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Serially passaged, conditionally reprogrammed nasal epithelial cells as a model to study epithelial functions and SARS-CoV-2 infection

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AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 2022年第4期322卷 C591-C604页

作者： Schmidt, Hanna Guthjahr, Lara Sauter, Alexander Zech, Fabian Nchioua, Rayhane Stenger, Steffen Frick, Manfred Kirchhoff, Frank Dietl, Paul Wittekindt, Oliver H. Ulm Univ Dept Pediat & Adolescent Med Med Ctr Ulm Germany Ulm Univ Inst Gen Physiol Ulm Germany Ulm Univ Inst Mol Virol Med Ctr Ulm Germany Ulm Univ Inst Med Microbiol & Hyg Med Ctr Ulm Germany

Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial dif-ferentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.

关键词： airway epithelial culture conditional reprogramming nasal epithelial cells SARS-CoV-2

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Significant functional differences in differentiated conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells

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JOURNAL OF CYSTIC FIBROSIS 2021年第2期20卷 364-371页

作者： Awatade, Nikhil T. Wong, Sharon L. Capraro, Alexander Pandzic, Elvis Slapetova, Iveta Zhong, Ling Turgutoglu, Nihan Fawcett, Laura K. Whan, Renee M. Jaffe, Adam Waters, Shafagh A. Univ New South Wales Fac Med Sch Womens & Childrens Hlth Sydney NSW Australia Univ New South Wales Mol & Integrat Cyst Fibrosis Res Ctr MiCF RC Sydney NSW Australia Sydney Childrens Hosp Sydney NSW Australia Sydney Childrens Hosp Dept Resp Med Sydney NSW Australia Univ New South Wales Biomed Imaging Facil Sydney NSW Australia Univ New South Wales Bioanalyt Mass Spectrometry Facil Sydney NSW Australia

Background: Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation. Methods: Nasal epithelial brushings from 14 individuals (CF=9;non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free "dual-SMAD inhibition"(SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult (TM)-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALI and SMADi(ALI) under basal and CFTR corrector treated (VX-809) conditions. Results: No significant difference in the structural morphology or baseline global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADi(ALI) compare to their CRCALI counterparts. Conclusion: Alteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges. (c) 2021 European Cystic Fibrosis Society. Published by Elsevier B.

关键词： In Vitro cell models CFTR Air liquid interface conditional reprogramming dual SMAD inhibition cystic fibrosis, companion diagnostic

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Modeling respiratory tract diseases for clinical translation employing conditionally reprogrammed cells

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Cell Insight 2024年第6期3卷 100201页

作者： Daneshdoust, Danyal He, Kai Wang, Qi-En Li, Jenny Liu, Xuefeng Comprehensive Cancer Center The Ohio State University Columbus OH United States Division of Medical Oncology Department of Medicine Wexner Medical Center The Ohio State University Columbus OH United States Department of Radiation Oncology Wexner Medical Center The Ohio State University Columbus OH United States Department of Pathology Wexner Medical Center Ohio State University Columbus OH United States Departments of Pathology Urology and Radiation Oncology Wexner Medical Center Ohio State University Columbus OH United States

Preclinical models serve as indispensable tools in translational medicine. Specifically, patient-derived models such as patient-derived xenografts (PDX), induced pluripotent stem cells (iPSC), organoids, and recently developed technique of conditional reprogramming (CR) have been employed to reflect the host characteristics of diseases. CR technology involves co-culturing epithelial cells with irradiated Swiss-3T3-J2 mouse fibroblasts (feeder cells) in the presence of a Rho kinase (ROCK) inhibitor, Y-27632. CR technique facilitates the rapid conversion of both normal and malignant cells into a “reprogrammed stem-like” state, marked by robust in vitro proliferation. This is achieved without reliance on exogenous gene expression or viral transfection, while maintaining the genetic profile of the parental cells. So far, CR technology has been used to study biology of diseases, targeted therapies (precision medicine), regenerative medicine, and noninvasive diagnosis and surveillance. Respiratory diseases, ranking as the third leading cause of global mortality, pose a significant burden to healthcare systems worldwide. Given the substantial mortality and morbidity rates of respiratory diseases, efficient and rapid preclinical models are imperative to accurately recapitulate the diverse spectrum of respiratory conditions. In this article, we discuss the applications and future potential of CR technology in modeling various respiratory tract diseases, including lung cancer, respiratory viral infections (such as influenza and Covid-19 and etc.), asthma, cystic fibrosis, respiratory papillomatosis, and upper aerodigestive track tumors. Furthermore, we discuss the potential utility of CR in personalized medicine, regenerative medicine, and clinical translation. © 2024 The Author(s)

关键词： Asthma conditional reprogramming Covid-19 Cystic fibrosis Lung cancer Preclinical models Respiratory diseases Respiratory papillomatosis Respiratory viral infections Translational medicine

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conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses

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Virologica Sinica 2020年第3期35卷 280-289页

作者： Xuefeng Liu Yuntao Wu Lijun Rong Department of Pathology Center for Cell ReprogrammingGeorgetown University Medical CenterWashingtonDCUSA Department of Oncology Lombardi Comprehensive Cancer CenterGeorgetown University Medical CenterWashingtonDCUSA National Center for Biodefense and Infectious Diseases School of Systems BiologyGeorge Mason UniversityManassasVA 20110USA Department of Microbiology and Immunology University of Illinoi at ChicagoChicagoIL 60612USA

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models(ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells(CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming(CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid(2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for airliquid interface(ALI) polarized 3 D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.

关键词： Normal cells Cell senescence conditional reprogramming Physiological conditions Functional models Air-liquid interface(ALI) Emerging viruses SARS-CoVs

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Culture and application of conditionally reprogrammed primary tumor cells

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Gastroenterology Report 2020年第3期8卷 224-233,I0002页

作者： Mengjun Zhong Liwu Fu State Key Laboratory of Oncology in South China Collaborative Innovation Center for Cancer MedicineGuangdong Esophageal Cancer InstituteSun Yat-sen University Cancer CenterGuangzhouGuangdongP.R.China

Cancer is still a major public-health problem that threatens human life worldwide and further study needs to be carried out in the basic and preclinical *** high-throughput sequencing technology and individualized precise therapy have made breakthroughs over the years,the high failure rate of clinical translational research has limited the innovation of antitumor drugs and triggered the urgent need for optimal cancer-research *** development of cancerous cell lines,patient-derived xenograft(PDX)models,and organoid has strongly promoted the development of tumor-biology research,but the prediction values are *** reprogramming(CR)is a novel cell-culture method for cancer research combining feeder cells with a Rho-associated coiled-coil kinase(ROCK)inhibitor,which enables the rapid and continuous proliferation of primary epithelial *** this review,we summarize the methodology to establish CR model and overview recent functions and applications of CR cell-culture models in cancer research with regard to the study of cancerbiology characterization,the exploration of therapeutic targets,individualized drug screening,the illumination of mechanisms about response to antitumor drugs,and the improvement of patient-derived animal models,and finally discuss in detail the major limitations of this cell-culture system.

关键词： conditional reprogramming primary tumor cell cancer research precise medicine

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Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings

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FASEB JOURNAL 2020年第1期34卷 386-398页

作者： Peters-Hall, Jennifer R. Min, Jaewon Tedone, Enzo Sho, Sei Siteni, Silvia Mender, Ilgen Shay, Jerry W. Univ Texas Southwestern Med Ctr Dallas Dept Cell Biol 5323 Harry Hines Blvd Dallas TX 75390 USA

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2 ' deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.

关键词： conditional reprogramming low oxygen ROCK inhibitor senescence telomerase

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Long-term differentiating primary human airway epithelial cell cultures: how far are we?

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CELL COMMUNICATION AND SIGNALING 2021年第1期19卷 63-63页

作者： Bukowy-Bieryllo, Zuzanna Inst Human Genet PAS Strzeszynska 32 PL-60479 Poznan Poland

Background: Human airway epithelial (HAE) cellular models are widely used in applicative studies of the airway physiology and disease. In vitro expanded and differentiated primary HAE cells collected from patients seem to be an accurate model of human airway, offering a quicker and cheaper alternative to the induced pluripotent stem cell (iPSCs) models. However, the biggest drawback of primary HAE models is their limited proliferative lifespan in culture. Much work has been devoted to understand the factors, which govern the HAE cell proliferation and differentiation, both in vivo and in vitro. Here, I have summarized recent achievements in primary HAE culture, with the special emphasis on the models of conditionally reprogrammed cells (CRC), which allow longer in vitro proliferation and differentiation of HAE cells. The review compares the CRC HAE technique variants (feeder culture or HAE mono-culture), based on recently published studies exploiting this model. The advantages and limitations of each CRC HAE model variant are summarized, along with the description of other factors affecting the CRC HAE culture success (tissue type, sampling method, sample quality). Conclusions: CRC HAE cultures are a useful technique in respiratory research, which in many cases exceeds the iPSCs and organoid culture methods. Until the current limitations of the iPSCs and organoid culture methods will be alleviated, the primary CRC HAE cultures might be a useful model in respiratory research. Plain English summary: Airway epithelium (AE) is a type of tissue, which lines the whole length of human airways, from the nose to the bronchi. Improper functioning of AE causes several human airway disorders, such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). Much work has been devoted to finding the best scientific model of human AE, in order to learn about its functioning in health and disease. Among the popular AE models are the primary in vitro cultured AE

关键词： Primary airway cell culture Air-liquid interface culture conditional reprogramming ROCK inhibitor SMAD inhibitor TGF-beta 1 inhibitor

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A 3D endometrial organotypic model simulating the acute inflammatory decidualisation initiation phase with epithelial induction of the key endometrial receptivity marker, integrin αVβ3

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HUMAN REPRODUCTION OPEN 2021年第4期2021卷 hoab034页

作者： Fraser, R. Smith, R. Lin, C-J Univ Edinburgh MRC Ctr Reprod Hlth Queens Med Res Inst 47 Little France Crescent Edinburgh EH16 4TJ Midlothian Scotland

STUDY QUESTION: Is it possible to develop a simplified physiological in vitro system representing the key cell-types associated with a receptive endometrial phenotype? SUMMARY ANSWER: We present a new concept to investigate endometrial receptivity, with a 3D organotypic co-culture model to simulate an early and transient acute autoinflammatory decidual status that resolves in the induction of a receptive endometrial phenotype. WHAT IS KNOWN ALREADY: Embryo implantation is dependent on a receptive uterine environment. Ovarian steroids drive postovulation structural and functional changes in the endometrium, which becomes transiently receptive for an implanting conceptus, termed the `window of implantation', and dysregulation of endometrial receptivity is implicated in a range of reproductive, obstetric, and gynaecological disorders and malignancies. The interactions that take place within the uterine microenvironment during this time are not fully understood, and human studies are constrained by a lack of access to uterine tissue from specific time-points during the menstrual cycle. Physiologically relevant in vitro model systems are therefore fundamental for conducting investigations to better understand the cellular and molecular mechanisms controlling endometrial receptivity. STUDY DESIGN, SIZE, DURATION: We conducted an in vitro cell culture study using human cell lines and primary human cells isolated from endometrial biopsy tissue. The biopsy tissue samples were obtained from three women attending gynaecological outpatient departments in NHS Lothian. The work was carried out between December 2016 and April 2019, at the MRC Centre for Reproductive Health, Queen's Medical Research Institute, University of Edinburgh. PARTICIPANTS/MATERIALS, SETTING, METHODS: An endometrial stromal cell (ESC) line, and endometrial epithelial cells (EECs) isolated from endometrial biopsy tissue and expanded in vitro by conditional reprogramming, were used throughout the study. Immun

关键词： endometrial receptivity window of implantation decidualisation endometrial organotypic model conditional reprogramming endometrial stromal cells endometrial epithelial cells progesterone oestrogen

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Fidelity of a PDX-CR model for bladder cancer

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2019年第1期517卷 49-56页

作者： Mondal, Abdul M. Ma, Ai-Hong Li, Guangzhao Krawczyk, Ewa Yuan, Ruan Lu, Jie Schlegel, Richard Stamatakis, Lambros Kowalczyk, Keith J. Philips, George K. Pan, Chong-Xian Liu, Xuefeng Georgetown Univ Med Ctr Dept Pathol Ctr Cell Reprograming Washington DC 20007 USA Univ Calif Davis Sch Med Dept Internal Med Div Hematol & Oncol Washington DC 95616 USA Wuhan Univ Renmin Hosp Dept Urol Washington DC USA Georgetown Univ Lombardi Comprehens Canc Ctr Washington DC USA MedStar Washington Hosp Ctr Dept Urol Washington DC USA MedStar Georgetown Hosp Dept Urol Washington DC USA MedStar Georgetown Hosp Dept Oncol Washington DC USA VA Northern Calif Hlth Care Syst Mather CA USA

Patient-derived xenografts (PDXs) are widely recognised as a more physiologically relevant preclinical model than standard cell lines, but are expensive and low throughput, have low engraftment rate and take a long time to develop. Our newly developed conditional reprogramming (CR) technology addresses many PDX drawbacks, but lacks many in vivo factors. Here we determined whether PDXs and CRCs of the same cancer origin maintain the biological fidelity and complement each for translational research and drug development. Four CRC lines were generated from bladder cancer PDXs. Short tandem repeat (STR) analyses revealed that CRCs and their corresponding parental PDXs shared the same STRs, suggesting common cancer origins. CRCs and their corresponding parental PDXs contained the same genetic alterations. Importantly, CRCs retained the same drug sensitivity with the corresponding downstream signalling activity as their corresponding parental PDXs. This suggests that CRCs and PDXs can complement each other, and that CRCs can be used for in vitro fast, high throughput and low cost screening while PDXs can be used for in vivo validation and study of the in vivo factors during translational research and drug development. (C) 2019 Elsevier Inc. All rights reserved.

关键词： conditional reprogramming Patient derived xenograft Cell line models Drug discovery Bladder cancer

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Personalized Drug Sensitivity Screening for Bladder Cancer Using conditionally Reprogrammed Patient-derived Cells

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EUROPEAN UROLOGY 2019年第4期76卷 430-434页

作者： Kettunen, Kimmo Bostrom, Peter J. Lamminen, Tarja Heinosalo, Taija West, Gun Saarinen, Irena Kaipio, Katja Rantala, Juha Albanese, Chris Poutanen, Matti Taimen, Pekka Univ Turku Inst Biomed Kiinamyllynkatu 10 FIN-20520 Turku Finland Turku Univ Hosp Dept Pathol Turku Finland Turku Univ Hosp Dept Urol Turku Finland Univ Turku Turku Finland Univ Turku Res Ctr Integrat Physiol & Pharmacol Inst Biomed Turku Finland Univ Turku Turku Ctr Dis Modeling Turku Finland Misvik Biol Turku Finland Georgetown Univ Lombardi Comprehens Canc Ctr Dept Oncol Washington DC USA Georgetown Univ Lombardi Comprehens Canc Ctr Dept Pathol Washington DC USA

Many patients with muscle-invasive bladder cancer (BC) are either ineligible for or do not benefit from cisplatin-based chemotherapy, and there is an unmet need to estimate individuals' drug sensitivities. We investigated the suitability of conditionally reprogrammed (CR) cells for the characterization of BC properties and their feasibility for personalized drug sensitivity screening. The CR cultures were established from six BC tumors with varying histology and stage. Four cultures were successfully propagated for genomic, transcriptomic, and protein expression profiling and compared to the parental tumors. Two out of four CR cultures (urothelial carcinoma and small cell neuroendocrine carcinoma [SmCC]) corresponded well to their parental tumors and underwent drug sensitivity screening to identify novel drugs for the respective tumors. Both cultures were sensitive to standard BC chemotherapy agents (eg cisplatin and gemcitabine) and to conventional drugs such as taxanes and inhibitors of topoisomerase and proteasome. The SmCC cells were also sensitive to statins (eg, atorvastatin and pitavastatin). In summary, after confirming their representativeness and origin, we conclude that CR cells are a feasible platform for personalized drug sensitivity testing and might thus add to the approaches used to personalize BC treatment strategies. Patient summary: We investigated the conditional reprogramming method for generating patient-derived bladder cancer cell cultures and studied their feasibility for planning personalized treatment strategies. (C) 2019 The Authors. Published by Elsevier B.V. on behalf of European Association of Urology.

关键词： Bladder cancer Chemotherapy conditional reprogramming Drug sensitivity testing Patient-derived cultures Small cell carcinoma

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