KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Secondary antibody anti-mouse-HRP | GE Healthcare | Cat# NA931; RRID:AB_772210 |
| Secondary antibody anti-rabbit-HRP | GE Healthcare | Cat# NA934; RRID:AB_772206 |
| anti-mouse IgG MicroBeads | Miltenyi | Cat# 130-048-402; RRID:AB_244361 |
| Rabbit monoclonal AcTUB | Abcam | Cat# ab179484 |
| Rabbit polyclonal AMY2A | SIGMA | Cat# A8273; RRID:AB_258380 |
| Mouse monoclonal anti-ACTB | Sigma | Cat# A5316; RRID:AB_476743 |
| Rabbit polyclonal C-peptide | Cell Signaling | Cat# 4593; RRID:AB_10691857 |
| Mouse monoclonal CA19-9 | Thermo | Cat# CA1003-200UL; RRID:AB_212741 |
| Rabbit monoclonal anti-CA2 | Abcam | Cat# ab124687; RRID:AB_10972000 |
| Rabbit monoclonal CDX2 | Cell Marque - RabMab | Cat# MU392-UC; RRID:AB_2335627 |
| Mouse monoclonal CFTR | R&D | Cat# MAB1660; RRID:AB_2260674 |
| Rabbit polyclonal CLDN1 | Abcam | Cat# ab15098; RRID:AB_301644 |
| Mouse monoclonal PE-conjugated CXCR4 antibody | Life Technologies | Cat# MHCXCR404; RRID:AB_10373097 |
| Mouse monoclonal APC-conjugated c-Kit antibody | Thermo | Cat# CD11705; RRID:AB_2536476 |
| Mouse monoclonal E-CAD | Dako/Agilent | Cat# M3612; RRID:AB_2341210 |
| Mouse monoclonal E-CAD | BD Bioscience | Cat# 610182; RRID:AB_397581 |
| Rabbit monoclonal E-CAD | Cell Signaling | Cat# 3195; RRID:AB_2291471 |
| Mouse monoclonal Gαs/olf (G-10) | Santa Cruz | Cat# sc-365855; RRID:AB_10842167 |
| Mouse monoclonal GCG | SIGMA | Cat# G2654; RRID:AB_259852 |
| Rabbit polyclonal GFP | Thermo | Cat# A-6455 RRID: AB_221570 |
| Mouse monoclonal GP2 | MBL International | Cat# D277-3, RRID:AB_10598500 |
| Rabbit polyclonal C-pep | Cell Signaling | Cat# 4593; RRID:AB_10691857 |
| Rat monoclonal CD31 | BD Bioscience | Cat# 557355; RRID:AB_396660 |
| Mouse monoclonal CTRC | Millipore | Cat# MAB1476; RRID:AB_2261190 |
| Rabbit polyclonal ERK | Cell Signaling | Cat# 9102; RRID:AB_330744 |
| Mouse monoclonal H-NUCL | Abcam | Cat# ab190710 |
| Rabbit monoclonal HA | Cell Signaling | Cat# 3724; RRID:AB_1549585 |
| Mouse monoclonal HNF1B | Abcam | Cat# ab236759 |
| Rabbit monoclonal Ki67 | Thermo | Cat# MA5-14520; RRID:AB_10979488 |
| Mouse monoclonal Ki67 | Dako/Agilent | Cat# M7240; RRID:AB_2142367 |
| Mouse monoclonal KRT7 | Dako/Agilent | Cat# M7018; RRID:AB_2134589 |
| Mouse monoclonal KRT8 | BD Bioscience | Cat# 345779; RRID:AB_2800363 |
| Mouse monoclonal KRT19 | Dako/Agilent | Cat# M0888; RRID:AB_2234418 |
| Rabbit polyclonal mCherry | Abcam | Cat# ab167453; RRID:AB_2571870 |
| Mouse monoclonal MUC1 | Santa Cruz | Cat# sc-7313; RRID:AB_626983 |
| Mouse monoclonal MUC5AC | Santa Cruz | Cat# sc-33667; RRID:AB_627973 |
| Rabbit monoclonal N-CAD | Cell Signaling | Cat# 13116; RRID:AB_2687616 |
| Rabbit polyclonal NANOG | Cell Signaling | Cat# 3580; RRID:AB_2150399 |
| Mouse monoclonal NKX6-1 | DSHB Hybridoma | Cat# F55A12; RRID:AB_532379 |
| Mouse monoclonal NKX6-1-APC | BD | Cat# 563338; RRID:AB_2738144 |
| Mouse monoclonal OCT4 | Santa Cruz | Cat# sc-5279; RRID:AB_628051 |
| Mouse monoclonal P15 | Santa Cruz | Cat# sc-271791; RRID:AB_10709436 |
| Rabbit monoclonal P16 | Cell Signaling | Cat# 80772; RRID:AB_2799960 |
| Rabbit monoclonal P21 | Abcam | Cat# ab109520; RRID:AB_10860537 |
| Mouse monoclonal P53 | Santa Cruz | Cat# sc-47698; RRID:AB_628083 |
| Rabbit monoclonal pERK | Cell Signaling | Cat# 4377; RRID:AB_331775 |
| Rabbit polyclonal PARP | Cell Signaling | Cat# 9542; RRID:AB_2160739 |
| Goat polyclonal PDX1 | R&D | Cat# AF2419; RRID:AB_355257 |
| Mouse monoclonal PDX1-PE | BD | Cat# 562161; RRID:AB_10893589 |
| Rabbit polyclonal PKC | Abcam | Cat# ab59364; RRID:AB_944858 |
| Rabbit monoclonal p-PKA substrates (RRXS*/T*) | Cell Signaling | Cat# 9624; RRID:AB_331817 |
| Rabbit monoclonal pRB | Cell Signaling | Cat# 8516; RRID:AB_11178658 |
| Mouse monoclonal RB | Cell Signaling | Cat# 9309; RRID:AB_823629 |
| Rabbit polyclonal SOX9 | Millipore | Cat# AB5535; RRID:AB_2239761 |
| Mouse monoclonal SSEA4 | Cell Signaling | Cat# 4755; RRID:AB_1264259 |
| Rabbit polyclonal Turbo GFP | Thermo | Cat# PA5-22688 RRID: AB_2540616 |
| Rabbit monoclonal VASP | Cell Signaling | Cat# 3132; RRID:AB_2213393 |
| Rabbit monoclonal VIM | Cell Signaling | Cat# 5741; RRID:AB_10695459 |
| Mouse monoclonal VINC | Sigma | Cat# V9264; RRID:AB_10603627 |
| Mouse monoclonal H2AX (pS139) Clone N1-431-APC | BD | Cat# 560447; RRID:AB_1645414 |
| Rabbit polyclonal Zeb1 | Santa Cruz | Cat# sc-25388; RRID:AB_2217979 |
| Mouse monoclonal ZO1 | Thermo | Cat# 33-9100; RRID:AB_2533147 |
| Antibody conditions for IHC/IF are listed in Suppl.Table6 | This paper | N/A |
| Bacterial and Virus Strains | ||
| Lentivirus hOKSM-dTomato | (Warlich et al., 2011) | N/A |
| Non-integrating Sendai virus (CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit) | Thermo | Cat# A16517 |
| One Shot™ ccdB Survival™ 2 T1R Competent Cells | Thermo | Cat# A10460 |
| Subcloning Efficiency DH5α Competent Cells | Thermo | Cat# 18265017 |
| Biological Samples | ||
| Human pancreatic ductal organoids from cadaveric organ donors | Tamara Madácsy, József Maléth (based on Boj et al. (2015)) | N/A |
| FPC patient keratinocytes | This paper | N/A |
| Human bone marrow stromal cells (HBMSCs) | Natasha Cherman (Pamela G. Robey) | N/A |
| Transformed human patient-derived organoids (Panc163) | Bruno Sainz, Patrick Hermann (Rubio-Viqueira et al., 2006) | N/A |
| Untransformed human patient-derived organoids from resection specimen | Maximilian Reichert, Zahra Dantes (Dantes et al., 2020) | N/A |
| Chemicals, Peptides, and Recombinant Proteins | ||
| 3,3,5-Triiodo-L-thyronine | Sigma | Cat# T0281; CAS: 5817-39-0 |
| A-83-01 | Tocris | Cat# 2939; CAS: 909910-43-6 |
| Activin A | PeproTech | Cat# 120-14; SDS: 25-120-14 |
| Carbonic anhydrase II from bovine erythrocytes | Sigma | Cat# C2273; CAS: 9001-03-0 |
| CHIR99021 | Axon MedChem | Cat# 1386; CAS: 252917-06-9 |
| Choleratoxin | Sigma | Cat# C9903; CAS: 131096-89-4 |
| Collagenase/Dispase | Roche | Cat# 11097113001 |
| Collagenase II | Life Technologies | Cat# 17101015 |
| Collagenase IV | Worthington | Cat# LS0004186; CAS: 9007-34-5 |
| Dexamethasone | Sigma | Cat# D1756; CAS: 50-02-2 |
| Dextran, Fluorescein 500,000 MW | Thermo | Cat# D7136 |
| Dispase | Sigma | Cat# D4693; CAS: 42613-33-2 |
| Dorsomorphin | Sigma | Cat# P5499; CAS: 866405-64-3 |
| Doxycycline hyclate (Dox) | Sigma | Cat# D9891; CAS: 24390-14-5 |
| EGF human | R&D | Cat# 236-EG-200 |
| Fatty acid free BSA | Proliant | Cat# 608068; CAS: 9048-46-8 |
| FGF2 | Novoprotein | Cat# C046 |
| FGF10 | R&D | Cat# 345-FG-250 |
| FGF-Basic | Thermo | Cat# PHG0360 |
| Forskolin (FSK) | Sigma | Cat# F 3917; CAS: 66575-29-9 |
| Gastrin I | Sigma | Cat# G9020; CAS: 10047-33-3 |
| KGF | PeproTech | Cat# 100-19; SDS: 25-100-19 |
| LDN-193189 (=DM3189) | Sigma | Cat# SML0559; CAS: 1062368-24-4 |
| MSC2530818 | Selleckchem | Cat# S8387; CAS: 1883423-59-3 |
| Matrigel Basement Membrane Matrix | Corning | Cat# 354234 |
| Matrigel Basement Membrane Matrix Growth Factor Reduced (GFR) | Corning | Cat# 354230 |
| Matrigel hESC-qualified Matrix | Corning | Cat# 354277 |
| N-acetyl-L-cysteine | Sigma | Cat# A7250; CAS: 616-91-1 |
| Nicotinamide (NA) | Sigma | Cat# N0636; CAS: 98-92-0 |
| Noggin murine | PeproTech | Cat# 250-38; SDS: 25-250-38 |
| Nu-Serum IV | Corning | Cat# 355104; |
| PKA inhibitor H 89 2HCl | Selleckchem | Cat# S1582; CAS: 130964-39-5 |
| Prostaglandin E2 | Tocris | Cat# 2296; CAS: 363-24-6 |
| R-Spondin 1 protein | R&D | Cat# 4645-RS |
| Retinoic acid (RA) | Sigma | Cat# R2625; CAS: 302-79-4 |
| ROCK inhibitor (Y-27632) | Abcam | ab120129; CAS: 129830-38-2 |
| SANT1 | Sigma | Cat# S4572; CAS: 304909-07-7 |
| TMT 10-plex | Thermo | Cat# A37725 |
| Wnt3a mouse | PeproTech | Cat# 315-20; SDS: 25-315-20 |
| XtremeGene 9 DNA transfection reagent | Roche | Cat# 6365787001 |
| ZnSO4 | Sigma | Cat# Z0251; CAS: 7446-20-0 |
| (-)-Indolactam V | STEMCELL Technologies | Cat# 72312; CAS: 90365-57-4 |
| 3-isobutyl-1-methylxanthine (IBMX) | Sigma | Cat# I5879; CAS: 28822-58-4 |
| Critical Commercial Assays | ||
| Active Ras Pull-Down and Detection Kit | Thermo | Cat# 16117 |
| AllPrep DNA/RNA/miRNA Universial Kit | Qiagen | Cat# 80224 |
| BCECF, AM | Thermo | Cat# B1170 |
| BP Clonase II enzyme mix | Life Technologies | Cat# 11789020 |
| cAMP-Gs HiRange Kit | Cisbio | Cat# 62AM6PEB |
| Click-iT EdU Alexa Fluor 647 Assay Kit | Life Technologies | Cat# C10635 |
| Gibson Assembly® Master Mix | New England BioLabs | Cat# E2611 |
| Human Comprehensive Cancer Panel | Qiagen | Cat# DHS-3501Z |
| LR Clonase II enzyme mix | Life Technologies | Cat# 11791100 |
| Maxwell RSC Blood Kit | Promega | Cat# AS1400 |
| Nextera XT Kit | Illumina | Cat# FC-131-1096 |
| NEBNext Ultra II FS DNA Library Prep Kit | NEB | Cat# E6177 |
| P3 primary cell 4D Nucleofector X Kit S | Lonza | Cat# V4XP-3032 |
| Senescence β-Galactosidase Staining Kit | Cell Signaling | Cat#9860S |
| SuperSignal West Dura Kit | Thermo | Cat# 34076 |
| Tissue Genomic DNA Purification Mini Prep Kit | Genaxxon | Cat# S5378.0050 |
| Qubit dsDNA HS reagents | Thermo | Cat# Q32851 |
| Deposited Data | ||
| Human reference proteome | UniProt | UP000005640 (downloaded 22.07.2013); https://www.uniprot.org/proteomes/UP000005640 |
| Human reference genome GRCh38.p13 | EMBL-EBI | GCF_000001405.39; https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.39/ |
| Raw and analyzed RNA data | This paper (ENA at EMBL-EBI) | ENA: PRJEB38015 |
| Raw and analyzed protein mass spectrometry data | This Paper (PRIDE) | PRIDE: PXD018785 |
| Pancreatic progenitor first gene set | (Xie et al., 2013) | DOI: 10.1016/j.stem.2012.11.023 |
| Pancreatic progenitor second gene set | (Gerrard et al., 2016) | E-MTAB-3928 |
| scRNA-seq data (first set) | (Enge et al., 2017) | GEO: GSE81547 |
| scRNA-seq data (second set) | (Baron et al., 2016) | GEO: GSE84133 |
| scRNA-seq data | (Qadir et al., 2020) | DOI: 10.1073/pnas.1918314117/-/DCSupplemental. |
| Trunk domain gene sets Trunk1/Trunk2 | (de Lichtenberg et al., 2018) | DOI: doi.org/10.1101/336305 |
| Trunk third gene set | (Krentz et al., 2018) | DOI: 10.1016/j.stemcr.2018.11.008 |
| Compiled reference gene sets applied in GSEA are listed in Suppl.Table7 | Indicated above | Indicated above |
| Experimental Models: Cell Lines | ||
| Human: HUES8 hESC line (NIH approval number NIHhESC-10-0021) | HSCI iPS Core, Harvard University, Cambridge, MA, USA | hES Cell Line: HUES-8; RRID:CVCL_B207 |
| HUES8-CDKN2Ako/ko (+/− KRASG12D) | This paper | N/A |
| HUES8-KRASG12D line | This paper | N/A |
| HUES8-GNASR201H line | This paper | N/A |
| HUES8-Luciferase line (Vector control) | This paper | N/A |
| Human: H1 ES cells | Wicell Research Institute, Madison, WI, USA | https://www.wicell.org/ |
| CoiPSC (Control iPSC) | This paper | N/A |
| MAS-GNASWT/R201C iPSCs | This paper | N/A |
| MAS-GNASWT/WT iPSCs | This paper | N/A |
| Rat embryonic fibroblast (REF) | This paper | N/A |
| Experimental Models: Organisms/Strains | ||
| NSG mice (strain: NOD.Cg-Prkdc<scid<tm1Wjl>/SzJ GVO) | Charles River | RRID:BCBC_4142 |
| Nod scid mice (strain: NOD.Cg-Prkdcscid/J) | The Jackson Laboratory | RRID:IMSR_JAX:001303 |
| Oligonucleotides | ||
| crRNA-CDKN2A exon2: GTAGGGGTAATTAGACACCT | This paper | N/A |
| crRNA-CDKN2A exon3: GTCTCGAGTCTATCGATATG | This paper | N/A |
| CDKN2AKO-external-fwd: GCGCTTGGATATACAGCAGTG | This paper | N/A |
| CDKN2AKO-external-rev: ACAGGAGCATCTCCCAACC | This paper | N/A |
| CDKN2AKO-internal-fwd: GGCATTGTGAGCAACCACTG | This paper | N/A |
| CDKN2AKO-internal-rev: CCTGTAGGACCTTCGGTGAC | This paper | N/A |
| Cloning primer for the “All-in-One piggyBac”-system are listed in Suppl.Table3 | This paper | N/A |
| CDKN2AiPSC-exon2-fwd: CCGCAGAAGTTCGGAGGATA | This paper | N/A |
| CDKN2AiPSC-exon2-rev: CTTTGGAAGCTCTCAGGGTACA | This paper | N/A |
| GNAS-exon8-fwd: CCAGACCTTTGCTTTAGATTGG | (Salinas-Souza et al., 2015) | N/A |
| GNAS-exon9-rev: CACAGCATCCTACCGTTGAAG | (Wood et al., 2017) | N/A |
| Primers for qPCR are listed in Suppl.Table5 | This paper | N/A or Qiagen Cat# |
| Recombinant DNA | ||
| pCAS9_GFP plasmid | Kiran Musunuru; (Ding et al., 2013) | Addgene plasmid #44719 |
| gRNA cloning vector | George Church; (Mali et al., 2013) | Addgene plasmid #41824 |
| pBabe-KRASG12D | Channing Der | Addgene plasmid # 58902 |
| pcDNA3.1+hsGNAS_EE(long)R201H | Franz. Oswald | N/A |
| pGL4.10[luc2] | Franz Oswald | N/A |
| pDONR201 | Thermo | Cat# 11798-014 |
| Destination vector PB-TAC-ERP2 | (Kim et al., 2016) | Addgene plasmid #80478 |
| PB-TAC-ERP2-(N-HA)KRAS_G12D | This paper | N/A |
| PB-TAC-ERP2-KRAS_G12D (w/o HA) | This paper | N/A |
| PB-TAC-ERP2-GNAS_R201H(EE) | This paper | N/A |
| PB-TAC-ERP2-Luc2 | This paper | N/A |
| Transposase-encoding vector | SBI Biosciences (Rao et al., 2016) | #PB200PA-1 |
| Software and Algorithms | ||
| Adobe Illustrator | Adobe | Creative Cloud |
| Adobe Photoshop | Adobe | Creative Cloud |
| AxioVision software | ZEISS | https://www.microshop.zeiss.com/de/de/system/software+axiovision-axiovision+basissoftware-axiovision+software/10221/ |
| “bwa mem” | (Heng, 2013) | Version 0.7.17 |
| CLC Genomic Workbench | Qiagen | Version 20.0.3 https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-clc-genomics-workbench/ |
| CRISPOR sgRNA prediction and designing platform | (Haeussler et al., 2016) | http://crispor.tefor.net/ |
| EnrichR webtool | Ma’ayan lab (Kuleshov et al., 2016) | https://amp.pharm.mssm.edu/Enrichr/ |
| Fiji | (Schindelin et al., 2012) | https://imagej.net/Fiji |
| “GATK” toolkit | (Poplin et al., 2018) | Version 4.1.4.1 |
| GraphPad Prism 8 | GraphPad Software, San Diego, California USA | www.graphpad.com |
| GSEA software | Broad Institute (Subramanian et al., 2005) | Version 4.0.3; https://www.gsea-msigdb.org/gsea/index.jsp |
| Imaris 8.1 software | Bitplane AG | https://imaris.oxinst.com/downloads |
| Interactive Genome Viewer (IGV) | Broad Institute (Robinson et al., 2011) | https://software.broadinstitute.org/software/igv/ |
| Maxquant software | (Cox and Mann, 2008) | Version v.1.5.7.4; https://www.maxquant.org/ |
| Modde | Umetrics, Sartorius | https://umetrics.com/product/modde-go |
| Olympus excellence software | Olympus | N/A |
| Phyton | Python Software Foundation | https://www.python.org/psf/ |
| Python package “scanpy” | (Wolf et al., 2018) | N/A |
| Python package “scran” | (Lun et al., 2016) | N/A |
| R | The R Project | Version 6.1.7601 https://cran.r-project.org/mirrors.html |
| R package “CopywriteR” | (Kuilman, 2020) | Version 2.16.0 |
| R package “DESeq2” | (Love et al., 2014) | Version 1.18.1 |
| R package “Limma” | (Ritchie et al., 2015) | https://www.bioconductor.org/packages/release/bioc/html/limma.html |
| R package “scBio” for Cell population mapping | (Frishberg et al., 2019) | https://github.com/amitfrish/scBio |
| R package “sva” | (Johnson et al., 2007; Leek et al., 2012) | Version 3.34.0 |
| “Sambamba” | (Tarasov et al., 2015) | Version 0.7.0 |
| Star aligner | (Dobin et al., 2013) | Version 2.6.1.c https://github.com/alexdobin/STAR |
| Trimmomatic | (Bolger et al., 2014) | Version 0.36 / 0.39 http://www.usadellab.org/cms/index.php?page=trimmomatic |
| Other | ||
| Biozero BZ-9000 microscope | Keyence | N/A |
| Dionex Ultimate 3000 HPLC system | Thermo | Cat# 6130-7-Sys-004 |
| Zeiss Axioscope2 | Zeiss | N/A |
| Ibidi-ibiTreat-precoated glass-bottom 24-well μ-plates | IBIDI | Cat# 82406 |
| Ibidi-ibiTreat-precoated 15-well polymer coverslip bottom μ slides | IBIDI | Cat# 81506 |
| Infinite M1000 pro | Tecan | N/A |
| Fusion SL system | VILBER | N/A |
| LSM780 NLO confocal microscope | Zeiss | N/A |
| LSM880 confocal microscope | Zeiss | N/A |
| LSR II flow cytometer | BD | N/A |
| NextSeq 500 Sequencing System | Illumina | Cat# 770-2013-053-F |
| Olympus CKX41 | Olympus | N/A |
| Olympus Cool LED PE-4000 illumination system | Olympus | N/A |
| Olympus IX73 | Olympus | N/A |
| Orbitrap Fusion Lumos Tribrid mass spectrometer | Thermo | Cat# IQLAAEGAAPFADBMBHQ |
| SteREO Discovery.V12 | Zeiss | N/A |
| 4D Nucleofector Core Unit | Lonza | Cat# AAF-1002B |
| 4D Nucleofector X Unit | Lonza | Cat# AAF-1002X |