Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells

Abstract The outbreak of COVID‐19 has become a serious public health emergency. The virus targets cells by binding the ACE2 receptor. After infection, the virus triggers in some humans an immune storm containing the release of proinflammatory cytokines and chemokines followed by multiple organ failure. Several vaccines are enrolled, but an effective treatment is still missing. Mesenchymal stem cells (MSCs) have shown to secrete immunomodulatory factors that suppress this cytokine storm. Therefore, MSCs have been suggested as a potential treatment option for COVID‐19. We report here that the ACE2 expression is minimal or nonexistent in MSC derived from three different human tissue sources (adipose tissue, umbilical cord Wharton`s jelly and bone marrow). In contrast, TMPRSS2 that is implicated in SARS‐CoV‐2 entry has been detected in all MSC samples. These results are of particular importance for future MSC‐based cell therapies to treat severe cases after COVID‐19 infection.

and deploy safe and effective vaccines. Currently, a number of COVID-19 vaccines have been rolled out in different countries or are in development. 3  Application of MSCs overexpressing heme-oxygenase-1 (HO-1) improved survival rate, attenuated lung pathological impairments and suppressed inflammatory reaction in a lipopolysaccharide (LPS)induced ARDS rat model. 8 Furthermore, inhibition of (repressor/activator protein) Rap1 in BM-MSCs decreased nuclear factor-kappa B (NF-κB) sensitivity to stress-induced proinflammatory cytokine production and reduced apoptosis. 9,10 Several clinical trials have preliminarily demonstrated the safety and efficacy of intravenous MSCs in patients with COVID-19-related lung diseases. 3,11,12 Conclusive interpretation of these clinical trials is difficult due to the unknown heterogeneity of the MSCs used.
Recently, intravenous administration of MSCs of unknown tissue origin to seven patients with different severities of ARDS resulting from COVID-19 showed in all patients an improvement after two days upon injection linked to an increase of circulating lymphocytes and CXCR3-negative regulated T cells and dendritic cells suggesting a switch from a proinflammatory to an anti-inflammatory state. 11 In another trial, double intravenous injections of 100 ± 20 × 10 6 UC-MSCs (subepithelial lining of an UC) were correlated with improvement of patient survival and a significant decrease of the cytokine storm. 12 The absence of serious adverse events directly related to UC-MSCs infusions suggest the potential safe use of MSCs for COVID-19 cell therapy. 12 Nevertheless, MSCs are highly heterogeneous, pleiotropic and sensitive to different microenvironments and secrete biologically active substances responsively. This characteristic has an impact on their differentiation ability and is an important limitation for their use in therapeutic applications.
Currently available data regarding the expression of ACE2 and TMPRSS2 in MSCs are discordant. Desterke et al. analysed available transcriptome datasets and concluded that both genes are significantly higher expressed in AD-MSCs and BM-MSCs compared to MSCs isolated from the umbilical cord or placenta. 13 In contrast, another study by Avanzini and coworkers showed the absence of expression of ACE2 and TMPRSS2 in MSCs derived from five different tissue sources (amniotic membrane of placenta, cord blood, cord tissue, adipose tissue and bone marrow). 14 The same study also showed that human MSCs are not permissive to SARS-CoV2 infection, 14 which is a prerequisite for their safe therapeutic use for treating COVID-19.
The aim of the present work was to evaluate whether human MSCs from different tissue sources (AD-MSCs, umbilical cord Wharton's jelly MSCs (WJ-MSCs) and BM-MSCs) express ACE2, TMPRSS2 and additional proviral and antiviral associated genes.

| Cell culture
Isolation procedure and MSC specific characterisation were performed as described (Kehl et al.). 4  Medium was changed every 3 days, and cells were passaged with 1× Accutase (Gibco, Life technologies) for 5 min at 37 °C when cells were about 80% confluent. Cells were incubated at 37 °C in an atmosphere with 95% humidity and 5% CO 2 . Table 1 displays an overview of all MSC donor characteristics.

| RNA Extraction
Total RNA was obtained using the RNeasy Mini Kit (Qiagen) and reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's protocol. qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and standard conditions on a QuantStudio 7 flex real-time PCR system (Applied Biosystems). Primers are listed in Table 2. The gene expression level analysis was normalized versus GAPDH and conducted in triplicate.

| Immunohistochemistry
Cells were fixed with 4% paraformaldehyde and incubated over night at 4 °C with primary antibodies (Table 3). Secondary antibodies were incubated for 1 h (Table 4), and pictures were taken and analysed with XX fluorescence microscope.

| RNA sequencing
Strand-specific mRNA-seq libraries for the Illumina platform were generated and sequenced by Eurofins GmbH.

| Bioinformatic processing and analysis
The

| RE SULTS
We have performed RNA sequencing on human MSCs isolated from three different tissue sources (Suppl. Material Table 1  disease, but clinical trials are urgently needed. Our findings emphasize a detailed characterisation before administration of these cells, pointing out tissue-specific as well as donor-specific variability.

ACK N OWLED G EM ENT
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declared no potential conflicts of interest.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
RNA sequencing data that support the findings of this study are available on request from the corresponding author [M.G.].