Pathophysiology of SARS-CoV-2
SARS-CoV-2 binds to ACE2 receptors to enter the target cell, where the virus replicates and subsequently infects other cells. The virus connects to ACE2 via a glycoprotein called peplomer. The lungs are the most affected as the enzyme ACE2 is abundantly present in the type II alveolar cells of the lungs. But these receptors are also present on heart, liver, kidney, intestines etc. When the virus enters the lungs, it infects the upper respiratory tract and then alveolar tissue, which may eventually progress to respiratory failure leading to death. As it enters blood circulation the virus spreads and may cause damage to other distant organs such as kidneys, heart and brain, gastrointestinal organs, liver etc. (14,15) Cytokine storm and pneumonia-associated hypoxia might also contribute to liver injury or even develop into liver failure in patients with COVID-19 who are critically ill. (16)
Cytokine storm in COVID-19
It has been reported that, dysregulated cytokine/chemokine responses and higher virus titers cause an inflammatory cytokine storm with lung immunopathological injury in COVID-19. Such Inflammation associated with the cytokine storm may begin at one local site and further spread throughout the body via the systemic circulation. (17) Similarly, infected patients have shown increased plasma concentrations of inflammatory cytokines, including interleukins (IL) 2, 6, 7, and 10, granulocyte-colony stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1 alpha (MIP1A), interferon-ˠ-inducible protein 10 (IP10)and TNF-α. (18)
Large amount of inflammatory cell infiltrations have been observed in lungs from severe COVID-19 patients, these aberrant pathogenic Th1 cells and inflammatory monocytes may enter the pulmonary circulation in huge numbers and play an immune damaging role to cause lung functional disability and quick mortality. In a study including 41 patients with COVID-19, Huang, et. al. demonstrated a cytokine profile that was like that of secondary hemophagocytic lymphohistiocytosis (sHLH), a hyper inflammatory condition triggered by viral infection (19). The patients admitted in the ICU showed higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma-induced protein 10 (IP10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP1A), and tumor necrosis factor alpha (TNFα). The role of a hyperinflammatory response underlying development of severe and critical illness was confirmed in another study by higher serum levels of C-reactive protein (CRP), interleukin-6 (IL-6) and Ferritin in patients that had died. (20) The dysregulated immune response results in endothelial injury and formation of micro and macro blood clots which together with pulmonary inflammation led to a diffuse alveolar damage, fibrin exudates and fibrotic healing in the lungs which further compromises the oxygen absorption causing oxygenation failure and acute respiratory distress syndrome (ARDS). This may also result in secondary pulmonary infections such as bacterial pneumonia. (21) Prevention or attenuation of cytokine storm is therefore crucial to lower COVID-19-induced mortality.
There are no approved treatments for Covid-19 but some medications including antiviral, anti-inflammatory have shown to be beneficial. There is an urgent need to explore treatment strategies that can prevent patients from worsening clinically and progressing to severe or critical stage. Due to its immunomodulatory properties, cell therapy has a potential to halt the progression of the disease and accelerate the recovery process.
To evaluate the safety and efficacy of cell therapy in COVID-19, we administered 10 patients with moderate illness with a mixture of 100 million MSCs derived from umbilical cord and placenta.
Rationale for use of MSCs derived from umbilical cord and placenta
Cellular therapy has been studied widely for treating various conditions, including pulmonary, immunological, haematological, cardiac, neurological, hepatic, endocrine, musculoskeletal, skin, and ophthalmological diseases. (22-28) Various clinical studies have demonstrated the safety and efficacy of umbilical cord and placental MSCs individually. (29-34) For this study, a mixture of umbilical cord and placenta derived MSCs was used since, a mixture results in an enhanced outcome combining the benefits of both types of cells. Invitro studies have shown that this mixture of cells secretes increased concentration of paracrine molecules such as IGF-1, KGF, VEGF, SDF-1α as compared to individual cell types which are responsible for stimulating lung repair, angiogenesis, stem cell recruitment, etc. Acute toxicity, chronic toxicity, genotoxicity, immunotoxicity, tumoregenic potential studies conducted for this mixture of cells showed that it was non-toxic, non- mutagenic, non-tumorogenic and had no immunotoxic effect on the organs of the immune system, and the cell parameters of peripheral blood and bone marrow. These cells are safe, effective and are easily available without any ethical barriers.
Immunomodulatory (paracrine) effects of MSCs counteracting the cytokine storm:
Umbilical cord cells also attenuate lung inflammation by a multitude of paracrine functions, including enhanced interleukin (IL)-10 expression and modulation via prostaglandin-E2 (PGE2), GM-CSF, IL-6 and IL-13. (35) Both umbilical cord and placenta derived stem cells secrete paracrine factors, including human angiopoietin-1 (Ang-1), Hepatocyte Growth Factor (HGF), insulin-like growth factor I (IGF-I), prostaglandin E2 (PGE2), transforming growth factor beta 1 (TGF-β1), vascular cell adhesion protein 1 (VCAM-1) and Vascular Endothelial Growth Factor (VEGF), in varying levels. Although they have different growth dynamics, stem derived cells show the highest secretion of Ang-1 and VEGF and the lowest secretion of TGF- β1, while umbilical cord derived cells show the highest secretion of IGF-I, PGE2 and TGF-β1, HGF and VCAM-1. (36)
Leng et al. 2020 have also demonstrated that anti-inflammatory and trophic factors like Transforming Growth Factor (TGF)-β, HGF, VEGF, Leukaemia Inhibiting Factor (LIF), Galanin (GAL), Nitric Oxide Associated protein 1 (NOA1), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Brain-derived Neurotrophic Factor (BDNF), and Nerve Growth Factor (NGF) are highly expressed in mesenchymal stem cells, further bolstering their immunomodulatory role. (37)
Differentiation of MSCs into AT2 cells and their immunity towards COVID-19:
The COVID-19 causing novel coronavirus 2019-nCoV uses the angiotensin converting enzyme II (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) receptor to enter host cells. (38) ACE2 is enriched in human alveolar cells (39) and TMPRSS2 is a known human airway and alveolar protease. (40) This may explain the rapid infection rate observed worldwide. It is shown that mesenchymal stem cells are ACE2 and TMPRSS negative, which indicates that these cells may not be susceptible to COVID-19. (37)
Surface Protein (SP) A and C are highly expressed in umbilical mesenchymal stem cells, indicating that these might differentiate to alveolar epithelial (AT2) cells. (37) These cells have also been shown to differentiate into a variety of alveolar cells and integrate into target tissue in rodent models. (35,41) The human placenta also enriches mesenchymal stem cells, which are multipotent progenitors. (42)
Antiviral effects of MSCs:
MSCs transplantation has shown significantly reduced mortality in H7N9 induced ARDS. (43) In severe influenza, MSCs’ can tackle inflammatory cytokine excess that leads to acute lung injury induced due to H5N1 infection. (44) MSCs also regulate inflammatory responses, improve alveolar fluid clearance, and maintain lung epithelial and endothelial integrity. (45,46) Moreover, KEGG pathway analysis has shown that these cells are closely involved in the antiviral pathways, specifically those related to Epstein-Barr, Hepatitis B, viral carcinogenesis and human T-cell leukaemia virus 1 infection. (47) They have also been implicated in several other pathways, namely herpes viral infections via the cGAS-STING pathway (48), Japanese encephalitis via up-regulation multiple pro-survival pathways (49), and a non-canonical PI3KNFκBpathway against latent HIV-1. (50)
Anti-inflammatory and immune regulatory ability:
MSCs have shown to induce immunomodulation primarily through paracrine signaling, stimulation of secretion of anti-inflammatory molecules such as Interleukin (IL)-10. Also, they increase the lymphocyte count thereby increasing their antiviral characteristic which in turn results in decreased C-reactive protein and pro-inflammatory cytokines including IL-6, TNFα, IL-8, etc. Studies have shown that the anti-inflammatory ability of MSCs can attenuate virus-induced lung injury and death in mice. (51,52) These cells also have a known safety and efficacy profile in ARDS, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), obstructive bronchiolitis (OB) and bronchopulmonary dysplasia. Cell therapy using MSCs has therefore been proposed as a suitable treatment approach and several clinical trials have begun.
The immune modulating action of stem cells is mediated by TLRs (TLR, mainly TLR3 and TLR4) present on the surface of MSCs. RNA viruses [act as Pathogen-associated molecular pattern (PAMP)] activate these TLRs, which leads to secretion of certain chemokines like MIP-1α and MIP-1β, RANTES, CXCL9, CXCL10, and CXCL11 etc, which leads to an anti-inflammatory response. (53) This response may be useful against the hyperimmune response/ cytokine storm observed in COVID-19. There has been hypersecretion of pro-inflammatory cytokines which includes IL-2, IL-6, IL-7, G-CSF, IP10, MCP1, MIP1A and TNFα which influences MSCs to release anti-inflammatory molecules (IL-10) with the release of soluble factors like transforming growth factor-β1 (TGF-β1) prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), indoleamine-pyrrole 2,3- dioxygenase (IDO), and nitric oxide (NO). (54) These in turn decrease the proliferation of activated T-cells and NO on the other hand causes cell cycle arrest by repressing the phosphorylation of signal transducers and transcription of STAT-5 in T-cells. Further, IDO secretion by MSCs leads to apoptosis of activated T-cells and converts tryptophan into kynurenine causing suppression of proliferation of effector T-cells. (55)
Regeneration and repair:
The abnormal immune response in SARS-CoV-2 infection leads to acute and chronic lung injury often involving fibrosis either as a cause or consequence. MSCs promote repair through paracrine signaling inducing secretion of growth factors such as Hepatocyte Growth Factor (HGF), transforming growth factor beta 1 (TGF-β1), Vascular Endothelial Growth Factor (VEGF) etc. which are necessary for tissue repair. Through their regenerative and reparative potential, they may protect alveolar epithelial cells, bring about recovery of pulmonary microenvironment, improve lung dysfunction and pulmonary fibrosis.
Multiple pro-survival pathways converge and death-inducing pathways are attenuated on a cellular level to bring about improvements in the lungs. Inhibition of pulmonary fibrosis and reduced collagen deposition by these cells (56-58) may lead to decreased edema and opacity in chest X-rays. Further, MSCs trapped in pulmonary circulation undergo differentiation over the long term to yield a multitude of alveolar cell types that integrate into the pulmonary tissue and improve lung function; (41,59) we may thus also expect improved tissue microarchitechture in the chest CT scans along with improved air exchange and lung function.
Antimicrobial properties
Along with immunomodulation and regeneration, MSCs also possess antimicrobial properties. Besides, these cells have shown to be ACE-2 negative and therefore cannot be infected by SARS-CoV-2. (60)
Angiogenesis
VEGF secreted by stem cells is a pro-angiogenic factor. Angiogenesis is critical for tissue regeneration. (61) In the lung, angiogenesis is crucial since the blood-air interface is the source of oxygenation and oxygen delivery to the body. Through secretion of VEGF, stem cells can help recovery from lung injury. Animal studies have shown that administration of VEGF improved aeration and prevented the development of respiratory distress syndrome and mortality in premature animals. (62)
Rationale for Intravenous injection
Safety and efficacy have been satisfactorily demonstrated in the intravenous administration of these cells in the human body for all the conditions mentioned above at different doses, reviewed exhaustively by Can et al. (22) Intravenous route of administration is considered to be an ideal approach given its broad biodistribution and easy access. It has been used as the route of cell delivery for a large number of preclinical and clinical studies. The first organ through which intravenously injected MSCs pass are the lungs. (63) Engraftment in the lungs is a very rapid event, cells can be detected already seconds or minutes after intravenous transplantation. (64,65) Cell fate tracking studies in sheep (66,67) and rats (68,69) have shown the superiority of the intravenous route for administering these cells, with the cells primarily distributed in the lungs. Studies have shown that the detainment of MSCs in the lungs is due to the combination of mechanical and physiological conditions and may be due to the small capillary size, the large capillary network and the strong adhesion properties of MSC. Cultured MSCs are more than 20 μm in diameter, which does not allow them to pass through the lungs as they are larger than the width of the micro-capillaries of the lungs. (70) These cells thus may improve the pulmonary microenvironment and lung function by differentiating into different types of alveolar epithelial cells as well as immunomodulation, are safely tolerated, and efficacious in improving tissue microarchitecture. These data taken together rationalize the intravenous administration of umbilical cord and placenta mesenchymal stem cells for the treatment of COVID-19
Clinical Evidence
The first study was conducted in China by Leng, et al to assess if cell therapy using MSC could improve outcome of patients with COVID-19. 7 patients, including 1 critically ill, 4 severe and 2 non-severe cases, received a single dose of MSCs (1×106 cells/kg body weight) by IV infusion; 3 severe cases forming the control group received placebo. Significant improvement in pulmonary function was noted in all patients in the MSC treatment group within 2 days. There were no MSC infusion associated adverse events. Two non-severe and one severe patient were discharged within 10 days following recovery. As compared to the control group, pro-inflammatory TNF-α significantly decreased while the anti-inflammatory IL-10 levels increased in the MSC treatment group. (37)
Another study including 31 patients with severe COVID-19 pneumonia demonstrated improved clinical outcomes following IV infusion of human umbilical cord-derived MSC (hUC-MSC) in a dose of 1 × 106 cells/kilogram of weight (71). The SARS-CoV-2 PCR test results turned negative in a mean time of 10.7 (4.2) days. Clinical data and laboratory parameters showed that UC-MSC therapy improved oxygenation and attenuated the hyper-inflammatory state in these patients.
Multiple case reports showing benefit of hUC-MSCs in patients with Covid 19 have been published. No adverse events were reported. Patients showed improved symptoms alongwith improved inflammatory markers. (72-74)
Sengupta et al. conducted a prospective non-randomized open-label cohort study using exosomes derived from allogenic BMMSCs in 24 confirmed COVID-19 patients. 83 % survival rate was observed with a recovery rate of 71 % (17/24), stability in 13 % (3/24) and mortality unrelated to treatment was 16 % (4/24). Laboratory investigations showed significant reduction in absolute neutrophil count with alleviated levels of acute phase reactants, C-reactive protein, downregulating cytokine storm and restoring immunity. (75)
Another cell-based therapy, derived from allogenic cardiospheres was assessed for its safety and effectiveness in 6 critically ill COVID-19 patients by Singh et al. All patients were on ventilatory support. All patients survived with 4 discharged and 1 on respiratory support compared to 18 % mortality in control group. Results were well correlated with diminished levels of ferritin and absolute lymphocyte counts, suggesting the role of cell-based therapies in modifying the immune responses. (76)
Clinical outcome of this study
Use of MSCs significantly decreased the time required for COVID-19 symptoms to resolve. (Table 2).
It has been reported that the median duration for symptom resolution of Covid 19 patients is16 days, (77) however, we noticed that all symptoms of our study group resolved within 10 days. Cough, sore throat, sputum, chest pain, loss of appetite, taste and smell, giddiness, resolved within 5 days. However, generalized fatigue, shortness of breath and requirement for supplemental oxygen was resolved within 10 days. 9 out of 10 patients were discharged within 9 days of their admission. Along with improved clinical symptoms, levels of inflammatory biomarkers such as C-reactive protein, interleukin 6, ferritin and D-dimer also improved in all patients after intervention. There was no deterioration observed in clinical and laboratory parameters. None of the patients progressed to severe stage of Covid. Improved oxygenation was recorded in all the patients which was demonstrated by improvement in the SpO2 / FiO2 ratio and PaO2 / FiO2 ratio. None of the patients showed any major or minor adverse events immediately after intervention or on follow up after 6 months. One patient with history of diabetes mellitus expired due to cardiac arrest after 3.5 months post intervention which was unrelated to cell therapy. Cardiac manifestations have been reported in patients recovering after COVID-19. (78) While patients with pre-existing cardiovascular disease and risk factors are more likely to experience cardiac sequelae, those with no cardiovascular history have also shown signs of cardiac complications because of COVID-19. Mechanisms responsible for cardiovascular sequelae in post COVID-19 may include direct viral invasion, downregulation of ACE2, inflammation and the immunologic response affecting the structural integrity of the myocardium, pericardium and conduction system. (79)
Consistent decline in disease severity within a short duration alongwith normalization of oxygen saturation can be attributed to the anti-inflammatory, immunomodulatory, angiogenic and anti-viral effects of MSCs. Administration of MSCs in moderate stage Covid patients resulted in prevention of the cytokine storm and halting the disease progression.
Radiological findings
Radiological investigations like Chest X-ray and Chest CT scan showed no adverse effects of the MSCs transplantation on lung tissue. In addition, no post-covid fibrosis was observed on Chest CT post 28 days of the treatment and Chest X ray post 6 months of the treatment.
On chest X-ray, 8 out of 10 patients showed significant bilateral lung infiltrates. 7 (87.5%) out of these 8 patients resolved completely in average 19 days. Improvement was noted in the radiographs at Day 7 in most patients. Warissara Kiththiworaphongkich, 2021 showed that the improvement was seen in X-rays post 13 days of the illness. (80) However, in this study improvement was seen post 7 days of treatment suggesting early resolution of lung pathology.
Temporal changes in the lung tissue involvement studied previously on a CT scan shows that CT scores peak during illness days 6-11. (81,82) In this study, 90% of the patients receiving MSCs transplantation showed reduction in the CT score at day 7 and the scores continued to reduce thereafter, suggesting better resolution of lung pathology post treatment.