37ºC with 5% CO₂ and 5% O₂ in a standard 95% humidified incubator.