Study design and participants
This study was a single-center open-label, individually randomized, standard treatment-controlled trial conducted at Huangshi Hospital of Traditional Chinese Medicine in Hubei Province from Feb 12 to March 25, 2020, and it was performed according to the Declaration of Helsinki and approved by the Ethics Committee of the Huangshi Hospital of Traditional Chinese Medicine (No. HSZYPJ-2020-009-01). Written informed consent was obtained from all patients or their representatives when data were collected retrospectively.
The diagnosis of COVID-19 was based on WHO interim guidance and a new coronavirus pneumonia diagnosis and treatment program (6th ed.) (in Chinese); the criteria for severe disease are A) an epidemiological history; B) etiological evidence (i.e., a positive SARS-CoV-2 nucleic acid test by the real-time reverse transcription polymerase chain reaction (RT-PCR) assay for SARS-CoV-2 RNA from the Chinese Center for Disease Control and Prevention following the protocol described previously[11, 21]); and C) CT imaging indicators of pneumonia. In addition, these factors should coincide with any of the following criteria: (a) respiratory distress, respiration rate (RR) ≥ 30 times/min; (b) oxygen saturation ≤ 93% in the resting state; and (c) PaO2/FiO2 ≤ 300 mmHg (1 mmHg = 0.133 kPa). In general, patients with severe COVID-19 whose clinical symptoms were not significantly alleviated under standard treatment for 7 to 10 days were recommended for participation in this pilot study. The patients were randomly divided into 2 groups: a standard treatment group (control group) and a standard treatment plus umbilical cord mesenchymal stem cell infusion group (hUC-MSC group). The standard treatment was as follows: (1) supplemental oxygen (noninvasive or invasive ventilation); (2) antiviral agents (abidor/oseltamivir); (3) antibiotic agents (moxifloxacin is taken orally; if there is clear evidence of bacteriological infection, the choice of antibacterial drugs is based on a drug sensitivity test), and (4) glucocorticoid therapy (1-2 mg/kg, less than a week). Exclusion criteria included the following: any kind of cancer, severe liver disease, known allergy or hypersensitivity to hUC-MSCs, and other conditions that the clinician deemed inappropriate for participation.
Cell preparation and transplantation
Clinical-grade hUC-MSCs were supplied, free of charge, by The Jiangsu Cell Tech Medical Research Institute and The Jiangsu Cell Tech Biotechnology Co. The product was registered and reviewed by the China Clinical Trial Center (Registration No. ChiCTR2000031494). It received approval from the Ethics Committee of Huangshi Hospital of Traditional Chinese Medicine (Approval Letter No. HSZYPJ-2020-009-01). Preparation was completed in a GMP laboratory. Cells at passages P3 to P5 were used and had the ISCT-recommended cell surface characteristics of MSCs, including expression (> 95%) of CD73, CD90, and CD105 and lack of cell surface presentation (< 2%) of CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR[22, 23]. Intravenous administration was used. Before the intravenous drip, the MSCs were suspended in 100 ml of normal saline, and the total number of transplanted cells was calculated as 2 x 106 cells/kg. The infusion was from the patients’ right cubital veins and lasted approximately 1 hour (35 drops/min).
Chest CT scans and imaging evaluation
All patients underwent chest CT scans from pretreatment to the follow-up period, and the characteristics of ground-glass opacity (GGO), consolidation, nodules, reticulation, interlobular septal thickening, crazy-paving pattern, linear opacities, subpleural curvilinear line, bronchial wall thickening, lymph node enlargement, pleural effusion and pericardial effusion were evaluated based on the Fleischner Society Nomenclature recommendations and similar studies.[24, 25] To quantify the extent of the lesions, a thin-section CT score was used. Each lobe was assigned a score as follows: 0, 0% involvement; 1, less than 5% involvement; 2, 5% to 25% involvement; 3, 26% to 49% involvement; 4, 50% to 75% involvement; and 5, greater than 75% involvement. A score of 0–5 was assigned to each lobe, with a total possible score of 0–25. [26, 27] The software Pulmonary Infection Assistant Diagnosis (V184.108.40.206, Dexin Medical Imaging Technology Co., Ltd., Xian City, Shaanxi Province, China) was applied for imaging evaluation.
Clinical outcome assessment
The patients were observed for 2 weeks after hUC-MSC infusion, and clinical symptoms, laboratory tests and radiological results were recorded and confirmed by experienced physicians. The primary clinical outcomes included the incidence of progression from severe to critical illness and the time to a clinical improvement of two points on a seven-category ordinal scale that has been used widely in clinical symptom assessment or discharge from the hospital. In our study, the NEWS2 score and seven-category ordinal scale were used to assess the clinical symptoms and conditions of the enrolled patients. The secondary clinical outcomes included patient status at days 7 and 14 assessed with a seven-category ordinal scale, hospital stay, changes in oxygenation index, hematological inflammatory factors and imaging.
Continuous variables with a normal distribution are expressed as the mean±standard deviation (SD); nonnormally distributed continuous variables are reported as the median (interquartile range, IQR). For the P value, the Mann-Whitney U test was used to analyze normally distributed continuous variables, and the Kruskal-Wallis test was used for nonnormally distributed data. Categorical variables were presented as percentages and analyzed by the Chi-square test or Wilcoxon rank-sum test. All statistical analyses were performed with Stata version 14.2 for Mac (StataCorp, College Station, TX), and a P value less than 0.05 was considered statistically significant.