key: cord-0774407-ol9b08az authors: Collins, Daniel.P.; Osborn, Mark.J.; Steer, Clifford.J. title: Differentiation of immortalized human MLPC to alveolar type 2 (AT2)-like cells: ACE-2 expression and binding of SARS-CoV-2 spike and spike 1 proteins date: 2021-08-17 journal: Cytotherapy DOI: 10.1016/j.jcyt.2021.07.017 sha: 558637fd3aa64afb4b869960d8755e709846c9e7 doc_id: 774407 cord_uid: ol9b08az Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the ACE-2 receptor to facilitate entry into respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. They express high levels of surface ACE-2 and thus are a leading target for primary infection by the SARS-CoV-2. This study describes a method to directly differentiate TERT-immortalized human cord blood-derived multilineage progenitor cells (MLPC) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, qRT-PCR, and binding of biotinylated SARS-CoV-2 spike (S and S1) proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. We defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. Our method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies. Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the ACE-2 receptor to facilitate entry into respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. They express high levels of surface ACE-2 and thus are a leading target for primary infection by the SARS-CoV-2. This study describes a method to directly differentiate TERT-immortalized human cord blood-derived multilineage progenitor cells (MLPC) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, qRT-PCR, and binding of biotinylated SARS-CoV-2 spike (S and S1) proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. We defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. Our method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies. This study describes a single-step differentiation procedure to generate long-lived alveolar type-2-like cells (AT2) from TERT-immortalized human MLPC. ACE-2 receptors on the AT2-like cells were able to specifically bind and internalize the SARS-CoV-2 spike protein. A protocol utilizing biotinylated spike proteins, streptavidin-alexa-594 and the AT2 was used to distinguish antibodies that were RBD specific. The AT2 could provide an accurate and reproducible in vitro tool for viral uptake and blocking studies. AT2-like cells differentiated from TERT-immortalized human MLPC expressed ACE-2, bound SARS-CoV-2 spike proteins and differentiated between RBD-specific and non-RBD-specific neutralizing antibodies. This long-lived cell line could provide a reliable tool to study spike protein binding and inhibition by antibodies or other potential therapeutics designed to inhibit infection by the SARS-CoV-2 virus. The SARS-CoV-2 is a highly transmissible virus responsible for the current COVID-19 pandemic, negatively impacting global health and economies (1) (2) (3) . High level transmission and mortality rates distinguish the SARS-CoV-2 virus from related corona viruses SARS-CoV (severe acute respiratory syndrome) and MERS-CoV (Middle East respiratory syndrome), however, pneumonia related symptoms including cough, fever, shortness of breath and fatigue are similar in all three infections (2, 4) . Differences in transmissibility and variable individual immune responses to the virus account for dramatically increased numbers of infections, prolonged periods of morbidity and higher mortality when compared to SARS-CoV and MERS- Viral entry into cells is the essential first step in infectivity and pathogenesis of SARS-CoV-2 (5, (9) (10) (11) . Studies have determined that a major portal of entry for the SARS-CoV-2 virus is the cell surface expressed protein angiotensin-converting enzyme-2 (ACE-2) (5, (9) (10) (11) . Attachment and entry of the virus via the ACE-2 receptor is mediated by spike proteins located on the surface of the virus (5, (9) (10) (11) . The spike protein is expressed as a trimer, with each monomer consisting of 2 distinct domains, the S1 and S2 (5, 9, 11) . The S1 domain contains the receptor-binding domain (RBD) responsible for the initial binding of the virus to ACE-2. The presence of TMPRSS2 proteases and lysosomal cathepsin proteins on the target cells are responsible for cleavage of the S1 and S2 domains which allow the S2 domain to mediate viral entry via membrane fusion (5) . The SARS-CoV virus also relies on the ACE-2 receptor for cellular invasion but differences in the RBD of the two viruses may contribute to the higher affinity of the SARS-CoV-2 spike protein binding to the ACE-2 and potentially the severity of infection (5, (12) (13) (14) . Inhibition of binding of the spike protein to the ACE-2 receptor has been identified as a target for the development of therapeutics against SARS-CoV-2 (10, 15) . Antibodies present in convalescent serum and monoclonal antibodies directed against the spike protein have been tested for therapeutic potential, but their potential effectiveness has yet to be fully determined. The lung epithelium is involved in many/most SARS-CoV-2 infections even those that are asymptomatic. Epithelial cells that line the alveoli and bronchia of the lungs express high levels of ACE-2 and, as such, are highly susceptible to viral attachment and entry (4) . Severe cases of pulmonary SARS-CoV-2 infection are associated with cell death and fibrosis of lung tissue. Alveolar type 2 (AT2) cells have multiple functions for proper alveolar functionality (16) . They act as stem cells for the regeneration of the lung epithelium by differentiating to alveolar type 1 (AT1) cells that line the alveoli and mediate gas exchange. They also produce the surfactants (lipids and proteins) that maintain the osmotic integrity of the alveoli. The regenerative properties of AT2 cells and their potential loss due to pathogenic processes such as SARS-CoV-2 infection makes them a desirable cell type for study. Commercial sources exist for lung epithelial cells, however, the utility of these cells is limited by their low in vitro longevity, limited expansion potential and high cost. Additionally, donor-to-donor differences of primary Small Airway Epithelial Cells (SAEpi) could affect functionality. We sought to define the process by which immortalized AT2 cells could be generated and propagated as a tool to study viral infection. A previous study using human umbilical cord blood-derived multilineage progenitor cells MLPC are multi-potent stem cells isolated from human umbilical cord blood. Umbilical cord blood was collected as part of an FDA submission to market PrepaCyte-CB, a product to MLPC were isolated as previously described (21) (22) (23) (24) . Briefly, after mixing and MLPC were immortalized by insertion of the gene for human telomerase reverse transcriptase (TERT), as previously described (23 As previously described (23) , clonal MLPC-TERT cell lines were developed by limited dilution cloning. One clone, E12-TERT (E12), exhibited the desired combined characteristics of immortality and differentiation beyond mesodermal outcomes. The E12 cells have been repeatedly cryo-preserved, thawed and expanded for over 14 years and were used in this study. Primary Small Airway Epithelial Cells (SAEpi) were obtained from ScienCell (Carlsbad, CA, cat #3230). Cells (5 x 10 5 ) were plated in a 75 cm culture vessel coated with poly-l-lysine. Cells were grown and expanded using 13 ml of small airway growth medium (SAGM, Lonza, Walkersville, MD, cat # 3118) with 3 medium exchanges per week. Figure 1A . All images were observed by separation of blue and red fluorescence and enumerated for expression of each marker as a percentage of blue nuclei also staining for the red fluorescence of each marker. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden Germany) and reverse transcribed using SuperScript™ IV VILO™ (ThermoFisher, Wcaltham, MA). TaqMan Gene expression was performed using ACE-2 (Hs01085331_m1), DPP4 (CD26) (Hs00897405_m1, EpCAM (Hs00158980_m1), SFTPC (Hs00951326_g1), KRT19 (Hs00761767_s1), TM4SF1 (Hs05418027_s1) and GAPDH (Hs02786624_g1) probes (ThermoFisher, Waltham, MA). Data were normalized to GAPDH. The ability of cells to bind SARS-CoV-2 spike and spike 1 proteins was analyzed by confocal microscopy using biotinylated spike proteins. Cells were prepared as described above and labeled with 250 ng of either biotinylated spike (RBD) (SPD-C8E9, Acrobiosystems, Newark, DE) or biotinylated spike 1 protein (SIN-C82E8, Acrobiosystems) for 30 minutes. Unbound spike proteins were removed by washing cells twice with 200 μl of PermaCyte medium. Bound spike proteins were visualized by staining with streptavidin-alexa 594 (S11227 Life Technologies). Cells were counterstained with DAPI to visualize the nucleus. This procedure would visualize binding to surface and cytoplasmic ACE-2. This procedure is outlined in Figure 1B . The ability of the biotinylated spike protein to be internalized was examined with E12 MLPC, E12 AT2, and primary SAEpi cells. Briefly, cells were plated in 16 well chamber slides, as described above. To each well, 250 ng of biotinylated spike protein was added and the cells were incubated for 2 hours at 37 o C. Unbound spike protein was removed by washing 3 times with PBS. Cells were then fixed with 1% formaldehyde for 1 hour at room temperature. The cells were then permeabilized by PermaCyte and stained with streptavidin-alexa 594. The nucleus of the cells was visualized with DAPI. This procedure would visualize the spike protein binding to surface receptors, but, the cytoplasmic staining would only be due to internalization of the spike protein. This procedure is outlined in Figure 1E . The specificity of biotinylated spike proteins binding to E12 AT2 cells was confirmed by blocking with a 5 molar excess of unlabeled spike protein. Cells were prepared as described for confocal analysis. Cells were incubated with 1.25 μg of unlabeled spike protein (Acrobiosystems, SPD-S52H6) for 1 hour. Without washing the unbound unlabeled spike protein, biotinylated spike and spike 1 proteins were added to the cells and incubated for 30 minutes. Cells were washed twice with PermaCyte medium to remove any unbound proteins. Visualization of bound biotinylated spike proteins was accomplished by secondary labeling with streptavidin-alexa 594. Cells were counterstained with DAPI to visualize nuclei. This procedure is outlined in Figure 1C . To examine the effects of neutralizing antibody on the binding of the spike proteins, commercially available neutralizing antibodies were obtained from Acrobiosystems (SAD-S35) and Novatein Biosystems (PR-nCOV-mABS1, Boston, MA). One μg of either antibody was preincubated with the spike protein for one hour prior to addition of the mixture to the cells prepared as previously described for binding of spike proteins. Visualization of the binding of biotinylated spike protein was accomplished by secondary staining with streptavidin-alexa 594. The nucleus of the cells was visualized with DAPI. This procedure is outlined in Figure 1D . The morphology of undifferentiated E12 MLPC, differentiated E12 AT2 cells and primary Small Airway Epithelial cells (SAEpi) are shown in Figure 2 . E12 MLPC were characterized by a spindle-shaped morphology and mononuclear. Differentiated E12 AT2 cells were round or cuboidal in morphology and either 1 or 2 nuclei per cells. Primary SAEpi also appeared as round to cuboidal in morphology with 1 to 2 nuclei. Under less than confluent conditions, some of the differentiated E12 AT2 and SAEpi cells expressed a more elongated and spindle-shaped morphology. Confocal microscopy results are summarized in Table 1 The The results shown in Figure 6 were normalized to GAPDH. Expression of the ACE-2 surface protein suggested that the AT2-like cells would be susceptible to binding by the SARS-CoV2 spike and spike 1 proteins. We tested this by binding Specificity of the binding of spike proteins was confirmed by blockade of biotinylated spike protein with a 5 molar excess of unlabeled spike protein and by neutralizing monoclonal human antibodies. As shown in Figure 8 , binding of biotinylated spike protein was essentially inhibited by preincubation with unlabeled spike protein and by neutralizing antibody from Acrobiosystems, but, was not by the antibody from Novatein Biosystems. Without further investigation, we posited that the Novatein antibody was specific for an epitope distinct from the RBD as both antibodies were shown to bind to the spike protein attached to paramagnetic beads. The focus of this study was to develop a cell line with the desired characteristics of AT2 cells and the ability to be cultured for extended periods of time and to be expanded extensively. In previous studies with E12-MLPC, differentiation of E12 cells into hepatocyte-like cells resulted in cells that were shown to be functionally immortalized (23) . As an alternative to primary alveolar cells, there have been efforts to develop alveolar cells and organoids through the differentiation of hPSC, which usually involved a multi-step process (26) (27) (28) . The first step involved commitment to anterior foregut endoderm, followed by We believe that E12 AT2-like cells can provide an important tool to study lung pathologies and specifically the binding of SARS-CoV-2 spike proteins to the ACE-2 receptor. 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Both cell types were 100% positive for surfactant protein C We would like to acknowledge the efforts of Peter W. Collins and Joel H. Hapke for contributions to tissue culture and analytical methodologies. Funding: CytoMedical Design Group, LLC (CMDG) provided funding for this study in the form of salaries for DPC and facility resources. Additionally, funding was provided by BioE, LLC.