Airway Fluids as a Resource for the Acquisition of Patients-Specific and Disease- Specific Airway Epithelium
Primary cell culture is a laboratory process that has been practiced for more than a century by which the cells of interest are isolated from tissues and grown under the appropriate conditions to achieve a certain cell number. In conducting airway, basal cells function as stem cells that self-renew and differentiate to maintain tissue homeostasis and to regenerate after injuries . Patient-specific and disease-specific airway basal cells provide valuable resources for human basic and translational research. Culture methods of primary human airway basal stem cells have reached remarkable achievements over the past several years [2-7]. Human airway tissues (rejected lungs for transplantation, surgical and bronchoscopy biopsies, postmortem tissues, etc.) are available for cell dissociation and subsequent culture. The existing method limits the easy establishment of a physiological cellular platform to tackle questions related to developmental and perinatal lung diseases. Infant tissues are difficult to access since invasive biopsy is unlikely and postmortem tissues are extremely rare. We reported previously that noninvasively obtained clinic fluids including induced sputum and bronchoalveolar lavage (BAL) were used to derive patient airway basal cells for a cystic fibrosis study . BAL can be obtained from pathologists as discarded samples after diagnosis. Induced sputa can be easily collected when patients visit their physicians’ offices. The article by the Lerou group and associates recently published in Pediatric Research  demonstrated that tracheal aspirate fluid from mechanically ventilated infants in the Neonatal Intensive Care Unit (NICU) can serve as a valuable alternative to tissues when generating neonatal airway basal cells. These cells are referred as neonatal tracheal aspirate-derived (nTAD) epithelial basal cell-like cells. The aspirates are collected from neonates intubated due to a variety of complications who are regularly suctioned to clear the tube. By using single-cell RNA-sequencing, we confirmed the presence of epithelial cells in tracheal aspirates based on epithelial gene signatures including epithelial cell adhesion molecule (EpCAM) and cytokeratin . Despite for presence of epithelial cell markers, we identified that the majority of cells in each sample are differentiated cells, mixed with a few basal stem cells. Since aspirates from intubated neonates are produced on a daily basis, a fast and effective way to grow out those basal stem cells is necessary. We did so by directly plating the filtered aspirates within hours after suctioning (any additional procedures including cell sorting would cause cell death and stress, resulting in decreased success with cell derivation). Based on our experience, the terminally differentiated airway epithelial cells and hematopoietic cells cannot attach to the plates and therefore are removed by medium change next day. Those transient amplifying cells can be eradicated from cell population after a couple of passages. The stem cells will then dominate the cell pool and continue propagating to produce a large quantity of cells . We demonstrated that by using this approach we have successfully established a repository of nTAD basal cell lines from infants ranging in gestational age from 24 weeks to full term and with a variety of clinical conditions . With this successful example, we expect that the similar approach can be deployed to derive primary airway cell lines from other clinical fluids such as nasopharyngeal fluids that are sucked from neonates immediately from birth in delivery rooms, as long as the fluids contain viable stem cells, thus providing an opportunity to generate airway basal cell lines from even healthy neonates.
There are two caveats for using fluids and the direct culture method to derive airway basal cells. One is that the regional information is lost, and all stem cells are expanded as a pool. Single cell analysis done directly on aspirates can be a parallel way to answer questions related to anatomic and regional cell heterogeneity in the airways of neonates. Another caveat is that a portion of basal cells might come from secretory cells. It has been reported in mice that a subset of club cells can undergo dedifferentiation to basal cells in vivo  and in vitro culture . However, once the differentiated club cells acquire basal cell signatures they are functionally and morphologically equivalent to original basal cells [5,9]. The dedifferentiation of human club cells to basal cells has not yet been reported in literature. We have detected club cell signatures in aspirates based on single cell sequencing data; therefore, we cannot exclude the possibility of dedifferentiation of club cells in our culture. However, based on previous results in mice we have reasons to believe that the “club cell originated basal cells” should not cause too much concerns.
Long-Term Expansion of nTAD Cells:Achieved with Rapamycin
Since only a small population of stem cells can be identified in aspirates, the application of nTAD cells in basic and translational research is dependent on the ability to expand them extensively. We have reported that ROCK inhibition and SMAD signaling inhibition maintains cell stemness to achieve serial expansion of adult epithelial basal cells of multiple organs including lung . These conditions also supported the initial outgrowth of basal cells from the aspirates. However, we identified that nTAD cells are easily deteriorated after a few passages unlike their adult counterparts. The mechanisms underlying the difference between adult and neonatal basal cell behaviors in culture are unclear. One of possibilities we can postulate is that the immaturity of neonatal cells makes them easy to differentiate or undertake morphological alteration (i.e. epithelial-mesenchymal transition or EMT). Likely one or more additional factors are needed to sustain long-term nTAD expansion. After searching for several possible candidates, we identified that rapamycin, an inhibitor for the mammalian target of rapamycin (mTOR) pathway, fits our purpose. The mTOR pathway has been well studied and is a critical pathway for tissue homeostasis, injury repair and regenerative medicine [10-14]. Inhibition of the mTOR pathway using rapamycin prevents epithelial stem cell senescence [15,16] and modulates the epidermal and tracheal basal cell pool in vivo [17,18]. Rapamycin has been used in culture to prolong the growth and proliferation of multiple epithelial stem cells and progenitors . In Lu et al., 2020, we provided evidence that rapamycin effectively sustains the cell growth of neonatal airway basal cells. In the presence of rapamycin, nTAD cells could be maintained in culture for at least 15 passages (or ~50 population doublings at 1:8-1:10 splitting ratio) . In addition, we used transcriptomic and protein analysis to show that rapamycin was responsible for preventing cellular differentiation pathways, in particular by upregulating RSK3. Interestingly, transcriptomic analysis also indicated that the suppression of epithelialmesenchymal transition is not implicated as a beneficial effect in cell cultures. Despite this rapamycin plays an essential role in EMT in numerous studies (Review in ).
Rapamycin must be included in the medium for continuous culture, withdrawal of this compound quickly causes the serial cell expansion to deteriorate. One may question that constitutive treatment with rapamycin could alter airway basal cell functions since mTOR and related pathways participate in pleiotropic cellular processes. Our work suggested that basal cells cultured in rapamycin retain characteristic morphology and stem cell biomarkers over passages . Air-liquid interface (ALI) culture further verified that basal cells cultured in rapamycin can effectively differentiate into a pseudostratified epithelium with appropriate cellular composition and epithelial barrier functions . These data in aggregate demonstrated that nTAD cells cultured in rapamycin genuinely recapitulate expected basal cell functions and are suitable for the study of lung development, stem cell function and diseases. Whether such cells can be also utilized for reparative cell therapy in infants with lung complications requires additional securitization to ensure their safety.
Prospective Application of nTAD Culture:Study Late Stage of Human Lung Development,Perinatal Diseases, and Beyond
The study of human lung development is important for understanding mechanisms underlying both congenital and acquired lung diseases that affect newborns and children . Animal genetic models have merit by providing insights for detailed cellular and molecular events across temporal progression of lung development [22,23]. However, mouse and human lungs differ drastically in anatomic architecture and timeframe of organogenesis [24,25]. This suggests that a human cellular model is critical and should be used as a gold standard to validate the mechanisms derived from mouse work and to develop the therapeutic drugs and strategies to treat infant diseases. Previously, the knowledge of human lung development and abnormalities mainly relied on histological and biomarker examination on embryonic and perinatal lung tissues. In recent years, human pluripotent stem cell (hPSCs) differentiation and the subsequently derived cells have provided valuable models for further understanding of human lung development and diseases (Review in [24,26,27]). Our ability to derive a large repository of perinatal airway basal cells provides us a unique opportunity to study late-stage human lung development. Lerou and colleagues have reported that the mesenchymal stromal cells (MSCs) derived from intubated aspirates maintained their developmental memory despite extensive expansion , setting the stage to use nTAD cells to study cellular phenotypes during lung development.
Another foreseeable application is that we can use nTAD cells to interrogate the similarities and differences of adult and infant airway epithelium responses to environmental stimuli and genetic mutations. It is well recognized that pediatric and adult asthmatic diseases exhibit different clinical manifestations and may be under regulation by distinct mechanisms [29-31]. In addition, respiratory viral infections often elicit different clinical outcomes in infants and adults. For example, human respiratory syncytial virus (RSV), the most important viral agent causing serious bronchiolitis in infants younger than 2, only causes mild symptoms in adults. Conversely, the current coronavirus disease 2019 (COVID-19) rarely induces lung symptoms in infants and young kids and the severity of disease increases with patients’ age (based on Data & Statistics | CDC). This age-dependent and virus-specific disease severity in infants/children and adults is intriguing. One of the mechanisms could lie in epithelial cells themselves including cellular architecture and composition, barrier function, transcriptome, susceptibility to infections, innate immunity. In the past, such comparative studies were not realizable due to the difficulty in procuring infant and pediatric airway basal cells. Now this type of study can be engaged by our groups with the new culture method. Meanwhile, we will continue our nTAD biobanking with the hope of utilizing them for many other valuable applications.
Competing Financial Interests
The author declaims no conflict of interest.
The author wishes to thank the Cystic Fibrosis Foundation Research Grant (H.M., MOU19G0), Hood Child Health Research Award (H.M.), Harvard Stem Cell Institute Seed Grant (H.M. SG-0120-19-00) to support the work of this manuscript.
2. Butler CR, Hynds RE, Gowers KH, Lee DD, Brown JM, Crowley C, et al. Rapid expansion of human epithelial stem cells suitable for airway tissue engineering. American Journal of Respiratory and Critical Care Medicine. 2016 Jul 15;194(2):156-68.
3. Gowers KH, Hynds RE, Thakrar RM, Carroll B, Birchall MA, Janes SM. Optimized isolation and expansion of human airway epithelial basal cells from endobronchial biopsy samples. Journal of Tissue Engineering and Regenerative Medicine. 2018 Jan;12(1):e313-7.
4. Hynds RE, Butler CR, Janes SM, Giangreco A. Expansion of human airway basal stem cells and their differentiation as 3D tracheospheres. InOrganoids 2016 (pp. 43-53). Humana, New York, NY.
5. Mou H, Vinarsky V, Tata PR, Brazauskas K, Choi SH, Crooke AK, et al. Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell stem cell. 2016 Aug 4;19(2):217-31.
6. Rayner RE, Makena P, Prasad GL, Cormet-Boyaka E. Optimization of normal human bronchial epithelial (NHBE) cell 3D cultures for in vitro lung model studies. Scientific Reports. 2019 Jan 24;9(1):1-0.
7. Sachs N, Papaspyropoulos A, Zomer-van Ommen DD, Heo I, Böttinger L, Klay D, et al. Long-term expanding human airway organoids for disease modeling. The EMBO Journal. 2019 Feb 15;38(4):e100300.
8. Lu J, Zhu X, Shui JE, Xiong L, Gierahn T, Zhang C, et al. Rho/SMAD/mTOR triple inhibition enables long-term expansion of human neonatal tracheal aspirate-derived airway basal cell-like cells. Pediatric Research. 2020 May 4:1-8.
9. Tata PR, Mou H, Pardo-Saganta A, Zhao R, Prabhu M, Law BM, et al. Dedifferentiation of committed epithelial cells into stem cells in vivo. Nature. 2013 Nov;503(7475):218-23.
10. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012 Apr 13;149(2):274-93.
11. Meng D, Frank AR, Jewell JL. mTOR signaling in stem and progenitor cells. Development. 2018 Jan 1;145(1).
12. Papadopoli D, Boulay K, Kazak L, Pollak M, Mallette F, Topisirovic I, et al. mTOR as a central regulator of lifespan and aging. F1000Research. 2019;8.
13. Wei X, Luo L, Chen J. Roles of mTOR signaling in tissue regeneration. Cells. 2019 Sep;8(9):1075.
14. Weichhart T. mTOR as regulator of lifespan, aging, and cellular senescence: a mini-review. Gerontology. 2018;64(2):127-34.
15. Houssaini A, Breau M, Kanny Kebe SA, Marcos E, Lipskaia L, Rideau D, et al. mTOR pathway activation drives lung cell senescence and emphysema. JCI Insight. 2018 Feb 8;3(3).
16. Iglesias-Bartolome R, Patel V, Cotrim A, Leelahavanichkul K, Molinolo AA, Mitchell JB, et al. mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell Stem Cell. 2012 Sep 7;11(3):401-14.
17. Castilho RM, Squarize CH, Chodosh LA, Williams BO, Gutkind JS. mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging. Cell Stem Cell. 2009 Sep 4;5(3):279-89.
18. Haller S, Kapuria S, Riley RR, O’Leary MN, Schreiber KH, Andersen JK, et al. mTORC1 activation during repeated regeneration impairs somatic stem cell maintenance. Cell Stem Cell. 2017 Dec 7;21(6):806-18.
19. Gidfar S, Milani FY, Milani BY, Shen X, Eslani M, Putra I, et al. Rapamycin prolongs the survival of corneal epithelial cells in culture. Scientific Reports. 2017 Jan 5;7(1):1-0.
20. Roshan MK, Soltani A, Soleimani A, Kahkhaie KR, Afshari AR, Soukhtanloo M. Role of AKT and mTOR signaling pathways in the induction of epithelialmesenchymal transition (EMT) process. Biochimie. 2019 Oct 1;165:229-34.
21. Herriges M, Morrisey EE. Lung development: orchestrating the generation and regeneration of a complex organ. Development. 2014 Feb 1;141(3):502-13.
22. Hogan BL, Barkauskas CE, Chapman HA, Epstein JA, Jain R, Hsia CC, et al. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell. 2014 Aug 7;15(2):123-38.
23. Morrisey EE, Hogan BL. Preparing for the first breath: genetic and cellular mechanisms in lung development. Developmental Cell. 2010 Jan 19;18(1):8-23.
24. Nikolic MZ, Sun D, Rawlins EL. Human lung development: recent progress and new challenges. Development. 2018 Aug 15;145(16).
25. Pan H, Deutsch GH, Wert SE, NHLBI Molecular Atlas of Lung Development Program Consortium. Comprehensive anatomic ontologies for lung development: A comparison of alveolar formation and maturation within mouse and human lung. Journal of Biomedical Semantics. 2019 Dec 1;10(1):18.
26. Miller AJ, Spence JR. In vitro models to study human lung development, disease and homeostasis. Physiology. 2017 Apr 12;32:246-260.
27. Snoeck HW. Modeling human lung development and disease using pluripotent stem cells. Development. 2015 Jan 1;142(1):13-6.
28. Spadafora R, Lu J, Khetani RS, Zhang C, Iberg A, Li H, Shi Y, Lerou PH. Lung-resident mesenchymal stromal cells reveal transcriptional dynamics of lung development in preterm infants. American Journal of Respiratory and Critical Care Medicine. 2018 Oct 1;198(7):961-4.
29. Bush A, Menzies-Gow A. Phenotypic differences between pediatric and adult asthma. Proceedings of the American Thoracic Society. 2009 Dec 15;6(8):712-9.
30. Cabana MD, Kunselman SJ, Nyenhuis SM, Wechsler ME. Researching asthma across the ages: insights from the National Heart, Lung, and Blood Institute’s Asthma Network. Journal of Allergy and Clinical Immunology. 2014 Jan 1;133(1):27-33.
31. Trivedi M, Denton E. Asthma in Children and Adults– What Are The Differences and What Can They Tell Us About Asthma?. Frontiers in Pediatrics. 2019;7:256.