Study design.
This prospective, single-blind and non-randomized controlled clinical study was screened for COVID-19 patients in The Sixth Hospital of Wuhan (Jianghan University affiliated Hospital) and Taikang Tongji (Wuhan) Hospital between March 5 and April 1, 2020. This project passed the academic review and ethical review in Shanghai East Hospital (East Hosipital affiliated to Tongji University) on January 26, 2020 and January 28, 2020 respectively, and get the projects approval of the Ministry of Science and Technology of the People’s Republic of China for ‘Emergency project on prevention and control of the epidemic caused by COVID-19’ on March 03, 2020. The Sixth Hospital of Wuhan and Taikang Tongji (Wuhan) Hospital had also passed the ethical review and participated in this study.
Participants.
Inclusion criteria: All patients with severe pneumonia (18-75 years old) caused by SARS-Cov-2 infection were selected according to the criteria of ‘Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 8)’. Confirmed patients at 18-75 years old were included if one meet any of the following criteria: an increased respiratory rate (≥30 times/min), an oxygen saturation≤93% when inhaled, or a PaO2 : FiO2 ≤ 300 mmHg. All patients were fully informed, agreed and signed informed consent.
Exclusion criteria:
1. Long term use of immunosuppressive drugs or organ transplantation; 2. T lymphocyte abnormality (the use of allogeneic may be considered, according to the clinical opinion), HIV positive; 3. High allergic constitution or severe allergic history, especially IL-2 allergic history; 4. Pregnant and lactating women; 5. Patients with a history of serious autoimmune diseases; those who are allergic to all biological agents in the treatment, such as IL-2; 6. Patients with serious complications: Patients with chronic cardiac insufficiency (NYHA cardiac function grade IV), chronic renal insufficiency (CKD stage 4 or above), chronic liver insufficiency (child Pugh score > 12), or malignant tumors. 7. There are other situations that the researcher thinks are not suitable for participating in this clinical study.
Randomization and masking.
Selected patients were divided into 3 groups. Principle investigators knew the group assignment for safety reasons, while participants, staff at the test site, computed tomography (CT) radiologists, and laboratory staff were unaware of the group assignment.
Procedures.
The day on which the patient was treated with UC-MSCs or placebo for the first time was defined as D0, and 30, 60 or 90 days after that were defined as D30, D60 or D90 respectively. Patients in control group (n=9) received intravenous infusion of placebo (100 ml normal saline containing 5% HAS) on the basis of the routine treatment that according to the treatment principle of severe and critical cases in “Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 8)”. Patients in MSC-1 group (n=11) received intravenous infusion of UC-MSCs (5×107 cells in 100 ml normal saline containing 5% HSA) for once on the basis of the routine treatment, and patients in MSC-2 group received intravenous infusion of UC-MSCs for 2 (n=10) or 3 (n=16) times, each with an interval of 5 days. The UC-MSC treatments was basically according to the clinical program in “Expert guidance on clinical research and application of stem cell treatment of COVID-19” which was joint published by the Stem Cell Biology Branch of Chinese Society for Cell Biology and Infectious Disease Branch of Chinese Medical Association. The vital signs after treatment were observed and recorded daily by the clinician. Each participant underwent a chest CT examination at D0、D30、D60 and D90 and recorded clinical scores and laboratory-related indicators.
HUC-MSCs were donations from healthy women who have undergone caesarean section, and have been approved by the ethics committee of Shanghai East Hospital, Shanghai, China. Full informed consents were obtained before delivery and donation. The manufacture and quality testing of the UC-MSC preparations used in this study were all carried out in the GMP lab of Shanghai East Hospital. Briefly, the Wharton’s Jelly was aseptically separated from the umbilical cord tissue and shredded in to small pieces with sterile scissors. After shredding, the tissue was cultured in α-MEM medium (Thermo) containing 5% serum substitute (UltraGRo-Advanced). After 7-10 days, the monolayer cells crawling around the tissue block were harvested and recorded as passage 0 (P0). Cells at P5 were used for clinical treatment and comprehensive quality testing. Qualified requirements for UC-MSC: 1) No gross agglomeration; 2) Cell survival rate ≥ 90%, showing logarithmic expansion; 3) No pathogenic microorganisms (bacteria, mycoplasma, syphilis, hepatitis B virus, hepatitis C virus, HIV, cytomegalovirus and fungus); 4) Endotoxin ≤0.5EU/mL; 5) characterization and purity: with CD73, CD90 and CD105 positive (≥95%), CD45, CD34, CD14 and HLA-DR negative (≤2%); 6) with the potential of adipogenesis, chondrogenesis and osteogenesis; 7) No tumorigenicity.
Fresh UC-MSCs were prepared once the informed consent was obtained. Qualified UC-MSCs were suspended in 100 ml normal saline containing 5% human serum albumin (HSA) and delivered to the bedside under 2-10℃ within 8 hours. In another case, cryopreserved UC-MSC preparations which were stored in 10 mL clinical grade cryopreservation solution under -196℃ were thawed, suspended in 100 ml normal saline containing 5% human serum albumin (HSA) and delivered to the bedside under 2-10℃ within 6 hours. UC-MSCs were infused into the patients by intravenous within around 90 min. Concerned that UC-MSC infusion may cause temporary obstruction of the pulmonary microcirculation or respiratory damage, blood pressure, heart rate, respiratory parameters and percutaneous oxygen saturation were close monitoring in real-time at the bedside before, during and 6 hours after UC-MSCs infusion.
CT image acquisition and image feature analysis.
Chest CT images were obtained from two scanners: SIEMENS SOMATOM go. Top&ALL and GE MEDICAL SYSTEMS Revolution CT with tube voltage at 130 kV or 120 kV and automatic tube current modulation at 100-400 mA. The reconstructed layer thickness is 0.8 mm or 0.6 mm, respectively. The interlayer spacing is 1.0 mm or 1.25 mm, respectively. All imaging features were reviewed and evaluated through Picture Archiving and Communication Systems (PACS, Tianjian, China) by three experienced radiologists (with 7, 8, and 28 years of chest CT diagnostic experience, respectively) with no knowledge of the group information.
CT manifestations include reticular pattern, fibrotic strips (irregular fibrotic streak shadow), halo sign, reversed halo sign, traction bronchiolectasis and structural distortion. All terms were described according to the Fleischner society glossary and the peer-reviewed literature on viral pneumonia. The distribution of lesions included peripheral, central and diffuse types. Lesions on the extra 1/3 of the lung was defined as peripheral type, lesions on the internal 2/3 was defined as central type, and lesions suffused through the lungs on both sides was defined as diffuse type. Plural effusion, mediastinal and hilar lymph nodes were also evaluated.
Imaging quantification.
UAI Discover-2019 nCoV (Shanghai United Imaging Intelligent Medical Co., Ltd) was used to quantitatively analyze the lung volume of patients with COVID-19. The software provided the function of segmenting lung lobes, lung segments or infection regions and quantitative analysis. After the DICOM format images of the chest CT were imported into the software, the software can quickly and automatically segment the normal areas and infection areas. Prior to this, the software has received a lot of training for COVID-19 cases in which each case was manually sketched by a senior radiologist. Based on these labeled data, the multiscale neural network model was trained to do the segmentation. In the application stage, the machine automatically extracts the lung lobe, lung segment and infected lesion area through the model after learning. The threshold range of -750 ~ -300 Hu or -300 ~ 50 Hu were defined as GGO or Consolidation[20, 21] respectively according to the segmentation results. Finally, the infection volume and percentage of the total lung, as well as the volume and percentage of the whole lung GGO, consolidation were automatically calculated by the machine (Figure 2).
Statistical analysis.
Numerical variables were expressed as median and interquartile spacing, and intergroup comparisons were performed using one-way ANOVA and Kruskal-Wallis H tests. Categorical variables calculate frequency, and chi-square test was used for comparison. Repeated measure ANOVA was used to compare the effects of treatment factors on chest CT parameters of COVID-19 patients between control and experimental groups, and pairwise comparison using LSD test. All the statistical analysis were completed in the SPSS.25 software. The generalized linear mixed effect model analysis of ordered multiple classification repeated measurement data was carried out using GLIMMIX program in SAS 9.4 software. Significant level P <0.05.