The novel coronavirus disease 2019 (COVID-19) has been a pandemic health issue since 30 January 2020 (1). Among those infected across the world, approximately 80% of the cases experienced mild disease. However, nearly 20% of infected patients progress to severe clinical courses, of which 5% are considered critical cases (2). In critically ill patients, mortality rates were superior to 50% (3, 4). The severity of COVID-19 infection varies among individuals (5) and is attributed to differences in the magnitude of the dysregulated cytokine responses to viral infection (6). Immunomodulatory therapies, namely, dexamethasone (7), tocilizumab (8), and barcitinib (9) have been applied to restore immune dysregulation processes and are effective in reducing mortality in some severe COVID-19 patients. However, despite the administration of immunotherapies, COVID-19 could lead to high mortality rates among patients admitted to intensive care units (ICU) with severe respiratory failure (10). To date, many emerging inhibitors of specific inflammatory pathways have been proposed (1). However, most treatments are waiting to be validated in large-scale trials.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with variable magnitudes of proliferation and activity. They are derived from various tissues and organs, including placenta choriodecidual-membranes (11, 12). The beneficial effects of MSCs in alleviating disease are attributed to their potential to modulate cytokine secretion, inflammatory cell migration, and immune function responses, and more specifically, their immunosuppressive properties against dendritic cells, NK cells, monocytes, macrophages, and T cells. Furthermore, this response is often associated with a concomitant increase in the regulatory T cell (Treg) fraction (13–17).

For treating pulmonary diseases, MSCs present a notable strength due to their ability to be retained in the lungs for a period ranging from hours to days (13). The therapeutic effects of MSC-based therapies in ARDS have been described in several experimental and clinical studies, including COVID-19 (18). A MSC clinical trial in severe COVID-19 patients conducted in the United States demonstrated benefits in attenuating clinical severity and associated cytokine changes (19). The immunomodulatory effects of MSCs in one case of COVID pneumonia with mild ARDS have been previously studied in China (20). Recent studies have also revealed improved clinical outcomes with MSC therapy, with reported reductions in mortality and morbidity rates amongst COVID-19 patients (21–23). However, little is known regarding the specific details of its immune-modulatory effect.

MSCs from placenta choriodecidual membranes (pcMSCs, Matriplax) consist of ex vivo culture-expanded human MSCs isolated from the placenta choriodecidual membranes of healthy adult mothers and have a high penetration capacity into lung tissue (12). pcMSCs suppress airway inflammation in asthmatic rats by increasing the number of Treg cells and are accompanied by a reduced presence of Th17 cells, macrophages, neutrophils, and eosinophils that infiltrate the lung. They have also been reported to improve survival and promote recovery in animal models with LPS-induced acute lung injury (ALI) (24, 25). Thus, treatment with pcMSCs is anticipated to be a feasible therapy for patients with severe or critically ill COVID-19. In this study, we evaluated the safety and clinical efficacy of pcMSC therapy in terms of lung injury scores, namely, the PaO2/FiO2 ratio, immune profiles, and serum inflammatory markers and cytokines in severe ARDS caused by COVID-19 pneumonia.

In this study, we showed that pcMSC treatment was safe and effective in patients with severe COVID-19 who developed ARDS. Nine of the 19 patients in the control group died by day 30, indicating a 47% mortality at our institution, which was consistent with the reported mortality rate of ~50% of critically ill COVID-19 patients (10), while none of the patients treated with pc-MSC died on day 28. It is interesting to note that 2 of the 5-treated patients did not require mechanical ventilation. These patients had identified risk factors for respiratory failure in severe COVID-19, such as higher BMI and a poor PaO2/FiO2 ratio.

All patients had elevated blood inflammatory markers, namely, CRP, D-dimer, LDH, and ferritin, as previously reported (26, 27), although the patients had received dexamethasone and tocilizumab as anti-inflammatory treatments. Treatment with pc-MSC suppressed the hyper-inflammatory state with markedly reduced ferritin, LDH, and CRP levels after 7 days.

SARS-CoV-2 RNAs act as pathogen-associated molecular patterns, detecting toll-like receptors, triggering downstream cascades in innate immune cells, resulting in the production of pro-inflammatory cytokines, such as IL-1β, IL-6, and IFN-γ that induce the synthesis of several liver defense proteins, namely, CRP, LDH, and ferritin (28). These multifunctional peptides, especially LDH and ferritin levels, are independent factors in predicting disease severity and mortality (29, 30). Ferritin, through NF-κB (31) creates a vicious loop by further increasing the production of pro-inflammatory cytokines, IL-1β, IL-6, and IFN-γ, contributing to the development of a cytokine storm syndrome (CSS). The reduction in serum ferritin levels after pcMSC treatment was associated with a reduction in IL-1β, IL-6, and IFN-γ serum levels, suggesting that pcMSC effectively broke the vicious cycle and attenuated the cytokine storm syndrome, resulting in a significant attenuation of the severity of COVID-19 pneumonia in terms of Murray’s lung injury scores.

MSC therapy offers a promising treatment option for severe lung diseases caused by autoimmune, sepsis, and COVID-19 infection (18–20). The underlying cellular and molecular mechanisms of MSC-mediated immunomodulation, although still elusive, are proposed to act via a synergy of cell contact-dependent mechanisms and soluble factors (13, 32), via cytokine-dependent and cytokine-independent functional changes of monocytes/macrophages, dendritic cells, T cells, B cells, and NKT cells (14–16, 33). Peripheral blood immune profiles with tSNE cell clusters revealed a significant change in monocytes, Th cells, B cells, Treg cells, and plasma/plasmablast cell subpopulations after pc-MSC treatment.

Monocytes and macrophages, being the most important innate immune cells, respond mainly to SARS-CoV-2 infections by producing pro-inflammatory mediators through ACE2-independent and ACE2-dependent pathways to remove pathogens and repair tissue injury. However, dysregulation of their functions can cause acute respiratory distress syndrome and cause damage to other vital organs, including the heart (34). During SARS-CoV-2 infection, the subpopulation of circulating CD14⁺CD16⁺ monocytes exhibits a notable contribution to the development of cytokine storms, orchestrating a high level of TNF-α, IL-10, and IL-6 production, which is ultimately related to clinical deterioration and thus, increases the likelihood of admission to the ICU (2, 35). The decrease in the absolute number of subpopulations CD14⁺ CD16⁺ may be attributed to the attenuation of hyper-inflammation responses in the pcMSC treatment group.

In severe COVID-19, lymphopenia (absolute counts <1,000 cells/μl) is considered an indicator of disease severity (36), therapeutic response (37), and disease outcome (38). High levels of pro-inflammatory cytokines such as TNF-α and IL-6 could induce lymphocyte deficiency (38). Our patients had absolute lymphocyte counts <1,000/μl before treatment. These measured lymphocyte numbers in the peripheral blood increased significantly after treatment, probably resulting from the inhibitory effect of pcMSC on pro-inflammatory cytokines such as IL-6.

Lymphocytes in the peripheral blood are critical to generating early control, viral clearance, and disease resolution after SARS-CoV-2 infection (39, 40). Regulatory T cells (Treg) play an important role in the prevention of excessive immune responses to SARS-CoV-2 infection (41). On analysis of the T lymphocyte subpopulations, our patients had initially lower counts of CD4⁺ and CD8⁺ T cells, but not in the proportion of CD4⁺, CD8⁺ NKT, or Treg cells, compared with age-matched healthy controls. Treatment with pcMSC significantly increased the proportion and absolute number of CD4⁺ T and Treg cells. Among CD4⁺ T cell subpopulations, memory T cells are crucial in viral clearance during events associated with re-infection. Although memory T cells were not identified as SARS-CoV2-specific, the presence of more memory T cells may suggest that adaptive immunity is being developed. MSCs facilitate Treg cell formation (14, 42) by inducing the formation of Tregs from conventional T cells (43, 44). The increase in Treg cells is considered responsible for the suppression of hyperinflammatory responses mediated by pcMSC in our patients. Furthermore, MSC treatment induces a switch from pro-inflammatory Th1 cells to anti-inflammatory Th2 cells (45). Increased serum levels of IL-5, IL-4, and IL-13 after pcMSC treatment may support this notion.

An effective immune response to the SARS-CoV infection depends on the activation of CD8⁺ cytotoxic T cells through the killing of virus-infected cells (46). However, although there was a trend for CD8⁺ T cells to increase with pcMSC treatment, statistical significance was not reached. Our data on the role of NK cells were limited. In one study, there were no differences in NK cell levels between responders and nonresponders before and after treatment (47). In another study, NKT cell levels showed an increasing trend among survivors, while in non-survivors, reductions were observed (48). In our study, there was no significant change in NK cells after pcMSC treatment.

B cell-mediated antibody responses through the coordination of other adaptive immune responses, namely, CD4⁺ and CD8⁺ T cells, are essential for effective control of COVID-19 infection (49). B cells contribute to the antiviral immune response by first rapidly releasing germline or near-germline antibodies from plasmablasts via an extrafollicular pathway. In patients with COVID-19 infection, there are increased portions of IgM⁺ memory B cells and CD27⁺CD38⁺ plasma/plasmablast cells circulate in the peripheral blood, compared to healthy controls, suggesting a protective memory response occurred (5). Upon appropriate stimulation of cytokines, B cells undergo class switching and/or enter germinal centers within secondary lymphoid organs to undergo affinity maturation. This maturation process produces long-lived plasma and memory B cells capable of responding to secondary challenges with homotypic or heterotypic antigenic challenges. pcMSC treatment significantly increased the proportion and absolute number of CD27⁺-switched activated B cells and concomitantly suppressed the proportion and absolute number of CD27⁺CD38⁺ plasma/plasmablast cells. These results suggest that pc-MSC treatment induced a shift of B cells from a protective memory state to an active response to COVID-19 infection by enhancing the generation of virus-specific immunoglobulin switched B cells or long-lived plasma cells (50).

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Taipei Medical University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

M-CC and KL wrote the manuscript. H-PK, Y-HH, C-MW and C-LC contributed to the design and implementation of the research. K-LC, ST, P-LW involved in clinical care and collecting clinical specimen. Y-RL, WC, Y-HH and M-JL carried out laboratory analysis. H-PK, C-LC and C-MW revised the manuscript and provided additional comments. All authors read and approved the final manuscript.

This study was supported by the Taiwan Ministry of Science and Technology grants, 109-2314-B-038-097 and 108-2314-B-038-134-MY2.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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