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EX VIVO BIOENGINEERING OF LUNG
Darcy Wagner, PhD
Lung Repair and Regeneration Group (Königshoff Laboratory)
Comprehensive Pneumology Center – Helmholtz Zentrum Munich
Weiss Laboratory
University of Vermont – Vermont Lung Center
The need for regenerating lungs ex vivo
• Many devastating lung diseases remain without a cure
• Chronic lung diseases predicted to increase
• COPD projected to be the third leading cause of death by 2030
• Lung transplant remains the only option
• There are not enough donor lungs to match demand
• Complicated by low transplant efficacy due to
• Acute and chronic rejection
• Required use of immunosuppressive drugs
• Only a 50% survival rate after 5 years
• New options for transplantation need to be explored
Engineering Approach to Restoring Lung Function
• Minimum requirements:
• Gas exchange (oxygen in,
carbon dioxide out)
• Filters (prevent particles and
pathogens from entering the
body)
• Other design requirements:
• Portable
• Long life cycle
http://www.swedish.org/Services/CancerInstitute/Services/Lung-Cancer/About-LungCancer#axzz2fSRKy5Dj
Mechanical Intervention:
Extracorporeal Membrane Oxygenation
ECMO does not meet ideal criteria
• Minimum requirements: √
• Gas exchange (oxygen in, carbon dioxide out) √
• Filters (prevent particles and pathogens from entering the body) √
• Other design requirements:
• Portable
• Long life cycle
• Bridge to transplantation
• Alternative options need to be explored
Ex vivo lung bioengineering
2008: First Clinical Success
Decellularization removes cells which largely causes immune rejection
Scaffold can be recellularized with the patients own stem cells
Minimizes the use of immunosuppressive drugs
To date…
• Simple airway structure supports and tracheas have been
transplanted
• Both synthetic and natural ECM scaffolds have been successfully
used
• Extremely early days
• Performed clinically only in the setting of compassionate use
• Patient survival exceeding one year in many cases
The reality for lung tissue…2015
Healthy 3D lung tissue slices can only be maintained for 5-7 days
ex vivo
Uhl et al, ERJ 2015
Current Approaches for Ex Vivo Lung Bioengineering
Scaffold
Cells
Primary (differentiated)
Stem
Progenitor
iPS
ESC
Decellularized
Mouse Lung
Synthetic material
+
Native Mouse Lung
or
Comparison of Current Tissue Engineering Scaffolds
Biologic Scaffold
Synthetic Scaffold
Differentiation and
engraftment cues
+
Largely retains native
integrin binding sites
- Lacks specific
integrin binding
sites
Immunogenicity
+
Antigen removal
during
decellularization
Manufacturability
+
Native architecture
largely retained
- Complex
architecture
-
Large variability
between donor
scaffolds
+ Precise control
possible (i.e..
repeatability)
-
Degradation with long
term storage
+ Improved storage
stability
Long term Storage
Unknown/variable
depending on
material
Whole lung decellularization removes cells while retaining
ECM proteins and native architecture
Key
fib=fibronectin
lam=laminin
elast=elastin
col I= collagen I
a= airway
bv= blood vessel
Bonenfant et al. Biomaterials 2013
Decellularized Mouse Lungs Retain Major Vascular and Airway
Routes
Acellular mouse lungs can be perfused
Acellular mouse lungs can be ventilated
Daly et al Tissue Engineering 2011
Orthotopically transplanted decellularized and
recellularized lungs can briefly function in vivo
Song et al., Nat Med 2010; Gilpin et al., Ann Thoraci Soc 2014
Factors affecting lung regeneration
• Major questions for
clinical implementation
• Scaffold source
• Cell source
• Ex vivo scheme
Wagner et al., Respirology 2013
WHAT CELL TYPES SHOULD BE USED IN
RECELLULARIZATION STRATEGIES?
Primary (differentiated)?
Stem?
Progenitor?
iPS?
ESC?
The lung is a complex organ with many different cell types
Where‘s the rest of me?
Vascular system
Cartilage system
Stromal support
Lymphatic system
Innervation
Immune system
ESC-derived Nkx2-1(TTF-1) Cell Growth and Differentiation in
Decellularized Lung Scaffolds and Slices
Day 0
Day 15
Tyler Longmire
Darrell Kotton MD
Boston University
T1α
10 day slice culture
Nkx2.1(TTF-1) DAPI
Longmire et al. Cell Stem Cell, 2012
DOES THE SCAFFOLD SOURCE OR COMPOSITION
INFLUENCE RECELLULARIZATION?
Different decellularization protocols result in different
scaffold composition and MMP activation
Wallis et al. Tissue Eng C 2012
Matrix-Bound HS Proteoglycans are necessary for directed
differentiation of ESC derived endoderm to airway epithelial cells
on acellular scaffolds
Shojaie et al. Stem Cell Reports 2015
Mouse Lungs Retain Characteristic of Age and Injury following
Decellularization
a= airway
bv= blood vessel
Sokocevic et al. (2013)
Biomaterials; 34:3231–45
Mass spectrometry proteomics can be used to analyze scaffold
composition following decellularization
Cytoskeletal
ECM
Cytosolic
Nuclear
1. Age+elastase (n=6)
2. Elastase (n=3)
3. Bleomycin (n=3)
4. Aged Mice (n=4)
5. Young Mice (n=6)
MembraneAssociated
1
2 3 4
5
Sokocevic et al. (2013) Biomaterials; 34:3231–45
Elastase-induced emphysematous changes significantly
impaired growth of C10 cells
Mouse bone marrow derived
mesenchymal stem cells (mMSC)
C10 – mouse alveolar epithelial
cells
Sokocevic et al. (2013)
Biomaterials; 34:3231–45
Summary of Cell Survival
Injury Model
mMSC
C10
Young
D28
D28
Aged
D28
D28
Elastase
D28
D14
Aged Elastase
D28
D3
Bleomycin
D28
D28
Summary I
• Decellularized mouse lungs retain architecture and proteins characteristic of
the different age and injury models
• Mass spectrometry is a powerful tool which can be used to detect differences
in residual proteins
• ECM proteins are largely retained following decellularization but cell-
associated proteins are also detected
• Age and injury seem to inhibit recellularization
Scaling up to produce acellular human lungs
• Major hurdles to
overcome:
• Size of scaffold
• Scaffold source
• Cell Numbers
• Cell Source/Type
• Factors for regeneration (cell
combinations, growth
factors, etc.)
• High throughput techniques
would accelerate progress
Wagner et al., Respirology 2013
Decellularized normal and emphysematous or fibrotic human
lungs retain characteristic gross and histologic appearances
Normal
IPF
Native
Decell
Wagner et al, Biomaterials 2014
Booth et al Am J Resp Crit Care Med 2012
Decellularized human lungs retain architecture characteristic of
lung disease
Wagner et al Biomaterials 2014 (1)
Thermographic analysis confirms preservation of
airway and vascular routes in acellular human lobes
FLIR Imaging
Wagner et al., Biomaterials 2014
Human lung origin significantly determines
residual proteins following decellularization
Spearman Rank
Correlations
Unique peptide hits
Wagner et al, Biomaterials 2014
Wagner et al., Biomaterials 2014
Wagner et al., Cell Mol Bioeng 2014
Previous state of the art: non specific injections or monolayer
seedings relies on stochastic binding events
Thin slice incubation/seeding:
Petersen et al, Science 2010
Booth et al., AJRCCM 2012
O’Neill et al., Ann Thorac Surg 2013
Gilpin et al., J Heart Lung Transplant 2014
Injections:
Nichols et al., Tissue Eng A 2013
Excision of small segments compromises the
integrity and function of lung pleura
pleura
Selecting a material for an artificial pleura
• Cytocompatible
• Adheres to acellular scaffold
• Mechanically stable to allow for inoculations
• Retains cells
• Cells do not preferentially adhere to the material
• Can be applied to the acellular lung in a nontoxic manner
Calcium alginate synthetic pleura permits
physiologic cellular inoculations
Vascular seeding(CBFs) Airway seeding (HBEs)
CBFs- human endothelial progenitor cells (courtesy of Mervin Yoder, Indiana University)
HBEs- human bronchial epithelial cells (courtesy of Albert van der Vliet, University of Vermont)
Wagner et al., Biomaterials 2014a
Human cells can be seeded into excised
alginate-coated segments of acellular human
lungs and cultured in thin slices
Wagner et al., Biomaterials 2014b
Acellular human emphysematous lungs do not support long term
viability as well as those from normal human lungs
Summary of Cell Survival in Slices
Cell Type
Normal
Emphysema
HBE
D21
D7
CBF12
D21
D7
HMSC
D21
D3
HLF
D28
D3
Cell types:
• HBE= human bronchial
epithelial cells
• CBF12= human
endothelial cell
(courtesy of Mervin
Yoder)
• HMSC= human
mesenchymal stem
cells
• HLF= human lung
fibroblasts
Wagner et al, Biomaterials 2014 (1)
Acellular human lungs can be used for high throughput studies as
either three-dimensional segments or in thin slices
Wagner et al., Cell Mol Bioeng 2014
Methacrylated alginate can be photocrosslinked on
acellular human lung
Wagner et al., Cell
Mol Bioeng 2014
Excised 3D Segments of Decellularized Human Lungs Can be
Ventilated and Used for High Throughput Screening
In collaboration with Rachael Oldinski,
UVM College of Engineering
Wagner et al., Cell Mol Bioeng 2014
Summary and Outlook
• Whole human lungs or individual lobes can be
decellularized
• Recellularization studies in human studies have thus far
been limited to proof of concept studies
• Scaffold is cytocompatible
• Scaffolds can be ventilated or perfused
• Use of high-throughput techniques may help expedite
path to clinic
• Cell type(s) need further exploration
• Ex vivo requirements for regeneration and schemes
remain unknown
Acknowledgements
Borok Lab
Zea Borok, MD
Beiyun Zhou, PhD
Weiss Lab
Daniel J. Weiss, MD, PhD
Nicholas Bonenfant
Zachary Borg
Elice Brooks
Elliot Marks
Amelia Payne
Charles Parsons, MD
Joseph Platz, MD
Patrick Saunders
Dino Sokocevic
Franziska Uhl, PhD
Basa Zvarova
Mervin Yoder, MD
UVM School of Engineering
Engineered Biomaterials
Research Laboratory (EBRL)
Rachael Oldinski, PhD
Spencer Fenn
UVM Department of Pathology
Yvonne Janssen-Heininger Lab
Yvonne Janssen-Heininger, PhD
Jos van der Velden, PhD
Albert van der Vliet, PhD
Funding
NIH RC4 (PI: DJ Weiss, MD, PhD)
NIH R21 (PI: DJ Weiss, MD, PhD)
NIH T32 Training Grant (PI: Charlie Irvin, PhD)
ATS Stem Cell Working Group
Follow us on Twitter
@RCMBStemCell
Königshoff Laboratory
Melanie Königshoff, MD, PhD
Franziska Uhl, PhD
Sarah Vierkotten, PhD
Rita Costa
Nadine Adam
Rabea Imker