Reagents:
Iscove’s Modified Dulbecco’s Medium(IMDM) and HiSep were from HiMedia Labs (Mumbai, India), Mesenchymal cell-specific fetal bovine serum (Mesen FBS) and 35 mm2 low adhesion plates for colony formation assays were from Stem Cell Tech (Vancouver, Canada); Dynabeads® CD34 Positive Isolation Kit was from Invitrogen/Thermo Fisher Scientific (MA); SCF, Granulocyte Monocyte Colony Stimulating factor (GM-CSF) and Interleukin-3 (IL-3) were from Peprotech (NJ, USA), Erythropoietin (EPO), TGFβ1, non-enzymatic cell dissociation solution, Cyclopiazonic Acid (CPA), Indolactam (+), Indolactam (-), monensin, antibody to cellular fibronectin (mouse monoclonal), were from Sigma-Aldrich (MO, USA), BAPTA AM [1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis(acetoxymethyl ester)], Thapsigargin, were from Molecular Probes/Invitrogen/Thermo Fisher Scientific (MA,USA), cyclic RGD peptide (AcPenRGDC-OH (Pen to C cyclised)), α4β1-specific peptide (1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys-OH), cell-permeant octapeptide PKC inhibitor (Myristoylated RKRTLRRL) and Fibronectin-adhesion-promoting peptide were from Bachem AG, Bubendorf, Switzerland, anti CD34 antibody (raised in mouse) was from BD Biosciences (CA, USA), and anti-Ki67 (raised in rabbit) antibody was from Abcam (Cambridge, UK) , FITC-conjugated anti-mouse and PE-conjugated anti-rabbit antibodies were from Chemicon (CA, USA), all tissue culture grade plastic ware was from Nunc/Thermo Fisher Scientific ( MA, USA) and microfuge tubes were from Eppendorf (Hamburg, Germany). Dynabeads® CD34 Positive Isolation Kit was from Invitrogen/Thermo Fisher Scientific (MA, USA).
Peptide sequences:
α5β1: H-*CRRETAWAC*-NH2 [ cyclised between C and C ]
αIIbβ3: H-*CNPRGD(109)RC*-NH2 [cyclised between C and C]
Most of the pharmacological reagents including all peptides were dissolved in DMSO, unless otherwise stated. Control BMSCs were treated with equal amounts of DMSO or its inactive control reagent dissolved in DMSO. The working concentration of all these reagents was determined by pre-screening them on BMSCs. The maximum tolerated non-toxic concentrations were used in the experiments.