Safety and feasibility of umbilical cord mesenchymal stem cells in patients with COVID‐19 pneumonia: A pilot study

Abstract Objectives We aim to explore the safety and feasibility of umbilical cord mesenchymal stem cells (UC‐MSCs) transplantation in patients with severe and critically severe coronavirus disease‐2019 (COVID‐19). Methods We conducted a small sample, single arm, pilot trial. In addition to standard therapy, we performed four rounds of transplantation of UC‐MSCs in sixteen patients with severe and critically severe COVID‐19. We recorded adverse events from enrolment to Day 28. We evaluated the oxygenation index, inflammatory biomarkers, radiological presentations of the disease and lymphocyte subsets count on the 7th day (D7 ± 1 day), the 14th day (D14 ± 1 day) and the 28th day (D28 ± 3 days). Results There were no infusion‐related or allergic reactions. The oxygenation index was improved after transplantation. The mortality of enrolled patients was 6.25%, whereas the historical mortality rate was 45.4%. The level of cytokines estimated varied in the normal range, the radiological presentations (ground glass opacity) were improved and the lymphocyte count and lymphocyte subsets (CD4+ T cells, CD8+ T cells and NK cells) count showed recovery after transplantation. Conclusions Intravenous transplantation of UC‐MSCs was safe and feasible for treatment of patients with severe and critically severe COVID‐19 pneumonia.

injury, secondary infection, leading to sepsis and multiorgan failure, which may lead to death, 1 any treatment that contributes to inhibiting the terrible cytokine storm will represent a major step forward.
Under this situation, stem cell therapy has become a promising therapeutic strategy due to its potential of self-renewal, multipotent differentiation, anti-inflammatory and immune regulatory functions.
Stem cells can be attracted to the site of injury to contribute to organ repair and can foster endogenous progenitor cell function in the lung. 2 Mesenchymal stem cells (MSCs) do not trigger a host response or cell rejection response due to their insensitivity to pro-inflammatory interferon-γ (IFN-γ)-induced human leukocyte antigen-II (HLA-II) expression, 3  Here, we conducted a pilot study to evaluate the feasibility and safety of intravenous infusion of umbilical cord MSCs (UC-MSCs) in severe and critically severe COVID-19 patients.

| Patients
Patients were enrolled from 7 February to 1 April 2020. We invited 40 patients, only 17 (42.5%) of them or their legal representatives signed the formed consents. Sixteen patients were enrolled, and a flow diagram is shown in Figure 1. In our protocol, the follow-up time would be 28 days in total. Due to the quarantine, the actual followup time was 39 days (7-67 days). Inclusion criteria and exclusion criteria were provided in supplementary file (Table 1).

| Cell preparation and transplantation
The clinical-grade UC-MSCs were supplied, for free, by Jilin Tuohua Biotechnology Co., Ltd. The cell product has been certified by the National Institutes for Food and Drug Control (Report number: SH201301098, SH201301175, SH201301317, SH201500350, SH201500351, SH201500477, SH201701982, SH201701983).
Before the intravenous drip, UC-MSCs were suspended in 50 mL of normal saline, and the total number of transplanted cells was 1 × 10 8 cells once. The patients would receive four rounds of transplantation in total, with one-day intervals in between. The transplantation was performed about 1.5 hours with a speed of 30-60 drops per minute.

| Outcome definitions
The primary outcome was the oxygenation index on D14. The secondary outcomes were as follows: (a) mortality on D28; (b) total length of hospital stay; (c) SARS-CoV-2 nucleic acid or antibody assay on D7, D14 and D28; (d) radiological presentations on D7, D14 and D28; (e) inflammatory biomarkers on D7, D14 and D28; (f) lymphocyte and its subsets count on D7, D14 and D28.

| Statistical analysis
All data were collected through EpiData and then imported into the SAS 9.4 statistical package. Figures were performed with GraphPad.v8 (GraphPad software). Categorical variables were described by frequency and proportions. Continuous variables were described by mean values when normally distributed or median and range when skewed distributed. The variables were tested using Fisher's exact or Student's t test, as appropriate. All descriptive statistical analysis was performed with SAS 9.4 (SAS Institute, Inc). All P values were 2-sided, and a P < .05 was considered statistically significant.

| Patients characteristics
A total of 16 patients with COVID-19 were enrolled and all finished UC-MSCs transplantation, of which nine patients were severe type and seven patients were critically severe type. The demographic information of the 16 patients is listed in Table 2.
For the small sample size of patients in each type, we just used a descriptive analysis.

| The safety and feasibility
No acute infusion-related or allergic reactions were observed within two hours after transplantation. Similarly, no delayed hypersensitivity or secondary infections due to UC-MSCs transplantation were detected after treatment. There were two severe adverse events (SAE) during the trial. These two patients suffered from bacterial pneumonia and septic shock, which were lethal complications of COVID-19. A previous report from Wuhan showed that bacterial pneumonia and septic shock occurred in 21.2% and 15% of ICU patients, respectively. 6 The two SAEs were considered to have no

| The secondary outcome
The mortality on D28 in total was 6.25%. One severe type patient died, and one critically severe type patient died. There was no statistical significance between severe type and critically severe type

| D ISCUSS I ON
We conducted a single arm, pilot trial of intravenous infusion of UC-MSCs in sixteen severe and critically severe COVID-19 patients, confirming its safety and feasibility, with a significant increase in oxygenation index and relatively low mortality. The improvement of radiological presentations, recovery of lymphocyte count and decrease of cytokine levels were also observed in our trial, making UC-MSCs transplantation a promising treating strategy.
The safety of MSC transplantation has been identified in previous clinical trials treating ARDS. 7,8 In recent studies about MSC Multiple clinical trials using stem cell therapy to treat the COVID- Region with COVID-19 ARDS admitted to ICU, the mortality was 26% and only 16% had been discharged. 10 In another series of patients in Milan, Italy, the mortality was 23% and 31% had been discharged. 11 In Vancouver, Canada, the mortality in ICU patients was 15.4%. 12 Earlier in Wuhan, the mortality of severe and critical patients (with the same diagnostic criteria) was 45.4%. 13 In our study, the mortality was about 6.25%. It is noteworthy that adults with COVID-19 often present with a profound decrease in lymphocyte count, including CD4 + and CD8 + T-cell subsets at the early stage of this disease. 13 In a subset of COVID-19, patients who progress to pneumonia, respiratory failure and death by the end of the first week showed an extreme rise in inflammatory cytokines including IL2, IL7, IL10 and TNF-α. 18