Umbilical cord mesenchymal stem cells for COVID‐19 acute respiratory distress syndrome: A double‐blind, phase 1/2a, randomized controlled trial

Abstract Acute respiratory distress syndrome (ARDS) in COVID‐19 is associated with high mortality. Mesenchymal stem cells are known to exert immunomodulatory and anti‐inflammatory effects and could yield beneficial effects in COVID‐19 ARDS. The objective of this study was to determine safety and explore efficacy of umbilical cord mesenchymal stem cell (UC‐MSC) infusions in subjects with COVID‐19 ARDS. A double‐blind, phase 1/2a, randomized, controlled trial was performed. Randomization and stratification by ARDS severity was used to foster balance among groups. All subjects were analyzed under intention to treat design. Twenty‐four subjects were randomized 1:1 to either UC‐MSC treatment (n = 12) or the control group (n = 12). Subjects in the UC‐MSC treatment group received two intravenous infusions (at day 0 and 3) of 100 ± 20 × 106 UC‐MSCs; controls received two infusions of vehicle solution. Both groups received best standard of care. Primary endpoint was safety (adverse events [AEs]) within 6 hours; cardiac arrest or death within 24 hours postinfusion). Secondary endpoints included patient survival at 31 days after the first infusion and time to recovery. No difference was observed between groups in infusion‐associated AEs. No serious adverse events (SAEs) were observed related to UC‐MSC infusions. UC‐MSC infusions in COVID‐19 ARDS were found to be safe. Inflammatory cytokines were significantly decreased in UC‐MSC‐treated subjects at day 6. Treatment was associated with significantly improved patient survival (91% vs 42%, P = .015), SAE‐free survival (P = .008), and time to recovery (P = .03). UC‐MSC infusions are safe and could be beneficial in treating subjects with COVID‐19 ARDS.

Diabetes Research Institute Foundation (DRIF); The Cure Alliance; North America's Building Trades Unions (NABTU) control group (n = 12). Subjects in the UC-MSC treatment group received two intravenous infusions (at day 0 and 3) of 100 ± 20 × 10 6 UC-MSCs; controls received two infusions of vehicle solution. Both groups received best standard of care. Primary endpoint was safety (adverse events [AEs]) within 6 hours; cardiac arrest or death within 24 hours postinfusion).
Secondary endpoints included patient survival at 31 days after the first infusion and time to recovery. No difference was observed between groups in infusion-associated AEs. No serious adverse events (SAEs) were observed related to UC-MSC infusions. UC-MSC infusions in COVID-19 ARDS were found to be safe. Inflammatory cytokines were significantly decreased in UC-MSC-treated subjects at day 6. Treatment was associated with significantly improved patient survival (91% vs 42%, P = .015), SAE-free survival (P = .008), and time to recovery (P = .03). UC-MSC infusions are safe and could be beneficial in treating subjects with COVID-19 ARDS. • UC-MSC treatment was associated with a significant decrease in a set of inflammatory cytokines involved in the COVID-19 "cytokine storm." • UC-MSC treatment was associated with significantly improved patient survival and time to recovery.
• The observed findings strongly support further investigation in a larger trial designed to estimate and establish efficacy.

Significance statement
This study was a double-blind, randomized, controlled, early  requiring hospitalization. 3,4 Severe COVID-19 is believed to result from hyperinflammation, overactive immune response triggering cytokine storm, and a prothrombotic state, collectively determined as immunothrombosis, all elicited by SARS-CoV-2 infection. [5][6][7][8] Subjects progressing to acute respiratory distress syndrome (ARDS) require high-flow oxygen therapy, intensive care, and frequently, mechanical ventilation. 3,4,[9][10][11][12] Mortality in patients with COVID-19 and ARDS was reported to be 52.4%. 10 There is an urgent need for novel therapies that can attenuate the excessive inflammatory response associated with the immunopathological cytokine storm and immunothrombosis, that can accelerate the recovery of functional lung tissue, and that can abate mortality in patients with severe COVID-19.
Mesenchymal stem cells, also known as mesenchymal stromal cells or medicinal signaling cells (MSCs), 13 have been shown to modulate overactive immune and hyperinflammatory processes, promote tissue repair, and secrete antimicrobial molecules. [14][15][16] These cells, with established safety profile when administered intravenously, 17 have been studied for treatment of autoimmune diseases (eg, type 1 diabetes 18,19 systemic lupus erythematous, 20 inflammatory disorders, 21 and steroid-refractory graft-vs-host-disease (GvHD). 22 MSCs have been reported to limit inflammation and fibrosis in the lungs, 23

| Participants
The trial was conducted in accordance with the principles of the Declaration of Helsinki and consistent with the Good Clinical Practice guidelines of the International Conference on Harmonisation.
Subjects diagnosed with COVID-19 ARDS were eligible for inclusion if they met the eligibility criteria listed in Table S1 within 24 hours of enrollment. The investigations were performed with informed consent.

| Randomization
Twenty-four subjects hospitalized for COVID-19 were randomized 1:1 to either UC-MSC treatment (n = 12) or to the control group (n = 12). Patients were assigned to treatment group using a stratified, blocked randomized design.
Additional details are provided in the supplemental online Methods.

| Blinding
The study was double-blinded: neither the patient nor the assessing physician was aware of treatment assignment, and the staff responsible for product administration were blinded to group assignment.

| UC-MSC investigational product
UC-MSCs were manufactured as previously described. 38

| Plasma preparation from peripheral blood
Whole blood was collected from randomized subjects at day 0 (immediately pretreatment) and at day 6 after treatment initiation. Whole blood was collected into EDTA-treated tubes, transferred on ice, and processed for plasma separation within 4 hours. Whole blood was centrifuged at 2000g for 15 minutes at 4 C. The plasma (top fraction) was collected, aliquoted into cryogenic tubes, and stored at −80 C until processing.

| Analysis of viral load by SARS-CoV-2 RT-PCR
The RealStar SARS-COV-2 RT-PCR kit (Altona Diagnostics GmbH, Hamburg, Germany) was used to detect the SARS-CoV-2-specific S gene and quantify the number of copies per mL of plasma. The assay was performed following the manufacturer's instruction, using plasma samples collected from the randomized subjects on day 0 and day 6. The fluorescent signals were visualized via a laser scanner equipped with a Cy3 wavelength (green channel) and converted to concentrations (pg/mL) using the standard curve generated per array.

| Statistical methods
Comparisons of AEs, SAEs, demographics, clinical characteristics, comorbidities, and concomitant treatments between the two groups were performed using Fisher's exact test and Wilcoxon two-sample tests for categorical and continuous variables, respectively. Survival, survival in absence of SAE (SAE-free survival), and time to recovery were estimated in each group with Kaplan-Meier survival estimates. Logrank tests were used to compare hazards between groups. For the analyses of viral load, P values were calculated using the Wilcoxon rank-sum test on SAS 9.4. The data were nonnormally distributed. For the analyses of inflammatory cytokines, chemokines, and growth factors, group data at a specific day were analyzed via nonparametric unpaired Mann-Whitney t test; for the analyses on longitudinal changes in each group, data at day 0 and day 6 were analyzed via nonparametric paired Wilcoxon t test.

| Clinical trial protocol
The clinical trial protocol is included in the Data S1.

| Participant flow
Participant flow is shown in Figure 1.

| Recruitment
From 25 April 2020, to 21 July 2020, a total of 28 subjects were enrolled. Four subjects were subsequently determined to be ineligible because of screen failure. Twenty-four subjects were randomized ( Figure 1). At enrollment, 11 subjects (46%) were receiving invasive mechanical ventilation, and 13 (54%) were on high flow oxygen therapy via noninvasive ventilation (including high flow nasal cannula, continuous positive airways pressure, or bilevel positive airways pressure) prior to initiation of treatment.
Demographics and baseline characteristics for enrolled subjects, along with stratification, randomization, and concomitant treatment information are presented in Table 1 and Table S2.
Two cases required special considerations. Subject #11 died for reasons unrelated to COVID-19 after failed endotracheal intubation. Therefore, this subject was considered as censored in the data analysis for time to COVID-19-related death and time to recovery outcomes. Subject #24 left the hospital against medical advice 11 days after second infusion and was thus considered as censored in the time to recovery analysis. This patient eventually recovered at home and was confirmed alive at 31 days after the first infusion.
A Data Safety Monitoring Board reviewed all safety data.
At the time of this writing, all subjects have been followed for 31 days after the first infusion, corresponding to 28 days after the second infusion.
Three subjects in each group were stratified to the mild-to-moderate ARDS severity stratum, and nine subjects in each group into the F I G U R E 1 Enrollment and randomization. UC-MSC, umbilical cord mesenchymal stem cell moderate-to-severe stratum. There were no significant differences in concomitant treatments between the groups (Table 1; Table S2). The only differences observed in baseline characteristics and comorbidities were in body mass index and obesity, which were higher in the UC-MSC treatment group (Table 1; Table S2). The analysis was by original assigned groups.
The viability of UC-MSCs (investigational product) at the time of product release for administration was found to be 96.2% ± 1.8% by trypan blue and 88.4% ± 7.6% by flow cytometry using fixable viability stain. Apoptosis, assessed by activated caspase-3, was found to be 2.4% ± 3.7%, by flow cytometry ( Figure S1).  Figure S1).

| Outcomes and estimations
A total of nine deaths were documented by day 28 after the second infusion. Two deaths occurred in the UC-MSC treatment group and seven deaths in the control group. One subject (Subject #11) in the UC-MSC treatment group died as a result of a failed endotracheal intubation. This outcome was deemed to be unrelated to the patient's COVID-19 disease. Therefore, data analyses for this subject were censored at the time of failed endotracheal intubation.
The details of all deaths are presented in Table S3.  Table 2.

| Adverse events
Subjects with AEs by relatedness to treatment c,d

| Primary endpoint
The primary endpoint was safety, defined as the occurrence of prespecified infusion-associated AEs within 6 hours after infusion in addition to cardiac arrest or death within 24 hours after infusion.
Prespecified infusion-associated AEs are outlined in Table 3. One subject in each group developed infusion-associated AEs. UC-MSC treatment was found to be safe, as it did not lead to an increase in prespecified infusion-associated AEs. In the UC-MSC treatment group, the only reported adverse event occurred in a subject with bradycardia, who experienced worsening of bradycardia and required transient vasopressor treatment. In the control group, all prespecified infusion-associated AEs occurred in the same subject, who experienced cardiac arrest 2 hours after infusion of vehicle solution. In each group, one subject developed infusion-associated AEs.

| Analysis of viral load in peripheral blood plasma
The median viral load at day 0 or day 6 did not differ significantly between the UC-MSC treatment and control group. The P values were .196 and .136 for day 0 and day 6, respectively ( Figure S2).

| Analysis of inflammatory cytokines and chemokines levels in peripheral blood plasma
The The vasopressor dose increase was ordered by the primary treating physician before the infusion started, but it was not given until hours later, after the infusion.  This study was not intended as an efficacy trial, but instead as an early phase study to establish safety. We relied on randomization to protect against imbalance in biasing preliminary estimates of efficacy.
Stratified, blocked randomization was employed to evenly represent ARDS severity and changing standard of care over time between groups. Even with blocked randomization, confounding may exist because, with small numbers, there is still potential for imbalance. Table 1 and Table S2 illustrate  and convalescent plasma. 43 The overall "signature" of the response in the UC-MSC treatment group is characterized by a reduction of the levels of key inflammatory molecules involved in the COVID-19 "cytokine storm," including IFNg, IL-6, and TNFa cytokines and RANTES chemokine. 50 In parallel, a reduction in GM-CSF was observed. GM-CSF is the main activator of the proinflammatory M1 macrophage phenotype; hence, its reduction could lead to macrophage polarization toward alternatively activated M2 macrophages. 51 The levels of PDGF-BB also resulted significantly reduced in the UC-MSC treatment group. Notably, PDGF-BB stimulates mesenchymal cell activation, airway smooth muscle cell proliferation and migration, lung fibroblast cytokine production, and activation of nociceptive neurons. [52][53][54] Hence, it is possible that the administration of allogeneic MSCs could accelerate the steps of tissue repair in the lungs, decreasing the need for further mesenchymal cell activation.
The positive response in subjects receiving UC-MSC treatment seems to be more closely associated to a decrease in inflammatory cytokines, rather than a change in viral load.
The observations made in this study could be of assistance for future studies in the field of COVID-19, ARDS, hyperinflammatory states, overactive immune responses, and autoimmunity. In addition, the preferential targeting of lung tissue after intravenous infusion could make UC-MSCs particularly appealing for ARDS secondary to trauma, microbial infection, and pulmonary GvHD.

| CONCLUSION
The results of this trial indicate that UC-MSC infusions in COVID-19 with ARDS are safe. Moreover, UC-MSC treatment was associated with a significant reduction in SAEs, mortality, and time to recovery, compared with controls.

ACKNOWLEDGMENTS
The authors wish to thank the North America's Building Trades

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
G.L.: conception/design, collection and/or assembly of data, data analy-

DATA AVAILABILITY STATEMENT
Individual participant data will be available. In particular, individual participant data that underlie the results reported in this article, after deidentification (text, tables, figures), will be available. Study protocol, statistical analysis plan, and analytic code will be available. Data will be available immediately after publication and ending 2 years after article publication. Data will be shared with investigators whose proposed use of the data has been approved by an independent review committee ("learned intermediary") identified for this purpose, to achieve aims in the approved proposal. Proposals may be submitted up to 24 months after article publication. After 24 months the data will be available in the University of Miami's data warehouse but without investigator support other than deposited metadata. Information regarding submitting proposals and accessing data may be found at http://www.