Engineering cancer stem-like cells from normal human lung epithelial cells.
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ABSTRACT:
SUBMITTER: Sasai K
PROVIDER: S-EPMC5381922 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair.
| S-EPMC5035637 | biostudies-literature
Project description:RationaleStem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.ObjectivesTo define a scalable cell culture system to deliver airway epithelium to clinical grafts.MethodsHuman respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.Measurements and main results3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model.ConclusionsOur method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.
| S-EPMC5003214 | biostudies-literature
Project description:Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.
| S-EPMC5755620 | biostudies-literature
Project description:BEAS-2B was originally established as an immortalized but non-tumorigenic epithelial cell line from human bronchial epithelium. Because of general recognition for its bronchial epithelial origin, the BEAS-2B cell line has been widely used as an in vitro cell model in a large variety of studies associated with respiratory diseases including lung carcinogenesis. However, very few studies have discussed non-epithelial features of BEAS-2B cells, especially the features associated with mesenchymal stem cells (MSCs), which represent a group of fibroblast-like cells with limited self-renewal and differentiation potential to various cell lineages. In this study, we compared BEAS-2B with a human umbilical cord-derived MSCs (hMSCs) cell line, hMSC1, which served as a representative of hMSCs in terms of expressing common features of hMSCs. It was observed that both BEAS-2B and hMSC1 shared the same expression profile of surface markers of hMSCs and exhibited similar osteogenic and adipogenic differentiation potential. In addition, like hMSC1, the BEAS-2B cell line exhibited suppressive activities on proliferation of mitogen-activated total T lymphocytes as well as Th1 lymphocytes, and IFNγ-induced expression of IDO1, all thus demonstrating that BEAS-2B cells exhibited an almost identical characteristic profile with hMSCs, even though, there was a clear difference between BEAS-2B and hMSCs in the effects on type 2 macrophage polarization. Most importantly, the hMSCs features of BEAS-2B were unlikely a consequence of epithelial-mesenchymal transition. Therefore, this study provided a set of evidence to provoke reconsideration of epithelial origin of BEAS-2B.
| S-EPMC6941928 | biostudies-literature
Project description:BackgroundSmall cell lung cancer (SCLC) is highly aggressive and is associated with a dismal prognosis. However, there are no clinically recognized biomarkers for early diagnosis. In this study, we used quantitative proteomics to build differential mitochondrial protein profiles that may be used for early diagnosis and investigated the pathogenesis of lung cancer.MethodsWe cultured SCLC cells (NCI-H446) and normal human bronchial epithelial cells (16-HBE); mitochondria were extracted and purified using differential and Percoll density gradient centrifugation. Subsequently, we used Western blot analysis to validate mitochondrial purity and labeled proteins/peptides from NCI-H446 and 16-HBE cells using relative and absolute quantification of ectopic tags. We then analyzed mixed samples and identified proteins using two-dimensional liquid chromatography-tandem mass spectrometry. Additionally, we performed subsequent bioinformatic proteome analyses using the programs ExPASy, GOA, and STRING. Finally, the relationship between ornithine aminotransferase expression and clinicopathological features in lung cancer patients was evaluated using immunohistochemistry.ResultsOne hundred and fifty-three mitochondrial proteins were differentially expressed between 16-HBE and NCI-H446 cells. The expression of 30 proteins between 16-HBE and NCI-H446 cells increased more than 1.3-fold. The upregulation of ornithine aminotransferase was associated with pathological grade and clinical tumor node metastasis stage.ConclusionOur experiment represented a promising method for building differential mitochondrial protein profiles between NCI-H446 and 16-HBE cells. Such analysis may also help to identify novel biomarkers of lung cancer.
| S-EPMC6209793 | biostudies-literature
Project description:BackgroundHuman embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.MethodsUndifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.ResultsExpression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and beta-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.ConclusionThese results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.
| S-EPMC2777141 | biostudies-literature
Project description:Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ?18 hours before radiation in epithelial cells (average dose modifying factor (DMF)?=?1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients.
| S-EPMC4275221 | biostudies-literature
Project description:The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.
| S-EPMC4101921 | biostudies-literature
Project description:To develop widely applicable diagnostic and potentially therapeutic approaches overcoming protein heterogeneity in human cancer, we have developed a technology to unbiasedly select high specificity compound(s) that bind any biomolecule (e.g., proteins, lipids, carbohydrates) presented on the cancer cell surface but not on normal cells. We utilized a peptidomimetic based on-bead two-color (OBTC) combinatorial cell screen that can detect differences between two cell surfaces at high accuracy by looking for beads (where each bead in the library had one peptide-peptoid hybrid on the surface) that only bound cancer but not normal cells. We screened a library of 393?216 compounds targeting HCC4017 lung adenocarcinoma cells (labeled in red) in the presence of HBEC30KT normal bronchial epithelial cells (labeled in green) derived from the same tissue of the same patient. This screen identified a peptide-peptoid hybrid called PPS1 which displayed high specific binding for HCC4017 cancer cells over HBEC30KT cells. Specificity was validated through on-bead, ELISA-like and magnetic bead pulldown studies, while a scrambled version of PPS1 did not show any binding. Of interest, the simple dimeric version (PPS1D1) displayed cytotoxic activity on HCC4017 cells, but not on normal HBEC30KT cells. PPS1D1 also strongly accumulated in HCC4017 lung cancer xenografts in mice over control constructs. We conclude that such combinatorial screens using tumor and normal cells from the same patient have significant potential to develop new reagents for cancer biology, diagnosis, and potentially therapy.
| S-EPMC4774050 | biostudies-literature
Project description:BackgroundWhole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs).MethodsWhole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position.ResultsActivin-A treatment, followed by transforming growth factor-β inhibition, induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways.ConclusionsDecellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation.
| S-EPMC4252658 | biostudies-literature