Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency

Podsumowanie

The present protocol describes the reprogramming of Pancreatic Ductal Adenocarcinoma (PDAC) and normal pancreatic ductal epithelial cells into induced pluripotent stem cells (iPSCs). We provide an optimized and detailed, step-by-step procedure, from preparing lentivirus to establishing stable iPSC lines.

Streszczenie

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Wprowadzenie

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies, and early diagnosis remains challenging due to the asymptomatic nature of the disease. The majority of PDAC patients are diagnosed at the advanced metastatic stage when very limited treatment options are available^1,2. This is mainly due to the lack of reliable biomarkers for the earlier stages, such as those that could be conveniently detected as proteins released into the bloodstream.

PDAC can disseminate very early during its progression, and a better prognosis has been linked to early cancer detection when PDAC is localized in the pancreas³. However, less than a tenth of PDAC patients are diagnosed with a favorable prognosis, allowing for surgical resection. Nonetheless, those few with resectable tumors are also prone to tumor recurrence within 12 months⁴.

In the past five decades, remarkable improvements have been made in surgical techniques, patient care, and treatment modalities^5,6. However, the 5-year survival rate in surgically resected PDAC patients has barely risen to 17%. Nonetheless, this is still better than that in non-resected patients, which has remained almost unchanged (0.9%)^4,7. Chemotherapy is the only other alternative PDAC treatment. Yet, this option is very limited as the great majority of PDAC patients exhibit strong resistance to chemotherapy medications such as Gemcitabine^7,8. Other drugs, such as Erlotinib, are only available to a small group of PDAC patients with specific mutations, most of whom show Erlotinib resistance⁹. The adverse side effects associated with chemotherapy in most PDAC patients are yet another disadvantage of this treatment¹⁰. Recently, promising strategies have shown that immune checkpoint inhibitors (ICIs) and small molecule kinase inhibitors (SMKIs) can be effective in treating PDAC, but durable responses to these targeted therapies remain limited to a minority of patients^11,12. Overall, the discovery of PDAC-specific early biomarkers can pave new avenues for early diagnosis and treatment.

PDAC develops from pancreatic intraepithelial neoplasms (PanIN) precursor lesions that result from non-invasive pancreatic duct epithelial proliferations^13,14. While the formation of PanIN is initiated by oncogene mutations such as KRAS, additional genetic and epigenetic alterations are required for the progression to PDAC. It has been projected that the progression of PanIN through the different stages into invasive PDAC takes about 10 years^13,15,16,17. This timeframe provides a great opportunity to benefit from early PDAC diagnosis. Therefore, extensive research has been carried out to establish tumor xenograft animal models and organoid cultures to study PDAC progression^18,19,20,21. These models have been very useful for studying the invasive stages of PDAC, although not the transition from the early PanIN phases. It is, therefore, important to develop experimental models that can recapitulate the early progression of PanIN stages to enable the discovery of early detection biomarkers.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) using the four transcription factors OCT4, SOX2, KLF4, and c-MYC (OSKM) has illustrated the extent of cellular plasticity²². Cancer cell plasticity has been well-documented, and reprogramming human cancer cells into iPSCs has been successfully used to reset cells to their original cellular state, removing many of the epigenetic insults that have accumulated during cancer progression^{23,24,25,26,27,28,29}. The possibility of using this reprogramming strategy to manipulate cancer cell identity has, therefore, presented great promise in treating cancer^30,31. Indeed, we have previously shown that the differentiation of iPSCs derived from PDACs can recapitulate PDAC progression through the early PanIN stages³². By identifying genes and pathways specific to the early-to-intermediate stages of PDAC, candidate biomarkers were identified that can be clinically used for early PDAC diagnosis^32,33. However, the biomarkers discovered using a single iPSC line showed limited coverage in the majority of PDAC patients³². The challenges of generating iPSC lines from other PDAC patients have halted the ability to discover more reliable biomarkers. This is due to many technical factors, including the heterogeneity of OSKM delivery, as only a small portion of human primary PDAC cells contained all four factors and responded successfully to reprogramming. Here, a detailed protocol is presented for reprogramming primary PDAC cells using a more efficient and consistent dual lentiviral delivery of OSKM.

Protokół

All experimental protocols were approved by the OHSU Institutional Review Board. All methods were carried out in accordance with relevant guidelines and regulations. All animal works for PDX tumors were performed with the OHSU Institutional Animal Use and Care Committee (IACUC) approval. This protocol was tested in Primary PDAC cells from patient-derived xenograft (PDX), BxPc3 cell line exhibiting epithelial morphology that was isolated from the pancreas tissue of a 61-year-old female patient with adenocarcinoma, the H6C7 immortalized epithelial cell line derived from normal human pancreatic duct epithelial, and primary human fibroblasts derived from skin biopsy of healthy individuals. Human PDAC specimens were obtained under the Oregon Pancreas Tissue Registry study (IRB00003609). Informed consent was obtained from all subjects. Human primary fibroblasts were derived in RBiomedical, Edinburgh, UK, from skin samples from anonymous donors undergoing routine surgery at the Edinburgh Royal Infirmary, Little France, UK, under their consent and ethical approval (09/MRE00/91).All lentivirus work has been carried out under Class 2 research activity (GM207/16.6) and approved by the Health and Safety Department at the University of Edinburgh and notified to the HSE competent authority of the Scottish Government. All reprogramming experiments using human pancreatic cells were carried out under the ethical approval of the School of Biological Sciences ethics committee at the University of Edinburgh (reference # asoufi-0002).

1. Preparation of lentiviruses

1. For lentivirus preparation, prepare high-quality packaging and expression plasmids (endotoxin-free) with concentrations between 1-2 µg/µL including psPAX2, pMDG³⁴, and two RES-containing bicistronic vectors; the pSIN4-EF1a-O2S encoding for OCT4 and SOX2 expression driven by EF-1α promoter, and pSIN4-CMV-K2M for KLF4 and c-MYC expression under the CMV enhancer/promoter³⁵ (see Table of Materials). Also, prepare pWPT-GFP plasmid to be used as a transfection control.
2. Thaw Human Embryonic Kidney cell line (293T) and culture in Glasgow's Minimum Essential Medium (GMEM), supplemented with 10% fetal calf serum (FCS), 1x non-essential amino acids, 1 mM sodium pyruvate and 1 mM glutamine at (see Table of Materials) 37 °C and 5% CO₂.
   NOTE: It is recommended to use 293T cells within four passages post thawing.
3. Seed 293T at a density of 3 million cells per 15 cm dish, 24 h before transfection. A total of three dishes are required. It is preferable to seed cells later in the afternoon ~16:00.
4. Next day (~16:00), when cells reach ~40%-50% confluency, prepare the following for three transfection reactions:
   NOTE: Always account for pipetting error by adding 10% volume extra.
   1. Label three 15 mL plastic tubes with the appropriate lentivirus name (pSIN4-EF1a-O2S, pSIN4-CMV-K2M, and pwPT-GFP control). Add 1.710 mL reduced serum medium (see Table of Materials) to each tube.
   2. Dilute 90 µL transfection reagent (see Table of Materials) in 1.710 mL reduced serum medium, mix by vertexing for 2 s, and incubate at room temperature for 5 min.
   3. Mix the packaging vectors; 5.1 µg of psPAX2 and 2.4 µg of pMDG (7.5 µg total).
   4. Add the packaging vector mixture to the transfection medium (from step 1.4.2) and vortex for 2 s.
   5. Add 7.5 µg of each reprogramming vector: pSIN4-EF1a-O2S and pSIN4-CMV-K2M and the pwPT-GFP control to the transfection mixture from step (1.4.4) and vortex for 2 s.
      NOTE: Use the expression vector: viral vector at a 1:1 ratio.
5. Incubate the transfection tubes for 15 min at room temperature.
6. Transfect the 293T cells with each lentivirus by directly adding the transfection-DNA mixture from step (1.4.5) to the media in a dropwise fashion. Swirl the dish to ensure even distribution across the entire surface.
7. Incubate the transfected cells in a 37 °C, 5% CO₂ incubator overnight.
   NOTE: Use virus-specific waste, a metal disposal bucket, double bag with autoclave bags, and take a liquid waste container for virus and add a disinfectant tablet. Dispose of all pipettes, tips, and tubes in a metal bucket. Dispose of old media into a virus-liquid waste container. Avoid using glass pipettes and glassware to eliminate accidental virus contamination.
8. After 14-16 h post-transfection, replace the medium with 30 mL fresh 293T medium.
9. Incubate the transfected cells at 37 °C, 5% CO₂ for 60-72 h post medium change. Observe cells daily and check transfection efficiency by GFP fluorescence.
   NOTE: Ideally, transfection efficiency should be >90% by GFP. For the other viruses, one should observe clear morphological changes of 293T cells, as they tend to become more round when producing virus particles.
10. Collect the media from each virus transfection culture into 50 mL tubes and spin down to clear out cell debris at 1932 x g for 10 min at 4 °C.
11. Filter each lentivirus supernatant through a 0.45 µM syringe filter to remove smaller debris and collect it into new 50 mL tubes.
12. Divide each lentivirus supernatant into 6 mL aliquots and snap freeze each in liquid nitrogen.
13. Store the lentivirus aliquots at -80 °C until ready to use.

2. Reprogramming lentivirus transduction

1. Thaw primary PDAC cells and culture in completely defined Keratinocyte Serum Free Medium (KSFM), supplemented with Bovine Pituitary Extract (BPE), human recombinant Epidermal Growth Factor (EGF) at 5 ng/mL, and Cholera toxin at 50 ng/mL in a 37 °C, 5% CO₂, and 5% O₂ (hypoxia) incubator (see Table of Materials).
2. Thaw BxPc3, a squamous pancreatic ductal adenocarcinoma cell line, and culture in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) at 37 °C, and 5% CO₂.
3. Thaw H6C7 cells, pancreatic ductal epithelial cells, and culture in KSFM supplemented with BPE and EGF at 5 ng/mL, at 37 °C and 5% CO₂.
4. Thaw Human fibroblast (hFib) and culture in Glasgow's Minimum Essential Medium (GMEM), supplemented with 10% FCS, 1x non-essential amino acids, 1 mM sodium pyruvate and 1 mM glutamine, and 0.05 mM (final) Beta-mercaptoethanol at 37 °C and 5% CO₂.
5. One day prior to lentivirus transduction (preferably late afternoon), prepare two wells of a 6-well plate containing 100,000 cells per well of each of PDAC,BxPc3, H6C7, and hFib cells. Infect one well with OSKM lentiviruses and use the second as an uninfected control.
   NOTE: Use a separate plate for each cell type.
6. The following day, in the afternoon (approximately 24 h later), ensure that the cell confluency reaches at least 70% before proceeding to the next lentivirus infection step.

3. Lentivirus transduction of PDAC

1. Defrost 5 mL of each lentivirus supernatant in a 37 °C virus incubator.
2. Prepare two 15 mL tubes, each containing 2 mL of pre-warmed PDAC culture medium.
3. To the first tube, add 6 mL of each reprogramming lentivirus (pSIN4-CMV-K2M and pSIN4-EF1a-O2S) and 12 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL, see Table of Materials) and mix.
4. To the second tube, add 2 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL).
5. Discard the medium from each well and wash once with PBS at room temperature.
6. Add the reprogramming infection media (tube 1) to the first well.
7. Add the mixture of tube 2 to the second well. This will be the uninfected control.
8. Incubate the PDAC cells in a 37 °C, 5% CO_2, and 5% O₂ (hypoxia) incubator overnight.
9. Next day, in the afternoon, discard the media from both wells and replace it with fresh PDAC culture medium.
10. Incubate the cells at 37 °C, 5% CO_2, and 5% O₂ (hypoxia) for 48 h.

4. Lentivirus transduction of BxPc3 cells

1. Defrost 3 mL of each lentivirus supernatant in a 37 °C virus incubator.
2. Prepare two 15 mL tubes, each containing 2 mL of BxPc3 culture medium.
3. To the first tube, add 3 mL of each reprogramming lentivirus (pSIN4-CMV-K2M and pSIN4-EF1a-O2S) and 6 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL) and mix.
4. To the second tube, add 2 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL).
5. Discard the medium from each well and wash once with PBS.
6. Add the reprogramming infection media (tube 1) to the first well.
7. Add the mixture of tube 2 to the second well. This will be the uninfected control.
8. Incubate the infected BxPc3 cells at 37 °C virus and 5% CO₂ overnight.
9. Next day, in the afternoon, discard the media from both wells and replace it with fresh BxPc3 culture medium.
10. Incubate the cells at 37 °C and 5% CO₂ for 48 h.

5. Lentivirus transduction of H6c7 cells infection

1. Defrost 4 mL of each lentivirus supernatant in a 37 °C virus incubator.
2. Prepare two 15 mL tubes, each containing 2 mL H6c7 culture medium.
3. To the first tube, add 4 mL of each reprogramming lentivirus (pSIN4-CMV-K2M and pSIN4-EF1a-O2S) and 8 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL) and mix.
4. To the second tube, add 2 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL).
5. Discard the medium from both wells and wash once with PBS.
6. Add the reprogramming infection media (tube 1) to the first well.
7. Add the mixture of tube 2 to the second well. This will be the uninfected control.
8. Incubate the infected H6C7 cells at 37 °C virus and 5% CO₂ overnight.
9. Next day, in the afternoon, discard the media from both wells and replace them with a fresh H6c7 culture medium.
10. Incubate the cells at 37 °C and 5% CO₂ for 48 h.

6. Lentivirus transduction of hFib cells

1. Defrost 2 mL of each lentivirus supernatant in a 37 °C virus incubator.
2. Prepare two 15 mL tubes, each containing 2 mL hFib culture medium.
3. To the first tube, add 2 mL of each reprogramming viruses (pSIN4-CMV-K2M and pSIN4-EF1a-O2S) and add 4 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL) and mix.
4. To the second tube, add 2 µL of 4.5 mg/mL polybrene (final 4.5 µg/mL) and mix.
5. Discard the medium from each well and wash once with PBS.
6. Add the reprogramming infection media (tube 1) to the first well.
7. Add the mixture of tube 2 to the second well. This will be the uninfected control.
8. Incubate the infected hFib cells at 37 °C and 5% CO₂ overnight.
9. Next day, in the afternoon, discard the media from both wells and replace it with fresh hFib culture medium.
10. Incubate the cells at 37 °C and 5% CO₂ for a further 48 h.
    NOTE: For efficient lentivirus transduction, titer the amount of lentivirus carefully, as this varies greatly between the different cell types. Multiple lentivirus transductions may be required for some cell types. PDAC cell lines require up to three doses, whereas one dose was enough for reprogramming BxPc3, H6C7, and hFib lines.

7. Preparation of iMEF feeder cells

1. Prepare 40 mL 0.2% gelatine solution by diluting 1% gelatine stock solution with PBS.
2. Coat four 6-well plates by covering each well with 2 mL of 0.2% gelatine solution.
3. Incubate the gelatine-coated plates at 37 °C and 5% CO₂ for at least 30 min.
4. Aspirate the excess gelatine solution and leave the plates to dry under the flow hood.
5. Thaw two vials (~ 4 million cells per vial) of irradiated mouse embryonic fibroblast (iMEFs) by swirling it in a 37 °C water bath.
   NOTE: To reduce the possibility of contamination, be careful to prevent water from splashing near the cap opening. Dry the vial with a paper towel and spray it with 70% ethanol.
6. In the tissue culture hood, pipette the content of each thawed iMEFs vial into one 15 mL tube containing 10 mL of pre-warmed hFib culture medium in a dropwise fashion and mix well by gently inverting the tube.
7. Spin down the cell suspension at 309 x g for 3 min at room temperature and remove the supernatant from each tube. Resuspend each cell pellet in 12 mL hFib medium.
8. Plate 2 mL of the cell suspension per well of the gelatine-coated 6-well plates. Evenly distribute the cells by gently shaking the plates in all directions.
9. Incubate the plates at 37 °C and 5% CO₂ overnight.
   NOTE: Prepare fresh iMEF plates 24 h before being used for the reprogramming experiment.

8. Transferring the infected cells onto the iMEF feeder layer

1. Discard media from the infected and non-infected PDAC, BxPc3, H6c7, and hFib cells. Wash each well twice with PBS at room temperature.
2. Dissociate the cells from the plate by adding 0.5 mL of trypsin to each well. Incubate PDAC cells in a 37 °C, 5% CO_2, and 5% O₂ (hypoxia) incubator for 15 min.
3. Incubate BxPc3 cells at 37 °C and 5% CO₂ for 5 min.
4. Incubate H6c7 and hFib cells at 37 °C and 5% CO₂ for 4 min.
5. Transfer the dissociated cell suspension from each well into 15 mL tubes labeled accordingly as infected or uninfected.
6. Harvest the cells by centrifugation as follows: for PDAC, spin the cell suspension at 300 x g for 5 min; for BxPc3, spin the cell suspension at 120 x g for 5 min; for H6C7, spin the cell suspension at 112 x g for 4 mins; for hFib, spin the cell suspension at 161 x g for 3 min. Perform all the centrifugations at room temperature.
7. Resuspend each cell type pellet in 1 mL of the appropriate culture media.
8. Wash iMEF plates prepared in step 7 with each cell type culture media twice. i.e., wash one plate with PDAC media, one with BxPc3 media, one with H6C7 medium, and the other with hFib medium.
9. Plate 50,000 infected cells per well in five wells of iMEF 6-well plate. Plate 50,000 uninfected control cells in the remaining one well of iMEF 6-well plate.
   NOTE: Optimise the number of infected cells to be plated from 1,000 to 50,000 cells per well.
10. Incubate PDAC cells at 37 °C, 5% CO₂, and 5% O₂ (hypoxia) overnight.
11. Incubate BxPc3, H6C7, and hFib cells at 37 °C and 5% CO₂ overnight.
12. Next day, prepare reprogramming medium as follows: add 100 mL knockout serum replacement (25% final), 5 mL of 200 mM glutamine (1 mM final), 5 mL of 100x non-essential amino acids (100 µM final), 1.5 mL of Beta-mercaptoethanol (0.1 mM final) to 400 mL Modified Eagle Medium (DMEM).
13. Store the reprogramming media in 100 mL aliquots at 4 °C for up to 4 weeks or at -20 °C for longer.
14. When ready to use, add 100 µL of 10 mg/mL basic fibroblast growth factor (bFGF) (10 ng/mL final) to one 100 mL reprogramming media aliquot.
15. Warm the reprogramming media in a 37 °C water bath.
16. Wash the cells growing on iMEFs feeders with pre-warmed reprogramming media twice.
    Add 2 mL of reprogramming medium to each well.
17. Transfer the reprogramming plates into a 37 °C, 5% CO₂and 3% O₂ (hypoxia) incubator.
    NOTE: The day when reprogramming media is added is considered day 1 of reprogramming.
18. Feed the cells daily with fresh reprogramming media until iPSC colonies start to appear.
    NOTE: Identify iPSC colonies by their ESC-like morphology (compact colonies with well-defined edges and comprised of cells with a high nucleus/cytoplasm ratio).
19. Monitor the iPSC colonies daily, and after an adequate number have been formed, passage them as a pool.
    NOTE: Ensure that the iPSC colonies do not touch each other, as this would result in differentiation and affect their long-term stability.
20. To establish clonal lines, passage the iPSC pool about 5 times, then pick robust colonies that maintain their ESC-like morphology.
21. To identify fully reprogrammed iPSC colonies, carry out live staining with TRA-160 (see Table of Materials), and harvest RNA from iPSC colonies for gene expression characterization.

9. Passaging of iPSC colonies

1. Prepare enough iMEF feeder plates 24 h before passaging the iPSC colonies as described in step 7.
2. Wash iMEF feeder plates twice with pre-warmed reprogramming media.
3. Add 2 mL of reprogramming media supplemented with ROCK inhibitor (Y2) (10 µM) to each well of iMEF plate.
4. Incubate the iMEF plates in a 37 °C, 5% CO_2, and 3% O₂ incubator until ready to use.
5. Prepare Ethylenediaminetetraacetic acid/ Phosphate Buffered Saline (EDTA/PBS) solution by diluting 0.5 M EDTA 1:1000 in PBS (0.5 mM final).
6. Replace the reprogramming culture media with 0.5 mL of EDTA/PBS in each well.
7. Incubate in a 37 °C, 5% CO_2, and 3% O₂ incubator or at room temperature, depending on the cell type. BxPc3 and PDAC iPSCs require 15 min incubation at 37 °C. H6C7 and hFib iPSCs need to be incubated at room temperature for 5 min.
8. Check the iPSCs cells under the microscope until they begin to uniformly dissociate from the feeder layer throughout the entire colony.
9. Collect the dissociated iPSC cell suspension into a 15 mL centrifuge tube. Spin down at 300 x g for 5 min at room temperature.
10. Resuspend the cell pellet in 1 mL of reprogramming media supplemented with Y2 (10 µM).
11. Plate the cells on iMEF feeder layer by transferring each 1 mL of iPSC cell suspension to three wells of the iMEF plate.
    NOTE: The split ratio may vary depending on the number of harvested iPSC colonies.
12. Incubate in a 37 °C, 5% CO₂ and 3% O₂ incubator.

10. Live staining of iPSC colonies with TRA-1-60

1. Prepare 0.5 mL per well of 4 µg/mL TRA-1-60 antibody in reprogramming media.
2. Discard reprogramming media and replace it with 0.5 mL antibody mixture in each well. Incubate in a 37 °C, 5% CO_2, and 3% O₂ incubator for 30 min.
3. Wash the cells twice with the reprogramming media.
4. Capture fluorescence images with a cell imaging system using the GFP filter.
5. Check the image quality by considering the negative control channel.

Wyniki

Representative images displaying the morphology of iPSC colonies derived from PDAC, BXPc3, H6C7, and hFib cells are shown in Figure 1. PDAC-iPSC colonies started to form on Day 25 of reprogramming. Robust iPSC colonies with a more established ESC-like morphology were identified on Day 40 of reprogramming (Figure 1). Similarly, the formation of BxPc3-iPSCs began on Day 23 and became more established by Day 35. H6C7-iPSC formation ...

Dyskusje

To facilitate the use of iPSC reprogramming for studying cancer progression, a robust protocol has been established for reprogramming pancreatic cancer cells. Reprogramming cancer cells into pluripotency has proven to be very challenging thus far, as only a few studies have successfully generated iPSCs from cancer cells^{32,36,37,38,39,}

Materiały

Name	Company	Catalog Number	Comments
2-Mercaptoethanol (50 mM)	Thermo Fisher	31350010
Alexa Fluor 488 anti- human TRA-1-60-R	BioLegend	330613
Bovine Pituitary Extract (BPE)	Thermo Fisher	13028014
BxPc3	ATCC	CRL-1687
Cholera Toxin from Vibrio cholerae	Merck	C8052-1MG
Collagen, Type I solution from rat tail	Merck	C3867
Completed Defined K-SFM	Thermo Fisher	10744-019
Corning Costar TC-Treated Multiple Well Plates	Merck	CLS3516
Corning syringe filters	Merck	CLS431231
Corning tissue-culture treated culture dishes	Merck	CLS430599
Day Impex Virkon Disinfectant Virucidal Tablets	Thermo Fisher	12328667
Dulbecco′s Phosphate Buffered Saline (PBS)	Merck	D8537
Fetal Calf Serum (FCS)	Thermo Fisher	10270-106
Fugene HD Transfection Reagent	Promega	E2312
Gelatin solution, Type B, 2% in H2O	Merck	G1393-100ML
Glasgow Minimum Essential Media (GMEM)	Merck	G5154
Human EGF Recombinant Protein	Thermo Fisher	PHG0311
Human FGF-basic (FGF-2/bFGF) (154 aa) Recombinant Protein, PeproTech	Thermo Fisher	100-18B
Human Pancreatic Duct Epithelial Cell Line (H6c7)	Kerafast	ECA001-FP
iMEF feeder cells	iXcells Biotechnologies	10MU-001-1V
Keratinocyte Serum Free Media (KSFM)	Thermo Fisher	17005-042
KnockOut DMEM	Thermo Fisher	10829018
KnockOut serum Replacement	Thermo Fisher	10828028
L-Glutamine (200 mM)	Thermo Fisher	25030-024
MEM Non-Essential Amino Acids Solution (100x)	Thermo Fisher	11140050
Millex-HP 0.45 μM syringe Filter Unit (Sterile)	Merck	SLHP033RS
Opti-MEM Reduced Serum Medium	Thermo Fisher	31985062
pMDG	AddGene	187440
Polybrene (Hexadimethrine bromide)	Merck	H9268-5G
pSIN4-CMV-K2M	AddGene	21164
pSIN4-EF2-O2S	AddGene	21162
psPAX2	AddGene	12260
pWPT-GFP	AddGene	12255
RPMI 1640 Medium (ATCC modification)	Thermo Fisher	A1049101
Sodym Pyruvate	Thermo Fisher	11360-039
Sterile Syringes for Single Use (60 mL)	Thermo Fisher	15899152
TrypLE Express Enzyme (1x), phenol red	Thermo Fisher	12605036
UltraPure 0.5M EDTA, pH 8.0	Thermo Fisher	15575020
Y-27632 (Dihydrochloride)	STEMCELL Technologies	72304