Fms like tyrosine kinase 3 flt3

Umbilical cord blood (UCB) was collected from the umbilical cord of full-term normal deliveries in collaboration with Milano Cord Blood Bank (IRCSS Ca’ Granda Foundation – Ospedale Maggiore Policlinico). The mononuclear cell fraction was obtained by density gradient centrifugation using Ficoll-Paque (Lymphoprep, Sentinel Diagnostics) and CD34⁺ HSPCs were immunomagnetically isolated using CD34 Microbead Kit (MiniMACS kit, Miltenyi Biotec). Flow-cytometric analysis allowed to assess the purity of sorted cell population, displaying 90% of CD34⁺ HSPCs and negligible presence of CD14⁺ (monocytes) and CD3⁺ (lymphocytes) cells (Additional file 1: Figure S1). Isolated CD34⁺ HSPCs were cultured in Stem Span medium (StemCell Technologies) supplemented with 20 ng/mL of interleukin (IL)‐6 (PeproTech), 20 ng/mL of IL‐3 (PeproTech), 50 ng/mL of fms‐like tyrosine kinase 3 (FLT3, PeproTech), and 50 ng/mL of stem cell factor (SCF, PeproTech). Cells were cultured in HG (30 mM of glucose, Sigma-Aldrich) or NG (30 mM of mannitol, Sigma-Aldrich) conditions for up to 20 days and treated or not with increasing concentration of LIRA (50 nM and 100 nM; MedCHemExpress) ± selective GLP-1R antagonist exendin (9-39) (150 nM EXE; MedCHemExpress).
Capan-1 cells (HTB-79^™), used as GLP-1R positive control, were purchased from ATCC and grown in RPMI medium supplemented with 20% FBS, as indicated by the supplier.

Mononuclear cells were isolated from CB (obtained from the Cleveland Blood Center on a contractual basis) by Ficoll-Hypaque Plus density centrifugation. CD34⁺ hematopoietic stem and progenitor cells (HSPCs) were purified by positive selection using the Midi-magnetic–activated cell sorting LS columns (130-042-401, Miltenyi Biotec). CD34⁺ cells were cultured in Iscove modified Dulbecco medium (IMDM) containing 20% BIT 9500 medium (Stem Cell Technologies) supplemented with SCF (100 ng/mL), Fms-like tyrosine kinase 3 (FLT-3; 10 ng/mL), IL-6 (20 ng/mL), and thrombopoietin (TPO; 100 ng/mL; these cytokines were purchased from PeproTech).