Safety and Long-term Improvement of Mesenchymal Stromal Cell Infusion in Critically COVID-19 Patients: A Randomized Clinical Trial

Carmen Kuniyoshi Rebelatto (  carmen.rebelatto@pucpr.br ) Ponti cal Catholic University of Parana: Ponti cia Universidade Catolica do Parana https://orcid.org/0000-0003-1407-0416 Alexandra Cristina Senegaglia Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Claudio Luciano Franck Hospital de Clinicas Debora Regina Daga Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Patrícia Shigunov Fundacao Oswaldo Cruz Marco Augusto Stimamiglio Fundacao Oswaldo Cruz Daniela Boscaro Marsaro Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Bruna Schaidt Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Andressa Micosky Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Ana Paula de Azambuja Hospital de Clinicas Cleverson Alex Leitão Hospital de Clinicas Ricardo Rasmussen Petterle Universidade Federal do Paraná: Universidade Federal do Parana Valderez Ravaglio Jamur Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Isadora May Vaz Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Antônio Paulo Mallmann Hospital Maternidade Brígida Hipólito Carraro Junior Hospital de Clinicas Eduardo Ditzel Hospital Pilar Paulo Roberto Slud Brofman Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana Alejandro Correa Pontifícia Universidade Católica do Paraná: Ponti cia Universidade Catolica do Parana

This study was a phase I/II, prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. The subjects were recruited from the Complexo Hospital de Clínicas, Universidade Federal do Paraná, a referral public hospital for the treatment of patients with COVID-19, and UC-MSCs were processed at Cell Core Technology (CCT) from the Ponti cia UniversidadeCatólica do Paraná (PUCPR). UCs were obtained from full-term neonates, and the mother of the donor signed an informed consent form approved by the institutional review board. Written informed consent was also obtained from the patients' parents for the collection and publication of clinical data ( Figure 1). The experimental design was conducted in accordance with the Helsinki Declaration for human studies and was approved by the Ethics Committee (CAAE: 30833820.8.0000.0020). This study was registered inthe Brazilian Registry of Clinical Trials (ReBEC), UTN code -U1111-1254-9819.Registered 31 October 2020 -Retrospectivelyregistered, https://ensaiosclinicos.gov.br/rg/RBR-3fz9yr.
Exclusion criteria were: use of any investigational products, previous or current history of malignancy under treatment; pre-existing thromboembolic disease; concomitant infection of human immunode ciency virus (HIV) or tuberculosis infection; pregnancy; pre-existing transplant or use of immunosuppressive therapy; inability to provide informed consent and greater than 72 hours of ICU admission.
The primary safety endpoints encompassed the occurrence of prespeci ed infusion-associated AEs within 24 hours after intravenous administration of UC-MSCs or placebo. According to the Common Terminology Criteria for Adverse Events version 5.0, it was assessed by recording all AEs based on duration, intensity, and possible association with the treatment under study. Investigators conducted assessments for the presence of any AEs from enrollment throughout the study.The secondary endpoint was exploratory e cacy de ned by clinical outcomes, changes in viral load, in ammatory, immunological and biochemical biomarkers and acute lung injury (ALI) score.
Seventeen eligible patients were enrolled in an approximate 1:2 randomization to IV of three doses of 5x10 5 cells/kg UC-MSCs,adosingintervalof 48 hours (n=11), or placebo (n=6). Concomitant corticosteroids and anticoagulants were allowed, and conventional treatment was performed together with the infusion of cells during the study period. All patients were assessed at baseline and the pre-established follow-up points on days 2, 4, 6 and 14, as well as at 2 and 4months post-infusion. These evaluations consisted of a clinical assessment, viral load, laboratory testing, including blood count, serologic, biochemical, cell subpopulation, cytokine and CT scan evaluations.

Advanced therapy product
The study was conducted following the Good Clinical Practice Guidelines for Advanced Therapy Products (ATP) (RDC 508/2021). One week before UC collection, healthy donors provided written informed consent, and serology for infectious diseases and RT-PCR for COVID-19 were performed.
UCs were obtained from full-term newborns by cesarean section and they were aseptically stored in sterile Iscove'smodi ed Dulbecco's medium (IMDM) (Gibco BRL, Grand Island, NY) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco BRL, Grand Island, NY). The umbilical cord was washed three times with phosphate buffered saline (PBS) (Gibco BRL, Grand Island, NY) and antibiotics, sectioned into small fragments (1-2 mm 2 pieces), and centrifuged at 280 gfor 10 minutes. After removing the supernatant fraction, the precipitate was washed with IMDMand centrifuged at 280 g for 10 minutes. The tissue was treated with 0.1% collagenase type II (Sigma, St Louis, MO, USA) at 37°C for 16 hours, washed, and further digested with 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) at 37°C for 15 minutes. Fetal bovine serum (FBS) (HyClone™, South Logan, USA) was added to the MSCs to neutralize the excess trypsin 57 .
Cells were plated in 75 cm 2 culture asks (Greiner Bio-One, Kremsmünster, AT) with IMDM supplemented with 20% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin and incubatedat 37°C and 5% CO 2 . At 96 hours, nonadherent cells were removed and washed with PBS, and the culture medium was replaced with fresh medium every three days. When the cell culture reached 70% to 80% con uence, cells were detached by treatment with 0,25% trypsin-EDTA and replated at a density of 8,000 cells/cm 2 into 150 cm 2 culture asks. At passage 3 (P3), cytogenetic analysis was performed. UC-MSCs were harvested and cryopreserved using a rate-controlled freezer at a nal concentration of 10% dimethyl sulfoxide (Origen, Texas, USA) and 90% FBS. Four days before infusion, cells were thawed and replated at a density of 8,000 cells/cm 2 into 150 cm 2 culture asks. When the number of cells was su cient for administration, con uent UC-MSCs were detached with 0,25% trypsin-EDTA and washed twice with saline, and samples were collected for quality control. This control includes viability and cell surface markers by ow cytometry 58 , cytogenetics analysis by GTG-banding method 59 , microbiological tests (Bact/Alert 3D, Biomerieux, Durham, USA), endotoxin (Endosafe ™ PTS, Charles River, Charleston, USA) and Mycoplasma (KIT MycoAlert ™ PLUS Mycoplasma Detection, Lonza, Rockland, USA), accordingto the manufacturer's instructions. These quality control tests were performed before each batch of cells was released. For infusion, 5×10 5 UC-MSCs/kg of body weight were resuspended in a nal volume of 30 mL of vehicle solution composed of saline (JP, Ribeirão Preto, Brazil), 5% AnticoagulantCitrate Dextrose (ACD) (JP, Ribeirão Preto, Brazil) and 20% albumin (BlauFarmacêutica, São Paulo, Brazil). The placebo group received a vehicle solution. Cells were infused between P3 and P5.

Multiparameter Flow Cytometry
Multiparametric ow cytometry was performed on all patients at baseline and on speci c days after infusion (days 2, 4, 6 and 14, as well as at 2 and 4 months). Absolute leukocyte counts were performed using a Sysmex XN-3000 counter at the time of MFC analysis.
Comercial antibodies were used to analyze the expression of the cell surface markers CD3, CD4, CD8, CD19, CD38, CD127, CD25, and HLA-DR (Becton Dickinson, San Diego, USA). Immunophenotypic characterization of peripheral blood (PB) lymphocytes was performed with conventional staining sample preparation techniques according to Kalina et al. 61 . A total of 1,000,000 cells/events per tube were acquired using a FACSCanto II® ow cytometer (Becton Dickinson, Franklin Lakes, USA) and In nicyt™ software (Cytognos, Salamanca, Spain-version 2.0) for ow cytometry analyses. The analysis protocol included removal of threshold debris, and lymphocytes were initially identi ed based on low frontal (FSC) and side scatter (SSC) and strong CD45 staining. The frequency and cell number of total, CD4+ and CD8+ T cells, as well as B (CD19+) and plasmablasts (CD19+CD38++) in patients, was determined using a Boolean strategy 62 . CD19-positive staining identi ed B cells, and strong CD38 positivity in B cells was used to identify the plasmablast population (CD19++CD38++). To identify T cell regulatory CD4 lymphocytes (Tregs),it was used CD25 positivity and CD127 negativity in the CD4 lymphocyte gate.
Analysis of in ammatory cytokines, chemokines, and growth factors in peripheral blood plasma Blood samples were collected in EDTA Vacutainer® tubes (BD Biosciences, Curitiba, PR) and immediately transported to the laboratory for processing. Plasma was obtained by centrifugation (1,600 g for 15 minutes at 4°C), divided into aliquots and stored at −80°C until analysis.
The BD™ Cytometric Bead Array Systromal(CBA Flex Set Systromal,BD Biosciences, San Diego, EUA) was used todetermine plasma levels of a set of in ammatory cytokines, chemokines, and growth factors, including granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin(IL) -2, IL-6, IL-7, IL-8, tumor necrosis factor(TNF)α,monocyte chemoattractant protein-1 (MCP1/CCL2) and macrophage in ammatory protein 1-alpha (MIP1a/CCL3),according to the manufacturer's recommendations.The assay was performed at preinfusion and on days 2, 4, 6 and 14, as well as 2 and 4monthspost-infusion. All samples were measured in duplicate. Standard curves for each cytokine were generated using the premixed lyophilized standards provided in the kits. The cytokine concentrations in samples were determined by measuringtheir uorescent intensities and referencingfrom the appropriate standard curve.Data were analyzed using the FlowJo™ v.10 software.

Statistics
In the descriptive analysis, it was used absolute and relative frequencies for categorical variables, and for quantitative variables,it was calculated the average values with theirrespective standard deviations. To investigate the change in response variable over time, it was used the framework of Gaussian copula marginal regression models 63 for longitudinal data analysis. First, it was select the probability distribution for the response variable from either Gaussian or gamma. Thereafter, it was select the available correlation structure: (i) independent, (ii) exchangeable, (ii) autoregressive of order 1 (AR1) and (iv) moving average of order 1 (MA1). Next, it was investigated the interaction effect between the group (placebo or UC-MSCs) and the evaluation times (time) that were treated as a factor with seven levels (baseline, 2, 4, 6 and 14 days, 2 and 4 months). When the interaction was not signi cant (p>0.05), it was used the additive effect on the linear predictor. On the other hand, when the interaction effect was signi cant, it was conducted a multiple comparison test, where the pvalues were obtained through Bonferroni correction 64 . It was adopted the Akaike (AIC) and Bayesian (BIC) information criteria and the maximized value of the log-likelihood function (logLik) to select the probability distribution for each response variable and the structure for the correlation matrix.The statistical data analysis was performed in R software version 4.0.3 65 using the R package GCMR 66 .

Results
Advanced Therapy Product In this study, samples from four UC donors were used, and all of them were negative for RT-PCRtestsforCOVID-19and serology for infectious diseases. In this clinical trial, fresh infused cells were used, and the interval between product release and patient IV infusion was up to three hours.
The ATP infused into the patients was negative for microbiological tests, and no clonal chromosomal abnormalities were observed. For each infusion, the average cell viability was 96.6±0.01, 95.4±0,03, and 95.5±0,02 for each infusion. Cell characterization was performed following the criteria de ned byInternational Society for Cellular Therapy (ISCT) Guidelines (An additional movie le shows this in more detail [see Additional Table 1). UC-MSCs show the potential to differentiate into osteoblasts, adipocytes, and chondroblasts, and the immunomodulation potential is higher than 50% ( Figure 2). The results from all evaluations were in conformation with those established by CCT/PUCPR.

Patient baseline characteristics and study population
A total of 17 patients were included in this study from 12 June to 13 July 2020. After randomization, 11 patients were included in the UC-MSC group, and 6 patients in the placebo group. In the UC-MSC group, one patient was excluded on the twelfth day because she did not undergo follow-up after cell infusion. At enrollment, all patients had ARDS and were in IMV in critical condition rated 6-7 on the WHO scale.
The average age of the UC-MSC group was 53±15.3 years, while the average age ofthe placebo group was 61,7±9.7 years. The baseline symptoms were fever, cough, nausea or vomiting, diarrhea, loss of taste or smell, shortness of breath, disorientation and confusion. No differences were observed when comparing the interval between symptom onset and hospital admission or the interval between symptom onset and rst cell injection. Two patients in the UC-MSC group had no basic chronic diseases, and all other patients had comorbidities when they were admitted to thehospital, such asdiabetes, hypertension, kidney disease, chronic obstructive pulmonary disease, schizophrenia and obesity. The patients received standard treatment with anticoagulants,steroids, and antibiotics if there was evidence of bacteriological infection. Two patients from the UC-MSC group were treated with antiviral drugs as a concomitant treatment.Patient baseline characteristics are demonstrated in Table 1. Laboratory tests, such as blood count and serologic, biochemical and cell subpopulations, were evaluated at baseline ( Table 2).All these parameters were considered response variables in the statistical analysis(An additional movie le shows this in more detail [see Additional Tables 2 and 3). In this study, ve patients from the UC-MSC group and one patient from the placebo group (35% of the patients) passed away, although no signi cant difference was observed between the groups. Five patients were male and one female, and their ages ranged from 41 to 78 years.
None of the deaths seemed to be related to UC-MSC infusion. The cause of death of ve patients was secondary to bacterial septic shock, and one patient died secondary to ARDS and multiorgan dysfunction syndrome. There was no association of mortalityand elderly patients.Five of the six patients had comorbidities such asobesity, diabetes, hypertension and schizophrenia, whichwas not associated with mortality. However, patients who presented dialysis kidney dysfunction during the course of the disease had higher associated mortality (p=0,029). The table 3 shows the details of death. Quanti cation of viral load was assessed in patient samples at baseline and 2, 4, 6, and 14 days after infusion with UC-MSCs or placebo ( Figure   3). The angular coe cient (slope) and R 2 value were established for each patient. Most patients had an angular coe cient below zero, which means that the viral load decreased over time ( Laboratory Assessments

Analysis of in ammatory markers
To determine the patients' in ammatory status, ferritin, C-reactive proteinand cytokine levels were analyzed. Ferritin values in the UC-MSC group were higher at baseline than at day 14 (p=0.03), 2 months (p=0.01) and 4 months (p=0.01). In the fourth month, there was a marked and statistically signi cant decrease in ferritin values and a return to normal levels. In the placebo group, the levels were always higher than the reference ranges. In the fourth month, there was an increase in the placebo group, opposite to the UC-MSC group (p=0.01). C-reactive protein, which is the main in ammatory marker in COVID-19 patients, showed a decrease in the comparison between baseline at 2 months (p=0.01) and baseline at 4 months (p=0.01) in the UC-MSC group. In the second month, the values were within normal levels, while in the placebo group, although the values were lower than those in the UC-MSC group, there were no differences over time, always maintaining levels above the reference ( Figure 4).
Conversely, IL-6 levels in the UC-MSC group showed differences between baseline and thefourteen day (p=0.02), second month (p=0.01) and fourth month (p=0.01). From the fourteen day onwards, IL-6 levels decreased signi cantly (fourteen day x second month, p=0.01; fourteen day x fourth month, p=0.01). During this same period, in the placebo group, the levels remained high, with no differences between evaluation time points. In the comparison between groups, at baseline (p= 0.01), day 2 (p=0.01) and day 4 (p=0.04), IL-6 values were higher in the UC-MSC group than in the placebo group. However, at month 4, there was a signi cant decrease in the UC-MSC group and an increase in the placebo group (p=0.01). The levels of IL-8 in the UC-MSC group until the fourteen day were always higher than those in the placebo group. In both groups, there was a large reduction in values at 2 and 4 months (UC-MSC group, baseline x 2 months, p=0.01; baseline x 4 months, p=0.01; placebo group, baseline x 2 months, p=0.01, baseline x 4 months, p=0.01). The UC-MSC group showed a decrease in MCP1 levels, with differences between baseline and thefourteen day (p=0.01), 2 months (p=0.01) and 4 months (p=0.01). Comparing groups, this cytokine level was higher in the UC-MSC group than in the placebo group at baseline (p=0.01), 2 days (p=0.01) and 4 days (p=0.01). However, from the sixth day to thefourth month, there was a decrease, with no differences in relation to the placebo group. Regarding the cytokines IL-6, IL-8 and MCP1-CCL2, all had higher levels in the UC-MSC group than in the placebo group until the fourth day. After this period, the levels decreased and were lower than those in the placebo group in the fourth month, suggesting that MSCs were effective in decreasing in ammation. The level of IL-7, which is a pleiotropic cytokine essential for lymphocyte survival and expansion, showed a decrease in the fourth month in the placebo group (baseline x 4 months, p=0.02), indicating a worsening for patients in this group ( Figure 6).
On the other hand, there was an increasing trend of anti-in ammatory cytokine IL-10 levels in the UC-MSC group at all evaluation times, although with no signi cant difference. A study with a larger number of patients will be necessary to con rm the possible stimulation of IL-10 production exerted by UC-MSCs, which reduces the in ammatory process. The results of in ammatory markers strongly suggest that UC-MSCs had an important anti-in ammatory action from the fourteen day of evaluation in most cases and that MSC immunomodulatory function contributed to the main e cacy outcome ( Figure 6).

Analysis of coagulationparameters
Coagulation markers were also evaluated, including D-dimer, platelets and neutrophils. With respect toD-dimer, both groups presented values above the reference in all evaluations. However, in the UC-MSC group, a decrease in D-dimer values was observed in the second month compared to baseline (p=0.01). At 2 months, values in the UC-MSC group were very close to the reference and signi cantly lower than the value in the placebo (UC-MSC x placebo, p=0.01). In the UC-MSC group, there was a decrease in the number of platelets in the comparison between baseline and 2 months (p=0.01) and 4 months (p=0.01).In the second and fourth months, platelets in the UC-MSC group were within the reference range, whereas in the placebo group, they were out of the reference range In the fourth month, there was a signi cant difference, with higher levels in the placebo group (p=0.01). A difference in the number of neutrophilsin both groups was also observed. The UC-MSC group had a lower number of neutrophils than the placebo group in the second month (p=0.03) and fourth month (p=0.01) after treatment. These results demonstrate that two months after cell infusion, there was a decrease in coagulation markers that could reduce the risk of thrombus formation compared to the placebo group ( Figure 4).

Analysis of cell subpopulation
In this trial, the main cell subpopulations were evaluated by ow cytometry. The total lymphocyte count at baseline was below the reference range in the UC-MSC group and signi cantly lower than that in the placebo group (p=0.01). From the sixth day, there was an increase in the lymphocyte count at UC-MSC group, with a return to the normal range and a difference between baseline and 2 months (p=0.04). In the placebo group, at all evaluation times, values were within reference. The numbers of TCD3 and TCD4 lymphocytes were also lower in the UC-MSC group than in the placebo group at baseline (p=0.02 and p=0.01, respectively) and on the secondday after infusion (p=0.04 and p=0.01, respectively), with no signi cant differences in the other evaluations. These results indicate that the placebo group was in better condition than the UC-MSC group at the beginning of the study. From the second month, an increase in the absolute TCD3 lymphocyte values was observed, with differences between baseline and 2 months (p=0.01) and 4 months (p=0.01) in the UC-MSC group. The same was observed in relation to TCD4 lymphocytes (baseline x 2 months, p=0.01; baseline x 4 months, p=0.01). In the placebo group, there were no differences over time. Values of NK cellsincreased signi cantly when comparing baseline and 2 months (p=0.01) in the UC-MSC group. These data indicate thatlymphopenia, which has important prognostic potential and is present in patients who need intensive treatment, was more common in patients in the UC-MSC group, and after cell infusion, there was an improvement in immune systromal function (Figure 4).
Even with a small number of individuals per group, the results presented thus far strongly suggest that treatments with MSCs signi cantly improve several of the patients' functional and in ammatory parameters.
The e cacy outcome -Hepatic, Cardiac, Kidney and Pulmonary Sequelae Months after COVID-19 infection, patients could still present some persistent symptoms that need to be monitored. Some biochemical tests to evaluate liver function, such as bilirubin, alanine aminotransferase (AST) and aspartate aminotransferase (ALT), showed no differences between groups. Regarding the function of the kidneys and heart, the levels of troponin I and creatinine were analyzed. Troponin I showed decreased levels in both groups. Values were within thenormal range at the second and fourthmonths after treatment (placebo group, baseline x 2 months, p=0.01; baseline x 4 months, p=0.01/UC-MSC group, 6 days x 2 months, p=0.04; 6 days x 4 months, p=0.01). Creatinine values indicative of renal function in the UC-MSC group were increased compared to those in the placebo group on days 4 (p=0.03), 6 (p= 0.01) and 14 (p= 0.02). On day 14, after three cell infusions, values were within the reference range, showing an improvement in renal function. In the second and fourthmonths, the UC-MSC group remained within the reference range, while there was an increase in levels in the placebo group above normal values, but there was no signi cant difference between groups.
In this trial, chest CT was used to detect lung damage in COVID-19 patients. All patients suffered from serious pulmonary damage and needed oxygen inhalation support during the course of disease. CT imaging results revealed bilateral, multilobular involvement as well as segmental consolidation and characteristics of pneumonia. Concerning chest CT abnormalities, there was no signi cant difference between groups (An additional movie le shows this in more detail [see Additional Table 4). However, there was a reduction in the extension of opacities related to COVID-19 in chest CT scans for both groups(An additional movie le shows this in more detail [see Additional Table 2). Visually, there was a higher degree of clearence in patients from the UC-MSC group than in the placebo group, with statistical analysis showing a signi cant difference in the degree of opaci cation in those patients when comparing baseline and 4 months (p=0.01) and 14 days and 4 months (p=0.01). Patients with pulmonary brosis were not observed (Figure 7).

Discussion
Severe SARS-CoV-2 infection induces a cytokine storm, leading to ARDS and MOD, which are very serious health conditions.Those For MSC therapy to be feasible in patients with critical illnesses such as COVID-19, cells must be obtainable within a very short time and in adequate numbers from a reproducible production process 68 . In this context, the use of allogeneic UC cells, available in a Biobank, allows postthaw, culture recoveryand infusion within 72 hours after the patient's inclusion in the study. Some studies 69, 70 show that MSCs need to restart their metabolism and other biochemical processes before infusion, recovered by a 24-72hours subculture, because thawed MSCs seem to be unresponsive directly after thawing. If MSCs are allowed to recover in culture, they restore their functionality. For this reason, in this trial, fresh cells were used in all infusions.
Sample randomization was conducted in this study to protect against imbalance in biasing caused by enrollment. However, with small numbers of patients, there is still potential for imbalance. Some differences between groups were observed at baseline, demonstrating that the UC-MSC group seemed to be more compromised than the placebo group.
All patients received standard treatment with steroids because it is recommended to use steroids in crically ill patients to inhibit the in ammatory response, especially for those requiring respiratory support 71 . Glucocorticoids, including dexamethasone, also reduce neutrophil extracellular trap (NET) formation, most likely by suppressing the expression of in ammatory mediators that activate neutrophils 72 . Heparin was also used as a standard treatment because it reduces NET formation 73 , as it has been shownto have therapeutic value in COVID-19 treatment 74 .
In this research, two patients in the UC-MSC group had no previous chronic diseases, while all other patients had comorbidities. This is in line with studies that show that the presence of underlying conditions such as cardiovascular disease, chronic pulmonary disease, and diabetes are risk factors that will require critical care 75 .
The interval between the rst infusion and hospital discharge was similar between groups (average of 16. The mortality rate in this study was 35%. Out of six patients, ve had at least one comorbidity, and the most common cause of death was secondary bacterial infection. A similar study carried out in Brazil showed that in the same period of time, the mortality rate in ICU patients in theSouth region was 53% 78 . Hashemian et al. 52 studied critically ill patients with severe hypoxemia who requiredMV, and observed a 45% mortality rate-most of which had signs of multiorgan failure or sepsis, and died 5-19 days after the rst infusion. Also Dilogo et al. 51 showed that the mortality rate was 65% in a group of intubated critically ill patients with COVID-19 in the ICU, and higher mortality was associated with patients who had two or more comorbidities. Another trial with critically ill patients was conducted by Adas et al. 50 , and they observed 33% mortality in patients who received MSC therapy. The most common cause of death was secondary infections due to bacteria, followed by myocardial infarction and thromboembolism. Data fromsuch studies reinforce that critically ill COVID-19 patients had small chances of survival. Gender was also shown to be related to mortality. The ACE2 gene localizes on the Xchromosome, and ACE2 levels in the blood are higher in males than in females as well as in patients with diabetes or cardiovascular disease [79][80][81][82][83][84][85][86][87][88][89] . In this study, it was observed that six patients (83.3%) were male. Therefore, male patients might be more likely to die from COVID-19 because of the high expression of ACE2.
A high mortalityrate of patients who presented kidney dialysis dysfunction was also observed 83 . The incidence of acute kidney injury (AKI) secondary to COVID-19 is high 84 . Kidney failure appears to occur late in the course of disease and is strongly associated with high mortality among hospitalized COVID-19 85,86 . Ghonimi et al. 87 reported a strong association between death related to COVID-19 infection in dialysis patients, which was also observed in the study of Costa et al. 88 , showing that COVID-19 patients with AKI who need dialysis had worse outcomes. Lino et al. 89 also showed that a worse prognosis is frequently associated with a more rapid evolution to intensive and respiratory care or even dialysis 88 .
According to Saleh et al. 53 , the optimal time for cell infusion is the second week of the disease, namely, the second phase, where there is hyperin ammation beginning on days 7 to 15. Zhu et al. 56 also suggested that MSCs can improve the outcome of patients with severe/critical symptoms more signi cantly than common/mild patients. In this trial, only critically ill patients were included, and the average interval between symptom onset and rst cell injection was 10.7 days for the UC-MSC group and 12.1 days for the placebo group. Possibly due to a combination of suitable patient and the ideal time points for patient treatment, an overall patient bene t after IV UC-MSC infusion was noticed.
Verifying the presence of genomic material of the virus in serum or plasma represents a useful approach to evaluate the impact of the extrapulmonary dissemination of viral material on disease severity and on the host response to the infection 90,91 . The systromalic dissemination of the virus or viral components is associated with the severity of COVID-19 and with a number of parameters indicating the presence of a dysregulated response to the infection 92 . In this study, in both groups, 14 days after infusion, there was a reduction in viral load over time, without signi cant differences. This is in line with Lanzoni et al. 46 , who showed no differences between the UC-MSC treatment and the control group. According totheauthors, UC-MSC treatment seems to be more closely associated with a decrease in in ammatory cytokines rather than a change in viral load.Leng et al. 39 also observed that critically severe patients became negative for hCoV-19 nucleic acid 13 days after transplantation.
In the acute phase reaction of an in ammatory process, there is a variation in the concentrations of various plasma proteins, including Creactive protein and ferritin. They are important biomarkers of in ammation in the context of COVID-19 progression because they are predictive of in-hospital 93,94 . Patients with the highest ferritin levels also presented signi cantly higher levels of C-reactive protein and serum creatinine 89 .
In this study, analysis of in ammatory markers showed that C-reactive protein and ferritin values, in the UC-MSC group, decreased in the second and fourthmonths compared with baseline. In the placebo group, the levels were always higher than the reference ranges. Those results are in accordance with studies that show that the in ammatory biomarkers were increased in COVID-19 patients 95,96 .
Critically ill COVID-19 patients have a CRS storm that involves elevated levels of circulating cytokines and immune-cell hyperactivation 97 . It occurs due to the combination of a defective (or delayed) rst line of defense, followed by persistent hypercytokinemia and a dysfunctional Tcell response. That results in impaired clearance of apoptotic cells or infected/activated macrophages, followed by multiple cytokine release, hemophagocytosis, coagulopathy, and ARDS 98-100 . The clinical manifestation is the sharp rise of a large number of cytokines within a short time frame. Liu et al. 101 identi ed that serum levels of IL-6 (>32.1 pg/mL), one of the mediators of hyperin ammation, have a signi cant correlation with the severity of COVID-19, and can be used to predict disease risk. As part of this study, analysis of plasma cytokine levels was performed, andincreased IL-6 levels were observed in both groups. However, in the UC-MSC group, there was a signi cant reduction from day 14 (mean 9.59 pg/mL in the second month and 3.70 pg/mL in the fourth month), while in the placebo group, the levels remained high (mean 45.97 pg/mL at the second month and 100.97 pg/mL at thefourth month). A gradual decline in IL-6 levels, as shown in the present study, might be a biologically relevant marker of the e cacy of UC-MSC treatment in patients with COVID-19.
Levels of IL-8 in the UC-MSC group until the fourteen day were always higher than those in the placebo group. In both groups, there was a large reduction in values at 2 and 4 months. IL-8 is known as a neutrophil chemotactic factor and plays a major role in the recruitment of neutrophils to the site of infection. Ma et al. 102 did not observe any association of IL-8 concentrations with the severity of COVID-19, butdid observe an association between IL-8 serum levels and the duration of illness in patients with severe COVID-19. Thus, IL-8 will be a signaling pathway in the evolution of COVID-19. This same result was obtained by Li et al. 103 , who showed that serum levels of IL-8 correlated to the overall clinical disease scores at different stages of the same COVID-19 patients. Hence, IL-8 may act as a biomarker for COVID-19 disease prognosis. The higher levels in the UC-MSC group enforced that patients in this group had a worse prognosis than those in the placebo group. When there was patient recovery, in the second month, there was a reduction in IL-8 levels in both groups.
The MCP-1-CCL2 chemokinehas a critical role in the process of in ammation, where it attracts or enhances the expression of other in ammatory factors/cells. It is a biomarker associated with the severity of COVID-19 disease and can be related to the risk of death in COVID-from the fourteen day to the fourth month, reaching levels with no differences in relation to the placebo group. Resultsreveal that there was decreased in ammation and clinical improvement in these patients after cell treatment.
There were no differences in IL-7 levels in the UC-MSC group at any evaluation time; on the other hand, in the placebo group, the IL-7 level was decreased in the fourth month, with a signi cant difference compared to baseline. IL-7 is a pleiotropic cytokine essential for lymphocyte survival and expansion. Most likely, for this reason, a recovery in the TCD3, TCD4 and NK lymphocyte numbers was not observed in the placebo group at different evaluation times. IL-7 promotes lymphocyte expansion and possibly reverses T-cell exhaustion and may be useful in restoring immune systemic homeostasis 105 . Studies show that IL-7 exerts antiapoptotic properties and induces potent proliferation of naive and memory T-cells, leading to replenishment of circulating TCD4+ and TCD8+ 106,107 .
At the in ammatory stage, there is a discharge of cytokines, chemokines and growth factors triggering neutrophil and monocyte recruitment 108 . Neutrophils and the imbalance between NET formation and degradation play a central role in the pathophysiology of in ammation, coagulopathy, organ damage, and immunothrombosis, which characterize severe cases of COVID-19 109  D-dimer, a brin degradation product, is also used as a biomarker for thrombotic disorders and has been identi ed as a potential indicator for prognosis in COVID-19 patients 113,114,115 . According to this study, both groups presented D-dimer values above the reference in all evaluations. However, in the UC-MSC group, a decrease in D-dimer values was observed in the second month. This indicates that cell infusion was effective in reducing D-dimer levels in the UC-MSC group, decreasing the risk of thrombosis formation in these patients.
Lymphopenia is a typical pro le in patients with COVID-19 116,117 and might be related to disease severity and mortality 118,119 ; therefore, it is very important to determine these parameters when evaluating critically ill patients. In this trial, the number of TCD3 and TCD4 lymphocytes was Tcells and fewer nonexhausted CD8+ Tcells than patients with mild COVID-19 122 . Several studies observed that lymphocyte count returned to the normal range in the experimental group, and the time was signi cantly faster after stromal cell infusion compared with the control treatment 48,123,124 .
The persistent follow-up of discharged patients with COVID-19 is essential to nd ways to improve quality of life and reduce morbidity and mortality by e cient prevention. In this study, some markers of cardiac and kidney function were evaluated,and a CTscan was performed for pulmonary evaluation. In this trial, no differences were observed in relation to troponinI levels, corroborating the results observed by Johnsen et al. 23 , who analyzed patients with long COVID-19 sequelae three months posthospitalization and observed no signs of cardiac dysfunction.
Huang et al. 25 showed that 13% of patients without acute kidney injury at the acute phase had a decreased glomerular ltration rate at follow-up, exhibiting an underestimation of patients with kidney dysfunction. Persistent impairment in renal function can occur following an episode of acute kidney injury, with the potential to progress to end-stage kidney disease with dialysis 125 , which highlights the importance of long-term follow-up. Creatinine values in the UC-MSC group were above the reference until thesecond month, followed by a reduction at the fourth month.
In the placebo group, there was an increase above the reference at the second and fourth months. It is probable that kidney lesions acquired during the disease's activity remain sequelae that may result in a slow and asymptomatic progression toward advanced stages and chronic kidneyfailure (CKD). Thus, patients who have recovered from COVID-19 who present proteinuria, hematuria, elevated creatinine and AKI should be monitored for CKD 126 . Increased creatinine values may also be associated with the patient's nutritional status; however, these patients underwent nutritional assessment at 2 and 4 months, and it was observed that all were in good nutritional status.
The bene ts of corticosteroid treatment for accelerating the recovery of lung injury,according to pulmonary function assessment and chest imaging in patients with COVID-19, are controversial 71,125,127 . Therefore, new strategies to avoid pulmonary sequelae need to be developed.
Once injected intravenously, a signi cant amount of MSCs accumulate in the lungs, and they secrete numerous factors that play an important role in immunomodulation, protect alveolar epithelial cells, restore thepulmonary alveolar niche, prevent brosis, and improve overall pulmonary function, which is a great bene t for treating severe pulmonary disease in 128 .In addition, lung function and chest CT changes may be impaired months after the infection 129 . Huang et al. 25 observed that a considerable proportion (22-56%) of patients had a pulmonary diffusion abnormality 6 months after symptom onset. In this trial,there was a decrease in lung lesion extension in the UC-MSC group after 4 months of follow-up. The improvement of pulmonary lesions directly affects the recovery of lung function and the remission of clinical symptoms 48 ; therefore, the results observed in this study could re ect reduced lung in ammation in the UC-MSC group mediated by immune regulation.
Throughout this trial, there were some limitations such as the patient assessment time, between the fourteen day and 2 month, was very long.
Many parameters may have improved before the 2 months, but the exact moment could not be observed. Sample randomization was conducted in this study; however, based on some in ammation markers andlymphocyte subpopulations, the UC-MSC group seemed to be more compromised than the placebo group at baseline. Although the sample size was not large enough to stratify subgroups, it was di cult to exclude bias. The emergency condition in ICUs did not allow us to carry out CT evaluations in all patients at different times.
The results of this study revealed that in the UC-MSC group, there was a reduction in the levels of ferritin, IL-6 and MCP1-CCL2 on the fourteen day. In the second month, a decrease in the levels of reactive C-protein was observed, as well as D-dimer and neutrophils and an increase in the numbers of TCD3, TCD4 and NK lymphocyteswere observed. A decrease in lung extension was observedinthe fourth month. The improvement in all the parameters was maintained until the end of patient follow-up. Those data show that UC-MSCs can play an important role both in the early stages, by preventing moresevere complicationsand in the chronic phase, with a reduction in sequelae.
COVID-19 is a complex multifactorial disease that makes treatment di cult using a single strategy. The promising long-term safety and e cacy results shown in this trial indicate that UC-MSCs could be used as adjunctive therapy for critically ill COVID-19 patients. UC-MSCs showed bene cial effects for patient recovery in the shortterm through a decrease inCRS by secreting anti-in ammatory factors, reducing risk of thrombosis and, in the longterm, viareduction in kidney and pulmonary sequelae based on tissue repair. The combination of immunomodulatory therapy based on UC-MSCs and antiviral drugs could help accelerate patient recovery, attenuating disease progression. Declarations collection; AM, performed the experiments and assembly of data; APA, ow cytometry; CAL, CT scan analysis; RRP, statistical analysis; VRJ, cytogenetic analysis; IMV, cytogenetic analysis; APM, umbilical cord selecting donors; HCJ, performed patient enrolment and intervention; ED, performed patient enrolment; PRSB conceived and designed experiments and analyze the data; AC, conceived and designed experiments, analyze the data and co-wrote the manuscript. All the authors reviewed and approved the nal manuscript. PRSB and AC: these authors contributed equally to this work.

Avaiability of data and materials
The majority of the data generated or analyzed during this study are included in this article.
Ethical Approval and consent to participate All procedures in this study were conducted in accordance with the Ethics Committee in Human Research of the PontifíciaUniversidadeCatólica do Paraná (PUCPR) (CAAE: 30833820.8.0000.0020) and Comissão Nacional de ÉticaemPesquisa (CONEP).Written informed consent was obtained from the patient for their anonymized information to be published in this article.

Competing interests
The author(s) declared no potential con icts of interest with respect to the research, authorship, and/or publication of this article.