Dataset Information

Establishing Proximal and Distal Regional Identities in Murine and Human Tissue-Engineered Lung and Trachea.

ABSTRACT: The cellular and molecular mechanisms that underpin regeneration of the human lung are unknown, and the study of lung repair has been impeded by the necessity for reductionist models that may exclude key components. We hypothesized that multicellular epithelial and mesenchymal cell clusters or lung organoid units (LuOU) could be transplanted to recapitulate proximal and distal cellular structures of the native lung and airways. Transplantation of LuOU resulted in the growth of tissue-engineered lung (TELu) that contained the necessary cell types consistent with native adult lung tissue and demonstrated proliferative cells at 2 and 4 weeks. This technique recapitulated important elements of both mouse and human lungs featuring key components of both the proximal and distal lung regions. When LuOU were generated from whole lung, TELu contained key epithelial and mesenchymal cell types, and the origin of the cells was traced from both Actin^GFP and SPC^GFP donors to indicate that the cells in TELu were derived from the transplanted LuOU. Alveolar epithelial type 2 cells (AEC2s), club cells, ciliated cells marked by beta-tubulin IV, alveolar epithelial type I cells, Sox-2-positive proximal airway progenitors, p63-positive basal cells, and CGRP-positive pulmonary neuroendocrine cells were identified in the TELu. The mesenchymal components of peribronchial smooth muscle and nerve were identified with a CD31-positive donor endothelial cell contribution to TELu vasculature. TELu successfully grew from postnatal tissues from whole murine and human lung, distal murine lung, as well as murine and human trachea. These data support a model of postnatal lung regeneration containing the diverse cell types present in the entirety of the respiratory tract.

SUBMITTER: Trecartin A

PROVIDER: S-EPMC5116661 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Establishing Proximal and Distal Regional Identities in Murine and Human Tissue-Engineered Lung and Trachea.

Trecartin Andrew A Danopoulos Soula S Spurrier Ryan R Knaneh-Monem Hanaa H Hiatt Michael M Driscoll Barbara B Hochstim Christian C Al-Alam Denise D Grikscheit Tracy C TC

Tissue engineering. Part C, Methods 20161101 11

The cellular and molecular mechanisms that underpin regeneration of the human lung are unknown, and the study of lung repair has been impeded by the necessity for reductionist models that may exclude key components. We hypothesized that multicellular epithelial and mesenchymal cell clusters or lung organoid units (LuOU) could be transplanted to recapitulate proximal and distal cellular structures of the native lung and airways. Transplantation of LuOU resulted in the growth of tissue-engineered ...[more]

PMID: 27796199

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Project description:The lung epithelium contains multiple distinct cell types which are maintained by regionally specific progenitor cell populations in both the airways and in the alveolar compartments. Each of the progenitor populations are known to have varying roles during homeostasis and have increasingly been shown to be deranged in lung diseases. The progenitor cells of the proximal lung epithelium are the basal cells (Krt5+/Krt14+/p63+) which sit at the basement membrane of the trachea, referred to as, proximal progenitors in this study. On the other hand, the distal alveolar epithelium is maintained by alveolar type II cells (Sftpc+), referred to as distal progenitors in this study. Several cell isolation methods are available to isolate each of these populations of proximal and distal progenitors but are traditionally performed separately on mice which is especially important in the context of disease. Methods which allow the isolation of the entire epithelial population from murine lungs, while retaining the capability to perform downstream studies such as organoid assays and air-liquid-interface, would open new possibilities for studying interactions between the proximal and distal airways in lung disease. However, an easy and reproducible method that allows isolation of both cell types simultaneously from an individual mouse has not been previously described. This is in part due to the technical and surgical challenges associated with each isolation method as well as, in part, due to a lack of cell surface markers that are specific and unique to each cell type that could allow their isolation from a single cell suspension of the entire epithelium. Additionally, transgenic models have been shown to be leaky and mark populations other than the target cells. Thus, classical methods that rely on positive or negative selection, coupled by surgical isolation remain the state of the art. In order to facilitate isolation of proximal and distal progenitors from an individual mouse, we developed a 3D printable Lobe Divider (3DLD) that allows for the reproducible separation of the trachea and lung lobes while allowing for controllable and temporal instillation of cell dissociation buffers for both cell isolation procedures. The efficacy of the 3DLD in isolating the two populations and the functionality of the cells isolated with the 3DLD was validated through multiple differentiation assays. Proximal progenitors were differentiated in 2D at air-liquid interface (ALI) culture for 28 days after monolayer formation for 7days. Distal progenitors isolated with the 3DLD or with the equivalent classical tracheal ligation method were differentiated in organoid culture in Matrigel for 14 days while at ALI. Total RNA was isolated from cell pellets of all cell types before differentiation, from ALI cultures of proximal cells, and organoids of distal cells isolated with 3DLD or classic method. Qiagen RNeasy micro kit (74004, QIAGEN, Sweden) was used to isolate total RNA following the manufacturer’s protocol. RNA concentrations were measured using NanoDrop 1000 and Qubit™ RNA HS Assay Kit (Q32852, Fisher Scientific). RNA quality was evaluated by the Agilent 2100 BioAnalyzer. Library preparation was done using the Illumina Stranded mRNA Prep Ligation (20040534, Illumina) with 14 cycles under final PCR amplification and samples were indexed with IDT for Illumina RNA UD Indexes Set B, Ligation (20040554, Illumina) for multiplexing. Clean up steps were automated using the King Fisher FLEX (18-5400620, Thermo Scientific). Library concentration was checked using the QuantIT 1X dsDNA HS Assay Kit (Q33232) and library size and quality were checked using the 5200 Fragment Analyzer™ 12-capillary (M3510AA, Agilent) using the DNF-930 dsDNA Reagent Kit, 75 bp – 20,000 bp(500 Samples) (Part # DNF-930-K0500, Agilent). A library input of 1.25 nM was sequenced using NovaSeq 6000 Sequencing System (20012850, Illumina).

Project description:The ideal construct for tracheal replacement remains elusive in the management of long segment airway defects. Tissue engineered tracheal grafts (TETG) have been limited by the development of graft stenosis or collapse, infection, or lack of an epithelial lining. We applied a mouse model of orthotopic airway surgery to assess the impact of three critical barriers encountered in clinical applications: the scaffold, the extent of intervention, and the impact of cell seeding and characterized their impact on graft performance. First, synthetic tracheal scaffolds electrospun from polyethylene terephthalate / polyurethane (PET/PU) were orthotopically implanted in anterior tracheal defects of C57BL/6 mice. Scaffolds demonstrated complete coverage with ciliated respiratory epithelium by 2 weeks. Epithelial migration was accompanied by macrophage infiltration which persisted at long term (>6 weeks) time points. We then assessed the impact of segmental tracheal implantation using syngeneic trachea as a surrogate for the ideal tracheal replacement. Graft recovery involved local upregulation of epithelial progenitor populations and there was no evidence of graft stenosis or necrosis. Implantation of electrospun synthetic tracheal scaffold for segmental replacement resulted in respiratory distress and required euthanasia at an early time point. There was limited epithelial coverage of the scaffold with and without seeded bone marrow-derived mononuclear cells (BM-MNCs). We conclude that synthetic scaffolds support re-epithelialization in orthotopic patch implantation, syngeneic graft integration occurs with focal repair mechanisms, however epithelialization in segmental synthetic scaffolds is limited and is not influenced by cell seeding. STATEMENT OF SIGNIFICANCE: The life-threatening nature of long-segment tracheal defects has led to clinical use of tissue engineered tracheal grafts in the last decade for cases of compassionate use. However, the ideal tracheal reconstruction using tissue-engineered tracheal grafts (TETG) has not been clarified. We addressed the core challenges in tissue engineered tracheal replacement (re-epithelialization and graft patency) by defining the role of cell seeding with autologous bone marrow-derived mononuclear cells, the mechanism of respiratory epithelialization and proliferation, and the role of the inflammatory immune response in regeneration. This research will facilitate comprehensive understanding of cellular regeneration and neotissue formation on TETG, which will permit targeted therapies for accelerating re-epithelialization and attenuating stenosis in tissue engineered airway replacement.

Dataset Information

Establishing Proximal and Distal Regional Identities in Murine and Human Tissue-Engineered Lung and Trachea.

Publications

Establishing Proximal and Distal Regional Identities in Murine and Human Tissue-Engineered Lung and Trachea.

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