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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Published: May 5th, 2016

DOI:

10.3791/54019

1Department of Physiology and Experimental Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

Directed differentiation of pluripotent cells to the lung lineage is dependent on precise signaling events in the microenvironment 1,2. Due to the dynamic nature of this process it has been challenging to mimic the precise events of lung organogenesis in vitro. Recent reports have used step-wise lineage restriction strategies with soluble growth factor supplementation of two-dimensional cultures to achieve lung differentiation3-8. In step-wise differentiation protocols, pluripotent cells, whether embryonic stem cells (ESC) or induced pluripotent stem cells, were first differentiated to the definitive endoderm germ layer. Endodermal cells....

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Animal experiments were carried out in accordance with the Animal Care Committee guidelines of the Hospital for Sick Children Research Institute.

1. Scaffold Preparation

  1. Decellularization of lungs
    1. Euthanize adult Wistar rats using CO2 chamber. Place animal in the chamber and start 100% CO2 exposure at a fill rate of 10-30% of chamber volume per minute.
      1. Observe animal for unconsciousness; this will occur after approximately 2-3 min. If unconsciousness does.......

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As outlined in this protocol, robust differentiation of definitive endoderm to mature airway epithelial cells can be achieved using extended culture of seeded cells on decellularized lung scaffold sections. It is recommended that decellularized scaffolds be characterized to ensure (1) host cells are completely removed, and (2) extracellular matrix proteins are preserved prior to using scaffolds for differentiation. Decellularization can be assessed using tissue staining with hematoxylin a.......

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The protocol described here generates mature ESC-derived airway epithelia using only natural lung scaffolds to direct differentiation with no other supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. No growth factor supplementation of base differentiation media is required. Previously published methods for generating stem cell-derived lung epithelial cells have used 2-dimensional strategies with growth factor supplementation to promote lineage restriction3,4,8,18,19. The.......

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We wish to thank Dr. Rossant and Dr. Bilodeau for the Nkx2-1mcherry ESC used in experiments depicted in Figures 1-3. FACS was performed in The SickKids-UHN Flow Cytometry Facility. This work was supported by operating grants from the Canadian Institutes for Health Research and an infrastructure grant (CSCCD) from the Canadian Foundation of Innovation.

....

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Name Company Catalog Number Comments
Reagents
Perfusion solution Sigma H0777 10U/mL heparin
Perfusion solution Gibco 14170112 dissolved in Hank's balanced salt solution (HBSS-)
Decellularization solution BioShop CHA003 8mM CHAPS 
Decellularization solution Sigma E9884 25mM EDTA 
Decellularization solution BioShop SOD002 1M NaCl
Decellularization solution Gibco 14190-144 dissolved in PBS
Benzonase nuclease Novagen 70664-3 90U/mL Benzonase nuclease 
Benzonase nuclease Gibco 14190-144 diluted in PBS
Antimicrobial solution  Gibco 15140 200U/mL penicillin streptomycin 
Antimicrobial solution  Gibco 15290 25μg/mL amphotericin B 
Antimicrobial solution  Gibco 14190-144 diluted in PBS
Trypsinization  Gibco 12605-028 TrypLE
Serum free differentiation media (SFDM) Gibco IMDM 2440-053, F12 11765-054 3:1 ratio of IMDM and Ham’s modified F12 medium
Serum free differentiation media (SFDM) Gibco 12587-010 B27 supplement (50x dilution) 
Serum free differentiation media (SFDM) Gibco 17502-048 N2 supplement (100x dilution) 
Serum free differentiation media (SFDM) Gibco 15260-037 0.05% (Fraction V) bovine serum albumin
Serum free differentiation media (SFDM) Gibco 35050-061 200mM Glutamax 
Serum free differentiation media (SFDM) Sigma M6145 4μM monothioglycerol
Serum free differentiation media (SFDM) Sigma A4403  0.05mg/mL ascobic acid
Endoderm induction R&D 338-AC/CF Activin A
Antibodies
CDH1 BD Biosciences 610181 Mouse, non-conjugated, 1:100
C-KIT BD Biosciences 558163 Rat, PE-Cy7, 1:100
CXCR4 BD Biosciences 558644 Rat, APC, 1:100
KRT5 Abcam ab24647 Rabbit, non-conjugated, 1:1000
NKX2-1 Abcam ab76013 Rabbit, non-conjugated, 1:200
Laminin Novus Biologicals NB300-144 Rabbit, non-conjugated, 1:200
SCGB1A1 Santa Cruz sc-9772  Goat, non-conjugated, 1:1000
SOX2 R&D Systems AF2018  Goat, non-conjugated, 1:400
TRP63 Santa Cruz sc-8431 Mouse, non-conjugated, 1:200
TUBB4A BioGenex MU178-UC Mouse, non-conjugated, 1:500
Goat IgG  Invitrogen A-11055 Donkey, Alexa Fluor 488, 1:200
Mouse IgG  Invitrogen A-21202 Donkey, Alexa Fluor 488, 1:200
Mouse IgG  Invitrogen A-31571 Donkey, Alexa Fluor 647, 1:200
Rabbit IgG  Invitrogen A-21206 Donkey, Alexa Fluor 488, 1:200
Rabbit IgG  Invitrogen A-31573 Donkey, Alexa Fluor 647, 1:200
Other Materials
Low adherent plates Nunc Z721050  Low cell binding plates,  6 wells  
Air-liquid interface membranes  Whatman 110614 Hydrophobic Nucleopore membrane, 8μm pore size
Vibratome Leica VT1200S  Leica Vibratome
Tissue Adhesive Ted Pella 10033 Pelco tissue adhesive

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