2.1 Cell Culture
Primary Human Bronchial Epithelial Cells (HBEpC) were obtained from PromoCell (Cat.# C-12640). HBEpC were isolated from the surface epithelium of human bronchi and stain positive for cytokeratin. HBEpC are useful for investigating the function and pathology of the respiratory system and were used as controls. Human pulmonary alveolar epithelial cells type I (AT1) were obtained from AcceGen Biotechnology (Cat. # ABC-TC3770) and cultured following the manufacturer’s guidelines and were used as controls.
Twenty-four lots of culture expanded human hUC-MSCs were utilized in this study, isolated from human umbilical cord tissue from normal, healthy births, voluntarily donated with a fully executed informed consent form. Sixteen of those lots were obtained from a biotechnology company that manufactures hUC-MSCs for use in clinical trials (Medistem Panama, City of Knowledge of the Republic of Panama, a laboratory licensed by the Panamanian Ministry of Health, following Good Tissue Practices 21 CFR 1271). The cell lots were passage 5 and were received frozen in dry shippers and stored at -150°C until studied. Manufacturing methodology is described in detail in publications of clinical trials that used these hUC-MSCs for treatment(33, 34).
For the other eight lots, umbilical cords were obtained from an FDA-registered tissue bank licensed by the AATB in the United States (FEI 300367004), according to current Good Tissue Practices from healthy, full-term, scheduled uncomplicated C-sections. Written informed consent was obtained from the donors. The isolation, selection and culture were performed by Aidan Research and Consulting LLC for research purposes only. The isolation process was done using the Umbilical Cord Dissociation Kit, human (Miltenyi Biotec Cat. # 130-105-737) following the manufacturer’s guidelines. When colony-forming units reached 70% of confluency, hUC-MSCs were selected using the MSC Phenotyping Kit, human (Miltenyi Biotec Cat. # 130-125-285) and >95% positive cells for CD73/CD105 were sorted using SH800 cell sorter (Sony Biotech). Cells were expanded through passage 5 and used for the measurements reported here.
The intended use of all cells was for research only and not for clinical use in human participants. Researchers did not have access to any identifying information of biospecimens. All cell lots used in this study met release criteria, namely: 75% viability and >95% positive for CD90, CD73, CD105 cell surface markers as determined by flow cytometry. These 24 lots were all used as samples for the subsequent experiments, and one of the Aidan Research and Consulting lots was transfected and used as a positive control.
2.2 Transient Transfection
For transfection, 6,000 hUC-MSCs per cm2 were plated in 100cm2 cell culture dishes. Once they reached 70% of confluency, 20µg of the ACE2 and TMPRSS2 dual expression vector pDUO2-hACE2-TMPRSS2 (InvivoGen Cat. # pduo2-hace2tpsa) was transfected using Lipofectamine™ Stem Transfection Reagent ( Thermo Fisher Scientific Cat. # STEM00015) following the manufacturer’s instructions. Forty-eight hours after transfection cells were either only fixated using Image-iT™ Fixation Kit (Thermo Fisher Scientific Cat. # R37602) for imaging or lysed using RIPA buffer (Thermo Fisher Scientific Cat. # 89901) with 1X protease and phosphatase inhibitor (Thermo Fisher Scientific Cat. # 78444) for Western Blot analysis.
2.3 Protein preparation and Western blot
Human lung homogenates were purchased from the OriGene tissue bank (CP565585, CP565542, CP565577, CP565443, CP565542 and CP565586). Whole protein was obtained by sonication of hUC-MSCs (transfected and non-transfected), AT1 and HBEpC scrapped with RIPA buffer supplemented with 1X protease and phosphatase inhibitor and quantified using the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific Cat. # A53226). 25 µg of protein was added to 4x LDS loading buffer and incubated at 50°C for 5 minutes. SDS-PAGE was performed with Criterion TGX Stain-free 4-20% Gel (Bio-Rad Cat. # 5678093) and transferred to a PVDF membrane using the iBlot™ 2 Gel Transfer Device (Thermo Fisher Scientific Cat. # IB21001). Membranes were blocked for 1 hour in StartingBlock™ T20 (TBS) Blocking Buffer (Thermo Fisher Scientific Cat. # 37543) at room temperature and incubated overnight at 4°C with 1:500 Rabbit anti-ACE2 (Thermo Fisher Scientific Cat. # MA5-32307), 1:500 primary antibody TMPRSS2 made in rabbit (Abcam Cat. # ab92323), and 1 hour at room temperature with 1:10,000 Mouse anti-GAPDH (Millipore, MAB374). Membranes were incubated in secondary antibodies, 1:5,000 Alexa Fluor 680 goat anti-rabbit (Thermo Fisher Scientific Cat. # A21076) and 1:5,000 Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific Cat. # A21202), for one hour at room temperature. Detection of relevant proteins and images were taken using iBright FL1500 Imaging System (Thermo Fisher Scientific). For relative quantification, the volume intensity of the bands was obtained using iBright software. The relative expression was calculated by dividing the values to GAPDH.
2.4 Quantitative real-time Polymerase chain reaction (qPCR)
Total RNA was isolated from cells using the Trizol™ Plus RNA Purification Kit (Thermo Fisher Scientific Cat. # 12183555) and DNA was removed using the TURBO DNA-free Kit Thermo Fisher Scientific Cat. # AM1907 from all hUC-MSC lots, AT1 and HBEpC. Additionally, RNA isolated from human lung tissues (OriGene Technologies; cat.#: CR559346, CR559185, CR560789, CR562469 and CR561266) was included as a positive control (n=5). All RNA extractions were then quantified using a Varioskan LUX™ (Thermo Fisher), and their integrity was checked using a 1% E-Gel™ EX Agarose Gel (Thermo Fisher Scientific Cat. # G401001). Subsequently, 1µg of purified RNA was reverse-transcribed to cDNA using the iScript™ cDNA Synthesis Kit (Biorad Cat. # 1708890) following the manufacturer protocol. Then, 20 ng of cDNA was amplified by qPCR using the TaqMan™ Fast Advanced Master Mix along with TaqMan™ Gene Expression Assays for ACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), and for the reference gene PPIA (Hs99999904_m1). All qPCR reactions were performed in triplicates on a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific). Raw cycle thresholds values were calculated using the QuantStudio™ Design and Analysis Software v.1.5.1 using automatic baseline settings and a threshold of 0.3. The relative expression of genes of interest was normalized to the expression of PPIA. A Mann-Whitney Rank Sum Test was used to calculate statistically significant differences between expression of ACE2 and TMPRSS2 in human lung RNA, AT1 cells, HBEpC and hUC-MSCs.
Cells were plated on 12-well glass-bottomed MatTek plates (P12G-1.5-14-F) and only fixed using an Image-iT™ Fixation Kit (Thermo Fisher Scientific Cat. #R37602) following the manufacturer instructions. Primary antibody ACE2 anti-mouse (Thermo Fisher Scientific Cat. # MA5-31395), in a dilution 1:50, was added to the indicated wells and left overnight at 4°C. The primary antibody TMPRSS2 made in rabbit (Abcam Cat. # ab92323), in a dilution 1:50 was used and incubated overnight at 4°C. Secondary antibodies Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific Cat. # A21202) and Cyanine3 goat anti-rabbit (Thermo Fisher Scientific Cat. # A10520), in a dilution 1:2000, were added to the wells and incubated for one hour at room temperature. Coverslips were mounted onto the slides using Prolong DAPI (Thermo Fisher Scientific Cat. # P36935) and images were taken using a Lionheart FX automated microscope (BioTek) using the same exposure settings in all the groups.
2.6 Flow Cytometry
Single cell suspensions of 24 hUC-MSC lots, one HBEpC and one AT1 were single stained with primary antibody TMPRSS2 made in rabbit (Abcam Cat. # ab92323) or Rabbit anti-ACE2 (InvitrogenThermo Fisher Scientific, Cat. # MA5-32307) with a secondary PE-Cy5.5 goat anti-rabbit antibody (InvitrogenThermo Fisher Scientific, Cat. # L42018). Non-stained controls and Non-Specific-Binding (stained only with PE-Cy5.5 goat anti-rabbit) were used to identify false positive populations and these values were subtracted from all the sample groups. The cells were resuspended in 300 µL of sorting buffer (0.05% FBS in PBS) and analyzed using the flow cytometry functions on a Sony SH800S Cell Sorter (SONY Biotechnology).
2.7 Statistical analysis
SigmaPlot 12.5 (Systat Software) was used for all statistical analyses. Means and standard errors of the means were calculated for all relevant quantities. One-way ANOVA (Mann-Whitney Rank Sum Test) was used for all comparisons. A p-value ≤ 0.05 was taken as the level for statistical significance.