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/Cancer-

Institute/Services/Lung-Cancer/About-Lung-

Cancer#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

Native Mouse Lung

Decellularized

Mouse Lung Synthetic material

Cells Primary (differentiated)

Stem

Progenitor

iPS

ESC +

Scaffold

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

Unknown/variable

depending on

material

Manufacturability +

-

Native architecture

largely retained

Large variability

between donor

scaffolds

-

+

Complex

architecture

Precise control

possible (i.e..

repeatability)

Long term Storage - Degradation with long

term storage

+ Improved storage

stability

Whole lung decellularization removes cells while retaining

ECM proteins and native architecture

Bonenfant et al. Biomaterials 2013

Key

fib=fibronectin

lam=laminin

elast=elastin

col I= collagen I

a= airway

bv= blood vessel

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

Tyler Longmire

Darrell Kotton MD

Boston University

Longmire et al. Cell Stem Cell, 2012

T1α Nkx2.1(TTF-1) DAPI

10 day slice culture

Day 0 Day 15

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

Membrane-

Associated

1 2 3

1. Age+elastase (n=6)

2. Elastase (n=3)

3. Bleomycin (n=3)

4. Aged Mice (n=4)

5. Young Mice (n=6) 4 5

Sokocevic et al. (2013) Biomaterials; 34:3231–45

Elastase-induced emphysematous changes significantly

impaired growth of C10 cells

Sokocevic et al. (2013)

Biomaterials; 34:3231–45

Mouse bone marrow derived

mesenchymal stem cells (mMSC)

C10 – mouse alveolar epithelial

cells

Injury Model mMSC C10

Young D28 D28

Aged D28 D28

Elastase D28 D14

Aged Elastase D28 D3

Bleomycin D28 D28

Summary of Cell Survival

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

Wagner et al, Biomaterials 2014

Normal IPF

Native

Decell

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

Wagner et al., Biomaterials 2014

FLIR Imaging

Human lung origin significantly determines

residual proteins following decellularization

Wagner et al, Biomaterials 2014

Spearman Rank

Correlations

Unique peptide hits

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

Airway seeding (HBEs) Vascular seeding(CBFs)

Wagner et al., Biomaterials 2014a

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)

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

Cell Type Normal Emphysema

HBE D21 D7

CBF12 D21 D7

HMSC D21 D3

HLF D28 D3

Wagner et al, Biomaterials 2014 (1)

Cell types:

• HBE= human bronchial

epithelial cells

• CBF12= human

endothelial cell

(courtesy of Mervin

Yoder)

• HMSC= human

mesenchymal stem

cells

• HLF= human lung

fibroblasts

Summary of Cell Survival in Slices

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

Wagner et al., Cell Mol Bioeng 2014

In collaboration with Rachael Oldinski,

UVM College of Engineering

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

UVM School of Engineering

Engineered Biomaterials

Research Laboratory (EBRL)

Rachael Oldinski, PhD

Spencer Fenn

Borok Lab

Zea Borok, MD

Beiyun Zhou, PhD

Acknowledgements

Funding NIH RC4 (PI: DJ Weiss, MD, PhD)

NIH R21 (PI: DJ Weiss, MD, PhD)

NIH T32 Training Grant (PI: Charlie Irvin, 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 Department of Pathology

Yvonne Janssen-Heininger Lab

Yvonne Janssen-Heininger, PhD

Jos van der Velden, PhD

Albert van der Vliet, PhD

Königshoff Laboratory

Melanie Königshoff, MD, PhD

Franziska Uhl, PhD

Sarah Vierkotten, PhD

Rita Costa

Nadine Adam

Rabea Imker

ATS Stem Cell Working Group

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