Mil 3

For evaluation of erythroid potential from progenitor populations, cells were seeded in Methocult M3436 and myeloid potential was evaluated with Methocult M3534. Colonies were scored after 8 days of culture. CFU-Mix colonies were scored in Methocult M3434 based on colonies containing red cells after 8 days of culture. Megakaryocytic potential was evaluated using the Megacult collagen-based assay. All these media were from StemCell Technologies, Vancouver, Canada. Megakaryocyte cultures were supplemented with 10ng/ml mIL-3, 20ng/ml hIL-6 (Peprotech), 50ng/ml hIL-11 (Peprotech), 50ng/ml hThpo (Peprotech), and megakaryocytes detected using acetylthiocholiniodide staining (Sigma) according to manufacturer’s instructions after 7-8 days culture. For evaluation of Mk potential from single GE⁺ and GE⁻ Lin⁻Sca-1⁺c-Kit⁺Flt3^hi cells, these cells were sorted into X-vivo15 medium containing glutamax and gentamycin (BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 10^-4M β-mercaptoethanol, 50ng/mL mSCF, 50ng/mL hFLT3 ligand (hFL; Immunex), 50ng/mL hThpo, 20ng/mL mIL-3 and 5U/ml hEPO (Roche). For each experiment, 240 cells were manually plated at 1 cell per well into 60 well Terasaki plates. Mk potential was evaluated 8 or 12 days after culture using an inverted microscope.