Dodging COVID-19 Infection: Low Expression and Localization of ACE2 and TMPRSS2 in Human Umbilical Cord-Derived Mesenchymal Stem Cells

Background


Background
With the worsening of the public health emergency caused by the global spread of the novel coronavirus disease 2019 (COVID-19) comes a pressing need to understand the molecular mechanisms driving the infection within the body (1). The spike glycoprotein of SARS-CoV-2, the virus responsible for COVID-19, allows for entry into cells via human angiotensin converting enzyme II (ACE2) once primed by the cellular serine protease TMPRSS2 (2)(3)(4). Therefore, cell membrane ACE2 and TMPRSS2 are integral components of viral transmission and spread (2).
In light of current treatments for severe cases of COVID-19 having had mixed results or serious adverse events (9)(10)(11), there remains a global interest to nd safe and effective avenues of treatment.
hUC-MSCs were used experimentally to treat seven patients with con rmed COVID-19 pneumonia in China (32). The pulmonary function of all patients signi cantly improved within two days of hUC-MSC transplantation and levels of TNF-α were signi cantly reduced. Additionally, the gene expression pro le in this study showed that MSCs were ACE2-and TMPRSS2-. However, the hUC-MSCs used for treatment were derived from only one donor, representing a limitation for a broader extrapolation of these results.
In this study, we investigated the expression of ACE2 and TMPRSS2 in hUC-MSCs lines derived from different donors using quantitative polymerase chain reaction (qPCR), Western Blot, immuno uorescence and ow cytometry. Human pulmonary alveolar cells type I, human lung homogenates, human lung RNA, and one lot of hUC-MSCs transfected with an ACE2 expression plasmid were used as controls.
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, fullterm, 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 con uency, 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 ow 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.
Detection of relevant proteins and images were taken using iBright FL1500 Imaging System (Thermo Fisher Scienti c). For relative quanti cation, the volume intensity of the bands was obtained using iBright software. The relative expression was calculated by dividing the values to GAPDH.

Quantitative real-time Polymerase chain reaction (qPCR)
Total RNA was isolated from cells using the Trizol™ Plus RNA Puri cation Kit (Thermo Fisher Scienti c Cat. # 12183555) and DNA was removed using the TURBO DNA-free Kit Thermo Fisher Scienti c 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 quanti ed using a Varioskan LUX™ (Thermo Fisher), and their integrity was checked using a 1% E-Gel™ EX Agarose Gel (Thermo Fisher Scienti c Cat. # G401001). Subsequently, 1µg of puri ed RNA was reverse-transcribed to cDNA using the iScript™ cDNA hUC-MSCs (n=24) had signi cantly lower (p=0.002) ACE2 expression relative to GAPDH compared with lung tissue homogenates (n=6) in Western blot analyses (Figure 1). A trend for lower expression of ACE2 in hUC-MSCs was observed when compared to hUC-MSCs transfected with ACE2 expressing plasmid (n=1). HEBpC and AT1 (n=1) also showed low expression of ACE2 but higher expression of TMPRSS2. TMPRSS2 levels in hUC-MSCs were signi cantly lower when compared to lung tissue (p=0.008).
Although expression was low, TMPRSS2 levels were variable between hUC-MSCs from different donors. ACE2 bands were observed to have a molecular weight of 120 kDa, while TMPRSS2 showed a thin band around 50 kDa. Expression of both proteins was normalized to GAPDH, which was observed at 37 kDa.

Low gene expression of ACE2 and TMPRSS2
Expression of ACE2 and TMPRSS2 was observed in all human lung RNA samples (n=5) while little to no expression was observed in hUC-MSC (n=24), AT1 (n=1) and HBEpC observed on qPCR. AT1 expressed less TMPRSS2 than the lung tissue and did not express ACE2. For each sample, relative expression of the genes was averaged to represent differences between the sample groups. Signi cant differences in the expression of ACE2 and TMPRSS2 were observed between the human lung RNA and hUC-MSCs (p ≤0.001 for both) according to the Mann-Whitney Rank Sum Test (Figure 2). Overall, expression of TMPRSS2 was higher in all groups.

ACE2 is not localized in the cell membrane of hUC-MSCs
Fluorescence microscopy was conducted to determine the presence and localization of ACE2 and TMPRSS2 in the membrane of the cells (HBEpC, AT1, hUC-MSC transfected with pDUO2-hACE2-TMPRSS2 plasmid, and hUC-MSCs). ACE2 was not observed in the cell membrane of hUC-MSCs ( Figure   3). However, the expression of TMPRSS2 was observed in the cell membrane in all the groups. hUC-MSCs transfected with the plasmid restored the expression of ACE2 in the cell membrane ( Figure 3).

Discussion
Mesenchymal stem cells (MSCs) are currently being considered as a potential treatment of COVID-19 and its associated complications (22,23). Given that ACE2 and TMPRSS2 are crucially involved in transmission and spread of the virus, we sought to investigate their expression in hUC-MSCs. In this study, we have demonstrated the negative expression of ACE2 and low expression of TMPRSS2 in 24 lots of hUC-MSCs derived from different donors. We investigated levels of gene expression and localization of the protein in the cells via different methods: gene expression was not found using qPCR and Western Blot in any of the 24 lots of hUC-MSCs compared to the positive control of human lung RNA; ACE2 was not detected in the hUC-MSCs membrane with immuno uorescence, and ow cytometry revealed that only 4.6% and 29.6% of MSCs positively expressed ACE2 and TMPRSS2, respectively. This broadens the novel ndings of Leng et al. regarding the expression of these speci c genes in hUC-MSCs(32) to a larger number of cell lots derived from different donors.
However, we clearly observed differences in the expression of TMPRSS2 at the protein level and localization in different cell types that do not correlate with the gene expression results reported in Leng et al.'s work and in another recent study (35). In addition to protein expression, localization, and cell population, the choice of antibody was of particular importance for the design of this experiment: certain antibodies were suitable for e.g. Western blot experiments, but unsuitable for immuno uorescence.
Choosing rabbit anti-ACE2 (# MA5-32307) allowed us to use a single antibody for Western blot, immuno uorescence, and ow cytometry, and levels of expression were corroborated with all techniques. Furthermore, levels of gene expression may vary depending on the tissue from which MSCs are derived: ACE2 levels in hematopoietic induced pluripotent stem cells (iPSCs), for example, would not correspond to those levels found in Wharton's jelly derived hUC-MSCs.
It is critical, when reporting in-vitro levels of expression of a particular protein, to correctly assess this with several techniques instead of relying solely on gene expression or mRNA levels. This is particularly important when ndings are intended to serve as a starting point for clinical applications. Levels in mRNA do not always correlate with levels in protein content (36)(37)(38)(39). A single method of measurement may therefore not be enough to fully investigate expression levels; we are here proposing that qPCR, Western blot, immuno uorescence and ow cytometry are to be used as complements of each other in detecting ACE2 and TMPRSS2 expression to have a well-rounded overview at the cellular level.
Lower expression or lack of expression of ACE2 and TMPRSS2 could have crucial implications for the design of future therapeutic options for COVID-19. Since the virus engages ACE2 as the entry receptor (40) and employs the cellular serine protease TMPRSS2 for the spike glycoprotein S priming (41,42), ACE2and low TMPRSS2 expressing cells should likely remain uninfected by the virus (Figure 5).
Demonstrating that hUC-MSCs do not appreciatively express ACE2 and express low levels of TMPRSS2, coupled with their immunomodulatory, anti-in ammatory and antimicrobial properties, could position them as a viable treatment option. After being administered to a COVID-19 patient, a large majority of hUC-MSCs should be able to exert their therapeutic action via the secretion of anti-in ammatory molecules while "dodging" the virus ( Figure 5). To date, MSCs have been used to treat pulmonary conditions such as idiopathic pulmonary brosis, acute respiratory distress syndrome and chronic obstructive pulmonary disease (14). MSC-derived exosomes were also able to revert pulmonary brosis in a mouse model (43). Likewise, trophic factors secreted by MSCs (MTF), which can be administered by inhalation, have shown therapeutic bene ts for pulmonary disease (44,45). In light of this, international trials are currently ongoing to treat COVID-19 with mesenchymal stem cells derived from a variety of tissues (46).
The intent of investigating the expression of ACE2 and TMPRSS2 was to determine the likelihood of hUC-MSCs becoming infected by SARs-Cov-2, as it would be counterproductive to supply the virus with a fresh in ux of susceptible cells. Quality control is of utmost importance when considering treatment with MSCs for COVID-19; in addition to investigating the infectivity potential of MSCs, attention should also be given to concerns about the variability of TF/CD142 expression among cells lots, which may trigger blood clotting and thromboembolism in this hypercoagulable pathology (47). High expression of TF/CD142 on cells used in the treatment of Covid-19 could lead to dire consequences if the cells enhanced the already present pro-thrombotic effects of the viral infection itself (48,49).
The extent of the ability of hUC-MSCs to mitigate the devastating cytokine storm observed in critically ill patients should be a next avenue in further research, particularly to ascertain which cytokines can be induced back to their normal levels in the presence of hUC-MSC secretions -this could lead to more targeted therapeutics. Since the levels of in ammatory cytokines and chemokines have been reported to be abnormally elevated in critically ill COVID-19 patients (5,32), the immunomodulatory effects of hUC-MSCs on these cytokines, and particularly in a setting of simultaneous overexpression, should be investigated promptly. Caution should be exerted, however, to administer hUC-MSCs outside of the clinically demonstrable cytokine storm, as MSC immunomodulatory effects could paradoxically aid the virus in earlier phases of the infection. Furthermore, AGTR2 could be a third marker of interest in addition to ACE2 and TMPRSS2, since its a nity to the spike protein of the virus appears to be higher than ACE2 (50). Over the course of this study, we began investigating AGTR2 expression in hUC-MSCs compared to controls, nding negative expression (data not shown). We hope to expand upon these ndings in future experiments.
Multiple repositories were contacted at the beginning of this study and alveolar cells type II were not available to us at the time, resulting in signi cant limitations for this study. The impossibility to obtain said alveolar cells for research in a reasonable timeframe meant choosing human lung and as pulmonary alveolar cells type I as controls in limited quantities. Alveolar cells type II would have been more ideal to replicate the more intrinsic mechanisms of SARS-CoV-2 infection, but we hope that these results may be extended to that type of cell when it becomes more readily available for research. Only twenty-four different donors were considered for this experiment, but we expect that these results could be replicated with a larger number of hUC-MSC lots. Finally, our current laboratory does not possess BSL-4 facilities that would allow culturing and testing the infection rate of SARS-CoV-2 in hUC-MSCs to verify our ndings.
Levels of ACE2 expression are of interest for COVID-19 research, and a recent report has indicated low ACE2 expression levels in MSCs derived from different tissues, though it is unclear how many cell lines were used in that particular context (35). We have studied twenty-four different hUC-MScs cell lines and have determined levels ACE2 and TMPRSS2 therein with several techniques, leading to our robust conclusion of no ACE2 expression and low TMPRSS2 infection in Wharton's jelly-derived hUC-MSCs.

Conclusions
hUC-MSCs have the potential to provide a safe, effective treatment for critically ill COVID-19 patients, which could help mitigate the devastating economic and public health consequences caused by the rapid worldwide spread of SARS-CoV-2. We have demonstrated negative expression of ACE2 and low expression of TMPRSS2, key proteins in the SARS-Cov2 infection process, in twenty-four lots of hUC-MSCs, and we hope that these results will encourage further research into hUC-MSCs for the treatment of the in ammatory effects of COVID-19 infection.

Declarations
Author contributions NHR and JHH contributed to the conception and design of the work. DB, LF, and AL contributed to the acquisition, analysis, and interpretation of data. All authors contributed to the writing of the manuscript. All authors read and approved the nal manuscript.
We would like to thank Ms. Dorita Avila for her help in the preparation of this manuscript, and Brenda Mendoza Flores for her assistance with immuno uorescence images.

Con ict of Interests
Neil H Riordan is a shareholder and CEO of Aidan Research and Consulting LLC. All other authors declare no con icts of interest.

Data Availability
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Ethics statement
The experiments involving human tissue were carried out in accordance with relevant guidelines and regulations from the Panamanian Ministry of Health, following Good Tissue Practices 21 CFR 1271 (related to sample screening and processing) and American Association of Tissue Banks (AATB) guidelines. The intended use of all cells was for research only and not for clinical use in human participants. No institutional committee reviewed this protocol, as this research was "not human-subject" research as de ned by 45 CFR 46.104(d)(4) (researchers did not have access to any identifying information of biospecimens).

Funding
This study was privately funded by Aidan Research and Consulting LLC.

Consent for Publication
Not applicable