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Gulberk Ozcebe S, Tristan M, Zorlutuna P. Adult human heart extracellular matrix improves human iPSC-CM function via mitochondrial and metabolic maturation. Stem Cells 2025; 43:sxaf005. [PMID: 39862185 PMCID: PMC12080356 DOI: 10.1093/stmcls/sxaf005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2024] [Accepted: 01/06/2025] [Indexed: 01/27/2025]
Abstract
Myocardial infarction can lead to the loss of billions of cardiomyocytes, and while cell-based therapies are an option, immature nature of in vitro-generated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) is a roadblock to their development. Existing iPSC differentiation protocols don't go beyond producing fetal iCMs. Recently, adult extracellular matrix (ECM) was shown to retain tissue memory and have some success driving tissue-specific differentiation in unspecified cells in various organ systems. Therefore, we focused on investigating the effect of adult human heart-derived extracellular matrix (ECM) on iPSC cardiac differentiation and subsequent maturation. By preconditioning iPSCs with ECM, we tested whether creating cardiac environments around iPSCs would drive iPSCs toward cardiac fate and which ECM components might be involved. We report novel high- and low-abundance proteomes of young, adult, and aged human hearts, with relative abundances to total proteins and each other. We found that adult ECM had extracellular galactin-1, fibronectin, fibrillins, and perlecan (HSPG2) which are implicated in normal heart development. We also showed preconditioning iPSCs with adult cardiac ECM resulted in enhanced cardiac differentiation, yielding iCMs with higher functional maturity, more developed mitochondrial network and coverage, enhanced metabolic maturity, and shift towards more energetic profile. These findings demonstrate the potential use of cardiac ECM in iCM maturation and as a promising strategy for developing iCM-based therapies, disease modeling, and drug screening studies. Upon manipulating ECM, we concluded that the beneficial effects observed were not solely due to the ECM proteins, which might be related to the decorative units attached.
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Affiliation(s)
- S Gulberk Ozcebe
- Bioengineering Graduate Program, University of Notre Dame, Notre Dame, 46556 IN, United States
- National Institute of Environmental Health Sciences (NIEHS), Durham, 27709 NC, United States
| | - Mateo Tristan
- Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, 46556 IN, United States
| | - Pinar Zorlutuna
- Bioengineering Graduate Program, University of Notre Dame, Notre Dame, 46556 IN, United States
- Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, 46556 IN, United States
- Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, 46556 IN, United States
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2
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Li K, He Y, Jin X, Jin K, Qian J. Reproducible extracellular matrices for tumor organoid culture: challenges and opportunities. J Transl Med 2025; 23:497. [PMID: 40312683 PMCID: PMC12044958 DOI: 10.1186/s12967-025-06349-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2025] [Accepted: 03/03/2025] [Indexed: 05/03/2025] Open
Abstract
Tumor organoid models have emerged as valuable 3D in vitro systems to study cancer behavior in a physiologically relevant environment. The composition and architecture of the extracellular matrix (ECM) play critical roles in tumor organoid culture by influencing the tumor microenvironment and tumor behavior. Traditional matrices such as Matrigel and collagen, have been widely used, but their batch-to-batch variability and limited tunability hinder their reproducibility and broader applications. To address these challenges, researchers have turned to synthetic/engineered matrices and biopolymer-based matrices, which offer precise tunability, reproducibility, and chemically defined compositions. However, these matrices also present challenges of their own. In this review, we explore the significance of ECMs in tumor organoid culture, discuss the limitations of commonly used matrices, and highlight recent advancements in engineered/synthetic matrices for improved tumor organoid modeling.
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Affiliation(s)
- Kan Li
- School of Basic Medical Sciences, Health Science Center, Ningbo University, Ningbo, 315211, China
| | - Yibo He
- Department of Surgical Oncology, Hangzhou Cancer Hospital, Hangzhou, Zhejiang, 310006, China
- Department of Breast Surgery, Affiliated Hangzhou First People'S Hospital, School of Medicine, Westlake University, Hangzhou, Zhejiang, 310006, China
| | - Xue Jin
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People'S Hospital (Affiliated People'S Hospital, Hangzhou Medical College), Hangzhou, Zhejiang, 310014, China
| | - Ketao Jin
- Department of Colorectal and Anal Surgery, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, Zhejiang, 310003, China.
| | - Jun Qian
- Department of Colorectal Surgery, Xinchang People'S Hospital, Affiliated Xinchang Hosptial, Wenzhou Medical University, Xinchang, Zhejiang, 312500, China.
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3
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Matějková J, Kaňoková D, Matějka R. Current Status of Bioprinting Using Polymer Hydrogels for the Production of Vascular Grafts. Gels 2024; 11:4. [PMID: 39851975 PMCID: PMC11765431 DOI: 10.3390/gels11010004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2024] [Revised: 12/23/2024] [Accepted: 12/24/2024] [Indexed: 01/26/2025] Open
Abstract
Cardiovascular disease is one of the leading causes of death and serious illness in Europe and worldwide. Conventional treatment-replacing the damaged blood vessel with an autologous graft-is not always affordable for the patient, so alternative approaches are being sought. One such approach is patient-specific tissue bioprinting, which allows for precise distribution of cells, material, and biochemical signals. With further developmental support, a functional replacement tissue or vessel can be created. This review provides an overview of the current state of bioprinting for vascular graft manufacturing and summarizes the hydrogels used as bioinks, the material of carriers, and the current methods of fabrication used, especially for vessels smaller than 6 mm, which are the most challenging for cardiovascular replacements. The fabrication methods are divided into several sections-self-supporting grafts based on simple 3D bioprinting and bioprinting of bioinks on scaffolds made of decellularized or nanofibrous material.
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Affiliation(s)
- Jana Matějková
- Department of Biomedical Technology, Faculty of Biomedical Engineering, Czech Technical University in Prague, 27201 Kladno, Czech Republic;
| | | | - Roman Matějka
- Department of Biomedical Technology, Faculty of Biomedical Engineering, Czech Technical University in Prague, 27201 Kladno, Czech Republic;
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Ahmadipour M, Prado JC, Hakak-Zargar B, Mahmood MQ, Rogers IM. Using ex vivo bioengineered lungs to model pathologies and screening therapeutics: A proof-of-concept study. Biotechnol Bioeng 2024; 121:3020-3033. [PMID: 38837764 DOI: 10.1002/bit.28754] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2023] [Revised: 02/19/2024] [Accepted: 05/12/2024] [Indexed: 06/07/2024]
Abstract
Respiratory diseases, claim over eight million lives annually. However, the transition from preclinical to clinical phases in research studies is often hindered, partly due to inadequate representation of preclinical models in clinical trials. To address this, we conducted a proof-of-concept study using an ex vivo model to identify lung pathologies and to screen therapeutics in a humanized rodent model. We extracted and decellularized mouse heart-lung tissues using a detergent-based technique. The lungs were then seeded and cultured with human cell lines (BEAS-2B, A549, and Calu3) for 6-10 days, representing healthy lungs, cancerous states, and congenital pathologies, respectively. By manipulating cultural conditions and leveraging the unique characteristics of the cell lines, we successfully modeled various pathologies, including advanced-stage solid tumors and the primary phase of SARS-CoV-2 infection. Validation was conducted through histology, immunofluorescence staining, and pathology analysis. Additionally, our study involved pathological screening of the efficacy and impact of key anti-neoplastic therapeutics (Cisplatin and Wogonin) in cancer models. The results highlight the versatility and strength of the ex vivo model in representing crucial lung pathologies and screening therapeutics during the preclinical phase. This approach holds promise for bridging the gap between preclinical and clinical research, aiding in the development of effective treatments for respiratory diseases, including lung cancer.
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Affiliation(s)
- Mohammadali Ahmadipour
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
- School of Medicine, Faculty of Health, Deakin University, Geelong, Victoria, Australia
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
| | - Jorge Castilo Prado
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Benyamin Hakak-Zargar
- School of Medicine, Faculty of Health, Deakin University, Geelong, Victoria, Australia
| | - Malik Quasir Mahmood
- School of Medicine, Faculty of Health, Deakin University, Geelong, Victoria, Australia
| | - Ian M Rogers
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
- Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
- Soham & Shaila Ajmera Family Transplant Centre, University Health Network, Toronto, Ontario, Canada
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Goltsis O, Bilodeau C, Wang J, Luo D, Asgari M, Bozec L, Pettersson A, Leibel SL, Post M. Influence of mesenchymal and biophysical components on distal lung organoid differentiation. Stem Cell Res Ther 2024; 15:273. [PMID: 39218985 PMCID: PMC11367854 DOI: 10.1186/s13287-024-03890-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Accepted: 08/21/2024] [Indexed: 09/04/2024] Open
Abstract
BACKGROUND Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. METHODS Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC). RESULTS We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. CONCLUSION Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.
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Affiliation(s)
- Olivia Goltsis
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada
- Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada
| | - Claudia Bilodeau
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Jinxia Wang
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada
| | - Daochun Luo
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada
| | - Meisam Asgari
- Faculty of Dentistry, University of Toronto, Toronto, ON, Canada
| | - Laurent Bozec
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
- Faculty of Dentistry, University of Toronto, Toronto, ON, Canada
| | - Ante Pettersson
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada
| | - Sandra L Leibel
- Department of Pediatrics, Rady Children's Hospital, San Diego, University of California, San Diego, La Jolla, CA, USA
| | - Martin Post
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada.
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
- Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
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Yang X, Zhao Y, Liu W, Gao Z, Wang C, Wang C, Li S, Zhang X. Single-cell transcriptomics reveals neural stem cell trans-differentiation and cell subpopulations in whole heart decellularized extracellular matrix. BIOPHYSICS REPORTS 2024; 10:241-253. [PMID: 39281200 PMCID: PMC11399890 DOI: 10.52601/bpr.2024.240011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2024] [Accepted: 04/16/2024] [Indexed: 09/18/2024] Open
Abstract
The whole heart decellularized extracellular matrix (ECM) has become a promising scaffold material for cardiac tissue engineering. Our previous research has shown that the whole heart acellular matrix possesses the memory function regulating neural stem cells (NSCs) trans-differentiating to cardiac lineage cells. However, the cell subpopulations and phenotypes in the trans-differentiation of NSCs have not been clearly identified. Here, we performed single-cell RNA sequencing and identified 2,765 cells in the recellularized heart with NSCs revealing the cellular diversity of cardiac and neural lineage, confirming NSCs were capable of trans-differentiating into the cardiac lineage while maintaining the original ability to differentiate into the neural lineage. Notably, the trans-differentiated heart-like cells have dual signatures of neuroectoderm and cardiac mesoderm. This study unveils an in-depth mechanism underlying the trans-differentiation of NSCs and provides a new opportunity and theoretical basis for cardiac regeneration.
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Affiliation(s)
- Xiaoning Yang
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Yuwei Zhao
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Wei Liu
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Zhongbao Gao
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Chunlan Wang
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Changyong Wang
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Siwei Li
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
| | - Xiao Zhang
- Beijing Institute of Basic Medical Sciences, Beijing 100850, China
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7
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Li X, Shan J, Chen X, Cui H, Wen G, Yu Y. Decellularized diseased tissues: current state-of-the-art and future directions. MedComm (Beijing) 2023; 4:e399. [PMID: 38020712 PMCID: PMC10661834 DOI: 10.1002/mco2.399] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2023] [Revised: 09/04/2023] [Accepted: 09/12/2023] [Indexed: 12/01/2023] Open
Abstract
Decellularized matrices derived from diseased tissues/organs have evolved in the most recent years, providing novel research perspectives for understanding disease occurrence and progression and providing accurate pseudo models for developing new disease treatments. Although decellularized matrix maintaining the native composition, ultrastructure, and biomechanical characteristics of extracellular matrix (ECM), alongside intact and perfusable vascular compartments, facilitates the construction of bioengineered organ explants in vitro and promotes angiogenesis and tissue/organ regeneration in vivo, the availability of healthy tissues and organs for the preparation of decellularized ECM materials is limited. In this paper, we review the research advancements in decellularized diseased matrices. Considering that current research focuses on the matrices derived from cancers and fibrotic organs (mainly fibrotic kidney, lungs, and liver), the pathological characterizations and the applications of these diseased matrices are mainly discussed. Additionally, a contrastive analysis between the decellularized diseased matrices and decellularized healthy matrices, along with the development in vitro 3D models, is discussed in this paper. And last, we have provided the challenges and future directions in this review. Deep and comprehensive research on decellularized diseased tissues and organs will promote in-depth exploration of source materials in tissue engineering field, thus providing new ideas for clinical transformation.
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Affiliation(s)
- Xiang Li
- Department of Orthopedic SurgeryShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
| | - Jianyang Shan
- Department of Orthopedic SurgeryShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
| | - Xin Chen
- Department of Orthopedic SurgeryShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
- College of Fisheries and Life ScienceShanghai Ocean UniversityShanghaiChina
| | - Haomin Cui
- Department of Orthopedic SurgeryShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
| | - Gen Wen
- Department of Orthopedic SurgeryShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
| | - Yaling Yu
- Department of Orthopedic SurgeryShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
- Institute of Microsurgery on ExtremitiesShanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
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8
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Petrella F, Cassina EM, Libretti L, Pirondini E, Raveglia F, Tuoro A. Mesenchymal Stromal Cell Therapy for Thoracic Surgeons: An Update. J Pers Med 2023; 13:1632. [PMID: 38138859 PMCID: PMC10744666 DOI: 10.3390/jpm13121632] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Revised: 11/14/2023] [Accepted: 11/21/2023] [Indexed: 12/24/2023] Open
Abstract
Stem cells are undifferentiated cells presenting extensive self-renewal features and the ability to differentiate "in vitro" and "in vivo" into a range of lineage cells, like chondrogenic, osteogenic and adipogenic lineages when cultured in specific inducing media. Two major domains of clinical applications of stem cells in thoracic surgery have been investigated: regenerative medicine, which is a section of translational research in tissue engineering focusing on the replacement, renewal or regeneration of cells, tissues and organs to re-establish damaged physiologic functions; drug loading and delivery, representing a new branch proposing stem cells as carriers to provide selected districts with anti-cancer agents for targeted treatments.
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Affiliation(s)
- Francesco Petrella
- Department of Thoracic Surgery, Fondazione IRCCS San Gerardo dei Tintori, 20900 Monza, Italy; (E.M.C.); (L.L.); (E.P.); (F.R.); (A.T.)
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Chan WS, Mo X, Ip PPC, Tse KY. Patient-derived organoid culture in epithelial ovarian cancers-Techniques, applications, and future perspectives. Cancer Med 2023; 12:19714-19731. [PMID: 37776168 PMCID: PMC10587945 DOI: 10.1002/cam4.6521] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Revised: 07/05/2023] [Accepted: 08/30/2023] [Indexed: 10/01/2023] Open
Abstract
Epithelial ovarian cancer (EOC) is a heterogeneous disease composed of different cell types with different molecular aberrations. Traditional cell lines and mice models cannot recapitulate the human tumor biology and tumor microenvironment (TME). Patient-derived organoids (PDOs) are freshly derived from patients' tissues and are then cultured with extracellular matrix and conditioned medium. The high concordance of epigenetic, genomic, and proteomic landscapes between the parental tumors and PDOs suggests that PDOs can provide more reliable results in studying cancer biology, allowing high throughput drug screening, and identifying their associated signaling pathways and resistance mechanisms. However, despite having a heterogeneity of cells in PDOs, some cells in TME will be lost during the culture process. Next-generation organoids have been developed to circumvent some of the limitations. Genetically engineered organoids involving targeted gene editing can facilitate the understanding of tumorigenesis and drug response. Co-culture systems where PDOs are cultured with different cell components like immune cells can allow research using immunotherapy which is otherwise impossible in conventional cell lines. In this review, the limitations of the traditional in vitro and in vivo assays, the use of PDOs, the challenges including some tips and tricks of PDO generation in EOC, and the future perspectives, will be discussed.
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Affiliation(s)
- Wai Sun Chan
- Department of Obstetrics and GynaecologyThe University of Hong KongPokfulamHong Kong SAR
| | - Xuetang Mo
- Department of Obstetrics and GynaecologyThe University of Hong KongPokfulamHong Kong SAR
| | | | - Ka Yu Tse
- Department of Obstetrics and GynaecologyThe University of Hong KongPokfulamHong Kong SAR
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10
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Hoffman E, Song Y, Zhang F, Asarian L, Downs I, Young B, Han X, Ouyang Y, Xia K, Linhardt RJ, Weiss DJ. Regional and disease-specific glycosaminoglycan composition and function in decellularized human lung extracellular matrix. Acta Biomater 2023; 168:388-399. [PMID: 37433361 PMCID: PMC10528722 DOI: 10.1016/j.actbio.2023.06.043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 06/16/2023] [Accepted: 06/28/2023] [Indexed: 07/13/2023]
Abstract
Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor β to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease.
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Affiliation(s)
- Evan Hoffman
- Larner College of Medicine, University of Vermont, 149 Beaumont Avenue, Health Science Research Facility (HSRF) 226, Burlington, VT 05405, USA
| | - Yuefan Song
- Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA
| | - Fuming Zhang
- Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA
| | - Loredana Asarian
- Larner College of Medicine, University of Vermont, 149 Beaumont Avenue, Health Science Research Facility (HSRF) 226, Burlington, VT 05405, USA
| | - Isaac Downs
- Larner College of Medicine, University of Vermont, 149 Beaumont Avenue, Health Science Research Facility (HSRF) 226, Burlington, VT 05405, USA
| | - Brad Young
- Larner College of Medicine, University of Vermont, 149 Beaumont Avenue, Health Science Research Facility (HSRF) 226, Burlington, VT 05405, USA
| | - Xiaorui Han
- Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA
| | - Yilan Ouyang
- Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA
| | - Ke Xia
- Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA
| | - Robert J Linhardt
- Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, NY, USA
| | - Daniel J Weiss
- Larner College of Medicine, University of Vermont, 149 Beaumont Avenue, Health Science Research Facility (HSRF) 226, Burlington, VT 05405, USA.
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11
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Goh SK, Bertera S, Richardson T, Banerjee I. Repopulation of decellularized organ scaffolds with human pluripotent stem cell-derived pancreatic progenitor cells. Biomed Mater 2023; 18. [PMID: 36720168 DOI: 10.1088/1748-605x/acb7bf] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Accepted: 01/31/2023] [Indexed: 02/02/2023]
Abstract
Diabetes is an emerging global epidemic that affects more that 285 million people worldwide. Engineering of endocrine pancreas tissue holds great promise for the future of diabetes therapy. Here we demonstrate the feasibility of re-engineering decellularized organ scaffolds using regenerative cell source. We differentiated human pluripotent stem cells (hPSC) toward pancreatic progenitor (PP) lineage and repopulated decellularized organ scaffolds with these hPSC-PP cells. We observed that hPSCs cultured and differentiated as aggregates are more suitable for organ repopulation than isolated single cell suspension. However, recellularization with hPSC-PP aggregates require a more extensive vascular support, which was found to be superior in decellularized liver over the decellularized pancreas scaffolds. Upon continued culture for nine days with chemical induction in the bioreactor, the seeded hPSC-PP aggregates demonstrated extensive and uniform cellular repopulation and viability throughout the thickness of the liver scaffolds. Furthermore, the decellularized liver scaffolds was supportive of the endocrine cell fate of the engrafted cells. Our novel strategy to engineer endocrine pancreas construct is expected to find potential applications in preclinical testing, drug discovery and diabetes therapy.
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Affiliation(s)
- Saik-Kia Goh
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States of America
| | - Suzanne Bertera
- Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA, United States of America
| | - Thomas Richardson
- Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, United States of America
| | - Ipsita Banerjee
- Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, United States of America.,Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States of America.,McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States of America
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12
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Júnior C, Ulldemolins A, Narciso M, Almendros I, Farré R, Navajas D, López J, Eroles M, Rico F, Gavara N. Multi-Step Extracellular Matrix Remodelling and Stiffening in the Development of Idiopathic Pulmonary Fibrosis. Int J Mol Sci 2023; 24:ijms24021708. [PMID: 36675222 PMCID: PMC9865994 DOI: 10.3390/ijms24021708] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Revised: 01/04/2023] [Accepted: 01/05/2023] [Indexed: 01/18/2023] Open
Abstract
The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young's Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.
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Affiliation(s)
- Constança Júnior
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- The Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
| | - Anna Ulldemolins
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- CIBER de Enfermedades Respiratorias, 28029 Madrid, Spain
- Institut d’Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain
| | - Maria Narciso
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- The Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
| | - Isaac Almendros
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- CIBER de Enfermedades Respiratorias, 28029 Madrid, Spain
- Institut d’Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain
| | - Ramon Farré
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- CIBER de Enfermedades Respiratorias, 28029 Madrid, Spain
- Institut d’Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain
| | - Daniel Navajas
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- The Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
- CIBER de Enfermedades Respiratorias, 28029 Madrid, Spain
| | - Javier López
- Institut Pasteur de Lille, U1019-UMR9017-CIIL-Centre d’Infection et d’Immunité de Lille, Université de Lille, CNRS, Inserm, CHU Lille, 59000 Lille, France
| | - Mar Eroles
- Aix-Marseille, CNRS, INSERM, LAI, Centuri Centre for Living Systems, 13009 Marseille, France
| | - Felix Rico
- Aix-Marseille, CNRS, INSERM, LAI, Centuri Centre for Living Systems, 13009 Marseille, France
| | - Núria Gavara
- Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain
- The Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
- Correspondence:
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13
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Shakir S, Hackett TL, Mostaço-Guidolin LB. Bioengineering lungs: An overview of current methods, requirements, and challenges for constructing scaffolds. Front Bioeng Biotechnol 2022; 10:1011800. [PMID: 36394026 PMCID: PMC9649450 DOI: 10.3389/fbioe.2022.1011800] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Accepted: 10/17/2022] [Indexed: 09/28/2023] Open
Abstract
Chronic respiratory diseases remain a significant health burden worldwide. The only option for individuals with end-stage lung failure remains Lung Transplantation. However, suitable organ donor shortages and immune rejection following transplantation remain a challenge. Since alternative options are urgently required to increase tissue availability for lung transplantation, researchers have been exploring lung bioengineering extensively, to generate functional, transplantable organs and tissue. Additionally, the development of physiologically-relevant artificial tissue models for testing novel therapies also represents an important step toward finding a definite clinical solution for different chronic respiratory diseases. This mini-review aims to highlight some of the most common methodologies used in bioengineering lung scaffolds, as well as the benefits and disadvantages associated with each method in conjunction with the current areas of research devoted to solving some of these challenges in the area of lung bioengineering.
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Affiliation(s)
- Shahad Shakir
- Department of Mechanical and Aerospace Engineering, Carleton University, Ottawa, ON, Canada
| | - Tillie Louise Hackett
- Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC, Canada
- Centre for Heart Lung Innovation, University of British Columbia, Vancouver, BC, Canada
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14
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Record Ritchie RD, Salmon SL, Hiles MC, Metzger DW. Lack of immunogenicity of xenogeneic DNA from porcine biomaterials. Surg Open Sci 2022; 10:83-90. [PMID: 36039075 PMCID: PMC9418979 DOI: 10.1016/j.sopen.2022.07.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Revised: 06/27/2022] [Accepted: 07/18/2022] [Indexed: 11/18/2022] Open
Affiliation(s)
- Rae D. Record Ritchie
- Cook Biotech, Inc, 1425 Innovation Place, West Lafayette, IN, USA 47906
- Corresponding author at: 1425 Innovation Place, West Lafayette, IN 47906. Tel.: + 1-765-497-3355; fax: + 1-765-497-2361.
| | - Sharon L. Salmon
- Department of Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Ave, MC-151, Albany, NY, USA 12208-3479
| | - Michael C. Hiles
- Cook Biotech, Inc, 1425 Innovation Place, West Lafayette, IN, USA 47906
| | - Dennis W. Metzger
- Department of Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Ave, MC-151, Albany, NY, USA 12208-3479
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15
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Singh G, Senapati S, Satpathi S, Behera PK, Das B, Nayak B. Establishment of decellularized extracellular matrix scaffold derived from caprine pancreas as a novel alternative template over porcine pancreatic scaffold for prospective biomedical application. FASEB J 2022; 36:e22574. [PMID: 36165227 DOI: 10.1096/fj.202200807r] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2022] [Revised: 08/25/2022] [Accepted: 09/16/2022] [Indexed: 11/11/2022]
Abstract
In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.
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Affiliation(s)
- Garima Singh
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, India
| | - Shantibhusan Senapati
- Tumor Microenvironment and Animal Models Laboratory, Institute of Life Sciences, Bhubaneswar, India
| | | | | | - Biswajit Das
- Tumor Microenvironment and Animal Models Laboratory, Institute of Life Sciences, Bhubaneswar, India
| | - Bismita Nayak
- Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, India
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16
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Lin Y, Wang D, Zeng Y. A Maverick Review of Common Stem/Progenitor Markers in Lung Development. Stem Cell Rev Rep 2022; 18:2629-2645. [DOI: 10.1007/s12015-022-10422-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/26/2022] [Indexed: 10/16/2022]
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17
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Bongolan T, Whiteley J, Castillo-Prado J, Fantin A, Larsen B, Wong CJ, Mazilescu L, Kawamura M, Urbanellis P, Jonebring A, Salter E, Collingridge G, Gladdy R, Hicks R, Gingras AC, Selzner M, Rogers IM. Decellularization of porcine kidney with submicellar concentrations of SDS results in the retention of ECM proteins required for the adhesion and maintenance of human adult renal epithelial cells. Biomater Sci 2022; 10:2972-2990. [PMID: 35521809 DOI: 10.1039/d1bm01017d] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
When decellularizing kidneys, it is important to maintain the integrity of the acellular extracellular matrix (ECM), including associated adhesion proteins and growth factors that allow recellularized cells to adhere and migrate according to ECM specificity. Kidney decellularization requires the ionic detergent sodium dodecyl sulfate (SDS); however, this results in a loss of ECM proteins important for cell adherence, migration, and growth, particularly glycosaminoglycan (GAG)-associated proteins. Here, we demonstrate that using submicellar concentrations of SDS results in a greater retention of structural proteins, GAGs, growth factors, and cytokines. When porcine kidney ECM scaffolds were recellularized using human adult primary renal epithelial cells (RECs), the ECM promoted cell survival and the uniform distribution of cells throughout the ECM. Cells maintained the expression of mature renal epithelial markers but did not organize on the ECM, indicating that mature cells are unable to migrate to specific locations on ECM scaffolds.
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Affiliation(s)
- Tonya Bongolan
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Jennifer Whiteley
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Jorge Castillo-Prado
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Amanda Fantin
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Brett Larsen
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Cassandra J Wong
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Laura Mazilescu
- Toronto General Hospital Research Institute, Toronto, ON, M5G 2C4, Canada.,Soham & Shaila Ajmera Family Transplant Centre, University Health Network, Toronto, ON, M5G 2C4, Canada
| | - Masataka Kawamura
- Toronto General Hospital Research Institute, Toronto, ON, M5G 2C4, Canada.,Soham & Shaila Ajmera Family Transplant Centre, University Health Network, Toronto, ON, M5G 2C4, Canada
| | - Peter Urbanellis
- Toronto General Hospital Research Institute, Toronto, ON, M5G 2C4, Canada.,Soham & Shaila Ajmera Family Transplant Centre, University Health Network, Toronto, ON, M5G 2C4, Canada
| | - Anna Jonebring
- Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, 431 83, Sweden
| | - Eric Salter
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Graham Collingridge
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
| | - Rebecca Gladdy
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada. .,Institute of Medical Science, University of Toronto, Toronto, ON, M5S1A8, Canada
| | - Ryan Hicks
- BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, 431 83, Sweden
| | - Anne-Claude Gingras
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada. .,Department of Molecular Genetics, University of Toronto, Toronto, ON, M5G1E2, Canada
| | - Markus Selzner
- Toronto General Hospital Research Institute, Toronto, ON, M5G 2C4, Canada.,Soham & Shaila Ajmera Family Transplant Centre, University Health Network, Toronto, ON, M5G 2C4, Canada
| | - Ian M Rogers
- Department of Physiology, University of Toronto, Toronto, ON, M5S 1A8, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada. .,Soham & Shaila Ajmera Family Transplant Centre, University Health Network, Toronto, ON, M5G 2C4, Canada.,Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, M5G1E2, Canada
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18
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Mohgan R, Candasamy M, Mayuren J, Singh SK, Gupta G, Dua K, Chellappan DK. Emerging Paradigms in Bioengineering the Lungs. Bioengineering (Basel) 2022; 9:bioengineering9050195. [PMID: 35621473 PMCID: PMC9137616 DOI: 10.3390/bioengineering9050195] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2022] [Revised: 04/21/2022] [Accepted: 04/26/2022] [Indexed: 12/25/2022] Open
Abstract
In end-stage lung diseases, the shortage of donor lungs for transplantation and long waiting lists are the main culprits in the significantly increasing number of patient deaths. New strategies to curb this issue are being developed with the help of recent advancements in bioengineering technology, with the generation of lung scaffolds as a steppingstone. There are various types of lung scaffolds, namely, acellular scaffolds that are developed via decellularization and recellularization techniques, artificial scaffolds that are synthesized using synthetic, biodegradable, and low immunogenic materials, and hybrid scaffolds which combine the advantageous properties of materials in the development of a desirable lung scaffold. There have also been advances in the design of bioreactors in terms of providing an optimal regenerative environment for the maturation of functional lung tissue over time. In this review, the emerging paradigms in the field of lung tissue bioengineering will be discussed.
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Affiliation(s)
- Raxshanaa Mohgan
- School of Pharmacy, International Medical University, Kuala Lumpur 57000, Malaysia;
| | - Mayuren Candasamy
- Department of Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur 57000, Malaysia;
| | - Jayashree Mayuren
- Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, Kuala Lumpur 57000, Malaysia;
| | - Sachin Kumar Singh
- School of Pharmaceutical Sciences, Lovely Professional University, Jalandhar-Delhi G.T Road, Phagwara 144411, India;
- Australian Research Centre in Complementary and Integrative Medicine, Faculty of Health, University of Technology Sydney, Sydney 2007, Australia;
| | - Gaurav Gupta
- School of Pharmacy, Suresh Gyan Vihar University, Jaipur 302017, India;
- Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 600077, India
- Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun 248007, India
| | - Kamal Dua
- Australian Research Centre in Complementary and Integrative Medicine, Faculty of Health, University of Technology Sydney, Sydney 2007, Australia;
- Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Sydney 2007, Australia
| | - Dinesh Kumar Chellappan
- Department of Life Sciences, School of Pharmacy, International Medical University, Kuala Lumpur 57000, Malaysia;
- Correspondence:
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19
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Ultrasound in cellulose-based hydrogel for biomedical use: From extraction to preparation. Colloids Surf B Biointerfaces 2022; 212:112368. [PMID: 35114437 DOI: 10.1016/j.colsurfb.2022.112368] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 01/12/2022] [Accepted: 01/23/2022] [Indexed: 02/07/2023]
Abstract
As the most abundant natural polymer on the pl anet, cellulose has a wide range of applications in the biomedical field. Cellulose-based hydrogels further expand the applications of this class of biomaterials. However, a number of publications and technical reports are mainly about traditional preparation methods. Sonochemistry offers a simple and green route to material synthesis with the biomedical application of ultrasound. The tiny acoustic bubbles, produced by the propagating sound wave, enclose an incredible facility where matter interact among at energy as high as 13 eV to spark extraordinary chemical reactions. Ultrasonication not only improves the efficiency of cellulose extraction from raw materials, but also influences the hydrogel preparation process. The primary objective of this article is to review the literature concerning the biomedical cellulose-based hydrogel prepared via sonochemistry and application of ultrasound for hydrogel. An innovated category of recent generations of hydrogel materials prepared via ultrasound was also presented in some details.
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20
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Unravelling the molecular mechanisms underlying chronic respiratory diseases for the development of novel therapeutics via in vitro experimental models. Eur J Pharmacol 2022; 919:174821. [DOI: 10.1016/j.ejphar.2022.174821] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 02/01/2022] [Accepted: 02/09/2022] [Indexed: 12/11/2022]
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21
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Ngan SY, Quach HT, Laselva O, Huang EN, Mangos M, Xia S, Bear CE, Wong AP. Stage-Specific Generation of Human Pluripotent Stem Cell Derived Lung Models to Measure CFTR Function. Curr Protoc 2022; 2:e341. [PMID: 35025140 DOI: 10.1002/cpz1.341] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture.
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Affiliation(s)
- Shuk Yee Ngan
- Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
| | - Henry T Quach
- Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Onofrio Laselva
- Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Medical and Surgical Sciences, University of Foggia, Foggia, Puglia, Italy
| | - Elena N Huang
- Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Maria Mangos
- Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, Canada
| | - Sunny Xia
- Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Christine E Bear
- Program in Molecular Medicine, Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Physiology, University of Toronto, Toronto, Ontario, Canada.,Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
| | - Amy P Wong
- Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
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22
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Moffat D, Ye K, Jin S. Decellularization for the retention of tissue niches. J Tissue Eng 2022; 13:20417314221101151. [PMID: 35620656 PMCID: PMC9128068 DOI: 10.1177/20417314221101151] [Citation(s) in RCA: 62] [Impact Index Per Article: 20.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Accepted: 05/01/2022] [Indexed: 12/25/2022] Open
Abstract
Decellularization of natural tissues to produce extracellular matrix is a promising method for three-dimensional scaffolding and for understanding microenvironment of the tissue of interest. Due to the lack of a universal standard protocol for tissue decellularization, recent investigations seek to develop novel methods for whole or partial organ decellularization capable of supporting cell differentiation and implantation towards appropriate tissue regeneration. This review provides a comprehensive and updated perspective on the most recent advances in decellularization strategies for a variety of organs and tissues, highlighting techniques of chemical, physical, biological, enzymatic, or combinative-based methods to remove cellular contents from tissues. In addition, the review presents modernized approaches for improving standard decellularization protocols for numerous organ types.
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Affiliation(s)
- Deana Moffat
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, NY, USA
| | - Kaiming Ye
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, NY, USA
- Center of Biomanufacturing for Regenerative Medicine, Binghamton University, State University of New York (SUNY), Binghamton, NY, USA
| | - Sha Jin
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, NY, USA
- Center of Biomanufacturing for Regenerative Medicine, Binghamton University, State University of New York (SUNY), Binghamton, NY, USA
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23
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Abstract
Organoids-cellular aggregates derived from stem or progenitor cells that recapitulate organ function in miniature-are of growing interest in developmental biology and medicine. Organoids have been developed for organs and tissues such as the liver, gut, brain, and pancreas; they are used as organ surrogates to study a wide range of questions in basic and developmental biology, genetic disorders, and therapies. However, many organoids reported to date have been cultured in Matrigel, which is prepared from the secretion of Engelbreth-Holm-Swarm mouse sarcoma cells; Matrigel is complex and poorly defined. This complexity makes it difficult to elucidate Matrigel-specific factors governing organoid development. In this review, we discuss promising Matrigel-free methods for the generation and maintenance of organoids that use decellularized extracellular matrix (ECM), synthetic hydrogels, or gel-forming recombinant proteins.
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Affiliation(s)
- Mark T Kozlowski
- DEVCOM US Army Research Laboratory, Weapons and Materials Research Directorate, Science of Extreme Materials Division, Polymers Branch, 6300 Rodman Rd. Building 4600, Aberdeen Proving Ground, Aberdeen, MD, 21005, USA.
| | - Christiana J Crook
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, City of Hope National Medical Center, 1500 Duarte Rd., Duarte, CA, 91010, USA
- Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 1500 Duarte Rd., Duarte, CA, 91010, USA
- Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, 1500 Duarte Rd., Duarte, CA, 91010, USA
| | - Hsun Teresa Ku
- Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, City of Hope National Medical Center, 1500 Duarte Rd., Duarte, CA, 91010, USA
- Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 1500 Duarte Rd., Duarte, CA, 91010, USA
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24
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Olesen K, Rodin S, Mak WC, Felldin U, Österholm C, Tilevik A, Grinnemo KH. Spatiotemporal extracellular matrix modeling for in situ cell niche studies. Stem Cells 2021; 39:1751-1765. [PMID: 34418223 DOI: 10.1002/stem.3448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 08/10/2021] [Indexed: 11/06/2022]
Abstract
Extracellular matrix (ECM) components govern a range of cell functions, such as migration, proliferation, maintenance of stemness, and differentiation. Cell niches that harbor stem-/progenitor cells, with matching ECM, have been shown in a range of organs, although their presence in the heart is still under debate. Determining niches depends on a range of in vitro and in vivo models and techniques, where animal models are powerful tools for studying cell-ECM dynamics; however, they are costly and time-consuming to use. In vitro models based on recombinant ECM proteins lack the complexity of the in vivo ECM. To address these issues, we present the spatiotemporal extracellular matrix model for studies of cell-ECM dynamics, such as cell niches. This model combines gentle decellularization and sectioning of cardiac tissue, allowing retention of a complex ECM, with recellularization and subsequent image processing using image stitching, segmentation, automatic binning, and generation of cluster maps. We have thereby developed an in situ representation of the cardiac ECM that is useful for assessment of repopulation dynamics and to study the effect of local ECM composition on phenotype preservation of reseeded mesenchymal progenitor cells. This model provides a platform for studies of organ-specific cell-ECM dynamics and identification of potential cell niches.
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Affiliation(s)
- Kim Olesen
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.,School of Bioscience, University of Skövde, Skövde, Sweden.,Polymer Chemistry, Department of Chemistry - Ångström Laboratory, Uppsala University, Uppsala, Sweden
| | - Sergey Rodin
- Department of Surgical Sciences, Division of Cardiothoracic Surgery and Anaesthesiology, Uppsala University, Akademiska University Hospital, Uppsala, Sweden
| | - Wing Cheung Mak
- Biosensors and Bioelectronics Centre, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden
| | - Ulrika Felldin
- Department of Surgical Sciences, Division of Cardiothoracic Surgery and Anaesthesiology, Uppsala University, Akademiska University Hospital, Uppsala, Sweden
| | - Cecilia Österholm
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
| | | | - Karl-Henrik Grinnemo
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.,Department of Surgical Sciences, Division of Cardiothoracic Surgery and Anaesthesiology, Uppsala University, Akademiska University Hospital, Uppsala, Sweden
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25
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Image-Based Method to Quantify Decellularization of Tissue Sections. Int J Mol Sci 2021; 22:ijms22168399. [PMID: 34445106 PMCID: PMC8395145 DOI: 10.3390/ijms22168399] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Revised: 07/20/2021] [Accepted: 08/02/2021] [Indexed: 01/17/2023] Open
Abstract
Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.
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26
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Archer F, Bobet-Erny A, Gomes M. State of the art on lung organoids in mammals. Vet Res 2021; 52:77. [PMID: 34078444 PMCID: PMC8170649 DOI: 10.1186/s13567-021-00946-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Accepted: 05/04/2021] [Indexed: 02/08/2023] Open
Abstract
The number and severity of diseases affecting lung development and adult respiratory function have stimulated great interest in developing new in vitro models to study lung in different species. Recent breakthroughs in 3-dimensional (3D) organoid cultures have led to new physiological in vitro models that better mimic the lung than conventional 2D cultures. Lung organoids simulate multiple aspects of the real organ, making them promising and useful models for studying organ development, function and disease (infection, cancer, genetic disease). Due to their dynamics in culture, they can serve as a sustainable source of functional cells (biobanking) and be manipulated genetically. Given the differences between species regarding developmental kinetics, the maturation of the lung at birth, the distribution of the different cell populations along the respiratory tract and species barriers for infectious diseases, there is a need for species-specific lung models capable of mimicking mammal lungs as they are of great interest for animal health and production, following the One Health approach. This paper reviews the latest developments in the growing field of lung organoids.
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Affiliation(s)
- Fabienne Archer
- UMR754, IVPC, INRAE, EPHE, Univ Lyon, Université Claude Bernard Lyon 1, 69007, Lyon, France.
| | - Alexandra Bobet-Erny
- UMR754, IVPC, INRAE, EPHE, Univ Lyon, Université Claude Bernard Lyon 1, 69007, Lyon, France
| | - Maryline Gomes
- UMR754, IVPC, INRAE, EPHE, Univ Lyon, Université Claude Bernard Lyon 1, 69007, Lyon, France
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27
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Gharenaz NM, Movahedin M, Mazaheri Z. Comparison of two methods for prolong storage of decellularized mouse whole testis for tissue engineering application: An experimental study. Int J Reprod Biomed 2021; 19:321-332. [PMID: 33997591 PMCID: PMC8106816 DOI: 10.18502/ijrm.v19i4.9058] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2020] [Revised: 07/04/2020] [Accepted: 09/26/2020] [Indexed: 11/24/2022] Open
Abstract
Background Biological scaffolds are derived by the decellularization of tissues or organs. Various biological scaffolds, such as scaffolds for the liver, lung, esophagus, dermis, and human testicles, have been produced. Their application in tissue engineering has created the need for cryopreservation processes to store these scaffolds. Objective The aim was to compare the two methods for prolong storage testicular scaffolds. Materials and Methods In this experimental study, 20 male NMRI mice (8 wk) were sacrificed and their testes were removed and treated with 0.5% sodium dodecyl sulfate followed by Triton X-100 0.5%. The efficiency of decellularization was determined by histology and DNA quantification. Testicular scaffolds were stored in phosphate-buffered saline solution at 4°C or cryopreserved by programmed slow freezing followed by storage in liquid nitrogen. Masson's trichrome staining, Alcian blue staining and immunohistochemistry, collagen assay, and glycosaminoglycan assay were done prior to and after six months of storage under each condition. Results Hematoxylin-eosin staining showed no remnant cells after the completion of decellularization. DNA content analysis indicated that approximately 98% of the DNA was removed from the tissue (p = 0.02). Histological evaluation confirmed the preservation of extracellular matrix components in the fresh and frozen-thawed scaffolds. Extracellular matrix components were decreased by 4°C-stored scaffolds. Cytotoxicity tests with mouse embryonic fibroblast showed that the scaffolds were biocompatible and did not have a harmful effect on the proliferation of mouse embryonic fibroblast cells. Conclusion Our results demonstrated the superiority of the slow freezing method for prolong storage of testicular scaffolds.
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Affiliation(s)
- Nasrin Majidi Gharenaz
- Anatomical Sciences Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Mansoureh Movahedin
- Anatomical Sciences Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Zohreh Mazaheri
- Basic Medical Science Research Center, Histogenotech Company, Tehran, Iran
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28
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Hedström U, Öberg L, Vaarala O, Dellgren G, Silverborn M, Bjermer L, Westergren-Thorsson G, Hallgren O, Zhou X. Impaired Differentiation of Chronic Obstructive Pulmonary Disease Bronchial Epithelial Cells Grown on Bronchial Scaffolds. Am J Respir Cell Mol Biol 2021; 65:201-213. [PMID: 33882260 PMCID: PMC8399573 DOI: 10.1165/rcmb.2019-0395oc] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling, and emphysema. Airway remodeling in patients with COPD involves both the airway epithelium and the subepithelial extracellular matrix (ECM). However, it is currently unknown how epithelial remodeling in COPD airways depends on the relative influence from inherent defects in the epithelial cells and alterations in the ECM. To address this, we analyzed global gene expression in COPD human bronchial epithelial cells (HBEC) and normal HBEC after repopulation on decellularized bronchial scaffolds derived from patients with COPD or donors without COPD. COPD HBEC grown on bronchial scaffolds showed an impaired ability to initiate ciliated-cell differentiation, which was evident on all scaffolds regardless of their origin. In addition, although normal HBEC were less affected by the disease state of the bronchial scaffolds, COPD HBEC showed a gene expression pattern indicating increased proliferation and a retained basal-cell phenotype when grown on COPD bronchial scaffolds compared with normal bronchial scaffolds. By using mass spectrometry, we identified 13 matrisome proteins as being differentially abundant between COPD bronchial scaffolds and normal bronchial scaffolds. These observations are consistent with COPD pathology and suggest that both epithelial cells and the ECM contribute to epithelial-cell remodeling in COPD airways.
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Affiliation(s)
- Ulf Hedström
- Department of Bioscience COPD/IPF, and.,Division of Lung Biology, Department of Experimental Medical Science, and
| | - Lisa Öberg
- Department of Translational Science and Experimental Medicine, Research and Early Development, Respiratory and Immunology, BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg, Sweden
| | | | - Göran Dellgren
- Transplant Institute and.,Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Martin Silverborn
- Transplant Institute and.,Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Leif Bjermer
- Division of Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund University, Lund, Sweden; and
| | | | - Oskar Hallgren
- Division of Lung Biology, Department of Experimental Medical Science, and.,Division of Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund University, Lund, Sweden; and
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Wanczyk H, Jensen T, Weiss DJ, Finck C. Advanced single-cell technologies to guide the development of bioengineered lungs. Am J Physiol Lung Cell Mol Physiol 2021; 320:L1101-L1117. [PMID: 33851545 DOI: 10.1152/ajplung.00089.2021] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Lung transplantation remains the only viable option for individuals suffering from end-stage lung failure. However, a number of current limitations exist including a continuing shortage of suitable donor lungs and immune rejection following transplantation. To address these concerns, engineering a decellularized biocompatible lung scaffold from cadavers reseeded with autologous lung cells to promote tissue regeneration is being explored. Proof-of-concept transplantation of these bioengineered lungs into animal models has been accomplished. However, these lungs were incompletely recellularized with resulting epithelial and endothelial leakage and insufficient basement membrane integrity. Failure to repopulate lung scaffolds with all of the distinct cell populations necessary for proper function remains a significant hurdle for the progression of current engineering approaches and precludes clinical translation. Advancements in 3D bioprinting, lung organoid models, and microfluidic device and bioreactor development have enhanced our knowledge of pulmonary lung development, as well as important cell-cell and cell-matrix interactions, all of which will help in the path to a bioengineered transplantable lung. However, a significant gap in knowledge of the spatiotemporal interactions between cell populations as well as relative quantities and localization within each compartment of the lung necessary for its proper growth and function remains. This review will provide an update on cells currently used for reseeding decellularized scaffolds with outcomes of recent lung engineering attempts. Focus will then be on how data obtained from advanced single-cell analyses, coupled with multiomics approaches and high-resolution 3D imaging, can guide current lung bioengineering efforts for the development of fully functional, transplantable lungs.
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Affiliation(s)
- Heather Wanczyk
- Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut
| | - Todd Jensen
- Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut
| | - Daniel J Weiss
- Department of Medicine, University of Vermont, Burlington, Vermont
| | - Christine Finck
- Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut.,Department of Surgery, Connecticut Children's Medical Center, Hartford, Connecticut
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30
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TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds. NPJ Regen Med 2021; 6:12. [PMID: 33674599 PMCID: PMC7935966 DOI: 10.1038/s41536-021-00124-4] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2020] [Accepted: 02/01/2021] [Indexed: 12/24/2022] Open
Abstract
The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.
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31
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Granath C, Noren H, Björck H, Simon N, Olesen K, Rodin S, Grinnemo KH, Österholm C. Characterization of Laminins in Healthy Human Aortic Valves and a Modified Decellularized Rat Scaffold. Biores Open Access 2020; 9:269-278. [PMID: 33376633 PMCID: PMC7757704 DOI: 10.1089/biores.2020.0018] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/11/2020] [Indexed: 01/13/2023] Open
Abstract
Aortic valve stenosis is one of the most common cardiovascular diseases in western countries and can only be treated by replacement with a prosthetic valve. Tissue engineering is an emerging and promising treatment option, but in-depth knowledge about the microstructure of native heart valves is lacking, making the development of tissue-engineered heart valves challenging. Specifically, the basement membrane (BM) of heart valves remains incompletely characterized, and decellularization protocols that preserve BM components are necessary to advance the field. This study aims to characterize laminin isoforms expressed in healthy human aortic valves and establish a small animal decellularized aortic valve scaffold for future studies of the BM in tissue engineering. Laminin isoforms were assessed by immunohistochemistry with antibodies specific for individual α, β, and γ chains. The results indicated that LN-411, LN-421, LN-511, and LN-521 are expressed in human aortic valves (n = 3), forming a continuous monolayer in the endothelial BM, whereas sparsely found in the interstitium. Similar results were seen in rat aortic valves (n = 3). Retention of laminin and other BM components, concomitantly with effective removal of cells and residual DNA, was achieved through 3 h exposure to 1% sodium dodecyl sulfate and 30 min exposure to 1% Triton X-100, followed by nuclease processing in rat aortic valves (n = 3). Our results provide crucial data on the microenvironment of valvular cells relevant for research in both tissue engineering and heart valve biology. We also describe a decellularized rat aortic valve scaffold useful for mechanistic studies on the role of the BM in heart valve regeneration.
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Affiliation(s)
- Carl Granath
- Division of Cardiothoracic Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
| | - Hunter Noren
- Cell Therapy Institute, Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, Davie, Florida, USA
| | - Hanna Björck
- Cardiovascular Medicine Unit, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
| | - Nancy Simon
- Cardiovascular Medicine Unit, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
| | - Kim Olesen
- Division of Cardiothoracic Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
- Department of Bioscience, University of Skövde, Skövde, Sweden
- Department of Chemistry, Ångström Laboratory, Uppsala University, Uppsala, Sweden
| | - Sergey Rodin
- Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
- Division of Cardiothoracic Surgery and Anesthesiology, Department of Surgical Sciences, Uppsala University, Akademiska University Hospital, Uppsala, Sweden
| | - Karl-Henrik Grinnemo
- Division of Cardiothoracic Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
- Division of Cardiothoracic Surgery and Anesthesiology, Department of Surgical Sciences, Uppsala University, Akademiska University Hospital, Uppsala, Sweden
| | - Cecilia Österholm
- Division of Clinical Genetics, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
- Address correspondence to: Cecilia Österholm Corbascio, PhD, Division of Clinical Genetics, Department of Molecular Medicine and Surgery, Karolinska Institutet, Solna, 171 64, Sweden
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Goh SK, Halfter W, Richardson T, Bertera S, Vaidya V, Candiello J, Bradford M, Banerjee I. Organ-specific ECM arrays for investigating Cell-ECM interactions during stem cell differentiation. Biofabrication 2020; 13. [PMID: 33045682 DOI: 10.1088/1758-5090/abc05f] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Accepted: 10/12/2020] [Indexed: 12/22/2022]
Abstract
Pluripotent stem cells are promising source of cells for tissue engineering, regenerative medicine and drug discovery applications. The process of stem cell differentiation is regulated by multi-parametric cues from the surrounding microenvironment, one of the critical one being cell interaction with extracellular matrix (ECM). The ECM is a complex tissue-specific structure which are important physiological regulators of stem cell function and fate. Recapitulating this native ECM microenvironment niche is best facilitated by decellularized tissue/ organ derived ECM, which can faithfully reproduce the physiological environment with high fidelity to in vivo condition and promote tissue-specific cellular development and maturation. Recognizing the need for organ specific ECM in a 3D culture environment in driving phenotypic differentiation and maturation of hPSCs, we fabricated an ECM array platform using native-mimicry ECM from decellularized organs (namely pancreas, liver and heart), which allows cell-ECM interactions in both 2D and 3D configuration. The ECM array was integrated with rapid quantitative imaging for a systematic investigation of matrix protein profiles and sensitive measurement of cell-ECM interaction during hPSC differentiation. We tested our platform by elucidating the role of the three different organ-specific ECM in supporting induced pancreatic differentiation of hPSCs. While the focus of this report is on pancreatic differentiation, the developed platform is versatile to be applied to characterize any lineage specific differentiation.
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Affiliation(s)
- Saik Kia Goh
- University of Pittsburgh, Pittsburgh, 15261, UNITED STATES
| | - Willi Halfter
- University of Pittsburgh, Pittsburgh, Pennsylvania, UNITED STATES
| | - Thomas Richardson
- Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, UNITED STATES
| | - Suzanne Bertera
- Allegheny Health Network, Pittsburgh, Pennsylvania, UNITED STATES
| | - Vimal Vaidya
- University of Pittsburgh, Pittsburgh, Pennsylvania, UNITED STATES
| | - Joe Candiello
- University of Pittsburgh, Pittsburgh, Pennsylvania, UNITED STATES
| | - Mahalia Bradford
- Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, UNITED STATES
| | - Ipsita Banerjee
- Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, UNITED STATES
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33
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Granato AEC, da Cruz EF, Rodrigues-Junior DM, Mosini AC, Ulrich H, Rodrigues BVM, Cheffer A, Porcionatto M. A novel decellularization method to produce brain scaffolds. Tissue Cell 2020; 67:101412. [PMID: 32866727 DOI: 10.1016/j.tice.2020.101412] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 06/30/2020] [Accepted: 07/17/2020] [Indexed: 12/13/2022]
Abstract
Scaffolds composed of extracellular matrix (ECM) can assist tissue remodeling and repair following injury. The ECM is a complex biomaterial composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans, secreted by cells. The ECM contains fundamental biological cues that modulate cell behavior and serves as a structural scaffold for cell adhesion and growth. For clinical applications, where immune rejection is a constraint, ECM can be processed using decellularization methods intended to remove cells and donor antigens from tissue or organs, while preserving native biological cues essential for cell growth and differentiation. Recent studies show bioengineered organs composed by a combination of a diversity of materials and stem cells as a possibility of new therapeutic strategies to treat diseases that affect different tissues and organs, including the central nervous system (CNS). Nevertheless, the methodologies currently described for brain decellularization involve the use of several chemical reagents with many steps that ultimately limit the process of organ or tissue recellularization. Here, we describe for the first time a fast and straightforward method for complete decellularization of mice brain by the combination of rapid freezing and thawing following the use of only one detergent (Sodium dodecyl sulfate (SDS)). Our data show that using the protocol we describe here, the brain was entirely decellularized, while still maintaining ECM components that are essential for cell survival on the scaffold. Our results also show the cell-loading of the decellularized brain matrix with Neuro2a cells, which were identified by immunohistochemistry in their undifferentiated form. We conclude that this novel and simple method for brain decellularization can be used as a scaffold for cell-loading.
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Affiliation(s)
- Alessandro E C Granato
- Department of Biochemistry, Neurobiology Lab, Escola Paulista de Medicina, Universidade Federal São Paulo, São Paulo, Brazil; Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
| | - Edgar Ferreira da Cruz
- Department of Medicine, Division of Nephrology, Universidade Federal de São Paulo, São Paulo, Brazil.
| | | | - Amanda Cristina Mosini
- Department of Biochemistry, Neurobiology Lab, Escola Paulista de Medicina, Universidade Federal São Paulo, São Paulo, Brazil
| | - Henning Ulrich
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | | | - Arquimedes Cheffer
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Marimelia Porcionatto
- Department of Biochemistry, Neurobiology Lab, Escola Paulista de Medicina, Universidade Federal São Paulo, São Paulo, Brazil
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34
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Cramer MC, Badylak SF. Extracellular Matrix-Based Biomaterials and Their Influence Upon Cell Behavior. Ann Biomed Eng 2020; 48:2132-2153. [PMID: 31741227 PMCID: PMC7231673 DOI: 10.1007/s10439-019-02408-9] [Citation(s) in RCA: 112] [Impact Index Per Article: 22.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Accepted: 11/08/2019] [Indexed: 01/16/2023]
Abstract
Biologic scaffold materials composed of allogeneic or xenogeneic extracellular matrix (ECM) are commonly used for the repair and remodeling of injured tissue. The clinical outcomes associated with implantation of ECM-based materials range from unacceptable to excellent. The variable clinical results are largely due to differences in the preparation of the material, including characteristics of the source tissue, the method and efficacy of decellularization, and post-decellularization processing steps. The mechanisms by which ECM scaffolds promote constructive tissue remodeling include mechanical support, degradation and release of bioactive molecules, recruitment and differentiation of endogenous stem/progenitor cells, and modulation of the immune response toward an anti-inflammatory phenotype. The methods of ECM preparation and the impact of these methods on the quality of the final product are described herein. Examples of favorable cellular responses of immune and stem cells associated with constructive tissue remodeling of ECM bioscaffolds are described.
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Affiliation(s)
- Madeline C Cramer
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
| | - Stephen F Badylak
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA.
- Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
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35
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Hussey GS, Nascari DG, Saldin LT, Kolich B, Lee YC, Crum RJ, El-Mossier SO, D'Angelo W, Dziki JL, Badylak SF. Ultrasonic cavitation to prepare ECM hydrogels. Acta Biomater 2020; 108:77-86. [PMID: 32268241 DOI: 10.1016/j.actbio.2020.03.036] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Revised: 03/24/2020] [Accepted: 03/25/2020] [Indexed: 10/24/2022]
Abstract
Hydrogels composed of extracellular matrix (ECM) have been used as a substrate for 3D organoid culture, and in numerous preclinical and clinical applications to facilitate repair and reconstruction of a variety of tissues. However, these ECM hydrogel materials are fabricated using lengthy methods that have focused on enzymatic digestion of the ECM with an acid protease in an acidic solution; or the use of chaotropic extraction buffers and dialysis procedures which can affect native protein structure and function. Herein we report a method to prepare hydrogels from ECM bioscaffolds using ultrasonic cavitation. The solubilized ECM can be induced to rapidly self-assemble into a gel by adjusting temperature, and the material properties of the gel can be tailored by adjusting ECM concentration and sonication parameters. The present study shows that ECM bioscaffolds can be successfully solubilized without enzymatic digestion and induced to repolymerize into a gel form capable of supporting cell growth. STATEMENT OF SIGNIFICANCE: ECM hydrogels have been used in numerous preclinical studies to facilitate repair of tissue following injury. However, there has been relatively little advancement in manufacturing techniques, thereby impeding progress in advancing this technology toward the clinic. Laboratory techniques for producing ECM hydrogels have focused on protease digestion methods, which require lengthy incubation times. The significance of this work lies in the development of a fundamentally different approach whereby an ECM hydrogel is rapidly formed without the need for acidic solutions or protease digestion. The ultrasonic cavitation method described herein represents a marked improvement in rheological properties and processing time over traditional enzymatic methods, and may lend itself as a platform for large-scale manufacturing of ECM hydrogels.
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Affiliation(s)
- George S Hussey
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA; Department of Surgery, School of Medicine, University of Pittsburgh, University of Pittsburgh Medical Center Presbyterian Hospital, 200 Lothrop Street, Pittsburgh, PA 15213, USA; ECM Therapeutics, Inc., 118 Marshall Dr., Warrendale, PA 15086, USA
| | - David G Nascari
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA
| | - Lindsey T Saldin
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA; Department of Surgery, School of Medicine, University of Pittsburgh, University of Pittsburgh Medical Center Presbyterian Hospital, 200 Lothrop Street, Pittsburgh, PA 15213, USA
| | - Brian Kolich
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA
| | - Yoojin C Lee
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA; Department of Bioengineering, University of Pittsburgh, 3700 O'Hara Street, Pittsburgh, PA 15261, USA
| | - Raphael J Crum
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA
| | - Salma O El-Mossier
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA
| | - William D'Angelo
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA; Department of Surgery, School of Medicine, University of Pittsburgh, University of Pittsburgh Medical Center Presbyterian Hospital, 200 Lothrop Street, Pittsburgh, PA 15213, USA
| | - Jenna L Dziki
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA; Department of Surgery, School of Medicine, University of Pittsburgh, University of Pittsburgh Medical Center Presbyterian Hospital, 200 Lothrop Street, Pittsburgh, PA 15213, USA; ECM Therapeutics, Inc., 118 Marshall Dr., Warrendale, PA 15086, USA
| | - Stephen F Badylak
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219-3110, USA; Department of Surgery, School of Medicine, University of Pittsburgh, University of Pittsburgh Medical Center Presbyterian Hospital, 200 Lothrop Street, Pittsburgh, PA 15213, USA; Department of Bioengineering, University of Pittsburgh, 3700 O'Hara Street, Pittsburgh, PA 15261, USA; ECM Therapeutics, Inc., 118 Marshall Dr., Warrendale, PA 15086, USA.
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36
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Ullah I, Busch JF, Rabien A, Ergün B, Stamm C, Knosalla C, Hippenstiel S, Reinke P, Kurtz A. Adult Tissue Extracellular Matrix Determines Tissue Specification of Human iPSC-Derived Embryonic Stage Mesodermal Precursor Cells. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2020; 7:1901198. [PMID: 32154066 PMCID: PMC7055561 DOI: 10.1002/advs.201901198] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Revised: 12/02/2019] [Indexed: 06/10/2023]
Abstract
The selection of pluripotent stem cell (PSC)-derived cells for tissue modeling and cell therapy will be influenced by their response to the tissue environment, including the extracellular matrix (ECM). Whether and how instructive memory is imprinted in adult ECM and able to impact on the tissue specific determination of human PSC-derived developmentally fetal mesodermal precursor (P-meso) cells is investigated. Decellularized ECM (dECM) is generated from human heart, kidney, and lung tissues and recellularized with P-meso cells in a medium not containing any differentiation inducing components. While P-meso cells on kidney dECM differentiate exclusively into nephronal cells, only beating clusters containing mature and immature cardiac cells form on heart dECM. No tissue-specific differentiation of P-meso cells is observed on endoderm-derived lung dECM. P-meso-derived endothelial cells, however, are found on all dECM preparations independent of tissue origin. Clearance of heparan-sulfate proteoglycans (HSPG) from dECM abolishes induction of tissue-specific differentiation. It is concluded that HSPG-bound factors on adult tissue-derived ECM are essential and sufficient to induce tissue-specific specification of uncommitted fetal stage precursor cells.
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Affiliation(s)
- Imran Ullah
- Berlin Institute of Health Center for Regenerative TherapiesCharité Universitätsmedizin BerlinAugustenburger Platz 113353BerlinGermany
| | - Jonas Felix Busch
- Department of UrologyCharité–Universitätsmedizin Berlin10117BerlinGermany
- Berlin Institute for Urologic Research10117BerlinGermany
| | - Anja Rabien
- Department of UrologyCharité–Universitätsmedizin Berlin10117BerlinGermany
- Berlin Institute for Urologic Research10117BerlinGermany
| | - Bettina Ergün
- Department of UrologyCharité–Universitätsmedizin Berlin10117BerlinGermany
- Berlin Institute for Urologic Research10117BerlinGermany
| | - Christof Stamm
- Berlin Institute of Health Center for Regenerative TherapiesCharité Universitätsmedizin BerlinAugustenburger Platz 113353BerlinGermany
- Deutsches Herzzentrum Berlin and German Center for Cardiovascular ResearchAugustenburger Platz 113353BerlinGermany
| | - Christoph Knosalla
- Deutsches Herzzentrum Berlin and German Center for Cardiovascular ResearchAugustenburger Platz 113353BerlinGermany
| | - Stefan Hippenstiel
- Department of Infectiology and PneumonologyCharité–Universitätsmedizin BerlinAugustenburger Platz 113353BerlinGermany
| | - Petra Reinke
- Berlin Institute of Health Center for Regenerative TherapiesCharité Universitätsmedizin BerlinAugustenburger Platz 113353BerlinGermany
| | - Andreas Kurtz
- Berlin Institute of Health Center for Regenerative TherapiesCharité Universitätsmedizin BerlinAugustenburger Platz 113353BerlinGermany
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37
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Uhl FE, Zhang F, Pouliot RA, Uriarte JJ, Rolandsson Enes S, Han X, Ouyang Y, Xia K, Westergren-Thorsson G, Malmström A, Hallgren O, Linhardt RJ, Weiss DJ. Functional role of glycosaminoglycans in decellularized lung extracellular matrix. Acta Biomater 2020; 102:231-246. [PMID: 31751810 PMCID: PMC8713186 DOI: 10.1016/j.actbio.2019.11.029] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2018] [Revised: 11/09/2019] [Accepted: 11/12/2019] [Indexed: 01/17/2023]
Abstract
Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFβ1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFβ1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. STATEMENT OF SIGNIFICANCE: Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.
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Affiliation(s)
- Franziska E Uhl
- University of Vermont, Larner College of Medicine, Burlington, VT, United States; Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Fuming Zhang
- Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Robert A Pouliot
- University of Vermont, Larner College of Medicine, Burlington, VT, United States
| | - Juan J Uriarte
- University of Vermont, Larner College of Medicine, Burlington, VT, United States
| | - Sara Rolandsson Enes
- University of Vermont, Larner College of Medicine, Burlington, VT, United States; Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Xiaorui Han
- Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Yilan Ouyang
- Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Ke Xia
- Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | | | - Anders Malmström
- Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Oskar Hallgren
- Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Robert J Linhardt
- Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Daniel J Weiss
- University of Vermont, Larner College of Medicine, Burlington, VT, United States.
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38
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Bilodeau C, Goltsis O, Rogers IM, Post M. Limitations of recellularized biological scaffolds for human transplantation. J Tissue Eng Regen Med 2019; 14:521-538. [PMID: 31826325 DOI: 10.1002/term.3004] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Revised: 11/12/2019] [Accepted: 11/14/2019] [Indexed: 12/15/2022]
Abstract
A shortage of donor organs for transplantation and the dependence of the recipients on immunosuppressive therapy have motivated researchers to consider alternative regenerative approaches. The answer may reside in acellular scaffolds generated from cadaveric human and animal tissues. Acellular scaffolds are expected to preserve the architectural and mechanical properties of the original organ, permitting cell attachment, growth, and differentiation. Although theoretically, the use of acellular scaffolds for transplantation should pose no threat to the recipient's immune system, experimental data have revealed significant immune responses to allogeneic and xenogeneic transplanted scaffolds. Herein, we review the various factors of the scaffold that could trigger an inflammatory and/or immune response, thereby compromising its use for human transplant therapy. In addition, we provide an overview of the major cell types that have been considered for recellularization of the scaffold and their potential contribution to triggering an immune response.
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Affiliation(s)
- Claudia Bilodeau
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Olivia Goltsis
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, Ontario, Canada.,Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
| | - Ian M Rogers
- Lunenfeld Research Institute, Mount Sinai Health, Toronto, Ontario, Canada
| | - Martin Post
- Translational Medicine Program, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.,Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
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39
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Young BM, Shankar K, Tho CK, Pellegrino AR, Heise RL. Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix. Acta Biomater 2019; 100:223-234. [PMID: 31593773 DOI: 10.1016/j.actbio.2019.10.009] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2019] [Revised: 10/03/2019] [Accepted: 10/03/2019] [Indexed: 12/28/2022]
Abstract
Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. STATEMENT OF SIGNIFICANCE: Efforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM. These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs.
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Affiliation(s)
- Bethany M Young
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States
| | - Keerthana Shankar
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States
| | - Cindy K Tho
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States
| | - Amanda R Pellegrino
- Department of Biomedical Engineering and Nursing, Duquesne University, 600 Forbes Ave, Pittsburg, Pennsylvania 15282, United States
| | - Rebecca L Heise
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States; Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, 1101 East Marshall St, Richmond, Virginia 23298, United States.
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40
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Szulc DA, Ahmadipour M, Aoki FG, Waddell TK, Karoubi G, Cheng HLM. MRI method for labeling and imaging decellularized extracellular matrix scaffolds for tissue engineering. Magn Reson Med 2019; 83:2138-2149. [PMID: 31729091 DOI: 10.1002/mrm.28072] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2019] [Revised: 10/15/2019] [Accepted: 10/20/2019] [Indexed: 11/08/2022]
Abstract
PURPOSE To develop a facile method for labeling and imaging decellularized extracellular matrix (dECM) scaffolds intended for regenerating 3D tissues. METHODS A small molecule manganese porphyrin, MnPNH2 , was synthesized and used to label dECM scaffolds made from porcine bladder and trachea and murine whole lungs. The labeling protocol was optimized on bladder dECM, and imaging on a 3T clinical scanner was performed to assess reductions in T1 and T2 relaxation times. In vivo MRI was performed on dECM injected in the rat dorsum to verify sensitivity of detection. Toxicity assays for cell viability, metabolism, and proliferation were performed on human umbilical vein endothelial cells. The incorporation of MnPNH2 and its long-term retention in dECM were assessed on transmission electron microscopy and ultraviolet absorbance of eluted MnPNH2 over time. RESULTS All tissues, including thick whole 3D organs, were uniformly labeled and demonstrated high signal-to-noise on MRI. A nearly 10-fold reduction in T1 was consistently obtained at a labeling dose of 0.4 mM, and even 0.2 mM provided sufficient contrast in vivo and ex vivo. No toxicity was observed up to 0.4 mM, the maximum tested. Binding studies suggested nonspecific association, and retention studies in the labeled whole decellularized lungs revealed less than 20% MnPNH2 loss over 30 days, the majority occurring in the first 3 days after labeling. CONCLUSION The proposed labeling method is the first report for visualizing dECM on MRI and has the potential for long-term monitoring and optimization of dECM-based organ tissue engineering.
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Affiliation(s)
- Daniel Andrzej Szulc
- Institute of Biomaterials & Biomedical Engineering, University of Toronto, Canada.,Ted Rogers Centre for Heart Research, Translational Biology & Engineering Program, Toronto, Canada
| | - Mohammadali Ahmadipour
- Institute of Biomaterials & Biomedical Engineering, University of Toronto, Canada.,Latner Thoracic Surgery Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Canada
| | - Fabio Gava Aoki
- Latner Thoracic Surgery Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Canada
| | - Thomas K Waddell
- Institute of Biomaterials & Biomedical Engineering, University of Toronto, Canada.,Latner Thoracic Surgery Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Canada.,Department of Mechanical and Industrial Engineering, University of Toronto, Canada
| | - Golnaz Karoubi
- Latner Thoracic Surgery Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Canada.,Department of Mechanical and Industrial Engineering, University of Toronto, Canada.,Ontario Institute for Regenerative Medicine, Toronto, Canada
| | - Hai-Ling Margaret Cheng
- Institute of Biomaterials & Biomedical Engineering, University of Toronto, Canada.,Ted Rogers Centre for Heart Research, Translational Biology & Engineering Program, Toronto, Canada.,Ontario Institute for Regenerative Medicine, Toronto, Canada.,Heart & Stroke/Richard Lewar Centre of Excellence for Cardiovascular Research, Toronto, Canada.,The Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, Canada
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41
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Lung bioengineering: advances and challenges in lung decellularization and recellularization. Curr Opin Organ Transplant 2019; 23:673-678. [PMID: 30300330 DOI: 10.1097/mot.0000000000000584] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
PURPOSE OF REVIEW Bioengineering the lung based on its natural extracellular matrix (ECM) offers novel opportunities to overcome the shortage of donors, to reduce chronic allograft rejections, and to improve the median survival rate of transplanted patients. During the last decade, lung tissue engineering has advanced rapidly to combine scaffolds, cells, and biologically active molecules into functional tissues to restore or improve the lung's main function, gas exchange. This review will inspect the current progress in lung bioengineering using decellularized and recellularized lung scaffolds and highlight future challenges in the field. RECENT FINDINGS Lung decellularization and recellularization protocols have provided researchers with tools to progress toward functional lung tissue engineering. However, there is continuous evolution and refinement particularly for optimization of lung recellularization. These further the possibility of developing a transplantable bioartificial lung. SUMMARY Bioengineering the lung using recellularized scaffolds could offer a curative option for patients with end-stage organ failure but its accomplishment remains unclear in the short-term. However, the state-of-the-art of techniques described in this review will increase our knowledge of the lung ECM and of chemical and mechanical cues which drive cell repopulation to improve the advances in lung regeneration and lung tissue engineering.
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42
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Ullah I, Abu-Dawud R, Busch JF, Rabien A, Erguen B, Fischer I, Reinke P, Kurtz A. VEGF – Supplemented extracellular matrix is sufficient to induce endothelial differentiation of human iPSC. Biomaterials 2019; 216:119283. [DOI: 10.1016/j.biomaterials.2019.119283] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Revised: 06/11/2019] [Accepted: 06/13/2019] [Indexed: 01/13/2023]
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Chiu YC, Shen YF, Lee AKX, Lin SH, Wu YC, Chen YW. 3D Printing of Amino Resin-based Photosensitive Materials on Multi-parameter Optimization Design for Vascular Engineering Applications. Polymers (Basel) 2019; 11:E1394. [PMID: 31450605 PMCID: PMC6780824 DOI: 10.3390/polym11091394] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Revised: 08/18/2019] [Accepted: 08/23/2019] [Indexed: 12/19/2022] Open
Abstract
Cardiovascular diseases are currently the most common cause of death globally and of which, the golden treatment method for severe cardiovascular diseases or coronary artery diseases are implantations of synthetic vascular grafts. However, such grafts often come with rejections and hypersensitivity reactions. With the emergence of regenerative medicine, researchers are now trying to explore alternative ways to produce grafts that are less likely to induce immunological reactions in patients. The main goal of such studies is to produce biocompatible artificial vascular grafts with the capability of allowing cellular adhesion and cellular proliferation for tissues regeneration. The Design of Experimental concepts is employed into the manufacturing process of digital light processing (DLP) 3D printing technology to explore near-optimal processing parameters to produce artificial vascular grafts with vascular characteristics that are close to native vessels by assessing for the cause and effect relationships between different ratios of amino resin (AR), 2-hydroxyethyl methacrylate (HEMA), dopamine, and curing durations. We found that with proper optimization of fabrication procedures and ratios of materials, we are able to successfully fabricate vascular grafts with good printing resolutions. These had similar physical properties to native vessels and were able to support cellular adhesion and proliferation. This study could support future studies in exploring near-optimal processes for fabrication of artificial vascular grafts that could be adapted into clinical applications.
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Affiliation(s)
- Yung-Cheng Chiu
- School of Medicine, China Medical University, Taichung 40447, Taiwan
- Department of Orthopedic Surgery, China Medical University Hospital, Taichung 40447, Taiwan
| | - Yu-Fang Shen
- Department of Bioinformatics and Medical Engineering, Asia University, Taichung 40447, Taiwan
- 3D Printing Medical Research Institute, Asia University, Taichung 40447, Taiwan
| | - Alvin Kai-Xing Lee
- School of Medicine, China Medical University, Taichung 40447, Taiwan
- 3D Printing Medical Research Center, China Medical University Hospital, Taichung 40447, Taiwan
| | - Shu-Hsien Lin
- 3D Printing Medical Research Center, China Medical University Hospital, Taichung 40447, Taiwan
| | - Yu-Chen Wu
- 3D Printing Medical Research Center, China Medical University Hospital, Taichung 40447, Taiwan
| | - Yi-Wen Chen
- 3D Printing Medical Research Center, China Medical University Hospital, Taichung 40447, Taiwan.
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40447, Taiwan.
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Obata T, Tsuchiya T, Akita S, Kawahara T, Matsumoto K, Miyazaki T, Masumoto H, Kobayashi E, Niklason LE, Nagayasu T. Utilization of Natural Detergent Potassium Laurate for Decellularization in Lung Bioengineering. Tissue Eng Part C Methods 2019; 25:459-471. [DOI: 10.1089/ten.tec.2019.0016] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Affiliation(s)
- Tomohiro Obata
- Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
- Medical-Engineering Hybrid Professional Development Center, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Tomoshi Tsuchiya
- Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
- Translational Research Center, Research Institute for Science and Technology, Tokyo University of Science, Chiba, Japan
| | - Sadanori Akita
- Department of Plastic Surgery, Reconstructive, and Aesthetic Surgery, School of Medicine, Fukuoka University, Fukuoka, Japan
| | - Takayoshi Kawahara
- Research and Development Department and Quality Assurance Department, Shabondama Soap Co., Ltd., Kitakyusyu City, Japan
| | - Keitaro Matsumoto
- Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
- Medical-Engineering Hybrid Professional Development Center, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Takuro Miyazaki
- Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Hiroshi Masumoto
- Biomedical Research Support Center, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Eiji Kobayashi
- Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke, Japan
- Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan
| | - Laura E. Niklason
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Takeshi Nagayasu
- Department of Surgical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
- Biomedical Research Support Center, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
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Wong AP, Shojaie S, Liang Q, Xia S, Di Paola M, Ahmadi S, Bilodeau C, Garner J, Post M, Duchesneau P, Waddell TK, Bear CE, Nagy A, Rossant J. Conversion of human and mouse fibroblasts into lung-like epithelial cells. Sci Rep 2019; 9:9027. [PMID: 31227724 PMCID: PMC6588580 DOI: 10.1038/s41598-019-45195-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2019] [Accepted: 05/31/2019] [Indexed: 01/08/2023] Open
Abstract
Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.
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Affiliation(s)
- Amy P Wong
- Program in Developmental & Stem Cell Biology, SickKids Research Institute, Hospital for Sick Children, Toronto, ON, Canada.
| | - Sharareh Shojaie
- Program in Translational Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Qin Liang
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Sunny Xia
- Program in Molecular Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Michelle Di Paola
- Program in Molecular Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Saumel Ahmadi
- Program in Molecular Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Claudia Bilodeau
- Program in Translational Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Jodi Garner
- Program in Developmental & Stem Cell Biology, SickKids Research Institute, Hospital for Sick Children, Toronto, ON, Canada
| | - Martin Post
- Program in Translational Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Pascal Duchesneau
- Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, and the McEwen Centre for Regenerative Medicine, Toronto, Canada
| | - Thomas K Waddell
- Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, and the McEwen Centre for Regenerative Medicine, Toronto, Canada
| | - Christine E Bear
- Program in Molecular Medicine, SickKids Research Institute, Hospital for Sick Children, Toronto, Canada
| | - Andras Nagy
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Janet Rossant
- Program in Developmental & Stem Cell Biology, SickKids Research Institute, Hospital for Sick Children, Toronto, ON, Canada. .,Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. .,Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Canada.
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Yeganeh B, Lee J, Ermini L, Lok I, Ackerley C, Post M. Autophagy is required for lung development and morphogenesis. J Clin Invest 2019; 129:2904-2919. [PMID: 31162135 DOI: 10.1172/jci127307] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Accepted: 04/12/2019] [Indexed: 12/30/2022] Open
Abstract
Bronchopulmonary dysplasia (BPD) remains a major respiratory illness in extremely premature infants. The biological mechanisms leading to BPD are not fully understood, although an arrest in lung development has been implicated. The current study aimed to investigate the occurrence of autophagy in the developing mouse lung and its regulatory role in airway branching and terminal sacculi formation. We found 2 windows of epithelial autophagy activation in the developing mouse lung, both resulting from AMPK activation. Inhibition of AMPK-mediated autophagy led to reduced lung branching in vitro. Conditional deletion of beclin 1 (Becn1) in mouse lung epithelial cells (Becn1Epi-KO), either at early (E10.5) or late (E16.5) gestation, resulted in lethal respiratory distress at birth or shortly after. E10.5 Becn1Epi-KO lungs displayed reduced airway branching and sacculi formation accompanied by impaired vascularization, excessive epithelial cell death, reduced mesenchymal thinning of the interstitial walls, and delayed epithelial maturation. E16.5 Becn1Epi-KO lungs had reduced terminal air sac formation and vascularization and delayed distal epithelial differentiation, a pathology similar to that seen in infants with BPD. Taken together, our findings demonstrate that intrinsic autophagy is an important regulator of lung development and morphogenesis and may contribute to the BPD phenotype when impaired.
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Affiliation(s)
- Behzad Yeganeh
- Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children
| | - Joyce Lee
- Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children.,Institute of Medical Science, and
| | - Leonardo Ermini
- Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children
| | - Irene Lok
- Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children
| | - Cameron Ackerley
- Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children.,Departments of Physiology and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Martin Post
- Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children.,Institute of Medical Science, and.,Departments of Physiology and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
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Okuyama H, Ohnishi H, Nakamura R, Yamashita M, Kishimoto Y, Tateya I, Suehiro A, Gotoh S, Takezawa T, Nakamura T, Omori K. Transplantation of multiciliated airway cells derived from human iPS cells using an artificial tracheal patch into rat trachea. J Tissue Eng Regen Med 2019; 13:1019-1030. [PMID: 30809958 DOI: 10.1002/term.2849] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Revised: 02/16/2019] [Accepted: 02/21/2019] [Indexed: 12/16/2022]
Abstract
Tracheal resection is often performed for malignant tumours, congenital anomalies, inflammatory lesions, and traumatic injuries. There is no consensus on the best approach for the restoration of tracheal functionality in patients with tracheal defects. Artificial grafts made of polypropylene and collagen sponge have been clinically used by our group. However, 2 months are required to achieve adequate epithelialization of the grafts in humans. This study aimed to investigate the feasibility of transplantation therapy using an artificial trachea with human-induced pluripotent stem cell (hiPSC)-derived multiciliated airway cells (hiPSC-MCACs). Collagen vitrigel membrane, a biocompatible and absorbable material, was used as a scaffold to cover the artificial trachea with hiPSC-MCACs. Analyses of hiPSC-MCACs on collagen vitrigel membrane were performed by immunocytochemistry and electron microscopy and by assessing ciliary beat frequency. Along with the artificial trachea, hiPSC-MCACs were transplanted into surgically created tracheal defects of immunodeficient rats. The survival of transplanted cells was histologically evaluated at 1 and 2 weeks after the transplantation. The hiPSC-MCACs exhibited motile cilia on collagen vitrigel membrane. The surviving hiPSC-MCACs were observed in the endotracheal epithelium of the tracheal defect at 1 and 2 weeks after transplantation. These results suggest that hiPSC-MCAC is a useful candidate for tracheal reconstruction.
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Affiliation(s)
- Hideaki Okuyama
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Hiroe Ohnishi
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ryosuke Nakamura
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Masaru Yamashita
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Yo Kishimoto
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ichiro Tateya
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Atsushi Suehiro
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Shimpei Gotoh
- Department of Respiratory Medicine, Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Toshiaki Takezawa
- Division of Biotechnology, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Ibaraki, Japan
| | - Tatsuo Nakamura
- Department of Bioartificial Organs, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Koichi Omori
- Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Allogenic tissue-specific decellularized scaffolds promote long-term muscle innervation and functional recovery in a surgical diaphragmatic hernia model. Acta Biomater 2019; 89:115-125. [PMID: 30851456 DOI: 10.1016/j.actbio.2019.03.007] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Revised: 02/22/2019] [Accepted: 03/05/2019] [Indexed: 01/08/2023]
Abstract
Congenital diaphragmatic hernia (CDH) is a neonatal defect in which the diaphragm muscle does not develop properly, thereby raising abdominal organs into the thoracic cavity and impeding lung development and function. Large diaphragmatic defects require correction with prosthetic patches to close the malformation. This treatment leads to a consequent generation of unwelcomed mechanical stress in the repaired diaphragm and hernia recurrences, thereby resulting in high morbidity and significant mortality rates. We proposed a specific diaphragm-derived extracellular matrix (ECM) as a scaffold for the treatment of CDH. To address this strategy, we developed a new surgical CDH mouse model to test the ability of our tissue-specific patch to regenerate damaged diaphragms. Implantation of decellularized diaphragmatic ECM-derived patches demonstrated absence of rejection or hernia recurrence, in contrast to the performance of a commercially available synthetic material. Diaphragm-derived ECM was able to promote the generation of new blood vessels, boost long-term muscle regeneration, and recover host diaphragmatic function. In addition, using a GFP + Schwann cell mouse model, we identified re-innervation of implanted patches. These results demonstrated for the first time that implantation of a tissue-specific biologic scaffold is able to promote a regenerating diaphragm muscle and overcome issues commonly related to the standard use of prosthetic materials. STATEMENT OF SIGNIFICANCE: Large diaphragmatic hernia in paediatric patients require application of artificial patches to close the congenital defect. The use of a muscle-specific decellularized scaffold in substitution of currently used synthetic materials allows new blood vessel growth and nerve regeneration inside the patch, supporting new muscle tissue formation. Furthermore, the presence of a tissue-specific scaffold guaranteed long-term muscle regeneration, improving diaphragm performance to almost complete functional recovery. We believe that diaphragm-derived scaffold will be key player in future pre-clinical studies on large animal models.
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Sedláková V, Kloučková M, Garlíková Z, Vašíčková K, Jaroš J, Kandra M, Kotasová H, Hampl A. Options for modeling the respiratory system: inserts, scaffolds and microfluidic chips. Drug Discov Today 2019; 24:971-982. [PMID: 30877077 DOI: 10.1016/j.drudis.2019.03.006] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 02/08/2019] [Accepted: 03/06/2019] [Indexed: 12/29/2022]
Abstract
The human respiratory system is continuously exposed to varying levels of hazardous substances ranging from environmental toxins to purposely administered drugs. If the noxious effects exceed the inherent regenerative capacity of the respiratory system, injured tissue undergoes complex remodeling that can significantly affect lung function and lead to various diseases. Advanced near-to-native in vitro lung models are required to understand the mechanisms involved in pulmonary damage and repair and to reliably test the toxicity of compounds to lung tissue. This review is an overview of the development of in vitro respiratory system models used for study of lung diseases. It includes discussion of using these models for environmental toxin assessment and pulmonary toxicity screening.
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Affiliation(s)
- Veronika Sedláková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; Division of Cardiac Surgery, Cardiovascular Tissue Engineering Laboratory, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa K1Y 4W7, Canada.
| | - Michaela Kloučková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic
| | - Zuzana Garlíková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; International Clinical Research Center, St Anne's University Hospital Brno, Pekařská 664/53, 656 91 Brno, Czech Republic
| | - Kateřina Vašíčková
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; International Clinical Research Center, St Anne's University Hospital Brno, Pekařská 664/53, 656 91 Brno, Czech Republic
| | - Josef Jaroš
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; International Clinical Research Center, St Anne's University Hospital Brno, Pekařská 664/53, 656 91 Brno, Czech Republic
| | - Mário Kandra
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; International Clinical Research Center, St Anne's University Hospital Brno, Pekařská 664/53, 656 91 Brno, Czech Republic
| | - Hana Kotasová
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic
| | - Aleš Hampl
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; International Clinical Research Center, St Anne's University Hospital Brno, Pekařská 664/53, 656 91 Brno, Czech Republic
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Abstract
Lung development is a complex process that requires the input of various signaling pathways to coordinate the specification and differentiation of multiple cell types. Ex vivo culture of the lung is a very useful technique that represents an attractive model for investigating many different processes critical to lung development, function, and disease pathology. Ex vivo cultured lungs remain comparable to the in vivo lung both in structure and function, which makes them more suitable than cell cultures for physiological studies. Lung explant cultures offer several significant advantages for studies of morphogenetic events that guide lung development including budding, branching, and fusion. It also maintains the native physiological interactions between cells in the developing lung, enabling investigations of the direct and indirect signaling taking place between tissues and cells throughout the developmental process. Studying temporal and spatial control of gene expression by transcriptional factors using different reporters to understand their regulatory function at different moments of development is another valuable advantage of lung explants culture.
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