Cellular immunotherapy for COVID-19-induced acute respiratory distress syndrome: Results of the CIRCA-19 phase 1 safety and phase 2 randomized controlled trials.

Cellular immunotherapy for COVID-19-induced acute respiratory distress syndrome: Results of the CIRCA-19 phase 1 safety and phase 2 randomized controlled trials - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice Stem Cell Reports . 2026 Mar 12;21(4):102854. doi: 10.1016/j.stemcr.2026.102854 Search in PMC Search in PubMed View in NLM Catalog Add to search Cellular immunotherapy for COVID-19-induced acute respiratory distress syndrome: Results of the CIRCA-19 phase 1 safety and phase 2 randomized controlled trials Shane W English Shane W English 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 2 Department of Medicine, University of Ottawa, Ottawa, ON, Canada Find articles by Shane W English 1, 2, 14 , Dean A Fergusson Dean A Fergusson 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 2 Department of Medicine, University of Ottawa, Ottawa, ON, Canada Find articles by Dean A Fergusson 1, 2, 14 , Manoj Mathew Lalu Manoj Mathew Lalu 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 3 Department of Anesthesiology and Pain Medicine, University of Ottawa, Ottawa, ON, Canada 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 5 Department of Cellular and Molecular Medicine, Ottawa of Ottawa, Ottawa, ON, Canada Find articles by Manoj Mathew Lalu 1, 3, 4, 5 , David W Courtman David W Courtman 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Find articles by David W Courtman 4 , Saad Khan Saad Khan 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Find articles by Saad Khan 4 , Mohamad Sobh Mohamad Sobh 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Find articles by Mohamad Sobh 1 , Irene Watpool Irene Watpool 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Find articles by Irene Watpool 1 , Josee Champagne Josee Champagne 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Find articles by Josee Champagne 1 , Samantha Hodgins Samantha Hodgins 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada Find articles by Samantha Hodgins 4 , Bernard Thébaud Bernard Thébaud 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 5 Department of Cellular and Molecular Medicine, Ottawa of Ottawa, Ottawa, ON, Canada Find articles by Bernard Thébaud 4, 5 , Karim Soliman Karim Soliman 6 Department of Critical Care, Lakeridge Health, Oshawa, ON, Canada 7 Department of Critical Care, Queen’s University, Kingston, ON, Canada Find articles by Karim Soliman 6, 7 , Michaël Chassé Michaël Chassé 8 Department of Medicine, Centre Hospitalier de l’Université de Montréal, Montréal, QC, Canada 9 Department of Medicine, Université de Montréal, Montréal, QC, Canada Find articles by Michaël Chassé 8, 9 , Claudia C dos Santos Claudia C dos Santos 10 The Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, 30 Bond Street, Toronto, ON, Canada 11 Institute of Medical Sciences and Interdepartmental Division of Critical Care, University of Toronto, Toronto, ON, Canada Find articles by Claudia C dos Santos 10, 11 , Marius A Möbius Marius A Möbius 12 Saxonian Center for Feto/Neonatal Health, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Saxony, Germany 13 Good Manufacturing Practice, Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden, Saxony, Germany Find articles by Marius A Möbius 12, 13 , Daniel Freund Daniel Freund 12 Saxonian Center for Feto/Neonatal Health, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Saxony, Germany 13 Good Manufacturing Practice, Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden, Saxony, Germany Find articles by Daniel Freund 12, 13 , Mario Rüdiger Mario Rüdiger 12 Saxonian Center for Feto/Neonatal Health, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Saxony, Germany Find articles by Mario Rüdiger 12 , Duncan J Stewart Duncan J Stewart 2 Department of Medicine, University of Ottawa, Ottawa, ON, Canada 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 5 Department of Cellular and Molecular Medicine, Ottawa of Ottawa, Ottawa, ON, Canada Find articles by Duncan J Stewart 2, 4, 5, 14, 15, ∗ Author information Article notes Copyright and License information 1 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 2 Department of Medicine, University of Ottawa, Ottawa, ON, Canada 3 Department of Anesthesiology and Pain Medicine, University of Ottawa, Ottawa, ON, Canada 4 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada 5 Department of Cellular and Molecular Medicine, Ottawa of Ottawa, Ottawa, ON, Canada 6 Department of Critical Care, Lakeridge Health, Oshawa, ON, Canada 7 Department of Critical Care, Queen’s University, Kingston, ON, Canada 8 Department of Medicine, Centre Hospitalier de l’Université de Montréal, Montréal, QC, Canada 9 Department of Medicine, Université de Montréal, Montréal, QC, Canada 10 The Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, 30 Bond Street, Toronto, ON, Canada 11 Institute of Medical Sciences and Interdepartmental Division of Critical Care, University of Toronto, Toronto, ON, Canada 12 Saxonian Center for Feto/Neonatal Health, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Saxony, Germany 13 Good Manufacturing Practice, Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Dresden, Saxony, Germany ∗ Corresponding author [email protected] 14 These authors contributed equally 15 Lead contact Received 2025 Dec 3; Revised 2026 Feb 11; Accepted 2026 Feb 11; Collection date 2026 Apr 14. © 2026 The Author(s) This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PMC Copyright notice PMCID: PMC13083786  PMID: 41825447 Summary The ability of immunomodulatory mesenchymal stromal cells (MSCs) to improve COVID-19-associated acute respiratory distress syndrome (ARDS) in clinical trials is uncertain. We assessed whether freshly cultured umbilical cord (UC)-derived MSCs improved outcomes in patients with severe COVID-19 ARDS. We enrolled 37 patients with severe COVID-19 ARDS: 15 in the phase 1 dose escalation and open label extension studies ( NCT04400032 ), and 22 patients in the phase 2b randomized clinical trial ( NCT04865107 ). Delivery of up to 270 × 10 6 MSCs in three divided daily doses was well tolerated and resulted in qualitative improvement in all clinical outcomes. Furthermore, MSCs resulted in resolution of lymphopenia, consistent with an important immunomodulatory effect, with significant improvement in patient reported quality-of-life measures (SF-36) at 6 months pointing to possible durable clinical effects. These findings suggest a potential benefit of freshly cultured, UC-MSCs in severe COVID-19 ARDS, associated with biological evidence of favorable immunomodulatory activity. Keywords: acute respiratory distress syndrome, COVID-19, SARS-CoV2, mesenchymal stem cells, cell therapy, immunomodulation, randomized clinical trial, lymphopenia Graphical abstract Open in a new tab Highlights • Freshly cultured MSCs were well tolerated in patients with severe COVID-19 ARDS • MSC administration resulted in qualitative improvement in all clinical outcomes • Quality-of-life measures (SF-36) were significantly improved in the MSC group • MSCs resulted in rescue of severe lymphopenia, a predictor of poor outcomes While underpowered, ∼40% improvements in all clinical endpoints, including the need for breathing support and survival, favored patients receiving freshly cultured mesenchymal stromal cells. This was associated with significant improvement in patient-reported quality-of-life measures at 6 months with complete recovery of severely reduced white blood cell counts, a well-known predictor of poor prognosis in patients with COVID-19-related lung injury. Introduction Since the emergence of the novel coronavirus SARS-CoV-2 virus in late 2019, COVID-19 rapidly evolved into a global pandemic, posing unprecedented challenges to healthcare systems worldwide. The virus has affected millions of people, resulting in significant mortality and morbidity. Pre-vaccine data suggested that 20% of symptomatic infections required hospitalization, and up to 25% of all patients admitted to hospital needed care in the intensive care unit (ICU) ( Niederman et al., 2020 ). About 5 million deaths world-wide were recorded in 2021 and 2022; however, this is likely an underestimate as the WHO has suggested that excess deaths due to COVID-19 were in fact over 14 million during this period ( Msemburi et al., 2023 ). Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in severe COVID-19 ( Siddiqi and Mehra, 2020 ) and is characterized by widespread hyperinflammation mediated by an intense innate immune response involving increases in proinflammatory macrophages and granulocytes, but associated with a profound lymphopenia, which predicts disease severity ( Tan et al., 2020 ). While a few pharmaceutical therapies have shown some benefit, otherwise promising pharmaceutical therapies have under-performed in trials yielding conflicting and, at best, modest results ( Borba et al., 2020 ; Wang et al., 2020 ). Mesenchymal stromal cells (MSCs) have emerged as a potential therapeutic option for ARDS due to their ability to modulate inflammation, mainly through paracrine mechanisms ( Crisan et al., 2008 ; da Silva Meirelles et al., 2008 ), as well as their demonstrated antibacterial properties ( Krasnodembskaya et al., 2010 ; Mei et al., 2010 ; Mezey and Nemeth, 2015 ). Additionally, MSCs have been shown to improve cellular energetics ( Islam et al., 2012 ; Jackson et al., 2016 ; Krasnodembskaya et al., 2010 ), promote tissue healing ( Fang et al., 2019 ), and vascular repair and have antithrombotic effects ( Netsch et al., 2018 ), all of which may make MSCs an especially useful therapy for COVID-19 ARDS ( Kaye, 2020 ). Our group was among the first to recognize the potential therapeutic benefits of MSCs in acute lung injury and ARDS ( Mei et al., 2007 ; 2010 ). Some early clinical studies have suggested potential efficacy of MSCs for the treatment of COVID-19 associated ARDS ( Atluri et al., 2020 ; Leng et al., 2020 ); however, two recent large trials using a cryopreserved MSC product did not show clinical benefit ( Bowdish et al., 2023 ; Matthay et al., 2025 ). These variable results may be related to the use of very different MSC products in different trials, the vast majority of which used high passage number, frozen cells that were immediately thawed prior to delivery ( Galipeau and Sensébé, 2018 ; Purdon and Glassberg, 2019 ). Few studies to date have assessed the safety and efficacy of a freshly cultured, non-frozen MSC product, which may have greater viability and potency for treatment of COVID-19-related ARDS ( Chinnadurai et al., 2016 ; Cottle et al., 2022 ; François et al., 2012 ). The Cellular Immunotherapy for COVID-19-induced ARDS (CIRCA-19) trials sought to investigate the safety and efficacy of freshly cultured umbilical cord-derived MSCs (UC-MSCs) in treating COVID-19-induced ARDS. We now report on the results of the Phase 1/2a Vanguard Trial, assessing safety and maximum feasible tolerated dose (MFTD) and the CIRCA-RCT, a phase 2, multicenter, double-blind, randomized placebo-controlled trial (RCT) efficacy trial. Results Patient characteristics The Vanguard Trial enrolled 15 patients; 9 patients in a phase 1 dose escalation study and 6 patients at the MFTD between October 2020 and March 2021 in the extension phase 2a trial. Enrollment in the phase 2 RCT began in April 2021. An additional 22 patients were consented and randomized to either 3 blinded doses of 90 million MSCs (cumulative dose of 270 million cells) ( N = 14) or blinded placebo ( N = 8) ( Figure 1 ). On May 30, 2022, enrollment was stopped due to the rapidly decreasing worldwide incidence of respiratory failure due to SARS-CoV-2 following the introduction of a successful vaccine and the emergence of the less virulent Omicron strains. Final follow-up of the RCT participants occurred in July 2023. The study cohort had a median age of 60 years (IQR 52, 68), was 45% female with a median APACHE II score of 15 (IQR 13, 20). Overall, the median time between the positive COVID-19 test and delivery of the study product was 6 days (IQR 3, 8). It is important to recognize that the majority of patients were receiving background immunomodulatory therapies at baseline including the IL-6 inhibitor, tocilizumab, and dexamethasone. Important baseline characteristics were balanced between the study arms and are presented in Table 1 . Figure 1. Open in a new tab CONSORT diagram for the CIRCA-19 RCT Table 1. Baseline demographics and clinical characteristics of all the enrolled participants in the Vanguard study and RCT Vanguard ( N = 15) RCT MSCs ( N = 14) Placebo ( N = 8) Demographics Age in years, median (IQR) 68 (63,70) 59 (50,66) 60 (53,71) Female sex 47% 50% 38% SOFA score, median (IQR) 4 (4,5) 4 (3,6) 5 (4,6) APACHE II score, median (IQR) 15 (14,17) 15 (14,19) 15 (13,22) Race, non-Caucasian 60% 43% 50% Days from COVID test to 1 st delivery, median (IQR) 8 (3,11) 4.8 (3,8) 6.5 (2,8) Co-morbidities Hypertension 53% 57% 50% Diabetes 47% 29% 38% Obesity 27% 36% 25% Chronic kidney disease 7% 21% 25% COPD 7% 14% 0% Respiratory status Intubated 20% 71% 75% P a O 2 /F i O 2 , median (IQR) 83 (69,110) 104 (90,134) 130 (94,179) FiO 2 , mean (SD) 79 (18) 64 (19) 68 (25) ARDS class Mild, P a O 2 /F i O 2 200–300 mmHg 0% 14% 13% Moderate, P a O 2 /F i O 2 100–200 mmHg 33% 36% 63% Severe, P a O 2 /F i O 2 <100 mmHg 67% 50% 25% Concurrent medications Anticoagulation Prophylaxis 93% 100% 100% Therapeutic 7% 14% 25% Antiviral agent Remdesivir 33% 21% 38% Immune modulation Tocilizumab 40% 64% 63% Dexamethasone 100% 100% 100% Open in a new tab The CIRCA-19 Vanguard Trial Three participants in each of 75-, 150-, and 270-million MSC dose panels received the study product. The cumulative MFTD was determined to be 270 million MSCs, delivered as infusions of 90 million cells on 3 consecutive days, and an additional 6 participants were enrolled at this dose. There were no reportable severe adverse events. Overall mortality at 28 days was 66% in the two lower dose panels and 43% in the 9 patients receiving the MFTD ( Figure 2 A). As well, the median oxygen free days by non-invasive ventilation (NIV) or high flow nasal cannula (HFNC) or mechanical ventilation at 28 days follow-up was 0 for the 6 patients enrolled in the two lower dose panels (IQR 0–23 and 0–17, respectively), and 18 (IQR 0–21) days in patients receiving the MFTD ( Figure 2 B). Figure 2. Open in a new tab Primary and secondary outcomes Effect of MSC treatment on measures of ARDS severity in the Vanguard Trial: (A) mortality at 28 days in the three dose panels ( n -value indicated above each bar); (B) oxygen (O2) free days in the Vanguard Trial. Primary endpoint in the RCT: (C) number of days free of free of oxygen by NIV/HFNC or mechanical ventilation IMV at 28 days. Secondary endpoints in the RCT: (D) number of days free of IMV at 28 days; (E) number of days not requiring ICU admission at 28 days; and (E) change in the PaO2/FiO2 (P/F) ratio from pre-infusion to day 4. The CIRCA-19 RCT Primary outcome The median number of days free of oxygen by NIV/HFNC or mechanical ventilation at 28 days follow-up was 17.5 (IQR 7.5–20.5) and 9.5 (IQR 0–20) days in the MSC and placebo groups, respectively ( p = 0.31) ( Figure 2 C) with a median difference (MD) of 8.0 days favoring the MSC group. Secondary outcomes Consistent nonsignificant clinical improvements were observed across all outcome measures at day 28 favoring the MSC group, including invasive mechanical ventilation (IMV)- and ICU-free days, incidence of new IMV, and P a O 2 /F i O 2 (P/F) ratio ( Figures 2 D–2F; Table 2 ). Moreover, quality-of-life (QOL), as measured by the SF-36 at 6 months post-enrollment, was consistently higher in patients receiving MSC therapy across all domains, with significant improvements in “role-emotional functioning” and “energy” domains and strong statistical trends the “physical functioning” and “emotional well-being” domains ( p = 0.06; Figure 3 ). Table 2. Primary and secondary efficacy outcomes Vanguard RCT N = 15 MSCs ( N = 14) Placebo ( N = 8) Median days free of advanced support O2 at day 28 (IQR) 17.7 (7.5,20.5) 9.5 (0,20) Mortality at day 28, n (%) 8 (53) 2 (14.3) 2 (25) Mortality at day 60, n (%) 8 (53) 3 (21) 3 (37.5) Ventilator-free days at day 28 (IQR) 0 (0,18) 14.5 (0,19.8) 0 (0,16.8) Incidence of new IMV (%) 6 (40) 2 (14) 2 (25) ICU-free days at day 28, median (IQR) 0 (0,18) 17.5 (7.5,20.5) 9.5 (0–20) P a O 2 /F i O 2 change, baseline to day 4, median (IQR) 15(–2, 53) 33 (13, 118) −12 (21.5, 43.5) SOFA score change, baseline to day 7, median (IQR) 0 (−1, 2) 0 (−2.5, 0.75) 0 (−1.0, 2.0) SF-36 score at 6 months, median (IQR) N/A 49(45,68) 23(22,29) Open in a new tab Figure 3. Open in a new tab Effect of MSC treatment on health-related QOL measured by the SF-36 Health Survey Questionnaire performed at 6 months post enrollment and based on 5 and 9 observations in the placebo and MSC groups, respectively, due to the death of 3 patients in each group ∗ p < 0.05, # 0.05 < p < 0.1. While not significant, the probability of survival over 1 year of follow-up again favored the MSC group ( Figure 4 A). Overall mortality was 14% and 25% in the MSC and placebo groups, respectively, at 28 days ( Figure 4 B) (relative risk [RR] = 0.57, 95% confidence interval [CI]: 0.10, 3.31) and 21% and 38%, respectively, at day 60 (RR = 0.38, 95% CI: 0.08, 1.82) ( Table 2 ; Figure 4 C). Figure 4. Open in a new tab Effect of MSCs on mortality (A) Kaplan Meier survival curves for groups randomized to placebo (blue) or MSC infusions (red). (B) 28 Days mortality for the participants receiving placebo or MSCs. (C) 1 Year mortality for the participants receiving placebo or MSCs. Biological assays The effect of MSC treatment on circulating levels of leukocytes is shown in Figure 5 . COVID-19 patients exhibited lymphopenia pre-infusion in both placebo and MSC groups ( Figure 5 A), a well-known indicator of disease severity and poor outcomes ( Chen and John Wherry, 2020 ; Shouman et al., 2024 ; Tan et al., 2020 ; Zhang et al., 2021 ). After MSC treatment, there was a robust and significant improvement in lymphocyte counts with resolution of lymphopenia, while lymphocyte counts remained low after placebo treatment. Baseline neutrophils counts were above the normal range, while monocyte levels were within the normal range ( Figures 5 B and 5C), and there were no differences between groups after MSC infusions. Figure 5. Open in a new tab Effect of MSC treatment on levels of circulating white blood cells (A) Lymphocytes; (B) Neutrophils; and (C) Monocytes. Shaded area between the dotted horizontal lines indicates the normal range. ∗ p < 0.05. As expected, levels of various proinflammatory cytokines were elevated in the placebo and MSC groups at baseline ( Figure S1 ). While there were no significant differences, IL-6 and IL-8 levels showed modest numerical decline over time after MSC treatment ( Figure S1 ); IFN-gamma levels, which can enhance MSC activity ( Krampera et al., 2006 ) and are positively correlated with better outcomes, ( Hu et al., 2020 ) tended to increase in the MSC group compared to placebo. MSC infusion had no effect on TNFα, IL-10, and ANGPT 1 and 2 levels, and there was a similar decline in IL-10 levels in both treatment groups over time. Biomarkers of disease severity were consistently elevated at baseline ( Figure S2 ) with similar reductions in fibrinogen, procalcitonin, and CRP levels over the 7 days follow-up in the two groups, whereas there was a nonsignificant increase in levels of D-dimers over the initial 3 days, which was more pronounced in the MSC group. Safety outcomes All MSC doses were successfully delivered in the RCT, with the exception of one patient who received a cumulative dose of 255 million cells, and there were no adverse events attributable to cell infusions. There were no reportable severe adverse events attributable to the study procedures or intervention. A priori established expected events were prospectively captured during both the Vanguard and RCT studies ( Table S1 ). Discussion Rather than repurposing a cell product developed for another indication, the CIRCA-19 program aimed to tailor an immunomodulatory MSC therapy to address the onslaught of severe COVID-19-related ARDS in the early stages of the pandemic, beginning with a phase 1 dose escalation study to define the MFTD for a subsequent randomized, double-blind placebo-controlled phase 2 trial. We developed a low passage, “freshly cultured” UC-MSC product by using continuous, overlapping MSC cultures within an ultraclean GMP cell manufacturing environment, such that cells were available whenever an eligible participant was identified. Also, to expedite this process, we utilized existing working cell banks of UC-MSCs that were available from the Center for Regenerative Therapies Dresden, Germany, as the starting material for our manufacturing process. Taken together, our results showed an excellent safety profile of the fresh MSC product, similar to other MSCs therapies ( Thompson et al., 2020 ; Thomspon et al., 2018 ). The continuous, staggered MSC culture strategy was designed to address the high burden of critically ill ARDS patients in the midst of the COVID-19 pandemic; however, this manufacturing approach was not economically feasible in the context of a more endemic ARDS setting due to the high cost of wasted cell products. Because of the evolution of the SARS-CoV2 virus to the less virulent Omicron strain and the development of effective vaccines, only 37 participants were ultimately enrolled into the CIRCA-19 trials; 22 of whom participated in the RCT. With less than half the target enrollment, we lacked sufficient power to perform an adequate statistical analysis. Nonetheless, consistent although mostly nonsignificant clinical improvements favoring the MSC group were observed for the primary endpoint of oxygen free days and all secondary outcomes, including survival and SF-36 QOL scores, which were statistically significant for some domains, supporting a potentially beneficial effect of freshly cultured UC-MSCs for the treatment of severe COVID-19 ARDS. This was seen despite the fact that almost two-thirds of participants were receiving immune modulatory therapy with the IL-6 antagonist, tocilizumab, and all patients were being treated with dexamethasone. The possible efficacy of MSC therapy was further supported by resolution of lymphopenia only after MSC treatment, which provides a plausible biological basis for the apparent clinical improvements. Moreover, the improvement in a validated QOL measure performed 6 months after the acute illness raises the possibility that MSCs may have a positive impact on the long-term consequences of SARS-CoV2 infection, which has been termed long COVID ( León-Moreno et al., 2023 ; Loke et al., 2021 ; Thomspon et al., 2018 ). The COVID-19 pandemic provided a unique opportunity to assess the efficacy of novel therapeutic strategies for severe ARDS. A number of MSC studies were performed across the globe using MSC-based therapies, with mixed results ( Curley et al., 2024 ). Many of the smaller RCTs reported some benefits, including improvement in survival ( Lanzoni et al., 2021 ) and various measures of oxygenation and functional recovery ( Adas et al., 2021 ; Lanzoni et al., 2021 ; Martínez-Muñoz et al., 2024 ; Shu et al., 2020 ; Soetjahjo et al., 2023 ; Zhu et al., 2021 ). In contrast, other studies failed to show benefit ( Gorman et al., 2023 ; Kaffash Farkhad et al., 2022 ; Monsel et al., 2022 ; Pochon et al., 2023 ) including two larger trials of over 100 participants. The Mesoblast trial of 222 participants was terminated prematurely because of futility ( Bowdish et al., 2023 ), although a post hoc analysis suggested improvement in older patents, whereas the STAT trial ( Matthay et al., 2025 ), which included 101 patients COVID-19 ARDS, showed no clinical benefit. However, cellular therapies are inherently complex, and all MSC products are not necessarily equivalent. Differences in the source of MSCs, their processing, storage, and delivery can have profound effects of the efficacy of a cell product ( Levy et al., 2020 ; Stewart and Fergusson, 2025 ). Cryopreservation is used in most allogeneic MSC trials to provide a convenient off-the-shelf product that can be rapidly thawed and delivered when needed. Of over 20 MSC studies for COVID-19 ARDS listed on ClinicalTrials.gov and other international registries, the vast majority used cryopreserved MSC products. Only one other trial used freshly cultured MSC ( Pochon et al., 2023 ), and this was a small study of patients with only mild to moderate ARDS showing significant improvement in PaO 2 /FIO 2 ratio in the MSC arm. Another study used a cryopreserved product that was recovered by 4 days of culture ( Rebelatto et al., 2022 ), but only effects on surrogate endpoints were reported, such as levels of D-dimers, which were improved in the MSC group. The potential superiority of fresh over cryopreserved MSCs has long been debated. Thawed, cryopreserved MSCs have been reported to exhibit impairment in immunomodulatory activity ( François et al., 2012 ). However, a systematic review of 18 preclinical studies comparing fresh vs. frozen MSCs showed rather modest differences in potency, in general favoring fresh MSCs ( Dave et al., 2022 ), but this analysis was limited by the small number of potency measures that were common between the different studies. Nonetheless, the use of cryopreserved MSCs is associated reduced cell viability ( Dave et al., 2022 ), which likely impacts therapeutic efficacy. Indeed, the negative outcome in a placebo-controlled randomized trial of systemically delivered allogeneic MSC in non-COVID ARDS was plausibly linked to low product viability at the time of release ( Matthay et al., 2019 ). However, there is also evidence that nonviable MSCs can have potent effects on tissue transcriptional response leading to important immune modulatory effects of systemic inflammation ( Galleu et al., 2017 ; Weiss et al., 2019 ). Therefore, in the absence of a well-designed clinical trial comparing fresh vs. cryopreserved MSCs, it is not possible to know the extent to which this variable could affect clinical outcomes. The dosing and delivery strategies may also affect the therapeutic efficacy of MSCs. Previous trials assessing MSC therapy for COVID-19 ARDS all utilized IV administration, and some studies, including our own used a repeat dosing strategy. The total cell dose varied from 1 to 4 million cells/kg (approximately 70–280 million cells), often divided up into 2 or 3 separate infusions separated by 1 or 2 days, very similar to the dosing strategy used in the CIRCA-19 trials, which used a cumulative cell dose of up to 270 million cells. Another distinguishing feature of the CIRCA program was that MSCs were isolated and expanded under hypoxic conditions at 5% O2, as opposed standard, normoxic conditions (i.e., 21% O2), which has been reported to enhance the activity of MSCs and increase their therapeutic potency ( Ejtehadifar et al., 2015 ; Yang et al., 2022 ). Finally, the degree of amplification of primary MSCs can markedly affect their biology and therapeutic potential ( Bain et al., 2014 ). Unlike commercial MSC products which are typically delivered at high passage number, we used low passage, primary MSC cultures that may better retain relevant anti-inflammatory and pathogen clearance activities. Levels of critical proinflammatory cytokines, such as IL-6, IL-8, and TNFα, were markedly elevated at baseline in both treatment arms in the CIRCA-19 RCT, consistent with the “hyperinflammatory” subtype described by latent class analysis in non-COVID ARDS ( Calfee et al., 2014 ). MSC administration was associated with non-statistically significant reductions in IL-6 and IL-8 plasma levels in patients receiving MSCs. IL-6 is a master cytokine mediating innate immune responses ( Tanaka and Kishimoto, 2014 ), and elevated IL-6 circulating levels in patients with COVID-19 play a critical role in the development of the hyperinflammatory state characteristic of this disease ( Liu et al., 2020 ). Indeed, IL-6 inhibitors have been shown to reduce inflammation and improve outcomes in severe COVID-19 ARDS ( Rosas et al., 2021 ; Xu et al., 2020 ). IL-8 promotes neutrophil-activation and coagulation ( van der Poll et al., 1990 ) and also plays an important role in the development of COVID-19-associated ARDS ( Cesta et al., 2021 ). In contrast, levels of interferon-gamma (IFNγ) were increased in patients receiving MSCs compared with controls, though these were not statistically significant. Recent reports suggest that IFN-y may be protective in SARS-CoV2 infection ( Hilligan et al., 2023 ) with low levels predicting need for hospitalization ( Cremoni et al., 2022 ) and likelihood of lung fibrosis ( Zhang et al., 2021 ). Moreover, IFN-y is a classical activator of MSCs enhancing their therapeutic efficacy ( Kanai et al., 2021 ). Finally, both treatment arms exhibited baseline lymphopenia, which is a well-documented predictor of poor outcomes in severe COVID ARDS ( Chen and John Wherry, 2020 ; Shouman et al., 2024 ; Tan et al., 2020 ; Zhang et al., 2021 ). While no improvement was seen in participants receiving the placebo product, MSC administration produced a robust and significant increase in lymphocyte counts, with normalization by day 3. Taken together, these biological findings suggest that MSCs can modify the circulating biomarker profile in patients with severe COVID-19 ARDS in a manner consistent with potentially beneficial immunomodulatory effects. While the CIRCA-19 trials benefited from a rigorous design, it was not possible to reach the enrollment target required by the predefined statistical analysis plan due to the marked decline in ICU admissions in the later stages of the pandemic. Nonetheless, we found that repeated delivery of freshly cultured, UC-derived MSCs appeared to result in consistent, though largely nonsignificant, changes in primary and secondary endpoints, all favoring the MSC treated group, which were supported by observed changes in biological indicators, in particular lymphocyte counts, consistent with a favorable immunomodulatory effect. While these findings need to be interpreted with caution, they suggest that a fresh, low passage MSC product may have salutary effects in patients with severe COVID-19 ARDS, thus representing a promising therapeutic product for future global pandemics. Methods Study design and participants The CIRCA-19 trials comprised two sequential, investigator-led multicenter trials: the phase 1 and 2a Vanguard Trial and a phase 2b RCT. The CIRCA-19 trials were approved by Health Canada and Research Ethics Boards at all participating institutions. Detailed methods of the two studies are available in the supplemental information . The Vanguard Trial was an open label, dose-escalating and safety trial using a 3+3+3 design conducted at three Canadian Hospitals with enrollment of 6 additional patients in an open label extension trial using the MFTD of fresh (non-frozen) UC-MSCs. The objectives of the Vanguard Trial were to determine the MFTD, assess the safety of increasing repeated doses of UC-MSCs, and verify surrogate and clinical outcomes. For the dose escalation, participants each received repeated unit doses of freshly cultured UC-MSCs delivered by IV infusion over 3 consecutive days (24 ± 4 h apart) according to the following dose-escalation schedule with three patients per dose panel: panel 1, 25 million cells/unit dose (cumulative dose [CD] 75 million MSCs); panel 2, 50 million cells/unit dose (CD 150 million MSCs); panel 3, up to 90 million cells/unit dose (CD: up to 270 million MSCs). This study was registered on ClinicalTrials.gov as NCT04400032 . The CIRCA19-RCT was a double-blind, allocation concealed, randomized, placebo-controlled trial carried out at five Canadian academic centers. The Ottawa Methods Center used a computer-generated randomization system to randomly assign study participants (2:1) to receive freshly cultured UC-MSCs or a matched placebo infusion using opaque bags and tubing to ensure blinding of the study team. The randomization used random permuted variable block sizes stratified by site and by need of mechanical ventilation or NIV/HFNC at baseline. Research coordinators used a web-based portal to confirm eligibility and then allocate study participants to their assigned treatment. Study participants and all trial personnel, including investigators and research coordinators, treating clinicians, nurses, and outcome assessors were blinded to the treatment assignment. This study was registered on ClinicalTrials.gov as NCT04865107 . For both the Vanguard and the RCT, eligible participants were adults (age ≥ 18) admitted to the ICU with COVID-19-associated ARDS requiring advanced respiratory support (see the “protocol” in supplemental for full criteria). Ethics approval and informed consent Ethics approval was maintained throughout the studies at each participating center and a 3-member international DSMB was established and provided safety oversight to both the Vanguard Trial and RCT. The phase 1 protocol, informed consent form(s), and all relevant participant materials were reviewed and approved by the Ottawa Health Research Institute Research Ethics Board (OHRI REB). The phase 2 protocol, informed consent form(s), and all relevant participant materials were reviewed and approved by the OHRI REB which served as the Research Ethics Board of Record under the Clinical Trials Ontario (CTO) Streamlined Research Ethics Review System, in compliance with the Tri-Council Policy Statement (TCPS 2). Written informed consent was obtained from all phase 1 and 2 trial participants (or their substitute decision maker) prior to the initiation of any study-related procedures, following a discussion of the trial objectives, intervention, potential risks and benefits, and participants’ rights. Both trials were conducted in accordance with the Declaration of Helsinki, International Council for Harmonisation Good Clinical Practice (ICH-GCP E6 [R2]) guidelines, and applicable Canadian regulatory requirements. Procedures Detailed descriptions of the study procedures and cell manufacturing processes are provided in the web supplement. In brief, MSCs were manufacturing using a two-stage process. The working cell banks were manufactured by the Center for Regenerative Therapies Dresden, Germany. These were then cryopreserved and shipped to the Cell Manufacturing Facility in Ottawa to be thawed and placed in staggered, continuous culture. As required, cultured MSCs were lifted between 3 and 5 days of culture (passage number 3), washed and suspended in Plasma-Lyte A containing 5% human albumin at 2.5 × 10 6 fresh UC-MSCs/mL to produce the final MSC drug product. For the RCT, a matching placebo product consisted of Plasma-Lyte A with 5% human albumin in an identical bag, comparable in volume and consistency to the UC-MSC product. The final cell product was delivered fresh (non-frozen) as 3 daily doses of up to 90 million cells/unit dose (cumulative dose: 75–270 million MSCs in the Vanguard and at the MFTD dose [up to 270 million MSCs] for the RCT). Study products (UC-MSCs or Plasma-Lyte A) were infused through a standard infusion pump at 1 mL/min delivered within 24 h of the participant enrollment. All other usual care therapies and interventions were left to the discretion of the treating ICU physician. Participants, treating physicians, nurses, researchers, outcome assessors, and biostatistician remained blinded to allocation and intervention. The treatment period began at the time of first administration of UC-MSCs or placebo (in the RCT) and terminated after completion of infusion of the third study treatment (3 days). Participants were followed up to 6 times after the last MSC infusion at 4, 7, 28, and 90 days, 6 months, and 1 year. RCT outcomes Primary efficacy outcome Number of days free of advanced support oxygen (HFNC/NIV or mechanical ventilation) at day 28. Importantly, this outcome accounts for mortality as participants that die within the 28 days are attributed a total of 0 free days out of a possible 28. Primary Feasibility Outcome: (1) participant enrollment across all 3 sites; (2) ability to manufacture and deliver 3 doses of fresh product to multiple sites. Secondary outcomes Clinical: (1) ICU mortality; (2) number of ventilator-free days at day 28; (3) incidence and duration of new IMV, where eligible, in the post-treatment period, defined as mechanical ventilation not present at time of enrollment but occurs subsequently; (4) change from baseline to day 4 in the P/F ratio (partial pressure of oxygen in arterial blood [PaO 2 ]/fraction of inspired oxygen [FiO 2 ], 1 day after last MSC infusion); (5) death at day 28; (6) ICU-free days at day 28; (7) organ failure severity as measured by SOFA Score Change (baseline to day 7); (8) patient reported outcomes-SF 36 at 6 months post-enrollment. Tertiary outcomes Safety events (adverse events [AEs] and serious adverse events [SAEs]), including allergic reactions and infusion-related reactions, were captured in accordance with Good Clinical Practice Guidelines and under the guidance of Health Canada. Statistical analysis For the RCT, we assumed that an additional 5 days free of advanced support oxygen by NIV/HFNC or mechanical ventilation within 28 days is a strong signal for clinical efficacy. Using a standard deviation of 5 free days, a power of 90%, an alpha of 0.05, and a 2:1 randomization ratio, requires a sample size of 48 patients. As the distribution of our primary outcome is expected to be non-parametric, the sample size was inflated by 15% (Lehmann method) ( Lehmann and D’Abrera, 2006 ), resulting in a planned total enrollment of 54 patients (36 UC-MSCs and 18 placebo). Baseline characteristics of trial participants are presented with median and interquartile range (IQR) for continuous variables or proportions for categorical variables with 95% CIs. Our primary outcome, number of days free of oxygen by NIV/HFNC, or mechanical ventilation was compared using the nonparametric Mann-Whitney U test. We also calculated mean and MDs with 95% CIs. Our secondary dichotomous efficacy outcomes were analyzed with unadjusted Chi-square tests as well as logistic regression to adjust for important prognostic risk factors. Unadjusted relative risks and 95% CIs were also calculated. For continuous outcomes, we compared groups using unadjusted appropriate parametric or nonparametric tests as well as generalized linear models to adjust for multiple covariates. The primary analysis was conducted with completed 28-day follow-up. Resource availability Lead contact Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Dr. Duncan J. Stewart ( [email protected] ). Materials availability N/A. Data and code availability The data supporting the findings of this study are not publicly available as they contain potentially identifiable information that could compromise the privacy of research participants given the small number of enrolled patients. However, select-derived data supporting the findings of this study will be made available from the corresponding author on request. Acknowledgments This study was supported by awards from the Canadian Institutes of Health Research (award #172654), the Canadian Stem Cell Network, the Ontario Research Fund (C-097-2425756-STEWART), and the German Federal Ministry for Education and Research (Cluster SaxoCell/03ZU1111IA). We would like to thank Ranjeeta Mallick of the Ottawa Methods Center for her expert statistical support. We would like to thank the Ottawa Methods Center for all their valuable support in trial management, and data collection and analysis. We are also like grateful to Yan Wang for her help with the manuscript preparation. Author contributions D.J.S., S.W.E., and D.A.F. had unrestricted access to all data and were involved in study design, acquisition of funding, as well as obtaining regulatory and ethics approvals, and overseeing the conduct of the trial, including data collection, curation and analysis, and preparing the first draft of the manuscript; D.A.F. was responsible for performing the statistical analyses; S.W.E. was also the lead investigator for the CIRCA-19 trials as well as Principal Investigator for the Ottawa site, responsible for patient recruitment; S.K. and S.H. were responsible for all aspects of cell manufacturing and preparation, qualification, release, and delivery of final study products; M.S., M.M.L., and B.T. contributed to protocol design and preparing funding application; I.W. was responsible for trial coordination and monitoring and was the Ottawa site coordinator; J.C. managed the preparation of funding applications, regulatory, and ethics submissions as well as day-to-day conduct of the study; K.S., M.C., and C.C.d.S. were responsible for patient recruit and the conduct of the at their respective sites; M.A.M., D.F., and M.R. were responsible for UC cell isolates and preparing the working MSC cell banks used for cell manufacturing. All authors contributed to the editing of the manuscript and agreed to submit the manuscript, read and approved the final draft and take full responsibility of its content, including the accuracy of the data and the fidelity of the trial to the registered protocol and its statistical analysis. Declaration of interests M.A.M. and D.F. are founders and co-owners of MDTB Cells GmbH, a spin-off company from the Technische Universität Dresden, producing mesenchymal cells for clinical and laboratory research purposes. M.A.M., D.F., and M.R. are inventors of granted patents DE102016114043 and US11535822 and pending patents CA3032048A1, EP3491113A1, and WO2018020008A1, which are all property of MDTB Cells GmbH. The sponsor, the Ottawa Hospital Research Institute, made no contribution to the development of the research and the manuscript. Published: March 12, 2026 Footnotes Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2026.102854 . 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Article plus supplemental information mmc4.pdf (4.8MB, pdf) Data Availability Statement The data supporting the findings of this study are not publicly available as they contain potentially identifiable information that could compromise the privacy of research participants given the small number of enrolled patients. However, select-derived data supporting the findings of this study will be made available from the corresponding author on request. 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