key: cord-0848268-z695bamh
authors: Avanzini, Maria A.; Mura, Manuela; Percivalle, Elena; Bastaroli, Francesca; Croce, Stefania; Valsecchi, Chiara; Lenta, Elisa; Nykjaer, Giulia; Cassaniti, Irene; Bagnarino, Jessica; Baldanti, Fausto; Zecca, Marco; Comoli, Patrizia; Gnecchi, Massimiliano
title: Human mesenchymal stromal cells do not express ACE2 and TMPRSS2 and are not permissive to SARS‐CoV‐2 infection
date: 2020-11-14
journal: Stem Cells Transl Med
DOI: 10.1002/sctm.20-0385
sha: 76b3619e915945e509f9a3a8bf52d9098ceb3960
doc_id: 848268
cord_uid: z695bamh

Anti‐inflammatory and immune‐modulatory therapies have been proposed for the treatment of COVID‐19 and its most serious complications. Among others, the use of mesenchymal stromal cells (MSCs) is under investigation given their well‐documented anti‐inflammatory and immunomodulatory properties. However, some critical issues regarding the possibility that MSCs could be infected by the virus have been raised. Angiotensin‐converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2) are the main host cell factors for the Severe Acute Respiratory Syndrome‐Coronavirus 2 (SARS‐CoV‐2) entry but so far it is unclear if human MSCs express or do not these two proteins. To elucidate these important aspects, we evaluated if human MSCs from both fetal and adult tissues constitutively express ACE2 and TMPRSS2 and, most importantly, if they can be infected by SARS‐CoV‐2. We evaluated human MSCs derived from amnios, cord blood, cord tissue, adipose tissue and bone marrow. ACE2 and TMPRSS2 were expressed by the SARS‐CoV‐2‐permissive human pulmonary Calu‐3 cell line but not by all the MSCs tested. MSCs were then exposed to SARS‐CoV‐2 wild strain without evidence of cytopathic effect. Moreover, we also excluded that the MSCs could be infected without showing lytic effects since their conditioned medium after SARS‐CoV‐2 exposure did not contain viral particles. Our data, demonstrating that MSCs derived from different human tissues are not permissive to SARS‐CoV‐2 infection, support the safety of MSCs as potential therapy for COVID‐19. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: Human mesenchymal stromal cells (hMSCs) are currently under investigation for the treatment of COVID‐19. However, the potential safety profile of hMSCs in this context has never been defined since none has described if they express ACE2 and TMPRSS2, the main host cell factors for SARS‐CoV‐2 entry, and if they can be infected by SARS‐CoV‐2. We provide the first evidence that ACE2 and TMPRSS2 are not expressed in hMSCs derived from both adult and fetal human tissues and, most importantly, that hMSCs are not permissive to SARS‐CoV‐2 infection. These results support the safety of MSCs as potential therapy for COVID‐19.

In December 2019, an outbreak caused by a novel coronavirus, later named SARS-CoV-2, occurred in China and rapidly spread throughout several other Countries becoming pandemic. 1 , the disease caused by SARS-CoV-2, mainly affects the respiratory system and can progress to respiratory distress syndrome (ARDS), a fatal condition in more than 50% of the cases. 1 Up to 30% of COVID-19 patients may develop cardiac damage due to acute coronary syndrome, septic heart, or acute myocarditis. [2] [3] [4] As there are no specific therapeutics for treating COVID-19, in particular for the most severe cases complicated by ARDS or acute fulminant myocarditis, new innovative therapeutic approaches are urgently needed. Anti-inflammatory drugs have been proposed as possible approaches and it has been suggested that immunosuppressive therapy may mitigate the manifestations of COVID-19. 5, 6 Mesenchymal stem cells (MSCs) possess immunomodulatory properties as demonstrated by numerous in vitro and animal model studies. These effects are mediated by cytokines and soluble factors able to modulate the systemic but also the tissue inflammatory response. 7 In particular, when administered intravenously, most MSCs lodge in the pulmonary vascular bed where they survive for at least a few days. 8 Importantly, several clinical studies, including phase III trials, documented their efficacy in the control of graft-vs-host disease in recipients of allogeneic hematopoietic stem cell transplantation, and there are evidences also in other immune-mediated disorders. 7 In addition, there are existing preclinical and a few preliminary feasibility and safety clinical studies supporting further investigation of cellbased therapies, particularly with MSC, or the MSC-derived secretome, for potential treatment of ARDS and acute myocarditis. [7] [8] [9] For all these reasons, it has been hypothesized that the administration of MSCs may be useful in the treatment of severe cases of COVID-19. A pilot study conducted in China on seven patients affected by SARS-CoV-2 pneumonia has reported feasibility and safety of MSC therapy, 10 and a total of 39 phase I/II trials testing MSC-therapy for COVID-19 are currently registered in ClinicalTrials.gov. However, infusion of MSCs in the presence of active viremia has raised some critical issues regarding the possibility that the virus may infect MSCs, causing not only lack of efficacy but also possible deleterious effects.

Angiotensin-converting enzyme 2 (ACE2) is the main host cell receptor for SARS-CoV-2 entry, and the virus uses the host cell transmembrane serine protease II (TMPRSS2) for Spike envelope protein priming. 11 It is known that ACE2 and TMPRSS2 are present on the surface of several human cells, such as alveolar cells and capillary endothelium, while immune cells, such as T and B lymphocytes, and macrophage are negative for ACE2. 12 Whether human MSCs of any origin constitutively express ACE2 and/or TMPRSS2 is so far unclear.

Leng et al 10 claimed the absence of ACE2 and TMPRSS2 expression on the umbilical cord-derived MSC infused in their study, even though a clear demonstration was not reported. In addition, SARS-CoV-2 could also use other, possibly unknown, receptors for cellular entry.

Accordingly, the aim of the present work was to evaluate whether human MSCs from various sources express ACE2 and TMPRSS2 and, most importantly, if they are permissive to SARS-CoV-2 infection.

An expanded method section is available as Supporting Information.

The MSC lines were previously isolated from amniotic membrane of human placenta, cord blood, cord tissue, bone marrow, and adipose tissue, expanded and characterized. [13] [14] [15] [16] [17] All the MSCs fulfill the criteria set by the ISCT. 18 To perform the experiments, we used passage 3 to 5 MSCs. Conditioned media were generated as 

Human mesenchymal stromal cells (hMSCs) are currently under investigation for the treatment of COVID-19. However, the potential safety profile of hMSCs in this context has never been defined since none has described if they express ACE2 and TMPRSS2, the main host cell factors for SARS-CoV-2 entry, and if they can be infected by SARS-CoV-2. This study provides the first evidence that ACE2 and TMPRSS2 are not expressed in hMSCs derived from both adult and fetal human tissues and, most importantly, that hMSCs are not permissive to SARS-CoV-2 infection. These results support the safety of MSCs as potential therapy for COVID-19.

MSCs, Calu-3, and VERO E6 were infected with 100 μL (100 TCID 50 / mL) of a previously titrated SARS-CoV-2 wild strain, isolated from an infected patient. The virus was incubated for 1 hour and then removed; the medium was changed every 3 days. Cells were scored every other day and for 1 week using a light microscope to detect the appearance of cell rounding, detachment, degeneration, and/or syncytium formation, called hereafter cytopathic effect (CPE). To verify if the cells tested can be infected and allow SARS-CoV-2 replication even in the absence of CPE, at day 7 from infection, supernatants from each MSC, Calu-3, and VERO E6 culture were collected and inoculated into VERO E6. CPE occurrence was monitored for 1 week.

All results are reported as mean ± SD and the data were analyzed with a one-way or two-way analysis of variance followed by Bonferroni all pair-wise multiple comparison test using the InStat software (GraphPad Software, Inc., San Diego, California; http://www. graphpad.com). P values less than .05 were considered statistically significant.

We assessed ACE2 and TMPRSS2 expression on human fetal MSCs MSCs infected in adhesion showed the typical spindle shape morphology with no signs of CPE ( Figure 3B and Table 1 ). Also, MSCs infected in suspension and seeded in 24 well plates were found adherent to plastic and showed no CPE starting from the day postinoculum up to 7 days (data not shown). Conversely, a 100% CPE was detected in the control VERO E6 cell line ( Figure 3B and Table 1 ). As expected, 21 we did not observe CPE in Calu-3 cells for the entire observation period ( Figure 3B and Table 1 ). At 7 days after infection, supernatants from all experiments were collected and tested in a reinoculation experiment in VERO E6 cells ( Figure 3A) . As expected, a typical CPE was evident in all the wells inoculated with supernatants collected from infected VERO-E6 and Calu-3 cultures ( Figure 3C and Table 1 ). On the contrary, none of the supernatant collected from the different MSC cell lines induced CPE, demonstrating the absence of viral replication inside the MSC lines and, consequently, the absence of SARS-CoV-2 infection ( Figure 3C and Table 1 ). 

MSCs by avian influenza or herpesviruses has been reported, 24,25 and a similar concern has been raised for the SARS-CoV-2. So far, evaluation of MSC infectiveness by coronaviruses, in particular by SARS-CoV-2, has not yet been investigated and described. There is only one study in which absence of the viral host cell factors ACE2 and TMPRSS2 expression on human umbilical cord blood-derived MSCs is claimed, but this cannot be considered a surrogate indicating a condition of refractoriness to infection. 10 Indeed, the presence on MSCs of a different receptor able to mediate viral entry cannot be ruled out.

Moreover, if all human MSC types express or not ACE2 is still a matter of debate and solid data are missing.

Using a variety of assays, we unequivocally demonstrated that human MSCs do express neither ACE2 nor TMPRSS2. Most importantly, using a direct infection assay, our study provides the first evidence that human MSCs derived from fetal and adult tissues are not permissive to SARS-CoV-2 infection. 

The data that support the findings of this study are available on request from the corresponding author.

https://orcid.org/0000-0001-7435-4328

Additional supporting information may be found online in the Supporting Information section at the end of this article. F I G U R E 1 hACE2 and TMPRSS2 expression in human MSCs from fetal and adult tissues. A, Relative quantification by RT-qPCR and the 2 −ΔΔCt method of ACE2 (left) and TMPRSS2 (right) mRNA levels in MSCs from cord blood (CB-MSC, n = 2 lines), cord tissue (CT-MSC, n = 2 lines), amnios (A-MSC, n = 4 lines), bone marrow (BM-MSC, n = 4 lines), and adipose tissue (AT-MSC, n = 2 RNA samples from the same line), compared with the Calu-3 cell line. Each sample was loaded in triplicate. Columns represent mean 2 −ΔΔCt values and SD. The GAPDH was used as a reference gene. *P < .05, **P < .01, ***P < .001 vs Calu-3. B, Representative Western blot of hACE2 and TMPRSS2 in cell lysates (left) and serum free 48 hours-conditioned media (right) of the different MSC types, and the positive control cell line Calu-3. Actin was used as loading control for cell lysates. The amount of conditioned media loaded for each sample was obtained from 1 × 10 5 cells. C, ACE2 protein quantification by ELISA assay in serum-free 48 hours-conditioned media generated by 1 × 10 5 MSCs. The bars represent the mean and SD of triplicates obtained from a single line of Calu-3 and 4 distinct lines of both A-MSCs and BM-MSCs. *P < .001 vs Calu-3. MSCs, mesenchymal stromal cells F I G U R E 2 MSC infection with a SARS-CoV-2 Spike pseudovirus. A-MSCs, BM-MSCs, and Calu-3 were transduced with a replicationdefective, green fluorescent protein (GFP)-tagged, pseudotyped retrovirus bearing the SARS-CoV-2 spike envelope protein (Spike pseudovirus; A, B), or the pantropic vesicular stomatitis virus glicoprotein (VSV-G; C). The green signal is present if cells are infected. Costaining for ACE2 (A) or TMPRSS2 (B) is shown in red. Nuclei were counterstained with the nuclear dye DAPI (Blue). Scale bar = 20 μm F I G U R E 3 MSC infection with SARS-CoV-2. A, Experimental design. First, MSCs from CB, CT, A, BM and AT, and the permissive cell lines VERO E6 and Calu-3 were incubated with SARS-CoV-2 wild-type strain. CPE defined as cell rounding, detachment, degeneration, and/or syncytium formation was assessed by microscopic analysis, and the cells maintained in culture for 7 days, changing the medium every three 3 days. Then, supernatants were collected and tested in a reinoculation experiment on VERO E6. B, Representative phase contrast images of VERO-E6, Calu-3, and hMSCs before (not infected-upper panels) and after exposure to SARS-CoV-2 (infected-lower panels). Scale bar = 200 μm. hMSCs and Calu-3 showed no signs of CPE and maintained their typical spindle-shaped and epithelial morphology, respectively, whereas VERO E6 displayed clear signs of CPE, since almost all the cells were round or fused into syncytia or detached. C, Representative phase contrast images of VERO E6 cells after exposure to supernatant generated by the infected cell lines respectively indicated on top of each frame. Scale bar = 200 μm. CPE, cytopathic effect; hMSCs, human mesenchymal stromal cells; MSCs, mesenchymal stromal cells

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