Rhtpo

Each cell was incubated in the culture medium without WEHI-CM for 48 h. For activity-based antibody selection, BaF3/MPL (1 × 10⁵ cells/mL) cells were incubated with rhTPO (R&D Systems) or antibodies. Cell proliferation was measured by the CellTiter® 96 AQueous One Solution Cell Proliferation Assay System (Promega, Madison, WI, US) with absorbance at 490 nm. To validate 2R13 agonistic activity, BaF3 (1 × 10⁵ cells/mL), BaF3/MPL (1 × 10⁵ cells/mL), and M07e (5 × 10⁵ cells/mL) were incubated with rhTPO (PeproTech, Rocky Hill, NJ, US) or 2R13. BaF3 and BaF3/MPL cell proliferation was measured by Cell Titer-Glo® Luminescent Cell Viability assay kit (Promega) using Victor3 V 1420 Multilabel Counter (Perkin Elmer Inc, Waltham, MA, US). M07e cell proliferation was measured by Cell Counting Kit-8 (GlpBio, Montclair, CA, US) with absorbance at 460 nm.

Sorted primary murine cKit⁺ and LSK cells were cultured in SFEMII media (Stemcell technology) supplemented with; rhFlt3/Flk-2 ligand (Stemcell technology), rhTPO (Stemcell technology), rhIL-6 (R&D system), rmIL-3 (R&D systems) and rmSCF (R&D systems) at a concentration of 20 ng/mL. UCB samples were provided by the Karolinska Hospital (Stockholm, Sweden) with informed consent from the parents and all investigation has been performed according ethical standards and to the declaration of Helsinki and to national and international guidelines. The experiments on the UCB samples were approved by The Regional Ethical Review Board in Stockholm (Dnr: 2012/480-31/1).
Lymphoprep solution (Invitrogen) was used to isolate mononuclear cell fraction and CD34⁺ cells were enriched by using CD34 microbead kit (Miltenyi Biotec). The CD34⁺ and sorted HSC UBC cells were expanded in SFEMII media supplemented with; rhIL-6, rhIL-3 (R&D systems), rhFl3/Flk-2 ligand, rhTPO and rhSCF (R&D systems) all in final concentrations of 20 ng/mL.