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PANCREAS Publication-derived

Cystic Fibrosis Pancreatic 2D Cell Cultures

Full SOP with Academic or a plan

Source Simsek et al., 2016 · Weill Cornell Medical College · 10.5966/sctm.2015-0276

Authors: Senem Simsek, Ting Zhou, Christopher L. Robinson, Su-Yi Tsai, Miguel Crespo, Sadaf Amin, Xiangyi Lin, Jane Hon, Todd Evans, Shuibing Chen Duration: 30 days 7 phases Cell source: Human ESC and Patient-Derived iPSC (Cystic Fibrosis)

Abstract

This protocol establishes an efficient strategy to direct human pluripotent stem cells (hESCs and CF-iPSCs) to differentiate into pancreatic ductal epithelial cells (PDECs) expressing functional CFTR protein. After purification, >98% of hESC-derived PDECs express functional CFTR. PDECs derived from CF-patient iPSCs recapitulate functional defects seen in patient disease, providing a 'disease-in-a-dish' platform for drug discovery.

Cell source
Human ESC and Patient-Derived iPSC (Cystic Fibrosis)
Application
Disease modeling and drug screening for cystic fibrosis pancreatic defects

Protocol overview

26 steps across 7 phases

Phase 1 · Cell Culture and Preparation Day 0
  1. a Culture hESCs and iPSCs on MEF feeders
Phase 2 · Definitive Endoderm (DE) Induction Day 0-1
  1. a Treat with CHIR99021 and Activin A
  2. b Change medium to Activin A in RPMI/FBS
  3. c Culture with FGF7
Phase 3 · Pancreatic Progenitor (PP) Induction Day 2-6
  1. a Culture with LDN193189, retinoic acid, and SANT-1
  2. b Culture with LDN193189, phorbol 12,13-dibutyrate, and ALK5 inhibitor
Phase 4 · Pancreatic Ductal Epithelial Cell (PDEC) Differentiation Day 6-23
  1. a Culture with FGF10
  2. b Culture with VPA and BMP4 for terminal differentiation
Phase 5 · CFTR+ PDEC Enrichment by Lentiviral Transduction and Selection Day 23-30 (approximately)
  1. a Transduce differentiated PDECs with CFTR-Puro-EF1a-ClopHensor lentivirus
  2. b Select with puromycin
  3. c Harvest enriched PDECs
Phase 6 · Chloride (Iodide) Efflux Assay for Functional CFTR Assessment Day 24+
  1. a Dissociate and replate PDECs onto imaging dishes
  2. b Pre-incubate in I2-free control buffer
  3. c Load cells with iodine
  4. d Measure I2 efflux
  5. e Optional: CFTR inhibitor control
  6. f Image analysis and data extraction
Phase 7 · iPSC Generation from Patient Fibroblasts (Optional CF Disease Model) Week 0-6
  1. a Transduce fibroblasts with reprogramming factors
  2. b Pick and expand colonies
  3. c Characterize pluripotency by immunocytochemistry
  4. d Confirm endogenous pluripotency gene expression by qRT-PCR
  5. e Validate CFTR mutations by genomic DNA sequencing
  6. f Confirm silencing of viral transgenes by qRT-PCR
  7. g Verify karyotype stability
  8. h Test pluripotency by in vitro differentiation (optional)
  9. i Test pluripotency by in vivo teratoma formation (optional)

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Attribution

This SOP was written by Organthis based on the published method in Simsek et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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