The blood samples were obtained from two healthy volunteers under the approval No. KEN1812-032 from the ethics committee of Okayama University Hospital. And the monocytes were purified with BD Vacutainer CPT (REF 362,753, Becton Dickinson and Company, NJ) and reprogrammed to iPSCs with Sendai virus vector, SeVdp (KOSM)302L which was a kind gift from Dr. Mahito Nakanishi, the National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan. The hiPSCs were maintained on mouse embryonic fibroblasts (MEF) in StemFit Basic 03 media (Ajinomoto Co, Japan) with 5 ng/ml fibroblast growth factor-2 (FGF2) (Katayama Chemical Industries, Japan). After 3–4 passages on MEF, hiPSCs were transferred to feeder-free conditions in dishes coated with Matrigel matrix (Corning, NY) with StemFit Basic 03 media and 5 ng/ml FGF2. The human pancreatic cancer cell line BxPC3 cells (ATCC, VA) were cultured in RPMI-1640 (Wako, Japan) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, MA). When the cells reached 70–80% confluence, the media were changed to RPMI-1640 containing 5% knockout serum replacement (Thermo Fisher Scientific, MA). After 48 h, the conditioned media was collected and centrifuged at 1000×g for 10 min, and the supernatants were then filtered through 0.45 μm filters (Sartorius, Göttingen, Germany). For the conversion, the hiPSCs were passaged to new Matrigel coated dishes with StemFit Basic 03 media and, the media were changed after 24 h to StemFit Basic 03 with 50% of CM and 10 μM AZD6244 (Selleck, Japan), 1 μM CHIR99021, 1000 U/mL leukemia inhibitory factor (Merck Millipore, MA) and 5 ng/ml FGF2. Half of the media was changed daily and when cells reached 70–80% confluence, they were passaged to new dishes. The conversion process was continued up to one month. The hiPSCs in day 0 and after four weeks of conversion were cultured on gelatin coated dishes in DMEM media (Wako, Japan) with 15% FBS. The primary cultures of cells from tumors were prepared as same as described previously (Hassan et al. 2022). After digestion, 3 ml of StemFit Basic 03 medium with 10% FBS was added to stop digestion. The cell suspension was then centrifuged at 4000×g for 5 min, the supernatant was discarded, cell pellets were resuspended in 2 ml of StemFit Basic 03 medium, and cells were then seeded into Matrigel coated dishes at a density of 1 × 106 cells/dish in StemFit Basic 03 medium. Blood samples were taken after obtained informed consent.

To evaluate the spheroid forming ability of cells, 1000 cells/well were seeded into 24-well ultra-low attachment plates (Sumitomo Bakelite, Japan) with StemFit Basic 03 medium. The plates were incubated at 37 °C for 10 days and then spheroids were photographed using an IX81 inverted microscope (Olympus, Japan).

One million cells were washed with cold PBS and incubated for 20 min on ice with a blocking buffer consisting of 0.2% BSA (Miltenyi Biotec, Germany) in PBS. Then, cells were incubated with primary antibodies; anti-CD44 monoclonal antibody (IM7) (Thermo Fisher Scientific, MA), anti-CD24 monoclonal antibody (SN3) (Thermo Fisher Scientific, MA), anti- EpCAM monoclonal antibody (E8Q1Z) (Cell Signaling Technology, MA), and anti-CD133 monoclonal antibody (A8N6N) (Cell Signaling Technology, MA). After incubation, cells were washed with PBS and incubated with secondary antibodies; goat anti-rabbit IgG (H+L) Alexa Fluor® 647 (Thermo Fisher Scientific, MA), goat anti-rat (IgG) PE (Abcam, Cambridge, United Kingdom) and goat anti-mouse IgG (H+L) Alexa Fluor® 488 (Thermo Fisher Scientific, MA) on ice for 20 min. Then, cells were washed and run on Accuri C6 Plus flow cytometer (BD Biosciences, NJ). The obtained data was analyzed with Flowjo software (FlowJo, LLC, ORE).

The NOD-SCID mice were purchased from Charles River, Japan and randomly divided into groups of three mice in each. The hiPSCs, hiBxcm1 and hiBxcm2 cells, were passaged, counted and 2 × 106 cells were prepared and injected into pancreases of mice. Mice were sacrificed after 5 weeks of injections and the tumors were excised. Details for animal experiments were in our previous report (Hassan et al. 2022). For serial transplantation of tumor tissues, the 4-week-old Balb/c nu/nu female mice (Charles River, Japan) were anesthetized for surgery. Then, a small cut in the skin was made, 3–4 mm tumor tissue pieces were prepared from tumors developed in pancreas of NOD-SCID mice, washed with PBS, transplanted into the cut area and skin was closed with sutures. Two months after transplantation, mice were sacrificed, and the tumors were excised. The animal experiments were conducted according to Okayama University guidelines under the approval No. 2020382 and 2020631 from the ethics committee for animal experiments of Okayama University.

Tumor tissues fixed in formalin and embedded in paraffin were sectioned by a microtome RM2255 (Leica, Germany). For the nuclear/cytoplasm staining, the sections were stained with hematoxylin and eosin Y (Sigma-Aldrich, MO).

Cells were suspended in 500 μl EBM2 media supplemented with human epidermal growth factor (EGF; 5 ng/mL), vascular endothelial growth factor (VEGF; 0.5 ng/mL), R3-insulin like growth factor-1 (20 ng/mL), ascorbic acid (1 μg/mL), hydrocortisone (0.2 μg/mL), human basic fibroblast growth factor (bFGF; 10 ng/mL), heparin (22.5 μg/mL) (Lonza, Switzerland) and 2% FBS, and were seeded at a density of 7.5 × 104 cells/well in 12-well plates coated with Matrigel. Twenty-four hours after seeding, tube structures formed by the cells were photographed with an IX81 inverted microscope.

Total RNA was extracted with TRIzol RNA isolation reagents (Life Technologies, CA, USA) and treated with DNase I (Promega, WI). Samples were subjected to sequencing with Novaseq6000 (Illumina, CA) and the obtained data were analyzed with Galaxy <usegalaxy.org> as descripted previously (Hassan et al. 2022). Briefly, differential expressed genes were identified with Cuffdiff tool and used as inputs to detect enriched pathways from Kyoto Encyclopedia of Genes and Genomes (KEGG) database using DAVID Bioinformatics Resources (Huang et al. 2009a, b). The integrated Differential Expression and Pathway analysis (iDEP) < http://bioinformatics.sdstate.edu/idep93/ > was used to generate the heat map and volcano plots (Ge et al. 2018).

Round coverslips (18 mm, Matsunami, Japan) were inserted into wells of 24-well plates, coated with Matrigel, and the cells were seeded at a density of 4 × 104 cells/well. After 24 h, cells were fixed, blocked, permeabilized and incubated with anti-CD44 monoclonal antibody, anti-CD24 monoclonal antibody (Thermo Fisher Scientific, MA), anti-CD133 monoclonal antibody, anti-NANOG monoclonal antibody, anti-ALDH1A1 monoclonal antibody (Cell Signaling Technology, MA) or anti-OCT3/4 monoclonal antibody (Santa Cruz Biotechnology, TX). Cells were then incubated with PE labeled anti-rat goat IgG, Alexa Fluor 488 labeled anti-mouse goat IgG, or Alexa Fluor 555 labeled anti-rabbit goat IgG (Thermo Fisher Scientific, MA). The VECTASHIELD Mounting Medium (Vector Labs, CA) was used to mount coverslips and slides were photographed using an FSX100 fluorescent microscope (Olympus, Japan).

Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane (Millipore Sigma, MA). After blocking with 5% skim milk, membrane was incubated overnight at 4 °C with primary antibodies against Erk1/2, phospho-Erk1/2 (Thr202/Tyr204), Akt, phospho-Akt (Ser473) (Cell Signaling Technology, MA) and β-actin (Wako, Japan). Then, membrane was incubated with either anti-mouse or anti-rabbit HRP–conjugated secondary antibodies, washed and visualized with the Western Lightning Plus ECL Substrate (Perkin Elmer, MA).

The Graphpad prism 9 software (GraphPad Software, SD) was used to analyze data. Each data was presented as mean ± standard deviation and two-tailed t-test was used to assess the significance. The p value ≤ 0.05 was considered significant.