Skip to content
← Back to browse
PANCREAS Publication-derived

Reconstituting development of pancreatic intraepithelial neoplasia from primary human duct cells

Source Lee et al., 2017 · Stanford University School of Medicine, Department of Developmental Biology · 10.1038/ncomms14686

👤 Jonghyeob Lee, Emily R. Snyder, Yinghua Liu, Xueying Gu, Jing Wang, Brittany M. Flowers, Yoo Jung Kim, Sangbin Park, Gregory L. Szot, Ralph H. Hruban, Teri A. Longacre, Seung K. Kim ⏱ 240 days 📋 9 phases 🧫 Human primary pancreatic duct cells (CD133+), healthy cadaveric donors

Abstract

This protocol describes genetic modification of primary human pancreatic duct cells via lentiviral KRAS[G12V] expression and CRISPR-Cas9-mediated inactivation of CDKN2A, SMAD4, and TP53 to generate immortalized spheres. After orthotopic transplantation into mouse pancreas, these cells develop PanIN-like lesions with histological and molecular features resembling native human PanINs, enabling investigation of pancreatic cancer precursor development.

Cell source
Human primary pancreatic duct cells (CD133+), healthy cadaveric donors
Application
Disease modeling - PanIN (pancreatic intraepithelial neoplasia) development and precursor lesion characterization

Protocol overview

52 steps across 9 phases

Primary Human Pancreatic Duct Cell Isolation and FACS Sorting Day 0-1
  1. 1 Cell dissociation and processing
  2. 2 Enzymatic dispersion with dispase
  3. 3 Cell filtering and preparation for FACS
  4. 4 FACS sorting for CD133+ ductal cells
Lentiviral Vector Construction and Viral Production Day 0-5
  1. 1 PCR amplification and cloning of KRAS[G12V]
  2. 2 Ligation of inserts into pCDH vectors
  3. 3 Construction of ERBB2-Puro lentiviral vector
  4. 4 Construction of lentiCRISPRv2 vectors for CDKN2A, SMAD4, TP53
  5. 5 Sequence verification of all constructs
  6. 6 Transfection of HEK 293T cells for lentiviral production
  7. 7 Lentiviral particle concentration and precipitation
Lentiviral Infection of Sorted CD133+ Cells Day 1-5
  1. 1 Cell preparation and suspension
  2. 2 Lentiviral infection combinations
  3. 3 Suspension infection and incubation
  4. 4 Post-infection washing and embedding in Matrigel
  5. 5 Media overlay and sphere culture initiation
Drug Selection and Sphere Expansion Day 5-30
  1. 1 Antibiotic selection for HPDE cells (if applicable)
  2. 2 Primary ductal sphere collection and passaging
  3. 3 Sphere collection and washing
  4. 4 Sphere trypsinization and cell counting
  5. 5 Replating of dispersed cells
  6. 6 Monitoring sphere growth and morphology
Genomic and Molecular Validation of Transduced Cells Day 14-30
  1. 1 Genomic DNA extraction
  2. 2 Transgene-specific PCR verification
  3. 3 Quantitative real-time PCR (qRT-PCR) for transgene expression
  4. 4 TIDE analysis for CRISPR-Cas9 indel efficiency
  5. 5 Off-target effect analysis (optional but recommended)
Sphere Clone Isolation Day 21-45
  1. 1 Selection and picking of individual spheres
  2. 2 First sphere expansion
  3. 3 Second sphere picking for clonal confirmation
Orthotopic Transplantation into Immunocompromised Mice Day 45-210
  1. 1 Cell preparation for transplantation
  2. 2 Cell resuspension in Matrigel-HBSS injection solution
  3. 3 Orthotopic injection into mouse pancreas
  4. 4 Post-operative animal care and monitoring
  5. 5 Tissue harvest at endpoint (4-6 months post-transplant)
Histology, Immunohistochemistry, and Pathological Analysis Day 210-240
  1. 1 Tissue fixation and processing
  2. 2 Hematoxylin and eosin (H&E) staining
  3. 3 Alcian blue staining for acid mucins
  4. 4 Immunohistochemistry for phospho-ERK
  5. 5 Immunohistochemistry for CK19 (pan-ductal marker)
  6. 6 Immunohistochemistry for human-specific markers (HuNu, mitochondria)
  7. 7 Immunohistochemistry for MUC5AC (mucin production)
  8. 8 Immunohistochemistry for NMU (biomarker validation)
  9. 9 Pathological classification of lesions
  10. 10 Mounting and microscopy
Molecular Profiling: RNA Extraction and RNA-Seq Day 45-180
  1. 1 RNA extraction from cultured hiPanIN clones
  2. 2 RNA-Seq library preparation
  3. 3 High-throughput sequencing
  4. 4 RNA-Seq data alignment and analysis
  5. 5 Gene set enrichment analysis (GSEA)
  6. 6 Data visualization and identification of biomarker candidates
  7. 7 Data deposition and availability

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Lee et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence