Double-blind, Randomized, Controlled, Clinical Trial to Assess the Efficacy of Allogenic Mesenchymal Stromal Cells in Patients With Acute Respiratory Distress Syndrome Due to COVID-19
Phase: PHASE2
Conditions: Acute Respiratory Distress Syndrome, COVID-19 Pneumonia
Sponsor: Cristina Avendaño Solá

 
                     
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EudraCT: 2020-002193-27 
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Double-blind, randomized, controlled, clinical trial to assess the 
efficacy of allogenic mesenchymal stromal cells in patients with 
acute respiratory distress syndrome due to COVID-19. 
 
 
 
Protocol Code: COVID-AT 
Phase: II 
 
Protocol Date: October 14th, 2020 
Version: 1.1 
EudraCT Number: 2020-002193-27 
 
Sponsor: Dr. Cristina Avendaño-Solà 
 
Principal Investigator: Dr. Rafael Duarte Palomino 
 
 
 
 
 
 
 
 
CONFIDENTIAL:  
The information and data included in this protocol is property of the Trial Steering 
Committee. No person is authorized to make such information public without the written 
permission of the Sponsor. These restrictions will also apply to all information that is 
provided to you in the future and that is considered privileged or confidential. This material 
may be disclosed and used by your team and associates, in so far as it may be necessary for 
the development of the clinical study. 
 
 

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2 
SIGNATURES PAGE  
 
SPONSOR 
Dr. Cristina Avendaño-Solá 
 
 
 
 
 
____________________________________  
(Signature) (Date)    
 
 
PRINCIPAL INVESTIGATOR  
Dr. Rafael Duarte Palomino 
 
 
 
 
 
____________________________________  
(Signature) (Date)  
 
 
I confirm that I have read the protocol and agree to conduct and carry out the study under the 
conditions described in this protocol as well as in accordance with the Guidelines on Good Clinical 
Practice and the Declaration of Helsinki. 
 
 
 
 

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KEY TRIAL CONTACTS 
 
Sponsor 
Cristina Avendaño Solá, MD, PhD 
Department of Clinical Pharmacology. 
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1. 28222 Majadahonda, Madrid 
Phone: 91 191 64 79 
E-mail: cavendano@salud.madrid.org 
 
Clinical Trial Steering 
Committee 
Cristina Avendaño Solá, MD, PhD 
Department of Clinical Pharmacology 
 
Rafael F. Duarte Palomino, MD, PhD, FRCP(Lon) 
Department of Hematology and GMP Unit  
 
Inés Lipperheide Vallhonrat, MD 
Department of Intensive Care Medicine 
 
Rosa Malo de Molina, MD 
Department of Respiratory Medicine 
 
María E. Martínez Muñoz, MD 
Department of Hematology and GMP Unit 
 
Concepción Payares-Herrera, MD, PhD 
Department of Clinical Pharmacology 
 
Antonio Ramos Martínez, MD, PhD 
Servicio de Medicina Interna – Enfermedades Infecciosas 
 
Juan José Rubio Muñoz, MD, PhD 
Department of Intensive Care Medicine 
 
Piedad Ussetti Gil, MD, PhD 
Department of Respiratory Medicine 
 
Rocío Zafra Pizarro, BSChem 
Department of Hematology and GMP Unit 

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Principal Investigators (PI) 
PI Project Coordinator: 
 
 
 
 
 
 
PI Intensive Care Unit: 
 
 
 
 
 
 
PI Respiratory Medicine: 
 
Rafael F. Duarte Palomino, MD, PhD, FRCP(Lon) 
Servicio de Hematología y Hemoterapia 
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 91 191 6662 
E-mail: rafael.duarte@salud.madrid.org  
Juan José Rubio Muñoz, MD, PhD 
Servicio de Cuidados Intensivos  
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916425 
E-mail: juanjose.rubio@salud.madrid.org  
Rosa Malo de Molina, MD 
Servicio de Neumología 
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916000 
E-mail: rmm02004@yahoo.es 
Pharmacovigilance 
Concepción Payares-Herrera, MD, PhD 
Servicio de Farmacología Clínica  
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916481 
E-mail: cpayares@idiphim.org 
 
Regulatory, 
Methodological and 
Statistical Aspects  
Concepción Payares-Herrera, MD, PhD 
Servicio de Farmacología Clínica  
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916481 
E-mail: cpayares@idiphim.org 
 
Project Management and 
Monitoring 
Concepción Payares-Herrera, MD, PhD 
Servicio de Farmacología Clínica  
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916481 
E-mail: cpayares@idiphim.org 
 
 
 
 

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LIST OF ABREVIATIONS 
 
Abbreviation 
Definition 
ACE2 
Angiotensin-Converting Enzyme 2  
ADR 
Adverse Drug Reaction 
AE 
Adverse Event 
AEMPS 
Agencia Española de Medicamentos y Productos Sanitarios (Spanish Medicines 
Agency) 
AESI 
Adverse Events of Special Interest 
ARDS 
Acute Respiratory Distress Syndrome 
BM 
Bone Marrow 
CEIm 
Comité de Ética de la Investigación con Medicamentos (Research Ethics 
Committee for Medicinal Products) 
COVID-19 
Coronavirus Disease 19 
CRF 
Case Report Form 
CRP 
C-reactive Protein 
ECMO 
Extracorporeal Membrane Oxygenation 
GMP 
Good Manufacture Practices  
ICU 
Intensive Care Unit 
IL 
Interleukin 
MSC 
Mesenchymal Stromal Cell 
PCR 
Polymerase Chain Reaction 
SAE 
Serious Adverse Event 
SOC 
Standard of Care 
SOFA 
Sequential Organ Failure Assessment 
SSF 
Saline Solution 
SUSAR 
Suspected Unexpected Serious Adverse Response 
VM 
Mechanical Ventilation 
WHO 
World Health Organization 
 
 
 
 
 
 

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TABLE OF CONTENTS 
1. TRIAL SYNOPSIS ........................................................................................................................................................... 7 
2. INTRODUCTION ......................................................................................................................................................... 13 
3. OBJETIVES AND ENDPOINTS ..................................................................................................................................... 16 
4. CLINICAL TRIAL DESIGN ............................................................................................................................................ 18 
4.1. TYPE OF STUDY AND OVERALL DESIGN ...................................................................................................................... 18 
4.2. STUDY PARTICIPATION DURATION ............................................................................................................................. 18 
4.3. EARLY TERMINATION OF THE TRIAL ........................................................................................................................... 18 
5. STUDY POPULATION ................................................................................................................................................. 19 
5.1. INCLUSION CRITERIA .............................................................................................................................................. 19 
5.2. EXCLUSION CRITERIA ............................................................................................................................................. 19 
5.3. SUBJECT WITHDRAWAL CRITERIA .............................................................................................................................. 19 
6. STUDY TREATMENT ................................................................................................................................................... 20 
6.1. INVESTIGATIONAL MEDICINAL PRODUCT AND DOSE .................................................................................................... 20 
6.2. DESCRIPTION OF TREATMENT .................................................................................................................................. 20 
6.2. 1. EXPERIMENTAL GROUP....................................................................................................................................... 20 
6.2.2. CONTROL GROUP: .............................................................................................................................................. 20 
6.2.3. STANDARD OF CARE: .......................................................................................................................................... 20 
6.3. TREATMENT ADMINISTRATION ................................................................................................................................ 20 
6.4. TREATMENT ADHERENCE ........................................................................................................................................ 21 
6.5. TREATMENT PREPARATION/STORAGE/ACCOUNTABILITY ............................................................................................... 21 
6.6. PACKAGING AND LABELLING .................................................................................................................................... 21 
6.7. CONCOMITANT TREATMENT ................................................................................................................................... 21 
7. MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING ....................................................................... 22 
8. ASSESMENTS AND PROCEDURES .............................................................................................................................. 23 
8.1. SCREENING PROCEDURES ....................................................................................................................................... 23 
8.2. VISIT 1 (TREATMENT)-DAY 0 .................................................................................................................................. 23 
8.3. EFFICACY ASSESSMENTS (DOUBLE-BLIND FOLLOW UP VISITS) ..................................................................................... 24 
8.4. SAFETY ASSESSMENTS (DOUBLE-BLIND AND/OR OPEN-LABEL PERIODS) ........................................................................ 24 
STUDY SCHEDULE .................................................................................................................................................... 25 
9. PHARMACOVIGILANCE ............................................................................................................................................. 26 
9.1. DEFINITIONS ......................................................................................................................................................... 26 
9.2. ADVERSE EVENTS .................................................................................................................................................. 27 
9.3. ASSESSMENT OF ADVERSE EVENTS ........................................................................................................................... 27 
9.4. CONDUCT IN CASE OF SEVERE ADVERSE EVENTS ........................................................................................................ 28 
9.5. SAFETY NOTIFICATION TO THE HEALTH AUTHORITIES .................................................................................................. 28 
10. ETHICAL ASPECTS .................................................................................................................................................... 29 
10.1. GENERAL CONSIDERATIONS ................................................................................................................................... 29 
10.2. INFORMED CONSENT............................................................................................................................................ 29 
10.3. CONFIDENTIALITY ................................................................................................................................................ 29 
11. FINANCIAL ASPECTS ................................................................................................................................................ 30 
11.1. INSURANCE POLICY .............................................................................................................................................. 30 
11.2. ECONOMIC COMPESATION .................................................................................................................................... 30 
12. STATISTICAL ANALYSIS ............................................................................................................................................ 31 
12.1. GENERAL APPROACH ............................................................................................................................................ 31 
12.2. SAMPLE SIZE CALCULATIONS ................................................................................................................................. 31 
12.3. POPULATIONS FOR ANALYSIS ................................................................................................................................. 31 
12.4. PRIMARY EFFICACY AND SAFETY ANALYSES .............................................................................................................. 32 
13. SUPPORTING DOCUMENTATION AND OTHER OPERTIONAL CONSIDERATIONS ................................................. 32 
13.1. DATA COLLECTION ............................................................................................................................................... 32 
13.2. CLINICAL MONITORING ........................................................................................................................................ 32 
13.3. COMMUNICATIONS AND PUBLICATIONS .................................................................................................................. 32 
13.4. OTHER OPERATIONAL CONSIDERATIONS ................................................................................................................. 32 
14. BIBLIOGRAPHY......................................................................................................................................................... 34 
15. ANNEXES .................................................................................................................................................................. 35 
 

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1. TRIAL SYNOPSIS 
 
Study Title 
Double-blind, randomized, controlled, clinical trial to assess the efficacy 
of allogenic mesenchymal stromal cells in patients with acute respiratory 
distress syndrome due to COVID-19.  
EudraCT Number 
2020-002193-27 
Rationale Summary   
COVID-19 patients present in many cases with acute respiratory distress 
syndrome (ARDS), a severe pulmonary complication characterized by a 
disruption of the alveolar-epithelial barrier, interstitial edema and 
inﬂammatory damage. COVID-19-associated ARDS leads to progressive 
acute respiratory failure, for which there is no specific treatment beyond 
supportive care and mechanical ventilation. Patient outcomes are dismal, 
with high mortality rates greater than 60% in patients who require 
admission to intensive care units (ICU).    
 
Mesenchymal stromal cells (MSC) have immunomodulatory and tissue-
regenerative properties for which they are widely used in cellular therapy. 
MSC are safe and effective in acute respiratory failure and ARDS of 
multiple causes, including promising preliminary evidence in ARDS in 
COVID-19. The Hospital Universitario Puerta de Hierro Majadahonda 
(HUPHM) has experience in the production and use of MSC in various 
immunomodulatory and regenerative indications, as well as in clinical 
investigation in COVID-19 and in the management of ARDS in these 
patients. Here, we present a summary of the rationale and justification to 
study the efficacy of MSC therapy in COVID-19 patients with ARDS 
through a double-blind, randomized, controlled clinical trial. 
 
- ARDS in COVID-19 patients: ARDS is a life-threatening pulmonary 
disorder that can associate with multiple causes. It characterizes by 
diffuse alveolar and capillary endothelial damage which lead to acute 
hypoxemia and non-cardiogenic pulmonary edema with bilateral 
radiographic opacities.  
▪ According to the largest reported series, ARDS occurs in 
approximately 30% of hospitalized COVID-19 patients (17%-42%), 
and in more than 90% of those critically ill.  
▪ The severity of ARDS can be measured by the pressure of arterial 
oxygen to fraction of inspired oxygen (PaO2/FiO2) ratio (Berlin 
criteria). Moderate and severe ARDS are defined by PaO2/FiO2 ratios 
of ≤200 mmHg and ≤100mmHg, respectively. Of note, if arterial 
blood gas measurement cannot be obtained, the pulse oximetric 
saturation SaO2/FiO2 ratio can be used as a reliable surrogate. 
▪ Despite improvement in ICU supportive care, to date, no 
pharmacologic therapy has effectively addressed the disease-specific 
pathophysiology or improved survival in these patients. Thus, 
reported mortality rates of COVID-19 in patients with moderate to 
severe ARDS remain very high at 69% (52%-91%). 
▪ In addition to a very high mortality, in many survivors the initial 
exudative phase is followed by a ﬁbrotic phase that causes long-term 
morbidity.    
 

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- MSC in ARDS: Multiple studies support the use of MSC in various forms 
of acute lung injury and ARDS.  
▪ Preclinical models have shown that MSC can be beneficial in acute 
lung injury, the preclinical correlate of ARDS. They include models 
with microbial, endotoxin and chemical agents, using various sources 
of MSC (bone marrow, adipose tissue and cord blood), delivered by 
either intravenous systemic or direct airway administration, using 
both fresh and cryopreserved MSC, usually as a single dose in a range 
from 5.0 x 104 to 3.6 x 107 cells/kg.  
▪ MSC have been shown to exert anti-inflammatory, anti-bacterial, 
and pro-angiogenic effects leading to reduced inflammation, 
enhancement of pathogen and fluid clearance, improvement of 
tissue repair and decreased mortality. These effects, and their 
impact on reduced mortality are maintained across the different 
preclinical models, MSC origin, method of preparation and timing and 
route of administration. 
▪ Clinical studies of MSC in ARDS with various designs, sample-sizes, 
MSC sources and doses have uniformly confirmed safety, with no 
adverse events, and the largest trials have suggested reductions in 
inflammatory markers, improvement in PaO2/FiO2 ratio and/or 
reduction in mortality with MSC. The largest clinical trial of MSC in 
ARDS in viral infections included 61 patients with ARDS caused by 
H7N9 influenza, showed a significant reduction of mortality in the 
experimental arm with 3-4 doses of 1 x 106 MSC/kg.  
 
- MSC in COVID-19 patients with ARDS: Preliminary experience has been 
reported on the use of MSC in a total of eight COVID-19 patients with 
moderate to severe ARDS:  
▪ Liang et al. treated a single case of a critically ill 65-year old woman 
with COVID-19 ARDS with three intravenous MSC infusions. The 
patient was extubated and off the ventilator within four days of the 
second MSC infusion, inflammation parameters improved, T Cell 
counts increased, and no related adverse events were observed. 
▪ Leng et al. treated seven patients with different degrees of COVID-
19 ARDS severity, including one critically severe case requiring 
invasive ventilation in ICU:  
• From a clinical perspective, out of three patients who received 
placebo as a control group, two deteriorated. However, all seven 
patients in the MSC experimental group, including the one 
classified as critically severe, showed clinical improvement within 
2-3 days of a single intravenous dose of 1x106 MSC/kg, in 
addition to no infusion reactions or severe related adverse events. 
• An important additional finding of this study of MSC in COVID-19 
is that gene expression profile of the infused MSC showed them 
not to express angiotensin-converting enzyme 2 (ACE2) or serine 
protease TMPRSS2, which are required for SARS-CoV-2 cell entry. 
Therefore, the absence of ACE2 and TMPRSS2 indicate that 
infused MSC in COVID-19 patients are free from SARS-CoV-2 
infection, which would have otherwise been one of the potential 
limitations to the success of this strategy. 
 

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- Summary of center’s experience and benefits and risks of the proposal:  
▪ The occurrence of ARDS and acute respiratory failure in COVID-19 
patients is common and represents a dramatic clinical scenario.  
• With the current absence of treatment options, beyond 
respiratory and supportive care, patient outcomes are dismal. 
• MSC offer immunomodulatory and tissue-regenerative properties, 
which make them a promising therapeutic option in this clinical 
setting.  
• In particular, MSC safety is uniformly shown in preclinical and 
clinical studies in ARDS and other indications.  
▪ HUPHM experience to carry out the proposed clinical trial: 
• The GMP Unit at the Department of Hematology produces 
autologous and allogeneic MSC derived from bone marrow (BM) 
for immunomodulatory and regenerative indications for more 
than a decade, it has participated in multiple national and 
international research projects and clinical trials, and is authorized 
for MSC production by the AEMPS (certificate of GMP 
compliance; renovated on 28/05/2019).  
• Currently, HUPHM has a surplus of MSC doses produced prior to 
the COVID-19 pandemic readily available to supply this trial. 
• In addition to production and use of MSC in Hematology, the 
investigators team includes the Departments of Clinical 
Pharmacology, Intensive Medicine, Respiratory Medicine and 
others. This team shares experience in the clinical management of 
COVID-19 patients during the current pandemic and in clinical 
investigation in this setting, including an on-going clinical trial 
(ConPLAS-19; 
CT 
Number: 
NCT04345523), 
which 
is 
complementary to the current proposal.  
 
In summary, in the midst of a worldwide pandemic of SARS-CoV-2, MSC 
represent a potential therapeutic option with a favorable benefit/risk 
balance for severe COVID-19 patients with ARDS for whom we currently 
have no available treatments. Thus, we propose to conduct a double-
blind, randomized, controlled, clinical trial to assess the efficacy of 
allogenic MSC in patients with ARDS due to COVID-19. 
Trial Design 
A double-blind, randomized, controlled, clinical trial to evaluate the 
efficacy and safety of MSC (mesenchymal stromal cells) intravenous 
administration in patients with COVID-induced ARDS compared to a 
control arm.  
All trial participants will receive SOC*.  
Randomization will be 1:1 between:   
⁃ Treatment arm: Allogenic MSC. 
⁃ Control arm: SSF + 4% albumin (same composition as the 
experimental treatment, without the MSC).  
 
*In the context of the current worldwide pandemic for which we have no 
approved medicines or vaccines, SOC can include medicines that are being used in 
clinical practice (e.g. lopinavir/ritonavir; hydroxy/chloroquine, tocilizumab, etc.). 
Center 
Hospital Universitario Puerta de Hierro Majadahonda  
Trial Participants 
Hospitalized adults (≥18 years old) with moderate to severe ARDS 
(according to the Berlin Definition) due to COVID-19 infection.     

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Planned Sample Size 
20 patients (1:1 ratio, for MSC:control arm). 
Follow-up duration 
12 months  
Trial Period  
16 months (3 months for recruitment + 12 months for follow-up + 1 
month for final analysis). 
⁃ Of note, the primary data analysis will be conducted when all patients 
reach day 28 after treatment. Prior to this analysis, a statistical plan will 
be in place with strict provisions to preserve data integrity according to 
ICH and other applicable guidelines. 
⁃ All patients will be followed until the end of study for safety purposes. 
Primary Objective 
⁃ To assess the efficacy of MSC versus a control arm as described in the 
primary endpoint.  
Secondary Objective 
⁃ To evaluate the effects of MSC on the secondary efficacy endpoints.  
⁃ To evaluate the safety and tolerability profiles of MSC. 
Exploratory Objectives 
⁃ To study soluble and cellular biomarkers that might be involved in the 
course of the disease and the response to the investigational product. 
Primary Endpoint 
⁃ Change in the PaO2/FiO2* ratio from baseline to day 7 of treatment 
administration, or to the last available PaO2/FiO2 ratio if death occurs 
before day 7. 
* If an arterial blood gas result cannot be obtained, the SaO2/FiO2 ratio 
could be substituted for the PaO2/FiO2 ratio [TW Rice, et al. Chest 2007]. 
Secondary Endpoints 
Clinical 
⁃ All-cause mortality on days 7, 14, and 28 after treatment.  
⁃ PaO2/FiO2 ratio at baseline and days 2, 4, 7, 14 and 28 after treatment. 
⁃ Oxygen saturation (by standardized measurement) at baseline, daily 
until day 14, and on day 28 after treatment.   
⁃ Time to PaO2/FiO2 ratio greater than 200 mmHg. 
⁃ Subjects’ clinical status on the WHO 7-point ordinal scale at baseline, 
daily until day 14, and on day 28 after treatment. 
⁃ Time to an improvement of one category from admission on the WHO 7-
point ordinal scale. 
⁃ Percentage of patients that worsen at least one category on the WHO 7-
point ordinal scale. 
⁃ Percentage of patients that improve at least one category (maintained 
48h) on the WHO 7-point ordinal scale. 
⁃ SOFA scale at baseline and days 2, 4, 7, 14 and 28 after treatment. 
⁃ Duration of hospitalization (days).  
⁃ Duration of ICU stay (days).  
⁃ Oxygen therapy-free days in the first 28 days after treatment.  
⁃ Duration of supplemental oxygen. 
⁃ Incidence of and duration of non-invasive and invasive mechanical 
ventilation in the first 28 days after treatment. 
⁃ Mechanical ventilation-free days in the first 28 days after treatment.  
⁃ Ventilation parameters. 
⁃ Incidence of new onset pulmonary fibrosis at 3 and 12 months after 
treatment, based on CT scan and pulmonary function tests.  
⁃ Survival at 3 and 12 months.  
 
Safety   
⁃ Cumulative incidence of serious adverse events (SAEs) and Grade 3 and 4 

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11 
adverse events (AEs).  
⁃ Cumulative incidence of adverse drug reactions (ADR) in the 
experimental treatment arm. 
⁃ Cumulative incidence of AEs of special interest. 
 
Analytical endpoints 
⁃ Levels of analytical markers (CRP, lymphocyte and neutrophil counts, 
lymphocyte subpopulations, LDH, ferritin, D-dimer, coagulation tests and 
cytokines...) at baseline and days 2, 4, 7, 14 and 28 after treatment. 
⁃ Other soluble and cellular biomarkers that might be involved in the 
course of the disease and the response to MSC. 
Main Inclusion Criteria 
 
 
1. Informed consent prior to performing study procedures (witnessed 
oral consent with written consent by representatives will be accepted 
to avoid paper handling). Written consent by patient or 
representatives will be obtained whenever possible.  
2. Adult patients ≥18 years of age at the time of enrolment. 
3. Laboratory-confirmed SARS-CoV-2 infection as determined by PCR, in 
oropharyngeal swabs or any other relevant specimen obtained during 
the course of the disease. Alternative tests (e.g., rapid antigenic tests) 
are also acceptable as laboratory confirmation if their specificity has 
been accepted by the Sponsor. 
4. Moderate to severe ARDS (PaO2/FiO2 ratio equal or less than 200 
mmHg) for less than 96 hours at the time of randomization.  
5. Patients requiring invasive ventilation are eligible within 72 hours from 
intubation. 
6. Eligible for ICU admission, according to the clinical team.  
Main Exclusion 
Criteria 
 
 
1. Imminent and unavoidable progression to death within 24 hours, 
irrespective of the provision of treatments (in the opinion of the 
clinical team). 
2. “Do Not Attempt Resuscitation” order in place.  
3. Any end-stage organ disease or condition, which in the investigator’s 
opinion, makes the patient an unsuitable candidate for treatment. 
4. History of a moderate/severe lung disorder requiring home-based 
oxygen therapy.  
5. Patient requiring ECMO, hemodialysis or hemofiltration at the time of 
treatment administration.  
6. Current diagnosis of pulmonary embolism.  
7. Active neoplasm, except carcinoma in situ or basalioma. 
8. Known allergy to the products involved in the allogenic MSC 
production process. 
9. Current pregnancy or lactation (women with childbearing potential 
should have a negative pregnancy test result at the time of study 
enrollment). 
10. Current participation in a clinical trial with an experimental treatment 
for COVID-19 (the use of any off-label medicine according to local 
treatment protocols is not an exclusion criteria).  
11. Any circumstances that in the investigator’s opinion compromises the 
patient’s ability to participate in the clinical trial. 
Investigational Product 
⁃ BM-derived allogenic MSC. 
Dose 
⁃ Patients receive a single dose of approximately 1x106 cells/kg on day 0. 

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Control arm 
⁃ Saline Solution + 4% albumin (same composition as the treatment under 
study, but without the MSC). 
 

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2. INTRODUCTION 
 
Study Rationale and Background  
 
COVID-19 is a new disease caused by a novel coronavirus (SARS-CoV-2) for which there is currently 
no vaccine or authorized therapeutic agents. COVID-19 patients present in many cases with acute 
respiratory distress syndrome (ARDS), a severe pulmonary complication characterized by a 
disruption of the alveolar-epithelial barrier, interstitial edema and inﬂammatory damage. COVID-19-
associated ARDS leads to progressive acute respiratory failure, for which there is no specific 
treatment beyond supportive care and mechanical ventilation. Patient outcomes are dismal, with 
high mortality rates greater than 60% in patients who require admission to intensive care units (ICU).    
 
Mesenchymal stromal cells (MSC) have immunomodulatory and tissue-regenerative properties for 
which they are widely used in cellular therapy. MSC are safe and effective in acute respiratory failure 
and ARDS of multiple causes, including promising preliminary evidence in ARDS in COVID-19. The 
Hospital Universitario Puerta de Hierro Majadahonda (HUPHM) has experience in the production 
and use of MSC in various immunomodulatory and regenerative indications, as well as in clinical 
investigation in COVID-19 and in the management of ARDS in these patients.  
 
Here, we present a summary of the rationale and justification to study the efficacy of MSC therapy in 
COVID-19 patients with ARDS through a double-blind, randomized, controlled clinical trial. 
 
ARDS in COVID-19 patients  
ARDS is a life-threatening pulmonary disorder that can associate with multiple causes. It 
characterizes by diffuse alveolar and capillary endothelial damage which lead to acute hypoxemia 
and non-cardiogenic pulmonary edema with bilateral radiographic opacities.  
• According to the largest reported series, ARDS occurs in approximately 30% of hospitalized 
COVID-19 patients (17%-42%), and in more than 90% of those critically ill.  
• The severity of ARDS can be measured by the pressure of arterial oxygen to fraction of 
inspired oxygen (PaO2/FiO2) ratio (Berlin criteria). Moderate and severe ARDS are defined by 
PaO2/FiO2 ratios of ≤200 mmHg and ≤100mmHg, respectively. Of note, if arterial blood gas 
measurement cannot be obtained, the pulse oximetric saturation SaO2/FiO2 ratio can be used 
as a reliable surrogate. 
• Despite improvement in ICU supportive care, to date, no pharmacologic therapy has 
effectively addressed the disease-specific pathophysiology or improved survival in these 
patients. Thus, reported mortality rates of COVID-19 in patients with moderate to severe 
ARDS remain very high at 69% (52%-91%). 
• In addition to very high mortality, in many survivors the initial exudative phase is followed by a 
ﬁbrotic phase that causes long-term morbidity.    
 
MSC in patients with ARDS 
Multiple studies support the use of MSC in various forms of acute lung injury and ARDS.  
• Preclinical models have shown that MSC can be beneficial in acute lung injury, the preclinical 
correlate of ARDS. They include animal and human lung explant models with microbial, 

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endotoxin and chemical agents, using various sources of MSC (bone marrow [BM], adipose 
tissue and cord blood), delivered by either intravenous systemic or direct airway 
administration, using both fresh and cryopreserved MSC, usually as a single dose in a range 
from 5.0 x 104 to 3.6 x 107 cells/kg.  
• MSC have been shown to exert anti-inflammatory, anti-bacterial, and pro-angiogenic effects 
leading to reduced inflammation, enhancement of pathogen and fluid clearance, 
improvement of tissue repair and decreased mortality. These effects, and their impact on 
reduced mortality are maintained across the different preclinical models, MSC origin, method 
of preparation and timing and route of administration. 
• Clinical studies of MSC in ARDS with various designs, sample-sizes, MSC sources and doses 
have uniformly confirmed safety, with no adverse events, and the largest trials have suggested 
reductions in inflammatory markers, improvement in PaO2/FiO2 ratio and/or reduction in 
mortality with MSC. The largest clinical trial of MSC in ARDS in viral infections included 61 
patients with ARDS caused by H7N9 influenza, showed a significant reduction of mortality in 
the experimental arm with 3-4 doses of 1 x 106 MSC/kg.  
 
MSC in COVID-19 patients with ARDS 
Preliminary experience has been reported on the use of MSC in a total of eight COVID-19 patients 
with moderate to severe ARDS:  
• Liang et al. treated a single case of a critically ill 65-year old woman with COVID-19 ARDS with 
three intravenous MSC infusions. The patient was extubated and off the ventilator within four 
days of the second MSC infusion, inflammation parameters improved, T cell counts increased, 
and no related adverse events were observed. 
• Leng et al. treated seven patients with different degrees of COVID-19 ARDS severity, including 
one critically severe case requiring invasive ventilation in ICU:  
o From a clinical perspective, out of three patients who received placebo as a control 
group, two deteriorated. However, all seven patients in the MSC experimental group, 
including the one classified as critically severe, showed clinical improvement within 2-3 
days of a single intravenous dose of 1x106 MSC/kg, in addition to no infusion reactions or 
severe related adverse events. 
o An important additional finding of this study of MSC in COVID-19 is that gene expression 
profile of the infused MSC showed them not to express angiotensin-converting enzyme 2 
(ACE2) or serine protease TMPRSS2, which are required for SARS-CoV-2 cell entry. Since 
one of the potential limitations to the success of this strategy would be the risk of 
infection of the administered MSC by the causative virus, the absence of ACE2 and 
TMPRSS2 indicate that infused MSC in COVID-19 patients are free from SARS-CoV-2 
infection. 
 
Summary of benefits and risks of the proposal 
• The occurrence of ARDS and acute respiratory failure in COVID-19 patients is common and 
represents a dramatic clinical scenario.  
• With the current absence of treatment options, beyond respiratory and supportive care, 
patient outcomes are dismal. 

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• MSC offer immunomodulatory and tissue-regenerative properties, which make them a 
promising therapeutic option in this clinical setting.  
• In particular, MSC safety is uniformly shown in preclinical and clinical studies in ARDS and 
other indications.  
 
Summary of HUPHM experience to carry out the proposed clinical trial: 
• The GMP Unit at the Department of Hematology produces autologous and allogeneic MSC 
derived from BM for immunomodulatory and regenerative indications for more than a 
decade, it has participated in multiple national and international research projects and clinical 
trials, and is authorized for MSC production by the AEMPS (certificate of GMP compliance; 
renovated on 28/05/2019).  
• Currently, HUPHM has a surplus of MSC doses produced prior to the COVID-19 pandemic 
readily available to supply this trial. 
• In addition to production and use of MSC in Hematology, the investigators team includes the 
Departments of Clinical Pharmacology, Intensive Medicine, Respiratory Medicine and others. 
This team shares experience in the clinical management of COVID-19 patients during the 
current pandemic and in clinical investigation in this setting, including an on-going clinical trial 
(ConPLAS-19; CT Number: NCT04345523), which is complementary to the current proposal.  
 
In summary, MSC represent a potential therapeutic option with a favorable benefit/risk balance for 
COVID-19 patients with ARDS, for whom we currently have no available treatments. Based on this 
unresolved therapeutic need, we propose to conduct a randomized, controlled clinical trial of MSC 
treatment in patients with severe COVID-19 pneumonia. 
 
 

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3. OBJETIVES AND ENDPOINTS 
 
The main objective of this study is to assess the clinical efficacy of allogenic MSC versus a control 
arm. 
 
PRIMARY 
 
OBJECTIVES 
ENDPOINTS (OUTCOME MEASURES) 
To evaluate the clinical efficacy of allogenic 
MSC versus placebo. 
 
• Change in the PaO2/FiO21 ratio from baseline to 
day 7 of treatment administration, or to the last 
available PaO2/FiO2 ratio if death occurs before 
day 7. 
SECONDARY 
OBJECTIVES 
ENDPOINTS (OUTCOME MEASURES) 
To evaluate effects of allogenic MSC on 
secondary efficacy endpoints as compared 
to Placebo as assessed by: 
 
Clinical aspects 
• Mortality: 
o 
15-day mortality 
o 
28-day mortality 
• Date and cause of death (if applicable). 
 
• Oxygenation: 
o 
Changes in PaO2/FiO2 ratio 
 
o 
Oxygenation-free days in the first 28 
days after treatment. 
 
 
• PaO2/FiO2 ratio1 at baseline and days 2, 4, 14 and 
28 after treatment. 
• Time to PaO2/FiO2 ratio greater than 200 mmHg 
• Oxygen saturation (by standardized 
measurement) at baseline, daily until day 14, and 
on day 28 after treatment.   
• Duration of supplemental oxygen (if applicable), 
and Oxygenation-free days  
• Mechanical Ventilation  
o 
Invasive ventilation-free days in the 
first 28 days after treatment. 
o 
Ventilatory parameters 
 
• Incidence of and duration of invasive and non-
invasive mechanical ventilation (if applicable). 
• Days without MV 
• Intubation/ extubating dates 
 
• Ventilatory parameters 
• Hospitalization 
o 
Duration of hospitalization (days) 
o 
Duration of ICU stay (days) 
 
• Duration of hospitalization. 
• Duration of ICU stay 
• Clinical severity 
o 
Clinical status on the WHO 7-point 
ordinal scale at baseline, daily until 
day 14 and on day 28 after 
 
• WHO Ordinal scale: 
1. Not hospitalized, no limitations on activities. 
2. Not hospitalized, limitation on activities. 
 
1 If an arterial blood gas result cannot be obtained, the SaO2/FiO2 ratio could be substituted for the 
PaO2/FiO2 ratio [TW Rice, et al. Chest 2007]. 

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treatment: 
o 
SOFA scale at baseline and on days 
2, 4, 7, 14 and 28 after treatment. 
3. Hospitalized, not requiring supplemental 
oxygen. 
4. Hospitalized, requiring supplemental oxygen. 
5. Hospitalized, on non-invasive ventilation or 
high flow oxygen devices. 
6. Hospitalized, on invasive mechanical 
ventilation or ECMO2. 
7.  Death. 
• Time to an improvement of one category from 
admission using an ordinal scale. 
• Percentage of patients that worsen at least one 
category  
• Percentage of patients that improve at least one 
category (maintained 48h).  
• SOFA scale 
• Pulmonary fibrosis  
o 
New onset pulmonary fibrosis at 3 
and 12 months after treatment, 
based on CT scan and pulmonary 
function tests 
 
• Incidence of and date of onset (if applicable). 
• Respiratory function tests 
• X-ray or CT scan  
• Survival 
o 
3-month survival 
o 
12-month survival 
 
• Date and cause of death (if applicable). 
• Laboratory parameters 
o 
Changes in serum levels of 
analytical markers at baseline and 
on days 2, 4, 7, 14 and 28 after 
treatment  
 
• Levels of CRP, lymphocyte and neutrophil counts 
lymphocyte subpopulations, D-Dimer, LDH, 
ferritin, coagulation tests and cytokines. 
To evaluate safety of allogenic MSC as 
compared to placebo 
Safety through day 28 and at 3 and 12 
months after treatment 
• Cumulative incidence of serious adverse 
events (SAEs)  
• Cumulative incidence of Grade 3 and 4 
adverse events (AEs)  
• Cumulative incidence of adverse reactions 
(ADR) in the experimental treatment arm 
• Cumulative incidence of adverse events of 
special interest (AESIs) 
 
• Serious adverse events (SAEs) 
 
• Grade 3 or 4 Adverse events (AEs) 
 
• Adverse drug reactions (ADR; experimental 
treatment arm) 
• Adverse events of special interest (AESIs)  
EXPLORATORY 
OBJECTIVES 
ENDPOINTS (OUTCOME MEASURES) 
To study soluble and cellular biomarkers 
that might be involved in the course of the 
disease and the response to the 
investigational product 
• Soluble and cellular biomarkers  
 
 
 
2 Extracorporeal membrane oxygenation 

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4. CLINICAL TRIAL DESIGN 
 
4.1. Type of Study and Overall Design 
This is a double-blind, randomized, controlled, clinical trial to evaluate the efficacy of allogenic MSC 
in patients with ARDS due to COVID-19. All trial participants will receive SOC and one of the 
following, according to treatment allocation (1:1):   
⁃ Experimental Treatment: Allogenic BM- derived MSC 
⁃ Control treatment (placebo): SSF+ 4% albumin (same composition as the experimental 
treatment, without the MSC). 
 
MSC will be administered intravenously as one single dose of 1x106 cells/kg on day 0. The control 
arm will receive matching placebo, which will consist of an equivalent volume of SSF and 4% 
albumin. All patients will receive the SOC as per clinical practice. In current the context of the 
worldwide COVID pandemic, there are no approved medicines or vaccines for the treatment of the 
COVID-19. Therefore, for the purpose of this study, SOC would also include any medicines that are 
being used in clinical practice (e.g. lopinavir/ritonavir; hydroxy/chloroquine, tocilizumab, etc.). 
 
For operational purposes, the study will be divided into two periods: 
- Period 1: from day 0 until day 28 after treatment. 
- Period 2: from day 29 until month 12 after treatment. 
 
While hospitalized, patients will be assessed on a daily basis from treatment administration until day 
14. Patients discharged before that timepoint will be regularly called on the phone or asked to 
attend face-to-face study visits. All patients will undergo a series of efficacy, safety, and laboratory 
assessments (see section 8). The main analysis will cover the period until day 28 after treatment 
(period 1). For safety purposes, patients will be followed-up for a year after treatment (period 2). 
The double-blind design will be maintained (for both, patient and evaluating clinician) until the 
communication of results by the implementation of a blinding plan for study treatment 
administration, and blinded clinical evaluations by the clinician. 
 
4.2. Study participation duration 
Duration of the recruitment: 3 months. 
Duration of the follow-up per patient: 12 months 
Total study duration (enrolment + follow-up + analysis): 16 months. 
 
The end of study is defined as the last visit of the last patient enrolled. Patients will complete their 
participation in the clinical trial at the end of the follow-up, planned at 12 months after treatment 
administration.  
 
4.3. Early termination of the trial 
The Sponsor could elect to close the study at any time. The Research Ethics Committee (CEIm) and 
the Competent National Authority must be informed within 15 days of early termination. The trial 
will be terminated prematurely in the following cases: 
• 
If adverse events occur that are so serious that the risk-benefit ratio is not acceptable. 
• 
If the number of dropouts is so high that proper completion of the trial cannot realistically 
be expected. 

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5. STUDY POPULATION 
5.1. Inclusion Criteria 
1. Informed consent prior to performing study procedures (oral consent will be accepted in 
order to avoid paper handling). Written consent by patient or representatives will be 
obtained whenever possible.  
2. Adult patients ≥18 years of age at the time of enrolment. 
3. Laboratory-confirmed SARS-CoV-2 infection as determined by PCR, in naso- or 
oropharyngeal swabs or any other relevant specimen obtained during the course of the 
disease. Alternative tests (e.g., rapid antigenic tests) are also acceptable as laboratory 
confirmation is their specificity has been accepted by the Sponsor. 
4. Moderate to severe ARDS (PaO2/FiO2 ratio equal or less than 200 mmHg) for less than 96 
hours at the time of randomization.  
5. Patients requiring invasive ventilation are eligible within 72 hours from intubation. 
6. Eligible for ICU admission, according to the clinical team.  
 
5.2. Exclusion Criteria 
1. Imminent and unavoidable progression to death within 24 hours, irrespective of the 
provision of treatments (in the opinion of the clinical team). 
2. “Do Not Attempt Resuscitation” order in place.  
3. Any end-stage organ disease or condition, which in the investigator’s opinion, makes the 
patient an unsuitable candidate for treatment. 
4. History of a moderate/severe lung disorder requiring home-based oxygen therapy.  
5. Patient requiring ECMO, hemodialysis or hemofiltration at the time of treatment 
administration.  
6. Current diagnosis of pulmonary embolism.  
7. Active neoplasm, except carcinoma in situ or basalioma. 
8. Known allergy to the products involved in the allogenic MSC production process. 
9. Current pregnancy or lactation (women with childbearing potential should have a negative 
pregnancy test result at the time of study enrollment). 
10. Current participation in a clinical trial with an experimental treatment for COVID-19 (the use 
of any off-label medicine according to local treatment protocols is not an exclusion criteria).  
11. Any circumstances that in the investigator’s opinion compromises the patient’s ability to 
participate in the clinical trial. 
 
5.3. Subject withdrawal criteria 
 
A patient may be removed/withdrawn from the study for the following reasons: 
• 
Patient withdraws consent or requests discontinuation from the study for any reason 
• 
Termination of the study 
• 
Lost to follow-up.  
• 
Investigator’s decision due to any medical reason for which continuing in the study is not 
considered an adequate option for the patient.  

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Patients will be able to withdraw from the study at any time. Patients will be listed as having 
withdrawn consent when they state that they no longer wish to participate in the study and do not 
authorize the Investigators to continue obtaining their outcome data.  
 
For patients lost to follow-up, every effort to contact them will be made, attempts made will be 
documented in their medical chart. 
 
Patients who withdraw from this study or are lost to follow-up after giving consent and 
administration of the study product, will not be replaced. The reason for patient discontinuation 
from the study will be recorded on the appropriate case report form (CRF). 
 
Treatment discontinuation is not expected, since MSC therapy will be administered as one single 
dose on day 0.  
 
 
 

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6. STUDY TREATMENT 
 
6.1. Investigational Medicinal Product and Dose 
 
The IMP, as described in the Investigator´s Brochure (IB), is composed of cultured MSC derived from 
BM and expanded under GMP conditions. For this study, the IMP used is part of a surplus of MSC 
from the GMP Unit of the Department of Hematology at HUPHM, which have been manufactured, 
preserved, and will be prepared for administration strictly following GMP guidelines. The dose will 
be approximately 1x106 cells/kg of recipient’s weight. 
 
6.2. Description of Treatment  
 
6.2.1. Experimental Group: 
• The patients in the experimental arm will receive a single dose of the IMP (20 mL with 
approximately 1x106MSC/kg), administered intravenously. Patients will receive the IMP 
as add-on to the SOC.  
• MSC are obtained by aspiration of approximately 30 mL of BM in the iliac crest of a 
donor, under general and local anesthesia and transferred to a bag containing culture 
medium (usually Dulbecco's Modified Eagle Medium-DMEM) and heparin. MSC are 
expanded in the GMP Unit using DMEM, supplemented with human platelet lysate (PL) 
and ciprofloxacin. Culture is performed in a humidified atmosphere with 5% CO2 at 37°C 
for a maximum of four passages. Twice a week the medium is renewed, and the level of 
confluence is evaluated to decide on the need for passaging.  
• MSC are collected following trypsinization and the final product is transferred to a sterile 
bag and carried to the Cryobiology Area (Processing Laboratory) in order to be processed 
for cryopreservation.  All MSC doses will be stored in liquid nitrogen (approximately at -
180ºC) until administration is required.  
 
6.2.2. Control Group: 
• The patients in the control arm will receive a single dose of 20 mL of SSF+4% albumin 
(same composition as the experimental treatment, without the MSC), as add-on to SOC. 
Packaging, labelling, distribution, and administration processes will be carried out in the 
same manner as those for the IMP. 
 
6.2.3. Standard of Care: 
• All patients will receive the SOC as per clinical practice. In the current context of the 
worldwide COVID pandemic, there are no approved medicines or vaccines for the 
treatment of the COVID-19. Therefore, for the purpose of this study, SOC would also 
include any medicines that are being used in clinical practice (e.g. lopinavir/ritonavir; 
hydroxy/chloroquine, tocilizumab, etc.). 
 
 
 
 

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6.3. Treatment Administration 
 
Distribution and administration of the IMP will be carried out according to the relevant standard 
operating procedures. These procedures are included in the center’s quality accreditation by JACIE 
(www.jacie.org). Briefly, administration will be performed intravenously, in approximately 3-5 
minutes with the aid of a syringe and a 3-way connector.  
 
6.4. Treatment adherence  
 
Treatment adherence will be ensured because the single dose of the experimental treatment and 
the control will be administered by a member of the investigator team on day 0. 
 
6.5. Treatment preparation and accountability 
 
MSC will be thawed in a water bath in the Processing Laboratory and without further manipulation 
will be transported to the clinical site in a temperature-controlled thermally insulated container. 
Distribution, and administration will be carried out according to the center’s standard operating 
procedures.  
The drug accountability will be performed at the Processing Laboratory. 
 
6.6. Packaging and labelling 
 
Packaging and labelling of the study drugs will be performed by the sponsor or its designee 
according GMP. The following information will be shown: 
- Name, phone, and address of the main contact person in the clinical trial. 
- Name of the IMP and comparator 
- Pharmaceutical form, route of administration, cell dose 
- Identification code of the therapeutic unit- Name of the study, protocol number 
- Patient’s identification (inclusion number 
- Investigator’s name 
- Regulatory text: “For biomedical research use only” 
- Storage conditions, expiration date. 
 
The label will be verified by the investigator responsible for the administration, by double-checking 
the correlation of the patient’s identification number, and date of birth, in order to guarantee that 
the product arrives to the corresponding patient.  
The placebo (SSF + 4% albumin) will be supplied in the same packaging as the IMP for study 
purposes. 
 
6.7. Concomitant Treatment  
 
All concomitant therapies will be permitted in order for the patients to receive full supportive care 
during the study, as appropriate. For the purpose of the study, relevant concomitant treatment 
received during the study, including those for COVID-19, will be recorded in the CRF.  

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7. MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
 
The assignment to treatment will be carried out randomly and blinded, with a 1:1 allocation. 
Randomization will be done through a centralized system embedded in the electronic CRF (eCRF). 
 
To ensure blinding, treatments will be prepared for administration at the GMP Unit and the 
administration of the treatment will be masked, not allowing the identification of the IMP.  
 
Prior to including the first patients, a blinding plan will be in place at the site, agreed by the sponsor 
and the site personnel involved in the treatment preparation. As an additional measure to preserve 
the blinding, the team member administering the treatments (unblinded) will be a different person 
from the blinded investigators that will conduct the study assessments. Any unblinded team 
members will not share any information that could allow the identification of the treatment 
allocation.  
 

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8. ASSESMENTS AND PROCEDURES 
 
8.1. Screening Procedures 
 
After informed consent is obtained, the following assessments will be performed to determine 
eligibility requirements, as specified in the selection criteria: 
• 
Laboratory-confirmed SARS-CoV-2 infection as determined by PCR in naso-/oropharyngeal 
swabs or any other relevant specimen obtained during the course of the disease. Alternative 
tests (e.g., rapid antigenic tests) are also acceptable as laboratory confirmation is their 
specificity has been accepted by the Sponsor. 
• 
Review of available imaging (X-ray and/or CT-scan), obtained during the course of the 
COVID-related hospitalization. 
• 
Focused medical history, including the following information: 
- Date of onset of COVID-19 symptoms (fever and/or other symptoms). 
- History of chronic medical conditions related to the selection criteria or known risk 
factors for COVID-19. 
- Date of onset of PaO2/FiO2 ratio equal or less than 200 mmHg (if arterial blood gas 
results cannot be obtained or are not available, the SaO2/FiO2 ratio could be used). 
• 
Confirm hospitalization/ICU admission period/duration due to COVID. 
• 
Confirm the initiation date of the invasive mechanical ventilation (if applicable) 
• 
Pregnancy test (only for women with childbearing potential). 
 
The overall eligibility of the subject to participate in the study will be assessed once all screening 
data are available. The screening process can be suspended prior to complete assessment at any 
time, if exclusions are identified by the research team. Study subjects who qualify will be 
immediately randomized and treatment should be administered within 24 hours after 
randomization. 
 
8.2. Visit 1 -Day 0 
 
Visit 1 and the screening visit could be done at the same time. For the purpose of the analyses, 
assessments and measurements performed as part of Visit 1 will be considered to be the baseline 
measurements. 
 
RANDOMIZATION 
Participants will be randomly assigned to one of the following options: 
• 
Experimental treatment: 1x106 cells/kg  
• 
Control (placebo): SSF+4% albumin   
Treatment assignment will be performed using a central randomization service implemented in 
the eCRF. 
 
PROCEDURES PRE-ADMINISTRATION 
The following assessments are performed before the administration of the assigned treatment: 
• 
WHO Ordinal scale. 

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• 
Vital signs including SpO2, temperature and PaO2/FiO2 ratio. 
• 
Targeted physical exam. 
• 
Routine blood samples (arterial blood gases, haematology, biochemistry, coagulation 
CRP, LDH, ferritin, IL-6, and D-dimer levels). 
• 
Review of relevant concomitant medications (including therapies related to COVID-19 
SOC). 
 
8.3. Efficacy Assessments  
During period 1, the following assessments will be performed: 
• 
Hospitalization. 
• 
ICU admission. 
• 
Ordinal scale (WHO 7-point ordinal scale). 
• 
Review of relevant concomitant medications. 
• 
Supplemental Oxygen requirement. 
• 
Type of device for Oxygen supplementation (e.g., mask with reservoir bag). 
• 
Non-invasive mechanical ventilation (via mask). 
• 
Mechanical ventilator requirement (via endotracheal tube or tracheostomy tube). 
• 
ECMO requirement. 
• 
SpO2. 
• 
Temperature 
• 
PaO2/FiO2 ratio. 
• 
Mechanical Ventilation parameters (when applicable). 
• 
SOFA scale. 
• 
Death. 
 
During period 2, the following assessments will be conducted: 
• 
Respiratory function tests. 
• 
Chest X-ray and/or CT-scan (according to clinical practice). 
• 
Lab tests (according to clinical practice). 
• 
Death (survival).   
 
8.4. Safety Assessments  
 
During the follow-up until the end of the study, the occurrence of adverse events and complications 
will be collected (i.e., SAEs, Grade 3 or 4 AEs, ADR [experimental treatment] and AESIs). 
• Physical examination (period 1): a symptom-directed (targeted) physical examination will be 
performed to evaluate for any possible adverse event.  
• Clinical laboratory evaluations (period 1): blood will be collected at the following time 
points: baseline and days 2, 4, 7, 14 and 28. 
 
Clinical laboratory parameters include CRP, lymphocyte count and subpopulations, neutrophils, LDH, 
D-Dimer, ferritin, IL-6, coagulation tests and cytokines. Soluble and cellular biomarkers will also be 
collected. During period 2, clinical laboratory evaluations will be conducted in accordance with local 
clinical practice.  

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STUDY SCHEDULE 
 
 
VISITS 
Screening1 
Day 01 
Follow-Up 
VISITS4 
Day 7 
Day 14 
Day 28 
3 m 
12 m 
(EOS) 
Day +/- Window  
−3 to 1 
0 
Daily* 
 
± 2 
± 3 
± 7 
± 15 
ASSESSMENTS/PROCEDURES 
 
 
 
 
 
 
 
 
Informed consent  
X 
 
 
 
 
 
 
 
 
 
 
Inclusion and exclusion 
criteria 
X 
X 
 
 
 
 
 
 
 
 
 
Demographics & Medical 
History  
X 
 
 
 
 
 
 
 
 
 
 
Pregnancy test  
X 
 
 
 
 
 
 
 
 
 
 
SARS-CoV-2 test review 
 X2 
 
 
 
 
 
 
 
 
 
 
X-ray review 
X2 
 
 
 
 
 
 
 
 
 
 
Randomization  
 
X 
 
 
 
 
 
 
 
 
 
Administration of study 
treatment  
 
X 
 
 
 
 
 
 
 
 
 
Clinical data collection   
 
X 
Daily* 
X 
X 
X 
 
 
Ordinal scale 
 
X 
Daily* 
X 
X 
X 
 
 
SOFA scale 
 
X 
Days 2  
and 4 
X 
X 
X 
 
 
Vital signs: SpO2 /Tª 
X 
X 
Daily* 
X 
X 
X 
 
 
Oxygen requirement and 
oxygenation parameters 
X2 
X 
Daily* 
X 
X 
X 
 
 
Mechanical ventilator 
requirement 
 
X 
Daily* 
X 
X 
X 
 
 
Mortality 
 
 
Daily* 
X 
X 
X 
X 
X 
Concomitant medication  
(Only related with COVID-19) 
 
X 
Daily* 
X 
X 
X 
 
 
AE /SAE (eCRF reporting)  
 
  X                 Daily* 
X 
X 
X 
X 
X 
Routine blood samples 
(Arterial blood gases, 
hematology, chemistry, 
coagulation tests) 
X2 
X 
Days 2  
and 4  
X 
X 
X 
X3 
X3 
Respiratory function tests3 
 
 
 
 
 
 
X 
X 
Chest X-ray and/or CT-scan3 
 
 
 
 
 
 
X 
X 
*Daily until hospital discharge; EOS: End of study; m: months; AE: adverse events; SAE: serious adverse event; eCRF: 
electronic case report form. 
1 Screening and visit 1 can be done at the same time; 2Review of clinical data, lab results or images performed during 
the course of the COVID episode. Results available closest to the screening date might be used; 3According to 
clinical practice. 

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9. PHARMACOVIGILANCE 
 
9.1. Definitions 
 
Adverse event (AE): Any untoward and unintended medical occurrence in a clinical trial participant 
who has been administered a medicinal product (investigational or non-investigational), and which 
does not necessarily have a causal relationship with the treatment.  
Any medical condition that is present at the time that the subject is screened will be considered as 
baseline/pre-existing and will not be reported as an AE. However, if the severity of any pre-existing 
medical condition increases, it should be recorded as an AE. 
Given the nature of severity of the underlying illness, subjects will have many symptoms and 
abnormalities in vitals and laboratory. Only Grade 3 and 4 AEs will be captured as AEs in this trial, 
according to the following classification: 
• Mild (Grade 1): Events that are usually transient and may require only minimal or no 
treatment or therapeutic intervention and generally do not interfere with the subject’s usual 
activities of daily living. 
• Moderate (Grade 2): Events that are usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, causing discomfort but 
poses no significant or permanent risk of harm to the research subject. 
• Severe (Grade 3): Events interrupt usual activities of daily living, or significantly affects 
clinical status, or may require intensive therapeutic intervention. Severe events are usually 
incapacitating. 
• Severe (Grade 4): Events that are potentially life threatening. 
 
Adverse Reaction (AR): All untoward and unintended responses to an investigational medicinal 
product related to any dose administered, including those related to the product administration 
process. 
 
Serious adverse event (SAE): any untoward medical occurrence or effect that at any dose: 
• 
Results in death 
• 
Is life-threatening 
• 
Requires inpatient hospitalization or prolongation of an existing inpatient hospitalization 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect  
• 
Is a suspected transmission of any infectious agent via a medicinal product 
• 
Is an important medical event that, when based upon appropriate medical judgment may 
jeopardize the subject and may require medical or surgical intervention to prevent one of 
the outcomes listed above in the definition for a serious adverse event.  
 
Unexpected adverse reaction: Any adverse reaction, the nature, or severity of which is not 
consistent with the applicable product information (e.g. IB or summary of the product characteristics 
(SmPC)) - and/or to the product administration process. 

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Suspected unexpected serious adverse reaction (SUSAR): all the SAEs that, according to the 
investigator’s evaluation, are considered to be related to the IMP and they are neither described nor 
contemplated in the IB of the IMP.  
 
9.2. Adverse Events  
 
The following adverse events will be recorded in the CRF of the study:  
• 
All serious adverse events. 
• 
Grade 3 and 4 adverse events. 
• 
All adverse reactions related to the investigational medicinal product. 
• 
All adverse events considered of special interest for this study, regardless of their 
seriousness. 
 
Occurrence of any of the above-mentioned events will be recorded from the date of receiving the 
allocated treatment onwards. 
The investigators will be instructed to actively monitor the adverse events of special interest, 
defined as any of the following: 
• 
Infusion-related reactions 
• 
Malignancies 
• 
Infections. 
 
9.3. Assessment of Adverse Events 
 
Severity 
The investigator is asked to evaluate the severity (or intensity)* of the adverse events observed in 
the person participating in the research and to note these in the CRF, either by using a scale for 
grading the adverse events (e.g. NCI-CTC classification for cancer trials), or by using more general 
terms such as: 
Severity* 
Characteristics 
Mild 
does not interfere with normal daily activity 
Moderate  
partial limitation of normal daily activity 
Severe  
limitation of normal daily activity 
*The criterion of severity must not be confused with the criterion of 
seriousness which is used as a guide in defining the declarative obligations. 
 
Seriousness 
Definitions of seriousness are described in the definitions section 9.1 of this protocol and are based 
on the regulatory definitions. 
 
Causality  
This is a clinical assessment of whether the adverse event is likely to be related to the IMP. The 
causality evaluation of the AEs will be made by the Investigator or designee. For the causality 
analysis, the following definitions will be used: 

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• 
Related – The event is known to occur with the study intervention, there is a reasonable 
possibility that the study intervention caused the AE, or there is a temporal relationship 
between the study intervention and event. Reasonable possibility means that there is 
evidence to suggest a causal relationship between the study intervention and the AE. 
• 
Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study 
intervention and event onset, or an alternate aetiology has been established. 
 
Expectedness 
The evaluation of expectedness will be based on the Reference Safety Information (RSI) for the IMP 
(included in IB). If the reaction is not described neither contemplated in the RSI, AR will be 
considered as a SUSAR. 
 
9.4. Conduct in case of SAEs 
 
In order to comply with current regulations, any SAE (regardless of causality) must be notified by the 
investigator to the Sponsor, as soon as he/she becomes aware of it and within a maximum period of 
24 calendar hours, sending a SAE form via e-mail or fax to:  
Servicio de Farmacología Clínica, Hospital Universitario Puerta de Hierro 
Email: farmacologia_clinica@idiphim.org; cpayares@idiphim.org 
 
The investigators will make an effort to complete all data included in the SAE form. However, if any 
of the SAE-related information is not available at the time of the initial submission, it may be 
supplemented later, in order not to delay the SAE notification. The initial submission should not be 
delayed when there is suspicion of a serious reaction.  
 
All SAEs occurring from the date of receiving the allocated treatment until de end of study must be 
notified.  
 
9.5. Safety Notifications to the Health Authorities 
 
All SUSAR will be notified to the AEMPS according to the procedures in the applicable laws. Non-fatal 
SUSARs will be notified within 15 days from the moment the Sponsor becomes aware of it, while 
deaths or life threating SUSARs will be notified with a 7-day timeframe. 
 
All SAE and SUSARs will be notified to the AEMPS and the CEIm in an annual safety report. 
 

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10. ETHICAL ASPECTS 
 
10.1. 
General Considerations 
 
The Clinical Trial will be conducted under conditions of respect for the fundamental rights of the 
person and the ethical postulates that affect biomedical research with human beings, following the 
international recommendations included in the Declaration of Helsinki, and their subsequent 
revisions. Likewise, the national recommendations will be followed in accordance with the 
guidelines of the Spanish Medicines Agency.  
 
Prior to conduction of the study, the sponsor will submit the project to the relevant Research Ethics 
Committee(s) and Regulatory Agency (AEMPS). During the conduct of this study, the investigators 
will strictly comply with the provisions of this protocol, fully completing the Clinical Case Form (CRF). 
 
10.2. 
Informed Consent 
 
Informed consent process that will be initiated prior to an individual agreeing to participate in a trial 
and continuing throughout the individual’s trial participation. Investigators will obtain the subject’s 
informed consent in accordance with applicable national legislation and the internationally accepted 
guidelines. Subjects will receive a concise and focused presentation of key information about the 
clinical trial, orally. Due to paper handling limitation in COVID wards, oral witnessed consent will be 
accepted before entering into the trial. Written consent will be obtained from the patient himself or 
acceptable representatives as soon as possible. The key information about the study will be 
organized and presented in lay terminology and language that facilitates understanding why one 
might or might not want to participate. 
 
10.3. 
Confidentiality 
 
Subject’s confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover clinical information relating to 
subjects, test results of biological samples and all other information generated during participation 
in the study. No identifiable information concerning subjects in the study will be released to any 
unauthorized third party. Subject confidentiality will be maintained when study results are published 
or discussed in conferences. 
 
The study monitor, other authorized representatives of the sponsor, representatives of the REC, 
and/or regulatory agencies may inspect all documents and records required to be maintained by the 
investigator, including but not limited to, medical records (office, clinic, or hospital) for the subjects 
in this study. The clinical study site will permit access to such records.  
 
All source records including electronic data will be stored in secured systems. All study data and 
research specimens that leave the site (including any electronic transmission of data) will be 
identified only by a coded number that is linked to a subject through a code key maintained at the 
clinical site. 

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11. FINANCIAL ASPECTS 
 
11.1. Insurance Policy 
 
According with current European legal and regulatory requirements regarding biomedical research, 
the Sponsor will take out, prior to study start, a liability insurance policy to cover the entire duration 
of the study. 
 
11.2. Economic Compensation 
 
No financial compensation is provided to the research team or patients. 
 
 
12.    STATISTICAL ANALYSIS 
 
12.1. General Aspects 
 
The statistical analysis will be carried out in accordance with the principles specified in the 
International Conference on Harmonization (ICH) Topic E9 (CPMP / ICH / 363/96)3. All statistical 
analyzes will be carried out in accordance with the intention-to-treat principle.  
 
12.2. Sample Size Calculation  
 
Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 9 subjects are necessary in 
first group and 9 in the second to recognize as statistically significant a difference greater than or 
equal to 0.4 units. The common standard deviation is assumed to be 0.3.  
 
12.3. Populations for Analysis  
 
There will the following analysis populations for this study:  
1) Full Analysis Set (FAS): All patients who are randomized into the study.  
2) Per Protocol (PP) Population: Those patients included in the FAS without major protocol 
deviations that might impact the study’s main assessments. These deviations will be 
assessed during the data review prior to database lock.  
3) The Safety population is defined as all randomized participants who received the 
investigational product. 
The precise reasons for excluding participants from each population will be fully defined and 
documented independently of the randomization codes during the data blinder review and before 
data lock. 
 
 
 
 
3  
CPMP/ICH/363/96. ICH E9 Statistical Principles for Clinical Trials. URL: 
http://www.ema.europa.eu/ema/pages/includes/document/open_document.jsp?webContentId=WC500002928, last access: 20-Mar-2020.  

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12.4. Primary Efficacy and Safety Analyses 
 
All statistical analyzes will be carried out in accordance with the intention-to-treat principle. The 
main analysis for efficacy will take place when all patients have completed their 28-day follow-up 
after treatment. Prior to this analysis and early during the recruitment phase, a statistical plan will 
be in place with strict provisions to preserve data integrity and will follow the general regulatory 
recommendations given in the ICHE9 guidance, and other applicable guidelines. 
 
For safety purposes, patients will be followed-up for a year after treatment. The double-blind design 
will be maintained (for both, patient and evaluating clinician) until the communication of the 
primary results by a blinding plan for study treatment administration, and blinded clinical 
evaluations by the clinician. 
 
Continuous variables will be expressed as mean and standard deviation and compared using the 
Student's T test; categorical variables will be compared with the Chi-square test or the Fisher exact 
test. Survival curves will be generated using the Kaplan-Meier method. The survival curves will be 
compared with the Mantel Cox log Rank test. Values of p less than 0.05 will be considered 
statistically significant. 
 
13. SUPPORTING DOCUMENTATION AND OTHER OPERATIONAL 
 
13.1. Data Collection  
 
Data collection will be performed, in accordance with Good Clinical Practices, under the 
responsibility of the local investigator. Study data will be collected and managed using REDCap 
electronic data capture tools. REDCap (Research Electronic Data Capture) is a secure, web-based 
software platform designed to support data capture for research studies, providing 1) an intuitive 
interface for validated data capture; 2) audit trails for tracking data manipulation and export 
procedures; 3) automated export procedures for seamless data downloads to common statistical 
packages; and 4) procedures for data integration and interoperability with external sources. 
The anonymity of the subjects will be ensured by identifying them with a code (e.g., their centre 
number and inclusion number). Persons working under the supervision of the sponsor or the 
coordinator are obliged to respect confidentiality. 
 
13.2. Clinical Monitoring 
 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial subjects are 
protected, that the reported trial data are accurate, complete, and verifiable. Clinical Monitoring 
also ensures conduct of the trial is in compliance with the currently approved protocol/ 
amendment(s), ICH, GCP, and with applicable regulatory requirement(s) and sponsor requirements. 
Monitoring for this study will be performed by the sponsor. Monitoring visits will include, but are not 
limited to, review of regulatory files, accountability records, CRFs, ICFs, medical and laboratory 
reports, site study intervention storage records, training records, and protocol and GCP compliance. 
The monitoring visits can be made online. 

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13.3. Communications and Publication  
 
The trial will be registered on the European Clinical Trials Register (EU CTR) and the US Clinical Trial 
register (clinicaltrials.gov).  
After completion of the study, a clinical study report will be prepared according to the ICH Guideline 
for Structure and Content of Clinical Study Reports (ICH E3). In addition, study results will be 
published in a scientific journal. There is a firm commitment from the Sponsor to publish and 
present data from this study, regardless of the results being positive or negative.  
 
13.4. Other Operational Considerations  
 
There will be a file of documentation for all the data, which will be kept intact during the time 
required by the regulatory provisions that are applicable. In any case, the confidentiality of the data 
and documents contained in the file will be ensured. 

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14. BIBLIOGRAPHY 
 
- ARDS Definition Task Force. Acute Respiratory Distress Syndrome: The Berlin Definition. JAMA. 
2012;307(23):2526-2533. 
- Arentz M, et al. Characteristics and Outcomes of 21 Critically Ill Patients with COVID-19 in Washington 
State. Arentz M, Yim E, Klaff L et al. JAMA. 2020; 323(16):1612-1614. 
- Asmussen S, et al. Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep 
model of bacterial pneumonia. Thorax 2014; 69: 819–25. 
- Chen N, et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia 
in Wuhan, China: a descriptive study. Lancet. 2020; 395(10223):507. 
- Chen J, et al. Clinical study of mesenchymal stem cell treating acute respiratory distress syndrome induced 
by epidemic Influenza A (H7N9) infection, a hint for COVID-19 treatment. Engineering (Beijing). 2020 Feb 
28. [Epub ahead of print] 
- Gupta N, et al. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival 
and attenuates endotoxin-induced acute lung injury in mice. J Immunol 2007, 179:1855–1863. 
- Huang C, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 
2020; 395 (10223):497.  
- Lee JW, et al. Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute 
lung injury in the ex vivo perfused human lung. Proc Natl Acad Sci USA 2009; 106: 16357–62. 
- Lee JW, et al. Therapeutic effects of human mesenchymal stem cells in ex vivo human lungs injured with 
live bacteria. Am J Respir Crit Care Med 2013; 187: 751–60. 
- Matthay MA et al. Treatment with Allogeneic Mesenchymal Stromal Cells for Moderate to Severe Acute 
Respiratory Distress Syndrome (START Study): A Randomised Phase 2a Safety Trial. Lancet Respir Med. 
2019 Feb;7(2):154-162. 
- McAuley DF, et al. Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance 
in human lungs rejected for transplantation. Am J Physiol Lung Cell MolPhysio. 2014; 306:809–815. 
- Mei SH, et al. Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells 
overexpressing angiopoietin 1. PLoS Med 2007, 4:e269. 
- Ortiz LA, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure 
and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A 2003, 100:8407–8411. 
- Rice TW, et al. Comparison of the SpO2/FIO2 ratio and the PaO2/FIO2 ratio in patients with acute lung 
injury or ARDS. Chest. 2007;132(2):410-7. 
- Rojas M, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell 
Mol Biol 2005, 33:145–152. 
- Wang D, et al. Clinical Characteristics of 138 Hospitalized Patients with 2019 Novel Coronavirus-Infected 
Pneumonia in Wuhan, China. JAMA. 2020; 323(11):1061-1069. 
- Wilson JG, et al. Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial. Lancet 
Respir Med 2015; 3: 24–32. 
- Wu C, et al.  Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With 
Coronavirus Disease 2019 Pneumonia in Wuhan, China. JAMA Intern Med. 2020; e200994. doi: 
10.1001/jamainternmed.2020.0994. 
- Xu J, et al. Prevention of endotoxin-induced systemic response by bone marrow-derived mesenchymal 
stem cells in mice. Am J Physiol Lung Cell Mol Physiol 2007, 293:L131–L141. 
- Yip HK, et al. Human Umbilical Cord-Derived Mesenchymal Stem Cells for Acute Respiratory Distress 
Syndrome. DOI: 10.1097/CCM.0000000000004285 
- Zheng G, Lanfang H, Tong H, et al. Treatment of acute respiratory distress syndrome with allogeneic 
adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study. Respiratory 
Research. 2014; 15:39. 
- Zhou F, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, 
China: a retrospective cohort study. Lancet. 2020; 395(10229):1054. 

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15. ANNEXES 
 
15.1.  ANNEX I. RESEACH TEAM INFORMATION 
 
PI Project Coordinator: 
Rafael F. Duarte Palomino, MD, PhD, FRCP(Lon) 
Servicio de Hematología y Hemoterapia 
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 91 191 6662 
E-mail: rafael.duarte@salud.madrid.org  
 
PI Intensive Care Unit: 
Juan José Rubio Muñoz, MD, PhD 
Servicio de Cuidados Intensivos  
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916425 
E-mail: jjrubiom@gmail.com 
 
PI Respiratory Medicine: 
Rosa Malo de Molina, MD 
Servicio de Neumología 
Hospital Univ. Puerta de Hierro Majadahonda 
C/ Manuel de Falla 1, 28222 Majadahonda, Madrid 
Phone: 911916000 
E-mail: rmm02004@yahoo.es 
 
15.2.  ANNEX II. GMP UNIT INFORMATION 
 
Unidad de Producción Celular (UPC) del Servicio de Hematología y Hemoterapia del Hospital 
Puerta de Hierro Majadahonda (HUPHM),  
Dirección: Joaquín Rodrigo, 2, Planta 1° Peine 7, 28222 Majadahonda, Madrid, Spain. 
Personas de Contacto:  Dr. Rafael Duarte Palomino  
                                          Dña. Rocío Zafra Pizarro 
Teléfono: +34 911916662 
                  +34 911917767 
Email: rduarte.work@gmail.com 
           rocio.zafra@salud.madrid.org 
 
Persona Cualificada: Dr. Rafael Duarte Palomino  
Tel. +34 911916662 
Email: rduarte.work@gmail.com