Research Progress of Mesenchymal Stem Cell Therapy for Severe COVID-19

Abstract

Corona virus disease 2019 (COVID-19) refers to a type of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Sixty million confirmed cases have been reported worldwide until November 29, 2020. Unfortunately, the novel coronavirus is extremely contagious and the mortality rate of severe and critically ill patients is high. Thus, there is no definite and effective treatment in clinical practice except for antiviral therapy and supportive therapy. Mesenchymal stem cells (MSCs) are not only characterized by low immunogenicity and homing but also have anti-inflammatory and immunomodulation characteristics. Furthermore, they can inhibit the occurrence and development of a cytokine storm, inhibit lung injury, and exert antipulmonary fibrosis and antioxidative stress, therefore MSC therapy is expected to become one of the effective therapies to treat severe COVID-19. This article will review the possible mechanisms of MSCs in the treatment of severe COVID-19.

Introduction

Corona virus disease 2019 (COVID-19) refers to a type of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which was first reported in Wuhan, Hubei Province, China, in December 2019 [1]. With the spread of SARS-CoV-2, cases of COVID-19 have been detected not only in other provinces of China but also other countries. In the early morning hours of January 31, 2020, Beijing time, the World Health Organization (WHO) announced that the outbreak of novel coronavirus pneumonia constituted a public health emergency of international concern [2]. By November 29, 2020, a total of 60 million confirmed cases have been reported worldwide, causing a total of 1.44 million deaths.

Thus, the WHO issued a statement that all countries should take strong containment measures to control and prevent the spread of COVID-19. In China, as an acute respiratory infectious disease, COVID-19 has been listed as a category B infectious disease stipulated in “Law of the Peoples' Republic of China on Prevention and Treatment of Infectious Diseases,” but has been prevented and controlled as a category A infectious disease. Patients with COVID-19 present with fever, fatigue, and dry cough as their main symptoms and a few of them exhibit nasal congestion and diarrhea, while severe patients develop dyspnea and hypoxemia 1 week later and can rapidly develop acute respiratory distress syndrome (ARDS), septic shock, metabolic acidosis, and coagulopathy, leading to very difficult clinical treatment [1,3]. More importantly, severe patients may have medium to low fever or even no obvious fever during the course of COVID-19.

In addition, one of the typical diagnostic features of severe patients is that lymphocytes are significantly decreased, but neutrophils are increased [3 –5] accompanied by bilateral ground glass opacity on chest computed tomography (CT) scans. Even more, levels of a lot of cytokines, such as interleukin-6 (IL-6), granulocyte colony-stimulating factor (GCSF), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and tumor necrosis factor-α (TNF-α), were significantly increased in intensive care unit (ICU) patients, indicating that a cytokine storm has occurred [6,7], leading to severe organ damage or even death. Although the research and case reports of COVID-19 have been increasing, no specific drugs or vaccines have been found against the virus until now.

Studies have shown that mesenchymal stem cells (MSCs) have been widely used to repair tissue damage [8 –10], inflammation [11,12], and autoimmune diseases [13 –15] due to their unique biological characteristics. A number of clinical research projects using MSCs to treat severe COVID-19 have been launched [16 –22]. This article will review the epidemiological characteristics and pathophysiological mechanisms of COVID-19, as well as possible mechanisms of MSCs, in the treatment of severe COVID-19.

Epidemiological Characteristics of COVID-19

Researchers sequenced the genomes of samples obtained from patients with unexplained new coronavirus pneumonia in Wuhan and discovered a coronavirus that had never appeared before. The WHO named it 2019 novel coronavirus (2019-nCoV) [23]. Subsequently, it was officially named SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV) in February 2020 [24].

As for the origin of the virus, the latest results indicate that bats are the most likely wild animals that carry SARS-CoV-2 [25], which belongs to the β-coronavirus genus [26]. The main source of SARS-CoV-2 infection is patients infected with the new coronavirus, and its main routes of transmission are respiratory transmission and close contact transmission along with aerosol transmission in closed environments. In addition, fecal–oral transmission may also occur, as reported by some studies [27,28]. According to the age and gender of infected persons nationwide, the population is generally susceptible, but elderly persons and those with certain underlying diseases are at greater risk. Most confirmed cases were with good prognosis, but a few confirmed patients, such as elderly patients and those with certain underlying diseases, can become critically ill with poor prognosis [7,29,30].

Pathophysiological Mechanisms of COVID-19

Studies have shown that angiotensin-converting enzyme 2 (ACE2) may be the receptor for SARS-CoV-2 to infect host cells, which is widely expressed in various human tissues, especially in the lungs, kidneys, heart, and islets [31]. SARS-CoV-2 binds to the ACE2 receptor of host cells through the densely glycosylated spike S protein, a class I trimeric fusion protein. When the S1 subunit binds to the host cell receptor, it destroys the stability of the trimer before fusion [26,32]. Furthermore, SARS-CoV-2 infection can cause multiple organ injury, mainly lung injury [33,34]. Chen et al. [3] reported that 17 COVID-19 patients of the 99 early cases developed ARDS, accompanied by respiratory failure, and eventually multiple organ failure, resulting in a total of 11 deaths.

In addition to the direct damage to lungs caused by SARS-CoV-2, it can also induce a cytokine storm, resulting in aggravation of inflammation [6]. A cytokine storm is an excessive immune response stimulated by viruses, bacteria, and external factors. Severe cytokine storm can produce significantly higher levels of proinflammatory cytokines through a specific feedback mechanism, including interferon (IFN), IL, chemokines, and TNF, which are responsible for multiorgan dysfunction [35,36]. Huang et al. [6] detected the expression of 27 inflammatory immune response cytokines in the plasma of 41 COVID-19 patients, including 13 ICU patients and 28 non-ICU patients. The results showed that the plasma concentrations of IL-1β, IL-7, IL-9, IL-10, basic fibroblast growth factor (bFGF), GCSF, IFN, IP-10, MCP-1, MIP-1α, MIP-1β, platelet-derived growth factor (PDGF), TNF-α, and vascular endothelial growth factor (VEGF) in patients were significantly increased compared with healthy adults, whereas the plasma concentrations of IL-2, IL-7, IL-10, GCSF, IP-10, MCP-1, MIP-1α, and TNF-α in ICU patients were significantly increased compared with non-ICU patients, indicating that the cytokine storm is closely related to the severity of COVID-19.

When the cytokine concentration increases, the immune cells will be overactivated, thereby resulting in multiple organ damage, especially to the lungs, and even ARDS, which can cause damage to COVID-19 patients such as sudden deterioration, severe and critical illness, or even death.

Limitations of Clinical Treatment for COVID-19

In the face of the sudden outbreak of SARS-CoV-2, there are no special therapies currently for patients other than supportive therapies. According to current research, the occurrence of COVID-19 is related to ACE2, so drugs targeting ACE2 can be used to treat COVID-19 [37 –39]. Because the SARS-CoV-2 strain is similar to SARS-CoV (nearly 79%) and Middle East respiratory syndrome coronavirus (MERS-CoV; nearly 50%) in genetic and transmission routes [26], drugs that are effective against MERS-CoV and SARS-CoV can be used for SARS-CoV-2, such as ribavirin (inhibit DNA and RNA viruses), lopinavir (antiviral protease inhibitor), and remdesivir (inhibit viral RNA polymerase) [40].

Moreover, there are also a number of other broad-spectrum antiviral drugs for the treatment of COVID-19 [41 –43]. Therefore, antimicrobial treatment can be applied appropriately, except for antiviral drugs in the clinic. However, for severe patients, in addition to antiviral therapy, the treatments for reducing excessive inflammation and lung damage are required to be discovered [6]. At present, appropriate glucocorticoids can help patients reduce excessive inflammation and modulate the body's immune response [44]. However, secondary infection and long-term complications may also occur when treating COVID-19 patients with high-dose corticosteroids [45].

Therefore, scholars are actively seeking new therapies to treat patients with severe COVID-19. It has been reported that advanced treatment, such as convalescent plasma and stem cell therapy, can improve the therapeutic effect of severe patients [18,46].

For stem cell therapy, MSCs have been widely used in the treatment of a variety of diseases, from basic research to clinical trials [47 –49], and their safety and efficacy have been demonstrated in many clinical trials [50 –52]. Currently, Shanghai University, Capital Medical University, the First Affiliated Hospital of Zhengzhou University, and other institutions are cooperating in the research on MSC transplantation for COVID-19 patients. Seven confirmed patients recruited in the Beijing You'an Hospital, China [18], including one critically severe patient, four severe patients, and two common patients, were enrolled for transplantation by 1 × 10⁶ MSCs per kg of body weight. No acute infusion-related or allergic reactions were observed within 2 h. All patients had no symptoms of fever, fatigue, shortness of breath, or hypoxemia after 2–4 days of MSC transplantation. Moreover, chest CT scans showed that the bilateral ground glass opacity and pneumonia infiltration levels were largely reduced on the 9th day and the nucleic acid tests of four patients were negative on the 13th day, while the rest of the patients also improved significantly, and no adverse reactions were observed, which proved the safety and reliability of MSC transplantation.

In addition, Sengupta et al. [17] explored the safety and efficacy of exosomes derived from bone marrow MSCs to treat 24 patients with severe COVID-19. No adverse events were observed within 72 h of exosome administration; levels of the acute phase reactants, C-reactive protein (CRP), ferritin, and D-dimer, were decreased significantly, while CD3⁺, CD4⁺, and CD8⁺ T lymphocyte counts increased sharply.

Although the expired treatment options remain to be explored, current data suggest that MSCs may be an option for the treatment of severe COVID-19.

Possible Mechanisms of MSCs in the Treatment of Severe COVID-19

In recent years, stem cells have attracted much attention in the field of medicine, and it is generally believed that they have the potential to treat several diseases [53 –59]. MSCs are pluripotent stem cells derived from the mesoderm and ectoderm, with the capacity for self-renewal and differentiation into multiple cell types [60,61]. They were first discovered in the bone marrow [62] and subsequently found in other organs, such as the umbilical cord, placenta, and adipose tissue [63 –66]. The International Association for Cell Therapy has defined the minimal identification criteria for MSCs as follows: cells that are adherent under standard culture conditions in vitro; cells that mostly express CD105, CD73, and CD90 (≥95% of the total cells); and cells that seldom express CD45, CD34, CD14, CD11b, CD79a, CD19, or HLA-II-type molecules (≤2% of the total cells). Moreover, they have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes under the induction medium in vitro [67].

Compared with embryonic stem cells, MSCs are easily available, more ethical, and easy to freeze and thaw under standard conditions in vitro, making clinical application more convenient and safer [68,69]. Furthermore, MSCs have not only low immunogenicity and homing properties but also anti-inflammatory and immunomodulatory properties [70]. At present, many clinical applications of MSCs in the treatment of severe COVID-19 patients have been carried out and have achieved inspiring therapeutic effects [16 –22]. In the next section, the mechanisms of MSCs in the treatment of severe COVID-19 are summarized as follows (Fig. 1).

FIG. 1.

Potential mechanisms of MSCs in the treatment of severe COVID-19. COVID-19, corona virus disease 2019; MSCs, mesenchymal stem cells.

Immunomodulatory effects of MSCs

When the immune system is overactivated by viral infection (such as SARS-CoV-2), autoimmune disease, or other factors, it will secrete a large amount of proinflammatory factors, resulting in cytokine storm production, so only one or more inflammatory factors are suppressed, which may not be an effective way to treat patients with severe COVID-19 [71]. It has been reported that the immune regulatory function of MSCs depends on the environment of host cells. When MSCs are exposed to the inflammatory microenvironment, they exert their immunosuppressive properties to inhibit the occurrence and development of cytokine storms, which is realized through the paracrine pathway of MSCs or direct interaction with immune cells [72 –74].

The involvement of MSCs in immune regulation is closely related to T cells, natural killer cells (NK cells), dendritic cells (DCs), B cells, and macrophages [75 –77]. MSCs can not only suppress the immune response by releasing soluble factors such as TGF-β1 and prostaglandin E2 (PGE2) to reduce the number of IL-2 receptors, leading to reduced proliferation of activated T cells [78], but also inhibit the proliferation and activation of T cells by programmed death-1 (PD-1), which is an important regulator of T cell activation and homeostasis. When exposed to proinflammatory cytokines, MSCs will secrete PD-1 ligands to inhibit CD4⁺ T cell activation and downregulate IL-2 expression [79].

Moreover, MSCs can inhibit the proliferation of NK cells induced by cytokines as well as prevent the cytotoxic function and production of cytokines. This inhibitory effect is related to sharp downregulation of the surface expression of activated NK receptors, such as NKp30, NKp44, and NKG2D [80]. In addition, MSCs can regulate the immune response by adjusting DC maturation and other functions [81]. Franquesa et al. [76] reported that MSCs derived from human adipose tissue exert an indirect effect on B cell proliferation through immunomodulation of T cells and a direct effect on B cells by inhibiting plasmablast differentiation and induction of IL-10-producing regulatory B cells. Some studies have shown that MSCs can induce macrophage differentiation through direct or indirect mechanisms to play an immunosuppressive role [82,83].

In clinical application of MSC therapy for COVID-19 3–6 days later, the excessive activation of CXCR3⁺CD4⁺ T cells, CXCR3⁺CD8⁺ T cells, and CXCR3⁺ NK cells disappeared, which further proved that MSCs can inhibit the activation of immune cell proliferation and play a role in immune regulation [18]. Moreover, Liang et al. [84] reported that MSCs combined with antibiotics and thymosin α1 were used to treat a severe COVID-19 patient, and many clinical indices and symptoms of the patient improved after intravenous injection of 5 × 10⁷ MSCs three times, with a 3-day interval. For instance, CT images showed remission of the inflammation symptoms; the counts of white blood cells, neutrophils, and lymphocytes returned to normal levels and so did the counts of CD3⁺, CD4⁺, and CD8⁺ T cells; the patient was subsequently transferred out of ICU; and the throat swab test reported negative 4 days later. These suggested that MSC therapy may be one of the ideal options for treating severe COVID-19 patients.

To sum up, MSCs play an immunosuppressive role in regulating the excessive immune response in patients with severe COVID-19.

Anti-inflammatory effects of MSCs

Because severe COVID-19 patients present with damaged organs, many inflammatory factors are released, including TNF-α, IL-1, IL-6, IL-12, IFN-α, IFN-β, MCP-1, and IL-8 [6,85]. Previous studies have shown that MSCs protect patients from excessive inflammatory invasion through a series of action modes [12]. First, MSCs express IL-1 receptor antagonists that downregulate IL-1 expression [86]. Second, a negative feedback loop is established. Macrophages secrete TNF-α to activate MSCs and secrete anti-inflammatory proteins with TSG-6 to inhibit the NF-κB signaling pathway, thereby regulating the cascade reactions of proinflammatory cytokines [87]. Third, a second negative feedback loop is established. Endotoxins, nitric oxide, or other possible damage-related molecular patterns from damaged tissues and macrophages activate MSCs to secrete PGE2, converting macrophages to the phenotype that secretes IL-10 [88].

One study showed that after intravenous injection of 1 × 10⁶ MSCs into septic mice, most MSCs were located in the lungs. Moreover, the serum IL-10 level was increased within 6–12 h, whereas the levels of IL-6 and TNF-α were significantly decreased [89]. In addition, MSCs significantly reduced the inflammatory factor level of TNF-α and increased the anti-inflammatory cytokine level of IL-10 in the clinical treatment of patients with severe COVID-19, which showed that MSC transplantation can suppress the excessive inflammation responses in severe patients [18].

Differentiation of MSCs into alveolar epithelial cells

Patients with COVID-19 developed diffuse lesions in bilateral lungs during the severe stage after having small lung nodules in the early stage, according to the chest CT scans [90,91]. Thus, lung damage gradually aggravated as the disease progressed, and some affected patients developed more severe pathological features of pulmonary fibrosis due to the SARS-CoV-2.

Studies have shown that MSCs have the potential of multidirectional differentiation, that is, they can be induced into a variety of cells in different environments [60]. Wang et al. [92] showed that when MSCs are cocultured with alveolar epithelial cells in vitro, they could be successfully differentiated into type II alveolar epithelial cells, which can not only repair the damaged alveolar structure but also effectively restore its function. Kun et al. [93] showed that exogenous BrdU-labeled MSCs could be transdifferentiated into type II alveolar epithelial cells with normal structure and function, which could improve the survival rate of alveolar epithelial cells. Moreover, a large number of animal experiments showed that MSCs had an expected therapeutic effect on lung injury caused by various causes [86,94,95], laying a foundation for the treatment of lung injury caused by SARS-CoV-2.

Antipulmonary fibrosis of MSCs

Pulmonary fibrosis can result from diseases caused by previous coronaviruses, such as SARS and MERS, or COVID-19 caused by SARS-CoV-2 [96 –99]. In addition to the alveolar damage, some severe COVID-19 patients also exhibit pulmonary fibrosis characteristics because of inflammation and cytokine imbalance caused by SARS-CoV-2, which may be due to overactivation of fibroblasts in the repair process of lungs and their conversion to myofibroblasts, resulting in the deposition of extracellular matrix [100,101]. Evidence shows that MSCs are able to migrate to damaged tissues, inhibit tissue fibrosis, and reduce tissue damage [102 –107]. The mechanisms by which MSCs might treat pulmonary fibrosis include paracrine effects, transdifferentiation into alveolar epithelial cells, anti-inflammatory and antioxidative stress, and immunomodulatory effects, among others.

The next section focuses on paracrine effects and antioxidative stress, as determined through in vitro and in vivo studies.

Paracrine effects of MSCs for ameliorating pulmonary fibrosis

In the past, researchers have reported that MSC-derived conditioned medium (MSC-CM) or MSC-derived exosomes (MSC-Ex) could exert antipulmonary fibrosis functions. Similarly, Mansouri et al. demonstrated that a single intravenous injection dose of purified exosomes derived from MSCs could effectively prevent and revert core features of bleomycin-induced pulmonary fibrosis, such as blunting collagen deposition, restoring lung architecture, and so on [108]. In addition, Shen et al. [109] demonstrated that MSC-CM was capable of inhibiting infiltration of inflammatory cells, inhibiting alveolar epithelial cell apoptosis, and decreasing collagen deposition for protecting against bleomycin-induced pulmonary fibrosis through a paracrine mechanism.

Furthermore, MSCs can secrete various cytokines, such as hepatocyte growth factor (HGF) [110] and keratinocyte growth factor (KGF), which can not only reduce the injury of alveolar epithelial cells but also inhibit the occurrence of pulmonary fibrosis. Li et al. [111] reported that in indirect coculture of bone marrow MSCs and silica-injured RLE-6TN cells (an ATII cell line stemmed from rat normal alveolar epithelium) for 48 h, the levels of hydroxyproline, collagen I, collagen III, and fibronectin (FN) were decreased compared with 6TN+silica group. Meanwhile, the expression of HGF, KGF, and bone morphogenetic protein (BMP-7) was significantly increased compared with 6TN+silica group. Thus, the possible mechanism of improving pulmonary fibrosis may be the paracrine effect of MSCs. In vivo studies by Cahill et al. [110] revealed that in bleomycin-induced murine models, early administration of MSCs reduced pulmonary fibrosis and was associated with reduced levels of the proinflammatory cytokine IL-1β, decreased levels of apoptosis, and significantly increased levels of HGF, but HGF knockdown in MSCs was unable to protect against pulmonary fibrosis. Therefore, these protective effects were, in part, mediated by HGF derived from MSCs.

Antioxidant mechanism

Pulmonary fibrosis is often accompanied by oxidative stress, and its oxidative effect is greater than that of the antioxidation effect, producing many intermediate oxidative products [112,113]. Furthermore, excessive oxidative stress is an important factor leading to pulmonary fibrosis [114]. In the virus-infected microenvironment of the lungs, the oxidative stress response may lead to activation and proliferation of fibroblasts for forming pulmonary fibrosis. Therefore, reducing excessive oxidative stress can prevent the occurrence and development of pulmonary fibrosis. MSCs have certain antioxidant and scavenging effects on oxygen free radicals, which are helpful to reduce pulmonary fibrosis [115]. Superoxide dismutase (SOD) is an important component of the antioxidant enzymatic defense system, which converts superoxide free radicals into H₂O₂ and the total antioxidant capacity (T-AOC) reflects the endogenous antioxidant capacity of the organs.

One study found that SOD activity and T-AOC in lung tissues of the MSC direct injection group were increased significantly compared with the bleomycin-induced group. Thus, the oxidative stress in pulmonary fibrosis could be significantly reduced by MSC injection [115]. Furthermore, Ni et al. [116] investigated the effects and underlying mechanism of MSC treatment on pulmonary fibrosis. The results showed that collagen deposition and hydroxyproline levels of lung tissues in the MSC group were reduced compared with the bleomycin-induced group. Meanwhile, the activity of SOD was significantly increased and the level of malondialdehyde (MDA) was decreased. Moreover, western blot analysis showed that protein expression levels of Nrf2 (a transcription factor considered as the master regulator of the antioxidant response) and Nrf2-mediated antioxidant stage phase II enzymes such as quinine oxidoreductase1 (NQO1), gamma-glutamylcysteine synthetase (γ-GCS), and heme oxygenase-1 (HO-1) were strikingly increased compared with the bleomycin-induced group. These results suggest that MSCs can reduce oxidative damage in pulmonary fibrosis through the Nrf2-ARE pathway.

Conclusion and Future Prospects

In view of the current understanding of the pathological mechanism of COVID-19 and the unique biological characteristics of MSCs (large-scale culture in vitro, differentiation potential, immune regulation, and anti-inflammatory and antifibrosis characteristics), the development of MSCs is a promising approach in view of the current SARS-CoV-2 pandemic and the lack of specific treatment for patients with severe COVID-19. At present, many research teams have tried to use MSC therapy to treat common or severe COVID-19 patients and achieved good clinical prospects according to relevant literature [16 –22,84,117] and the website of ClinicalTrials.gov (Table 1).

Table 1.

Clinical Trial of Mesenchymal Stem Cells for Corona Virus Disease 2019

No.	Title/trial No.	Intervention	Results	Country	Status	Phase
1	Transplantation of ACE2-mesenchymal stem cells improves the outcome of patients with COVID-19 pneumonia	MSC source: unknown; dose: 1.0 × 10⁶ cells/kg, a single infusion; and transplantation route: intravenous	TNF-α ↓; IL-10, VEGF ↑; ground glass opacity and pneumonia infiltration ↓; the oxygen saturations rose to ≥95% at rest; and no delayed hypersensitivity or secondary infections were detected.	China	Completed	Unknown
2	Clinical remission of a critically ill COVID-19 patient treated by human umbilical cord mesenchymal stem cells	MSC source: allogeneic human umbilical cord; dose: 5.0 × 10⁷ cells/patient, three times; and transplantation route: intravenous	The concentrations of serum bilirubin, CRP, ALT, and AST ↓; the white blood cell count and neutrophil count decreased to the normal level; the lymphocyte count increased to the normal level; CD3⁺ T cells, CD4⁺ T cell, and CD8⁺ T cells increased to normal levels; the pulmonary inflammation ↓; negative throat swab; and no obvious side effects.	China	Completed	Unknown
3	Intravenous infusion of human umbilical cord Wharton's jelly-derived mesenchymal stem cells as a potential treatment for patients with COVID-19 pneumonia	MSC source: Wharton's jelly-derived human umbilical cord; dose: 1.0 × 10⁶ cells/kg, a single infusion; and transplantation route: intravenous	CRP, IL-6, and TNF-α ↓; CD3⁺, CD4⁺, and CD8⁺ T cells ↑; ground glass opacity and pneumonia infiltration ↓; the oxygen saturation rose to 98% at rest; negative throat swab; and no delayed hypersensitivity or secondary infections were detected.	China	Completed	Unknown
4	Exosomes derived from bone marrow mesenchymal stem cells as treatment for severe COVID-19	MSC source: bone-marrow; dose: 15 mL of ExoFlo was added to 100 mL of normal saline, a single infusion; and transplantation route: intravenous	CRP, ferritin and D-dimer ↓; CD3⁺, CD4⁺, and CD8⁺ T cells ↑; average PaO₂/FiO₂ ratio ↑; 71% of the patients recovered after a mean of 5.6 days; 16% of the patients expired; and 13% of the patients remained critically ill.	United States	Completed	Unknown
5	Adipose-derived mesenchymal stromal cells for the treatment of patients with severe SARS-CoV-2 pneumonia requiring mechanical ventilation. A proof-of-concept study	MSC source: adipose tissue; dose: most patients received median number of 0.98 × 10⁶ cells/kg, twice; and transplantation route: intravenous	CRP, ferritin, LDH, IL-6 ↓; B lymphocytes, CD4⁺, and CD8⁺ T cells ↑; D-dime and fibrinogen in most patients ↓; did not develop a thromboembolic event; and no adverse events.	Spain	Completed	Unknown
6	Clinical study using mesenchymal stem cells for the treatment of patients with severe COVID-19	MSC source: menstrual blood; dose: 1.0 × 10⁶ cells/kg, three times; and transplantation route: intravenous	CRP, IL-6, FiO₂, and neutrophils ↓; lymphocytes and CD4⁺ T cells ↑; lung exudate lesions were adsorbed; SaO₂ and PO₂ improved; and negative throat swab.	China	Completed	Unknown
7	Human umbilical cord-derived mesenchymal stem cell therapy in patients with COVID-19: a phase 1 clinical trial	MSC source: human umbilical cord; dose: 3.0 × 10⁷ cells/patient, three times; and transplantation route: intravenous	IL-6, CRP, ALT, creatinine, SF, and platelets improved; no serious adverse events associated with UC-MSCs; lung exudate lesions were adsorbed; PaO₂/FiO₂ improved; and all patients recovered.	China	Completed	Phase 1
8	Safety and feasibility of umbilical cord mesenchymal stem cells in patients with COVID-19 pneumonia: a pilot study	MSC source: umbilical cord; dose: 1.0 × 10⁸ cells/patient, four times; and transplantation route: intravenous	IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and CRP recovered to normal levels; CD4⁺ T cells, CD8⁺ T cells, and NK cells recovered to normal levels; ground glass opacity improved; oxygenation index improved; and no infusion-related or allergic reactions were found.	China	Completed	Unknown
9	Repair of Acute Respiratory Distress Syndrome by Stromal Cell Administration (REALIST) (COVID-19) (NCT03042143)	MSC source: umbilical cord; dose: 1.0 × 10⁸ cells/patient, four times; and transplantation route: intravenous	Unknown	United Kingdom	Completed recruiting	Phase 1; Phase 2
10	Mesenchymal Stem Cell Treatment for Pneumonia Patients Infected With COVID-19 (NCT04252118)	MSC source: unknown; dose: 3.0 × 10⁷ cells/patient, three times; and transplantation route: intravenous	Unknown	China	Recruiting	Phase 1
11	Clinical Research Regarding the Availability and Safety of UC-MSC Treatment for Serious Pneumonia and Critical Pneumonia Caused by the 2019-nCOV Infection (NCT04269525)	MSC source: umbilical cord; dose: 9.9 × 10⁷ cells/patient, four times; and transplantation route: intravenous	Unknown	China	Recruiting	Phase 2
12	A Pilot Clinical Study on Aerosol Inhalation of the Exosomes Derived From Allogeneic Adipose Mesenchymal Stem Cells in the Treatment of Severe Patients With Novel Coronavirus Pneumonia (NCT04276987)	MSC source: adipose; dose: 2.0 × 10⁸ nanovesicles derived from MSCs/3 mL/day for 5 consecutive days; and transplantation route: aerosol inhalation	Unknown	China	Completed	Phase 1
13	A Phase II, Multicenter, Randomized, Double-blind Placebo-controlled Trial to Evaluate the Efficacy and Safety of Human Umbilical Cord-derived Mesenchymal Stem Cells in the Treatment of Severe COVID-19 Patients (NCT04288102)	MSC source: human umbilical cord; dose: 4.0 × 10⁷ cells/patient, three times; and transplantation route: intravenous	Unknown	China	Completed	Phase 2
14	Treatment of COVID-19 Patients Using Wharton's Jelly Mesenchymal Stem Cells (NCT04313322)	MSC source: cord tissue of newborns; dose: 1.0 × 10⁶ cells/kg, three times; and transplantation route: intravenous	Unknown	Jordan	Recruiting	Phase 1
15	Cell Therapy Using Umbilical Cord-derived Mesenchymal Stromal Cells in SARS-CoV-2-related ARDS (NCT04333368)	MSC source: umbilical cord; dose: 1.0 × 10⁶ cells/kg, three times; and transplantation route: intravenous	Unknown	France	Recruiting	Phase 1; Phase 2
16	Clinical Research of Human Mesenchymal Stem Cells in the Treatment of COVID-19 Pneumonia (NCT04339660)	MSC source: human umbilical cord; dose: 1.0 × 10⁶ cells/kg, a single infusion, depending on the condition to be given again at an interval of 1 week; and transplantation route: intravenous	Unknown	China	Recruiting	Phase 1; Phase 2
17	Umbilical Cord-derived Mesenchymal Stem Cells for COVID-19 Patients With Acute Respiratory Distress Syndrome (ARDS) (NCT04355728)	MSC source: umbilical cord heparin along with best supportive care; dose: 1.0 × 10⁸ cells/patient, twice; and transplantation route: intravenous	Unknown	United States	Completed	Phase 1; Phase 2
18	MSC therapy for SARS-CoV-2-related acute respiratory distress syndrome (NCT04366063)	MSC source: unknown; dose: 1.0 × 10⁸ cells/patient or 1.0 × 10⁸ cells+extracellular vesicles derived from MSCs/patient, twice; and transplantation route: intravenous	Unknown	Iran	Recruiting	Phase 2; Phase 3
19	Clinical Trial to Assess the Safety and Efficacy of Intravenous Administration of Allogeneic Adult Mesenchymal Stem Cells of Expanded Adipose Tissue in Patients with Severe Pneumonia Due to COVID-19 (NCT04366323)	MSC source: allogeneic adipose; dose: 8.0 × 10⁷ cells/patient, twice; and transplantation route: intravenous	Unknown	Spain	Active, not recruiting	Phase 1; Phase 2
20	MSCs in COVID-19 ARDS (NCT04371393)	MSC source: unknown; dose: 2.0 × 10⁶ cells/kg, twice; and transplantation route: intravenous	Unknown	United States	Active, not recruiting	Phase 3
21	Clinical use of stem cells for the treatment of COVID-19 (NCT04392778)	MSC source: umbilical cord; dose: 3.0 × 10⁶ cells/kg, three times; and transplantation route: intravenous	Unknown	Turkey	Recruiting	Phase 1; Phase 2
22	MSCs for acute respiratory distress syndrome due to COVID-19 (NCT04416139)	MSC source: unknown; dose: 1.0 × 10⁶ cells/kg, a single infusion; and transplantation route: intravenous	Unknown	Mexico	Recruiting	Phase 2
23	Efficacy of Intravenous Infusions of Stem Cells in the Treatment of COVID-19 Patients (NCT04437823)	MSC source: human umbilical cord; dose: 5.0 × 10⁵ cells/kg, three times; and transplantation route: intravenous	Unknown	Pakistan	Recruiting	Phase 2
24	Mesenchymal Stem Cell Infusion for COVID-19 Infection (NCT04444271)	MSC source: bone marrow; dose: 2.0 × 10⁶ cells/kg, twice; and transplantation route: intravenous	Unknown	Pakistan	Recruiting	Phase 2
25	Therapeutic study to evaluate the safety and efficacy of DW-MSCs in COVID-19 patients (NCT04535856)	MSC source: unknown; dose: 5.0 × 10⁷ or 1.0 × 10⁸ cells/patient, a single infusion; and transplantation route: intravenous	Unknown	Indonesia	Active, not recruiting	Phase 1
26	Cord blood-derived mesenchymal stem cells for the treatment of COVID-19-related acute respiratory distress syndrome (NCT04565665)	MSC source: cord blood; dose: MSCs, single infusion, depending on physician discretion to be given again within 7 days; and transplantation route: intravenous	Unknown	United States	Recruiting	Phase 1

2019-nCoV, 2019 novel coronavirus; ACE2, angiotensin-converting enzyme 2; ARDS, acute respiratory distress syndrome; COVID-19, corona virus disease 2019; CRP, C-reactive protein; IFN, interferon; IL, interleukin; MSC, mesenchymal stem cell; NK, natural killer; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor; UC-MSCs, umbilical cord mesenchymal stem cells; VEGF, vascular endothelial growth factor.

In MSC treatment of common/severe COVID-19 and other viral infections [118,119], intravenous infusion was applied in most clinical trials, except for only one clinical trial currently using nebulized inhalation. Some COVID-19 clinical trial results showed a single intravenous injection dose of (1–3) × 10⁶ cells per kg of body weight, with single or multiple (two to four times) infusions, which were decided by the symptoms or signs of COVID-19 patients. The negative conversion of viral nucleic acid was used as the primary end point, and the improvement of some indices such as the oxygenation index (PaO₂/FiO₂), inflammatory factors, and CT was used as the secondary end point in the treatment of COVID-19. The window period of MSC transplantation is defined as the time when symptoms or signs of COVID-19 patients are worsening after symptomatic treatment [18].

Chen et al. [118] reported that H7N9-induced ARDS patients received three to four intravenous injections, with a single infusion of 1 × 10⁶ MSCs per kg of body weight, in the early stage or the late stage of H7N9 infection. The results showed that MSCs can significantly reduce the mortality rate of H7N9-induced ARDS. Shi et al. [119] assessed the safety and initial efficacy of umbilical cord mesenchymal stem cell (UC-MSC) intravenous infusion in patients with acute-on-chronic liver failure (ACLF)–hepatitis B virus (HBV) infestation at 0.5 × 10⁶ cells per kg of body weight, three times, at 4-week intervals. The results showed that UC-MSC infusion significantly increased the survival rate of HBV-ACLF patients, reduced the model of end-stage liver disease (MELD) scores, and improved the liver function. However, in the practical application of MSCs in treatment of severe COVID-19, the following aspects should be paid attention to.

First of all, because of the limited number of clinical trials of MSCs for severe COVID-19, placebo-controlled, randomized, large-sample clinical trials are needed to support whether MSCs can become a standard therapy for severe COVID-19. Second, the dose, route, frequency, and stage of MSC engraftment, as well as standardized protocols for MSC transplantation, are required to be explored.

Third, further research must be intensively explored regarding limitations of MSC therapy in general and for viral infections. The general limitations include (1) the genetic stability of MSCs: the genetic stability of MSCs was mainly affected by multiple passages, hypoxic culture conditions, and cryopreservation [120,121]. Chromosomal aberrations were detected earlier in the subculture of MSCs under hypoxic conditions than under normal oxygen conditions. Moreover, the frequency of karyotype aberrations increased with the increase of passage time, and the risk increased after the fourth passage [120]. A significant number of nonclonal chromosomal aberrations, including monosomies and structural changes, were apparent after cryopreservation of MSCs [121]. Therefore, as far as possible, we conducted clinical trials using MSCs within four generations under normal oxygen conditions before cryopreservation. (2) MSCs spontaneously transform into other cells, even malignant cells: due to potential differentiation, MSCs can spontaneously transform into other cells through long-term culture, most of which can not only differentiate into harmless cells such as osteoblasts, cardiomyocytes, and adipocytes but may also differentiate into malignant cells [122,123]. (3) Risk of zoonoses associated with cell culture reagents: fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) are the common reagents for culturing and cryopreservation of MSCs in vitro, but can result in negative effects, such as zoonotic contamination diseases and toxic side effects, even hypersensitivity reactions, in clinical treatment. Therefore, the alternative medium supplements of FBS, such as human blood components of autologous or allogeneic donors, have been studied for clinical use [124,125]. Furthermore, serum-free culture systems containing optimal growth factors and alternative cryopreservation methods to DMSO for MSC culture are under study [68].

In addition to these, the limitation for viral infections (especially for lung injury) includes the formation of pulmonary embolism and thrombosis. Studies have shown that a large number of MSCs are trapped in lungs after infusion at first, followed by their relocation to other organs. In the model of ischemia/reperfusion-induced lung injury treated by MSCs, pulmonary thrombosis and embolism were found in various sizes in the pulmonary artery, including the large pulmonary artery, medium pulmonary artery, and small pulmonary arteriole, suggesting that MSCs were prone to forming aggregates in pulmonary microcirculation and exacerbate post-transplantation embolic events [126]. Furthermore, accumulating clinical evidence has shown that intravascular delivery of MSCs can cause substantial vascular obstruction events [127]. The same complications may occur when treating COVID-19 with MSCs, so it is necessary to closely monitor the postinput circulation indicators and find out the possible risks immediately.

Last but not least, MSC products, including MSC exosomes and cytokines or genetically modified MSCs, may provide new therapeutic options for severe COVID-19. Studies have found that MSCs can be used as vectors to express exogenous genes such as HGF to exert a better inhibiting effect on pulmonary fibrosis [107,128]. Finally, the paracrine mechanism of MSCs to alleviate pulmonary fibrosis caused by severe COVID-19 must be interpreted clearly, including specific cytokine signaling pathways to prevent the progress of pulmonary fibrosis, as few cytokines secreted by MSCs have been identified as antifibrotic factors.

In general, there is no specific vaccine for COVID-19 during the current pandemic. By studying the mechanism of MSCs and assessing the pathogenesis of COVID-19, MSC therapy is expected to be one of the best choices for treatment of severe COVID-19.

Footnotes

Author Disclosure Statement

The authors declare no conflicts of interest.

Funding Information

This work was supported by the Jilin Provincial Subject under grant no. 20190201010JC.

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