Immortalized Human Pancreas-Derived Mesenchymal Stromal Cells
Human pancreatic specimens, acquired post-pancreatectomy from a pancreatic cancer patient, were transported in alpha MEM (Paneco, Russia) with 15% FBS (Biosera, France), 2% penicillin/streptomycin (Gibco, USA), and 300 KIU/ml aprotinin. Following established protocols (Mehigan et al. 1980; Ricordi et al. 1988; Stull et al. 2012), specimens were dissected, rinsed in HBSS (Paneco, Russia), and incubated in 200 IU/ml collagenase (Paneco, Russia) for 30 minutes at 37 with agitation. After digestion, the material was centrifuged, the supernatant discarded, and the pellet layered onto 10 ml Ficoll 1.077 (Paneco, Russia) for separation. The islets were isolated from the supernatant through a 70 m strainer and cultured in DMEM (Paneco, Russia) with supplements until 8090% confluency. hPD-MSCs were immortalized by stable expression of hTERT. The lentiviral plasmid construct encoding hTERT was described previously (Evtushenko et al. 2021). The hTERT fragment was delivered by lentiviral transduction with polybrene (Serva, Germany). Positive selection, employing puromycin (Sigma-Aldrich, USA) was executed over a two-passage duration one week post-transduction. One million cells were collected, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germany). The library was prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA).