Safety and long-term improvement of mesenchymal stromal cell infusion in critically COVID-19 patients: a randomized clinical trial

Abstract

Background: COVID-19 is a multisystem disease that presents acute and persistent symptoms, the postacute sequelae (PASC). Long-term symptoms may be due to consequences from organ or tissue injury caused by SARS-CoV-2, associated clotting or inflammatory processes during acute COVID-19. Various strategies are being chosen by clinicians to prevent severe cases of COVID-19; however, a single treatment would not be efficient in treating such a complex disease. Mesenchymal stromal cells (MSCs) are known for their immunomodulatory properties and regeneration ability; therefore, they are a promising tool for treating disorders involving immune dysregulation and extensive tissue damage, as is the case with COVID-19. This study aimed to assess the safety and explore the long-term efficacy of three intravenous doses of UC-MSCs (umbilical cord MSCs) as an adjunctive therapy in the recovery and postacute sequelae reduction caused by COVID-19. To our knowledge, this is one of the few reports that presents the longest follow-up after MSC treatment in COVID-19 patients.

Methods: This was a phase I/II, prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. Seventeen patients diagnosed with COVID-19 who require intensive care surveillance and invasive mechanical ventilation-critically ill patients-were included. The patient infusion was three doses of 5 × 105 cells/kg UC-MSCs, with a dosing interval of 48 h (n = 11) or placebo (n = 6). The evaluations consisted of a clinical assessment, viral load, laboratory testing, including blood count, serologic, biochemical, cell subpopulation, cytokines and CT scan.

Results: The results revealed that in the UC-MSC group, there was a reduction in the levels of ferritin, IL-6 and MCP1-CCL2 on the fourteen day. In the second month, a decrease in the levels of reactive C-protein, D-dimer and neutrophils and an increase in the numbers of TCD3, TCD4 and NK lymphocytes were observed. A decrease in extension of lung damage was observed at the fourth month. The improvement in all these parameters was maintained until the end of patient follow-up.

Conclusions: UC-MSCs infusion is safe and can play an important role as an adjunctive therapy, both in the early stages, preventing severe complications and in the chronic phase with postacute sequelae reduction in critically ill COVID-19 patients. Trial registration Brazilian Registry of Clinical Trials (ReBEC), UTN code-U1111-1254-9819. Registered 31 October 2020-Retrospectively registered, https://ensaiosclinicos.gov.br/rg/RBR-3fz9yr.

Keywords: COVID-19; Cell therapy; Mesenchymal stromal cells; Postacute sequelae.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2022. The Author(s).

Figures

Fig. 1

Flow chart for patient enrollment, intervention and follow-up

Fig. 2

Characterization and quality control for UC-MSC. A Representative image of cell differentiation. (a, c, e) Control cells; (b) Cells differentiated into adipocytes characterized by the presence of lipidic vacuoles stained with Oil Red O; (d) Cells differentiated into osteoblasts characterized by the presence of calcium deposits stained with Alizarin Red S (red); (f) Presence of vacuoles around young chondrocytes and proteoglycan in the matrix. B Representative histograms of UC-MSC surface markers, cell viability and apoptosis/necrosis. The isotype control is shown as a red line histogram. C UC-MSC karyogram after cell expansion. Normal karyotype: 46, XX. D Representative histograms from the lymphocyte inhibition assay. MSCs were cultivated with PHA stimulated CD3 + lymphocytes labeled with CFSE. (a) CD3 + lymphocytes not labeled with CFSE; (b) CFSE-labeled CD3 + lymphocytes; (c) CD3 + lymphocytes labeled with CFSE and stimulated with PHA (1 µg/µL); (d) MSCs were cultivated with CD3 + lymphocytes labeled with CFSE 1:10; (e) MSCs were cultivated with CD3 + lymphocytes labeled with CFSE 1:5; (f) MSCs were cultivated with CD3 + lymphocytes labeled with CFSE in a 1:2 ratio

Fig. 3

Viral load from day 1 (baseline) to 14 days after UC-MSC treatment. A–D Graphs representing relative viral quantification. Viral load was determined based on the relative expression of the viral gene RdRP in relation to the human POLR2B gene (normalizer). Viral gene expression gradually decreased in patient samples from day 1 (baseline) to day 14 (A, B). C, D After linearizing the data by the natural logarithm (ln (x)) and obtaining the linear equation, the angular coefficient of the viral load line (slope) and the coefficient of determination (R2) were established. C UC-MSC and D Placebo. The angular coefficient (slope) and R2 of each patient are plotted in (E). Average of the angular coefficient of each group (F). Mean with SEM; Student's unpaired t test analysis. UC-MSC, umbilical cord mesenchymal stromal cell; ns, not significant

Fig. 4

Coagulation parameters and inflammatory markers. Comparison between UC-MSC (red line) and placebo (blue line) groups over time. The bars show standard deviations (SD). *p ≤ 0.05 for comparison between groups (black asterisk). Statistical differences inside groups, longitudinal follow-up, were depicted in the graphs (UC-MSC—red line, placebo—blue line). *p ≤ 0.05 and **p ≤ 0.01 for longitudinal follow-up (red and blue asterisks). A D-dimer, B Platelets, C Neutrophils, D Ferritin and E C-Reactive Protein. UC-MSCs, umbilical cord mesenchymal stromal cells

Fig. 5

Profile of plasma cytokines, chemokines and growth factors in the patients during different clinical stages. Comparison between groups (UC-MSCs vs Placebo) and evaluated time points (baseline, days 2, 4, 6, 14 and 2 and 4 months). A Heatmap jointly comparing all analytes evaluated over the treatment time, either with cell therapy (UC-MSCs group) or placebo. Graphs show the results of some analytes separately, comparing the UC-MSC (red line) and placebo (blue line) groups over time. The bars show standard deviations (SD), and the broken line is the trend line fitted to the data. B Granulocyte–Macrophage Colony-Stimulating Factor (GM‐CSF); C Interleukin 2 (IL-2); D Macrophage Inflammatory Protein 1-Alpha (MIP1a/CCL3); E Tumor Necrosis Factor (TNF); F Interleukin 6 (IL-6); G Interleukin 8 (IL-8); H Interleukin 10 (IL-10); I Monocyte chemoattractant protein-1 (MCP1-CCL2); J Interleukin 7 (IL-7). Abbreviations: UC-MSCs, Umbilical Cord Mesenchymal Stromal Cells. *p ≤ 0.04 and **p ≤ 0.02 for comparison between groups. Statistical differences inside groups, longitudinal follow-up, were not depicted in the graphs but indicated in the main text

Fig. 6

Analysis of cell subpopulation. A Total lymphocytes, B CD3 T lymphocytes, C CD4 T lymphocytes, D Natural killer. Comparison between UC-MSC (red line) and placebo (blue line) groups over time. The bars show standard deviations (SD). *p ≤ 0.05 for comparison between groups (black asterisk). Statistical differences inside groups, longitudinal follow-up, were depicted in the graphs (UC-MSC—red line, placebo—blue line). *p ≤ 0.05 and **p ≤ 0.01 for longitudinal follow-up (red and blue asterisks). Flow cytometry strategy for lymphocyte analysis, total lympohocytes, CD3 T, CD8 T and CD4 T lymphocytes. E Lymphocyte gate FSC/SSC low; F Strong CD45 +  + ; G CD3 positive; H CD4/CD8 dotplot; I CD4 positive. UC-MSCs, umbilical cord mesenchymal stromal cells; SSC, side scatter; FSC, forward scatter

Fig. 7

Cardiac and kidney function markers. Comparison between UC-MSC (red line) and placebo (blue line) groups over time. The bars show standard deviations (SD). *p ≤ 0.05 for comparison between groups (black asterisk). Statistical differences inside groups, longitudinal follow-up, were depicted in the graphs (UC-MSC—red line, placebo—blue line). *p ≤ 0.05 and **p ≤ 0.01 for longitudinal follow-up (red and blue asterisks). A Troponin I and B Creatinine. UC-MSCs, umbilical cord mesenchymal stromal cells

Fig. 8

Representative images from chest CT at the level of the lower lobes in patients from the treatment and control groups. Red arrows point to ground-glass opacification, blue arrows to peripheral consolidations, and the yellow arrow to crazy-paving, all of which are typical features of COVID-19. It is possible to note the higher degree of clearance in patients from the treatment group (first line) compared to the control group (last line). UC-MSC, umbilical cord mesenchymal stromal cell