Thrombopoietin tpo

Patients’ bone marrow cells harvested under general anesthesia, were separated with lymphoprep (Eurobio, Oslo, Norway) and centrifuged in order to collect mononuclear cells (MNC). Patient’s MPB was collected by apheresis. The positive selection of CD34+ cells from MNC or from MPB was performed using immunomagnetic beads and an immunomagnetic enrichment device (CliniMACs, Miltenyi Biotec, Bergish – Gladbach, Germany). Purified CD34+ cells were seeded on cell culture bags pre-coated with clinical grade Retronectin™ (Takara Bio Co) in serum free medium (X-Vivo 20, Biowhittacker/Lonza) and clinical grade stem cell factor (SCF) (300 ng/ml), FLT3-L (300 ng/ml), thrombopoietin (TPO) (100 ng/ml) and IL-3 (20 ng/ml) (all from Peprotech). After 24 hours of prestimulation, cells were transduced twice with LV-w1.6.WASp (1×10⁸ig/ml) each time for 18 hours. At the end of the transduction procedure, washed cells were resuspended in 4% human serum albumin and transferred in a sterile bag for infusion to the patient. Aliquots of cells were further cultured for 14 days to measure stable proviral integration by qPCR and WASp expression by flow cytometry.

Purified human CD34+ progenitor cells were derived from GCSF–treated peripheral blood cells of healthy donors. These cells were grown at 37°C with 5% CO2 in serum-free medium consisting of Iscove’s modified Dulbecco’s medium (IMDM) with 1-thioglycerol, BIT9500 supplement (BITS) (Stem Cell Technologies), BSA (Sigma-Aldrich), and the indicated cytokines (PeproTech). The cells initially underwent 72 h of expansion with 100 ng/ml SCF (PeproTech), 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3 ligand) (PeproTech), 100 ng/ml thrombopoietin (TPO) (PeproTech), and 50 ng/ml IL-3 (PeproTech). After expansion cells were then seeded in erythroid differentiation medium, which contains recombinant human erythropoietin at 4.5 U/ml (Procrit; Amgen), 10 ng/ml SCF and BIT9500 supplement. Human bronchial epithelial cells (Beas2B) were grown at 37°C with 5% CO2 in DMEM medium with 10% FBS. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies, reverse transcriptase, and real-time PCR SosoFast reagents were purchased from Bio-Rad Laboratories (Hercules, CA). Amicon Ultra centrifuge filters were purchased from EMD Millipore (Billerica, MA). Anti-IL-33 antibody was from R&D Systems (Minneapolis, MN). Anti-LarminA/C antibody was from Santa Cruz (Dallas, TX). Anti-GAPDH and β-actin antibodies and RBC lysis buffer were from Sigma (St. Louis, MO).