EX VIVO BIOENGINEERING OF LUNG

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1 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

2 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

3 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 Institute/Services/Lung-Cancer/About-Lung- Cancer#axzz2fSRKy5Dj

4 Mechanical Intervention: Extracorporeal Membrane Oxygenation

5 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

6 Ex vivo lung bioengineering

7 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

8 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

9 The reality for lung tissue 2015

10 Healthy 3D lung tissue slices can only be maintained for 5-7 days ex vivo Uhl et al, ERJ 2015

11 Current Approaches for Ex Vivo Lung Bioengineering Cells Primary (differentiated) Stem Progenitor ips ESC + Synthetic material Scaffold or Decellularized Mouse Lung Native Mouse Lung

12 Comparison of Current Tissue Engineering Scaffolds Differentiation and engraftment cues Biologic Scaffold + Largely retains native integrin binding sites Synthetic Scaffold - Lacks specific integrin binding sites Immunogenicity + Antigen removal during decellularization Manufacturability + - Native architecture largely retained Large variability between donor scaffolds Long term Storage - Degradation with long term storage - + Unknown/variable depending on material Complex architecture Precise control possible (i.e.. repeatability) + Improved storage stability

13 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

14 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

15 Orthotopically transplanted decellularized and recellularized lungs can briefly function in vivo Song et al., Nat Med 2010; Gilpin et al., Ann Thoraci Soc 2014

16 Factors affecting lung regeneration Major questions for clinical implementation Scaffold source Cell source Ex vivo scheme Wagner et al., Respirology 2013

17 WHAT CELL TYPES SHOULD BE USED IN RECELLULARIZATION STRATEGIES? Primary (differentiated)? Stem? Progenitor? ips? ESC?

18 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

19 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 Nkx2.1(TTF-1) DAPI 10 day slice culture Longmire et al. Cell Stem Cell, 2012

20 DOES THE SCAFFOLD SOURCE OR COMPOSITION INFLUENCE RECELLULARIZATION?

21 Different decellularization protocols result in different scaffold composition and MMP activation Wallis et al. Tissue Eng C 2012

22 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

23 Mouse Lungs Retain Characteristic of Age and Injury following Decellularization a= airway bv= blood vessel Sokocevic et al. (2013) Biomaterials; 34:

24 Mass spectrometry proteomics can be used to analyze scaffold composition following decellularization Cytoskeletal ECM Cytosolic Nuclear Membrane- Associated Age+elastase (n=6) 2. Elastase (n=3) 3. Bleomycin (n=3) 4. Aged Mice (n=4) 5. Young Mice (n=6) Sokocevic et al. (2013) Biomaterials; 34:

25 Elastase-induced emphysematous changes significantly impaired growth of C10 cells Mouse bone marrow derived mesenchymal stem cells (mmsc) C10 mouse alveolar epithelial cells Summary of Cell Survival Injury Model mmsc C10 Young D28 D28 Aged D28 D28 Elastase D28 D14 Aged Elastase D28 D3 Bleomycin D28 D28 Sokocevic et al. (2013) Biomaterials; 34:

26 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 cellassociated proteins are also detected Age and injury seem to inhibit recellularization

27 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

28 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

29 Decellularized human lungs retain architecture characteristic of lung disease Wagner et al Biomaterials 2014 (1)

30 Thermographic analysis confirms preservation of airway and vascular routes in acellular human lobes FLIR Imaging Wagner et al., Biomaterials 2014

31 Human lung origin significantly determines residual proteins following decellularization Spearman Rank Correlations Unique peptide hits Wagner et al, Biomaterials 2014

32 Wagner et al., Biomaterials 2014 Wagner et al., Cell Mol Bioeng 2014

33 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

34 Excision of small segments compromises the integrity and function of lung pleura pleura

35 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

36 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

37 Human cells can be seeded into excised alginate-coated segments of acellular human lungs and cultured in thin slices Wagner et al., Biomaterials 2014b

38 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)

39 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

40 Methacrylated alginate can be photocrosslinked on acellular human lung Wagner et al., Cell Mol Bioeng 2014

41 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

42 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

43 Acknowledgements 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 UVM School of Engineering Borok Lab Zea Borok, MD Beiyun Zhou, PhD Mervin Yoder, MD 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)

44 ATS Stem Cell Working Group Follow us on Königshoff Laboratory Melanie Königshoff, MD, PhD Franziska Uhl, PhD Sarah Vierkotten, PhD Rita Costa Nadine Adam Rabea Imker