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PANCREAS Publication-derived

Directed Differentiation of Pluripotent Stem Cells to Pancreatic Ductal Epithelial Cells for Cystic Fibrosis Disease Modeling

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

👤 Senem Simsek, Ting Zhou, Christopher L. Robinson, Su-Yi Tsai, Miguel Crespo, Sadaf Amin, Xiangyi Lin, Jane Hon, Todd Evans, Shuibing Chen ⏱ 30 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Simsek et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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