Characterization of Idiopathic Pulmonary Fibrosis Mesenchymal Stem Cells

Tissue fragments were cultured with MSCGM medium (Lonza, Basel, Switzerland) [43 (link)]. MSCs isolated from patients affected by idiopathic pulmonary fibrosis were named IPF-MSCs, while MSCs isolated from control lung of patients with adenocarcinoma were defined C-MSCs. According to the criteria by Dominici [20 (link)], cells were characterized by testing the plastic adherence, the immunophenotype and the multipotency as previously described [21 (link),44 (link)]. Finally, the expression of genes related to stemness (OCT4, SOX2, NANOG, KFL4) was analyzed by qRT-PCR. Cell pellet was collected from 3 × 10⁵ cells after 72 h of culture and total RNA was extracted with Total RNA Purification Plus Kit (Norgen, Biotek Corp, Schmon Parkway, Thorold, ON, Canada). cDNA synthesis was performed using 5× All-In-One RT MasterMix (Applied Biological Materials, Richmond, BC, Canada). After amplification qRT-PCR was carried out with SsoFast EvaGreen Supermix (Bio-Rad, Milano, Italy) and fluorescence and melting curves were acquired. The parameter threshold cycle (Ct) was defined as the cycle number at which the first detectable increase above the threshold in fluorescence was observed. All samples were tested in duplicate with the housekeeping genes GAPDH for data normalization. mRNA expression was calculated by the 2^−ΔΔCt method [45 (link)]. The primer sequences are summarized in Table 3.