Stem cell therapy for COVID‐19, ARDS and pulmonary fibrosis

Abstract Coronavirus disease 2019 (COVID‐19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). COVID‐19 mainly causes damage to the lung, as well as other organs and systems such as the hearts, the immune system and so on. Although the pathogenesis of COVID‐19 has been fully elucidated, there is no specific therapy for the disease at present, and most treatments are limited to supportive care. Stem cell therapy may be a potential treatment for refractory and unmanageable pulmonary illnesses, which has shown some promising results in preclinical studies. In this review, we systematically summarize the pathogenic progression and potential mechanisms underlying stem cell therapy in COVID‐19, and registered COVID‐19 clinical trials. Of all the stem cell therapies touted for COVID‐19 treatment, mesenchymal stem cells (MSCs) or MSC‐like derivatives have been the most promising in preclinical studies and clinical trials so far. MSCs have been suggested to ameliorate the cytokine release syndrome (CRS) and protect alveolar epithelial cells by secreting many kinds of factors, demonstrating safety and possible efficacy in COVID‐19 patients with acute respiratory distress syndrome (ARDS). However, considering the consistency and uniformity of stem cell quality cannot be quantified nor guaranteed at this point, more work remains to be done in the future.


| INTRODUC TI ON
In December 2019, an outbreak of unidentifiable pneumonia cases was first officially reported in Wuhan, China. It was subsequently confirmed that the pneumonia is an acute respiratory infectious disease caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel β-coronavirus which had never been reported before. 1,2 As the global epidemic grew and spread rapidly, the World Health Organization (WHO) officially named the new type of disease as coronavirus disease 2019 .  was confirmed to be more contagious than either the severe acute respiratory syndrome (SARS) or Middle East respiratory syndrome (MERS), with a confusing manifestation ranging from asymptomatic patients to severely ill patients with acute respiratory distress syndrome (ARDS) and pulmonary fibrosis, amongst several potential health problems, and has had disastrous consequences for public health management. It can encode 29 proteins and has 79% homology to the SARS virus sequence. The spike glycoprotein (S protein) on its surface is an essential structural protein that mediates its invasion into human cells. Through the host cell receptor-angiotensin converting enzyme 2 (ACE2), SARS-CoV-2 adsorbs onto and enters the host, then replicates, assembles, and releases a large number of viral particles. 3 ACE2 is expressed on the surface membrane of alveolar, tracheal and bronchial epithelial cells in the lung, and monocytes and macrophages in the immune system. It can also be expressed in the heart, kidney and intestines. ACE2 lowers blood pressure and regulates the renin-angiotensin system by inactivating angiotensin II (Ang II) produced by ACE, and serves as a crucial regulator of pulmonary oedema. SARS-CoV-2 utilizes a highly glycosylated homotrimeric S protein to enter the host cell, and its affinity for ACE2 is 10-20 times that of SARS virus, thus enhancing its transmissibility. 4 In fact, although the fatality rate of COVID-19 is lower than SARS (9.6%) and MERS (34.4%), its higher infectious rate has led to a much wider outbreak with significantly more complications in epidemic prevention and control, due to a large number of asymptomatic and mild patients ( Figure 1).

| The novel coronavirus and its infection pathways
For patients with symptoms, the incubation period (time from exposure to onset of symptoms) has a wide range but averages to ~4-5 days. 5 The most common symptoms include dry cough, fever and shortness of breath. Other common symptoms are myalgia, fatigue, sore throat, nausea, vomiting, diarrhoea, conjunctivitis, anorexia and headache (cdc.gov/coronavirus/2019-ncov/hcp/ clinical-guidance-management-patients.html). For a small number of severely ill patients, the disease begins to worsen about 5-10 days F I G U R E 1 COVID-19 pathogenic progression after the onset of symptoms, and complications such as acute respiratory distress syndrome (ARDS) and other end-organ failures could occur. 6 The mortality rate is significantly higher amongst elderly adults over 65. Adults with underlying cardiovascular disease, respiratory disease, endocrine metabolic disease, diabetes or a weakened immune system are the most vulnerable to serious complications of COVID-19. 7

| Cytokine release syndrome
One of the reasons for aggravated severe illness in COVID-19 patients aggravation is the excessive immune response associated with the cytokine release syndrome (CRS), which in turn leads to lung tissue damage, repair imbalance and respiratory failure. The patient could also eventually die from multiple organ failure. 8  Extremely high concentrations of IL-6, GCSF, CRP and TNF-α have also been recorded in COVID-19 patients. 10 The excessive inflammatory CRS could also promote thrombosis and deaths by thromboembolism in critically ill patients. [11][12][13] At present, there is no specific treatment for CRS. In clinical practice, glucocorticoid injections for systemic immune suppression and cytokine inhibitors have been the primary methods. However, the use of glucocorticoids in viral pneumonia carries additional risks of steroid treatment sequelae such as diabetes and osteonecrosis, so there has been controversy in the academic community.

| Acute respiratory distress syndrome
ARDS refers to acute progressive hypoxic respiratory failure caused by various pulmonary and extrapulmonary pathogenic factors other than cardiogenic. 14 According to the Berlin definition, patients with less severe hypoxaemia (as defined by a ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen of 300 or less) are considered to have acute lung injury (ALI), and those with more severe hypoxaemia (as defined by a ratio of 200 or less) are considered to have the ARDS. 15,16 ARDS is a continuous pathological process, and its early stage is ALI. The main manifestations are sudden progressive dyspnoea, varying degrees of cough, less sputum, late cough and bloody sputum. Arterial hypoxaemia is a characteristic feature, which is irresistible to oxygen therapy. If PaCO2 increased, it indicated that the patient is in critical condition. Early chest X-ray is often negative, and then, interstitial pulmonary oedema occurs, manifested as two lungs scattered in different sizes and fuzzy edge of the patchy increased density shadow. Pulmonary interstitial fibrosis may occur in the late stage. Multiple organ failure may occur after the disease develops. 15

| Pulmonary fibrosis
Although many patients who develop ARDS survive the acute phase of the disease, and might even be discharged, a large proportion of them die subsequently from progressive pulmonary fibrosis. 17 Dysregulation of matrix metalloproteinases in the inflammatory phase of ARDS could lead to a complex combination of epithelial and endothelial damage, and thus uncontrolled fibrosis. 18 Continuous and aberrant activation of epithelial cells could lead to cellular senescence and overactive secretion of pro-fibrotic growth factors, chemokines, vascular inhibitors and procoagulant mediators. These factors are collectively referred to as senescence-associated secretory phenotype (SASP) factors. 19 SASP factors can lead to abnormal wound healing, which is characterized by a dysregulated crosstalk between epithelial cells and mesenchymal cells, and the consequent accumulation of myofibroblasts. Fibroblasts and myofibroblasts in fibrotic lungs exhibit markers of stress and senescence, including resistance to apoptosis and excessive production of extracellular matrix components. 20 The resultant increase in matrix stiffness could affect the microenvironment and thus the crosstalk between fibroblasts and epithelial cells, resulting in irreversible damage and fibrosis. 21

| Pathological anatomy
Studies have found that COVID-19 can cause multiple organ and tissue damage, especially in the respiratory system. 22,23 The tracheal and bronchial mucosa exhibited hyperaemia and increased secretions. 13 Although the pathological characteristics of lung lesions caused by SARS-CoV-2 are similar to SARS, there were also significant differences. Parenchymal areas contain diffuse alveolar injury and exudative inflammation. 24 The alveolar cavity is often filled with serum, fibrin exudate and extensive transparent hyaline membrane formations, as observed in autopsies. 22,[25][26][27] White blood cells that infiltrate the alveoli are mainly monocytes and macrophages. Type II lung alveolar cell proliferation and focal lung cell shedding can be observed. Pulmonary interstitial fibrosis is frequently observed in cases with a long duration of the disease.
COVID-19 can also affect multiple organs with varying degrees of acute damage. SARS-CoV-2 was also detected in the lymph nodes, spleen, heart, liver, gallbladder, kidney, stomach, breast, skin and testis, through qRT-PCR-based viral nucleic acid detection, electron microscopy and immunohistochemical staining. 23 A study noted lesions in the lymphoid hematopoietic organs. 28 Lymphocytes in the spleen and lymph nodes, especially CD4 + and CD8 + T cells, were significantly reduced. Lymphocyte degeneration, necrosis and macrophage proliferation were frequently observed. The myocardium also exhibits cellular degeneration, occasional necrosis, interstitial oedema, and mild infiltration of monocytes, lymphocytes and/or neutrophils. Hepatocyte degeneration, spot necrosis, and small, bridging or large necrosis of neutrophil infiltration are found in the liver. In the kidney, hyperaemia, segmental hyperplasia or necrosis, and protein exudation in the glomerulus were observed. Sometimes pancreatic islet cell degeneration and lysis are detected. The oesophagus, stomach and intestinal mucosal epithelium showed varying degrees of degeneration, necrosis and exfoliation. The testes also showed different degrees of reduction and damage of spermatogenic cells.
Brain congestion and oedema, some neuronal degeneration, and ischaemic changes were also detected.

| P OTENTIAL MECHANIS MS OF S TEM CELL THER APY IN COVID -19
Stem cells are endowed with the properties of self-renewal and multi-lineage differentiation potential, thus making them an attractive modality for cell therapy in the clinic. However, due to many ethical and legal restrictions, clinical development and progression of stem cell therapies have been relatively slow. 29 Because adult stem cells are exempt from the aforementioned ethical and legal restrictions, while possessing excellent tissue repair capabilities, usage of adult stem cells has been more popular than embryonic or pluripotent stem cells in the clinic. 30 Accumulating studies have shown that stem cell therapy is becoming one of the emerging treatment strategies for several refractory diseases with F I G U R E 2 The potential mechanisms of MSCs therapy for COVID-19. MSCs have great therapeutic potential in immunomodulation and tissue repair through secretion of soluble paracrine protein factors and exosomes. MSCs can regulate the functions of a variety of immune cells, secrete several cytokines, promote tissue repair and regeneration, and may play important therapeutic roles in patients with COVID-19. MSCs: mesenchymal stem cells; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; KGF, keratinocyte growth factor; FGF, fibroblast growth factor; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α; MSC-exo, exosomes no known treatments, including viral infections. 31 Newly emerging viral pandemic, which could cause multi-organ damage and for which there are no particular therapies, drugs or vaccines available, is especially amenable to stem cell therapy. With the COVID-19 pandemic, stem cell therapies and especially mesenchymal stem cell (MSC)-related therapies have demonstrated their therapeutic potential for newly emerging diseases with no available treatments ( Figure 2).

| MSC-related cells
MSCs are derived from the mesoderm and ectoderm of early embryonic development. They express specific cell surface markers such as CD73, CD90, CD105, CD29, CD44, CD146 and CD166, while being negative for CD45, CD31 and CD34. [32][33][34] MSCs are also known as mesenchymal stromal cells, and these matrix-derived cells are capable of self-renewal and differentiation into chondrocytes, osteoblasts and adipocytes. 35 MSCs were initially found and isolated from the bone marrow (BM), but were subsequently also discovered in various tissues such as the adipose fat pads, dental pulp, umbilical cord and placenta. 36 Currently, MSCs from different tissues are being tested for their therapeutic effects in COVID- 19. 37 MSCs express low levels of human leucocyte antigen (HLA) class I molecules, and do not express HLA class II molecules or costimulatory molecules such as CD40, CD40L, CD80 and CD86.
This expression profile allows MSCs to escape the cytotoxic effects of lymphocytic T cells, B cells and NK cells, were thus termed 'immune-privileged' cells. [38][39][40][41] In addition, MSCs possess immunomodulatory and anti-inflammatory effects, and can detect microenvironmental injury signals to direct pro-regenerative signalling processes, 42-44 thus making them attractive candidates for therapeutic use in various diseases. For clinical allotransplantation, their hypo-immunogenicity and short lifespan in vivo make them especially suitable for clinical research. Therefore, MSCs are a promising tool for the treatment of disorders involving immune dysregulation and extensive tissue damage, as is the case with COVID-19, and multiple clinical trials have been launched. 45 Immunity-and matrix-regulatory cells (IMRCs) are a new type of hESC-derived MSC-like cells that resemble MSCs in their capacity for self-renewal and tri-lineage mesenchymal differentiation.
Moreover, compared to standard adult MSCs, they show enhanced immunomodulatory and anti-fibrotic functions, and significantly extended lifespans in vitro for consistent quality in production. A recent report showed that intravenously delivered IMRCs could home into the lungs and inhibit both pulmonary inflammation and fibrosis after bleomycin-induced acute lung injury in mouse models in vivo. 46 Moreover, a pilot study for compassionate use of IMRCs showed that they could ameliorate the ARDS in two severely ill COVID-19 patients. IMRCs' hyper-immunomodulatory function, pro-regenerative paracrine signals and functional inhibition of TGF-β1-induced fibrosis were potential mechanisms for amelioration of pulmonary injury. Clinical trials for IMRCs are now in progress as well.

| Immunomodulatory function of MSCs
MSCs have been widely used in basic research and clinical studies on immune-mediated inflammatory diseases, such as graftversus-host disease (GvHD), Crohn's disease, inflammatory bowel disease, rheumatoid arthritis and ARDS. MSCs' properties in immunomodulatory and anti-inflammatory signalling make them uniquely suited for these complex multifactorial diseases, including COVID-19. 28 As such, several clinical trials have launched for these diseases. 47,48 MSCs can activate immune regulatory responses through interactions with a wide repertoire of immune cells and participate in both innate immunity and adaptive immunity regulation. 49 Below, we outline some of the host immune cells that MSCs interact with, either by direct contract or indirectly through paracrine secretion of various cytokines ( Figure 1) to modulate the immune cells. 50,51 imbalance. [56][57][58][59] In fact, in severely ill COVID-19 patients, the number of CD4 + and CD8 + T cells in the peripheral blood is often significantly reduced, while the overall immune system is abnormally activated and dysregulated by the cytokine storm during ARDS, suggesting a severe immune imbalance. Therefore, the immunomodulatory effects of MSCs and IMRCs on T cells may have potential therapeutic significance for patients with COVID-19 and ARDS.

| Antigen-presenting cells
MSCs can interfere with the antigen presentation functions, differentiation and maturation of dendritic cells (DC), thereby reducing DC activation and inflammatory factor secretion. 60 MSCs regulate the differentiation of CD11c + B220-DC precursors into regulatory DCs via prostaglandin E2 and PI3K signalling. 61 MSCs vigorously promote the proliferation of mature DCs and drive mature DCs to transdifferentiate into a novel regulatory DC population to escape their apoptotic fate. 62 In addition, MSCs can prevent DCs from secreting IFNγ and promoting T-cell expansion in tumours. 63 Since SARS-CoV-2 infection also results in DC reduction, 64 MSCs could rescue DCs for the treatment of COVID-19.
Macrophages are the other major antigen-presenting cell type, and they are one of the cell types considered to play an essential role in ARDS. 65 MSCs can regulate macrophage polarization via secretory exosomes to suppress chronic inflammation and promote tissue healing after injury. 66 MSCs also secrete the TSG-6 factor and IL-10 to inhibit NF-κB signalling and other pro-inflammatory pathways, thereby driving the polarization of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages. 54,67 Thus, MSCs could regulate macrophage polarization and their related signalling molecules, to modulate ARDS, the anti-viral immunity, and tissue healing in COVID-19 patients. 68

| Neutrophils
Neutrophils can kill pathogens (bacteria, fungi and viruses) through an oxidative burst of reactive oxygen species (ROS) and phagocytosis, and they are recruited early to sites of infection to perform their defensive functions. 69 Paradoxically, it has been reported that excessive neutrophil recruitment might exacerbate COVID-19 immunopathology. 70 Clinical studies have found that the number of neutrophils in the bronchoalveolar lavage fluid of ARDS patients is positively correlated with the severity of COVID-19 and the cytokine storm. 71 In fact, the neutrophil to lymphocyte ratio (NLR) can be used as an independent risk factor for severe disease in COVID-19 patients. 72 Intravenous injection of bone marrow mesenchymal stem cells (BMSCs)-derived exosomes into severe COVID-19 patients with ARDS can significantly reduce the production of neutrophils by 32%, thereby reducing their NLR levels and improving their clinical oxygenation index. 73

| Other immune cells
MSCs can also inhibit excessive proliferation of B cells, prevent their differentiation into plasma cells and reduce excessive levels of immunoglobulin secretion by downregulating the expression of Blimp-1. 55 After MSC treatment, overactivated CXCR3 + NK cells also disappear in 3-6 days, showing that MSCs have a potential regulatory effect on NK cells as well. 74

| Tissue repair and regeneration capabilities of MSCs
Severely ill COVID-19 patients often present severe pneumonia, respiratory failure, ARDS and pulmonary fibrosis. During this complex inflammatory pathogenic process, 55,56 the integrity of the lung alveolar capillary membrane is gradually destroyed, contributing to the formation of pulmonary oedema, lung tissue degeneration and fibrosis.
MSCs can secrete a variety of growth factors and cytokines to improve the microenvironment of the lung tissue and promote endogenous lung repair, with potential benefits for COVID-19 patients (Figure 2). For example, MSCs can promote cell proliferation and tissue damage repair by secreting hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and fibroblast growth factor (FGF). 75 MSCs secrete HGF through extracellular vesicles, reduce inflammatory damage and increase autophagy, thereby retaining or restoring the alveolar epithelium and the pulmonary vascular endothelial lining. 76,77 The HGF, KGF and angiopoietin-1 secreted by MSCs also possess pro-angiogenic, anti-inflammatory and pro-proliferation effects. 42 It has been reported that MSCs can reduce apoptosis of the alveolar epithelial cells and endothelial cell by secretion these three growth factors. [77][78][79] The VEGF and FGF secreted by MSCs can also promote lung tissue repair. 80,81 In addition, MSCs can reduce the levels of pro-fibrotic factors to The MSC-like IMRCs could also reverse pulmonary fibrosis by overexpressing the matrix metalloproteinase MMP1 and reducing collagen I levels during fibrogenesis induced by TGF-β1. 84 MSCs can also promote the repair of other damaged tissues in COVID-19 patients. Pathological results show that SARS-CoV-2 virus can also affect the kidneys, causing severe acute tubular necrosis. 85 Studies have shown that MSCs secrete cytokines to activate a variety of repair mechanisms in acute kidney injury, including anti-inflammatory, anti-apoptotic and pro-angiogenic pathways, thereby promoting the repair of kidney injury. 86,87 MSCs can also treat COVID-19-related intestinal injury through mucosal repair and epithelial regeneration. 88  Follow-up after treatment is strictly required according to the clinical protocol guidelines.

| Overview
Clinical trials for stem cell therapies against COVID-19 were searched by using the terms 'COVID-19' and 'stem cells' in the ClinicalTrials.gov  (Table S1). All observational studies and 6 withdrawn clinical studies were excluded from the list. Eventually, 88 clinical trials related to stem cells were found to be registered in different countries. In these clinical studies, the therapeutic efficacy (60 trials) and the safety (32 trials) of stem cells and their derivatives for treating COVID-19 were being investigated.

| Indications and phases
In total, 88 trials were found to be registered to investigate the

| Source and dosing of stem cells
The source of stem cells used in these trials is a major point of variability amongst the searched studies ( Figure 3C Cell dose and proposed regimens also varied greatly amongst studies. While the MSC infusion dosage ranged over an order of magnitude between 0.5 × 10 6 and 10 × 10 6 cells/kg (Table S1), the most commonly used infusion dosage was 1 × 10 6 cells/kg. In some studies, stem cells are infused regardless of the weight of the patient.
In these cases, the proposed infusion dosage ranged from 1.5 × 10 7 to 75 × 10 7 cells per round regardless of the weight of the patient, with 10 × 10 7 cells per round as the most common dose (Table S1).
The highest dose of 75 × 10 7 MSCs per round was used for the 'extracorporeal stromal cell therapeutics' against COVID-19-related acute kidney injury (NCT04445220). Of note, higher cell doses will likely bring higher treatment risks. Therefore, it is necessary to find a balance between therapeutic efficacy and safety concerns.

| Route and frequency of administration
Intravenous injection of MSCs may produce a first-order lung effect, 92 which leads to significant cell retention in the lungs, thus providing an advantage for lung tissue repair in COVID-19, ARDS and pulmonary fibrosis. Therefore, most of the ongoing clinical trials proposed to perform intravenous cell infusion (65 out of 88; Figure 3F).
Three studies focused on the administration of MSCs-derived exosomes via the inhalation route. Intramuscular injection of MSCs was used in one study (NCT04389450). The 'extracorporeal stromal cell therapeutics' was used in COVID-19 subjects with acute kidney injury in a study (NCT04445220). However, in 18 studies, the route of MSCs administration was not clearly stated.
Although a single round of MSC infusion, as proposed in 20 out of 88 trials, has been shown to provide therapeutic benefits, more than one round may be required to induce complete tissue repair or even to maintain therapeutic benefits ( Figure 3G). In some studies, the mentioned MSC doses would be injected two (14 out of 88), three (17 out of 88), four (7 out of 88) and even five rounds (3 out of 88) with short time intervals of 2 or 3 days ( Figure 3G; Table S1).

| The safety and efficacy of stem cell therapy in COVID-19
Recently, some studies have been published to report the safety and efficacy of stem cell therapy for COVID-19 (Table 1). In these pub-

| MSCs
The first study for stem cell treatment of COVID-19 by Dr Zhao improved. Additionally, the chest CT scans indicated that the patients' bilateral lung exudate lesions were adsorbed after MB-MSC infusion.

MSC-like stem cells derived from hESCs
Despite their safety and efficacy, clinical applications of primary

| FUTURE PROS PEC TS
Of knows yet when the current COVID-19 crisis will end, and when the next crisis will come.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
YT, JW and JH conceived the project and supervised the manuscript.
ZL, SN, B.G and TG contributed equally to this work and wrote the manuscript with help from all the authors. ZL, SN, BG, TG, LW, YW, L.W., YT, JW and JH participated in the experiments and data analysis.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the