key: cord-0726994-u7vd2tmj authors: Kaushal, S.; Khan, A.; Deatrick, K.; Ng, D. K.; Snyder, A.; Shah, A.; Caceres, L. V.; Bacallao, K.; Bembea, M.; Everett, A.; Zhu, J.; Kaczorowski, D.; Madathil, R.; Tabatabai, A.; Rosenthal, G.; Brooks, A.; Longsomboon, B.; Mishra, R.; Saha, P.; Desire, Y.; Saltzman, R.; Hankey, K. G.; Arias, S. A.; Ayoade, F.; Tovar, J. A.; Lamazares, R.; Gershengorn, H. B.; Magali, F. J.; Loebe, M.; Mullins, K.; Gunasekaran, M.; Karakeshishyan, V.; Jayaweera, D. T.; Atala, A.; Ghodsizad, A.; Hare, J. M. title: Intravenous Mesenchymal Stem Cells in Extracorporeal Oxygenation Patients with Severe COVID-19 Acute Respiratory Distress Syndrome date: 2020-10-20 journal: nan DOI: 10.1101/2020.10.15.20122523 sha: 7d493ad7ea2bc4f7be42e4e37da8e91b2b110e6e doc_id: 726994 cord_uid: u7vd2tmj Background: There is an ongoing critical need to improve therapeutic strategies for COVID-19 pneumonia, particularly in the most severely affected patients. Adult mesenchymal stem cell (MSC) infusions have the potential to benefit critically ill patients with acute respiratory syndrome SARS-COV-2 infection, but clinical data supporting efficacy are lacking. Methods: We conducted a case-control study of critically ill patients with laboratory-confirmed COVID-19, severe acute respiratory distress syndrome (ARDS). To evaluate clinical responsiveness in the most critically ill patient we examined outcomes in a sub-group of those requiring extracorporeal membrane oxygenation (ECMO) support. Patients (n=9) were administered with up to 3 infusions of intravenous (IV) MSCs and compared to a local ECMO control group (n=31). The primary outcome was safety, and the secondary outcomes were all-cause mortality (or rate of hospital discharge), cytokine levels, and viral clearance. Findings: MSC infusions (12 patients) were well tolerated and no side effects occurred. Of ECMO patients receiving MSC infusions, 2 out of 9 died (22.2%; 95%CI: 2.8%, 60.0%) compared with a mortality of 15 of 31 (48.4%; 95%CI: 30.2%, 66.9%; p = 0.25) in the ECMO control group. Isolated plasma exosomes containing the SARS-COV-2 Spike protein decreased after MSC infusions between day 14 or 21 after administration (p=0.003 and p=0.005, respectively) and was associated with a decrease in COVID-19 IgG Spike protein titer at same time points (p = 0.006 and p=0.007, respectively). Control ECMO patients receiving convalescent plasma did not clear COVID-19 IgG over the same time frame. Interpretation: Together these findings suggest that MSC IV infusion is well tolerated in patients with a broad range of severity including the most severe COVID-19 ARDS requiring ECMO. These data also raise the possibility that MSCs, in addition to exerting an immunomodulatory effect, contribute to viral clearance and strongly support the conduct of randomized placebo-controlled trial. 3 Introduction: The emergence and spread of the novel coronavirus disease 2019 (COVID-2019) has led to an unprecedented global pandemic (1) . As of September 11, 2020, COVID-19 has resulted in over 28.5 million cases and ~900,000 deaths worldwide, and at least 6.4 million cases and ~190,000 deaths in the United States. The mortality largely results from the development of a severe acute respiratory distress syndrome (ARDS), causing severe fatal hypoxemia, and multisystem organ failure (2) . The virus affects the respiratory system by binding to the angiotensin converting enzyme 2 (ACE-2) receptor, which is constitutively expressed in alveolar cells and vascular endothelium (3) . Infected patients have a nearly universal development of a severe proinflammatory state, reflected by elevated levels of C-Reactive Protein, Interleukin-6, ferritin, and other cytokines (4-6) (7). Traditional interventions for inflammatory syndromes have had limited efficacy, and there are no therapeutic agents which prevent or treat ARDS in COVID-19 patients, although remdesivir has now entered clinical practice based on studies showing reduced hospitalization days (2, 8, 9) . Recently, a Wuhan study reported that out of 201 hospitalized patients, 42% developed ARDS which resulted in a 52% hospital mortality (10) . A recent pooled analysis reported as high as 71% mortality in COVID-19 ARDS patients, and a reported 94% mortality in extracorporeal membrane oxygenation (ECMO) COVID-19 patients (11) . To reduce mortality in the most severe COVID-19 ARDS patients, mitigating the cytokine storm represents a key strategy. However, many anti-inflammatory drugs have serious sideeffects including the potential to offset immune mechanisms aimed at decreasing the viral load. Accordingly, an immunomodulatory therapy, such as the MSC, which also possesses anti-viral properties would be highly valuable in the anti-covid19 armamentarium. Mesenchymal stem cells (MSCs) possess unique and powerful immunomodulatory characteristics and effectively block cytokine release syndrome in laboratory models (12) (13) (14) . Recently in China, intravenous adult MSCs have been reported to improve 7 COVID-19 patients . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 5 Since the MSCs were administered based on compassionate use, there was no randomization. However, we established a control group for comparison of outcomes and clinical characteristics between the MSC-administered subjects and other critically ill ARDS ECMO patients hospitalized at our local institution with similar baseline characteristics. For our institution, we retrospectively characterized all ECMO COVID-19 patients admitted during the same time period from April 1, 2020 to September 1, 2020. Allogeneic MSCs were derived from healthy bone-marrow donors and the samples were >70% viable at the time of intravenous injection. Patients on ECMO received infusion of allogeneic MSCs via the ECMO circuit return cannula (positioned in the right atrium, with the majority of blood directed across the tricuspid valve and into the pulmonary circulation) and non-ECMO patients received the MSCs intravenously through a central venous catheter. Standard of care infusion was provided for all ARDS patients on or off ECMO. Clinical information for the 12 patients before and after MSC infusion and non-MSC administered control group admitted at the same time was obtained from a review of the hospital electronic medical system and include the following: demographic data, days of admission from symptom onset, and presenting symptoms; data about various infusions, including mechanical ventilation, ECMO support, antiviral therapies, medications, and steroids; clinical data, including PAO2/FiO2, Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe illness), laboratory data, including blood cultures, white blood cell count, chemistry panels assessing liver and kidney function, viral PCR load, inflammatory factors Creactive protein (CRP; mg/dL), IL-6 (pg/mL), ferritin (ng/mL) and procalcitonin (ng/mL); data from . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 20, 2020. 6 chest imaging studies; and information on complications, such as ARDS, ECMO, MSC infusion reactions, ventilation, bacterial pneumonia, and multiple organ dysfunction syndrome. Since the ECMO blood flow remained constant during support, the oxygen delivery provided by the ECMO circuit remained constant for these patients. Therefore, increasing PAO2/FiO2 ratios or the need to reduce sweep gas flow for these patients is related to improvement in their native lung function. Plasma biomarkers were measured at baseline and post-infusion at selective timepoints. IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12-p70, IL-13, TNF-α, and VEGF-A concentrations(pg/mL) were measured using commercial assay (Meso Scale Discovery, Gaithersburg, MD). To study the imprecision and variability of the biomarker measurements, intraand inter-assay coefficients of variation (CV) were determined. Intra-assay CVs tested the variability of biomarker measurements performed in the same sample on the same assay plate. Inter-assay CVs tested the variability of biomarker measurements performed in the same sample on different assay plates often used to measure long-term imprecision. When measuring biomarkers with multiplex assays, generally CV's <15% are targeted and CVs <5% are considered excellent. For the preliminary analysis, all intra-assay CVs were less than 10% and inter-assay CVs for these biomarkers were <10%. Sera collected from severe COVID-19 patients following MSC IV administration infusion were utilized to detect and quantify SARS-CoV-2 spike antigen containing exosomes by immunoblot. Exosomes were isolated using ultracentrifugation and the presence of exosomes specific marker CD9 was validated using immunoblot as previously published (17) (18) (19) . Briefly, Total exosome protein (30 μg) was resolved in polyacrylamide gel electrophoresis, and the proteins were transferred into a polyvinylidene difluoride (PVDF) membrane. The PVDF . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 7 membrane was blocked with 5% non-fat milk prepared in 1x phosphate buffered saline (PBS) and was probed with exosome-specific marker CD9 (312102, BioLegend) and SARS-CoV-2 spike (GTX632604, GeneTex) were used as primary antibodies and goat-anti-mouse conjugated with horse radish peroxidase (7076, Cell Signaling Technology) were used as secondary antibody. The blots were washed with 1x PBS-Tween (Thermo Fisher Scientific), developed using chemiluminescent HRP substrate (WBKLS0500, Millipore Sigma, Burlington, MA), and exposed using Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE). The intensity of SARS-CoV-2 spike antigen was quantified using ImageJ software and normalized with CD9. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 20, 2020. . https://doi.org/10.1101/2020.10.15.20122523 doi: medRxiv preprint 8 To assess temporal changes in cytokines associated with MSC infusions, we constructed graphs anchoring at the baseline cytokine level (on the x-axis) which was defined as the sample obtained immediately prior to the first MSC infusion. We plotted cytokine levels 3 days after the first infusion, 3 or 4 days after the second MSC infusion, and 2 days after the third infusion on the y-axis. These days were selected based on availability of data and represent reasonably similar durations from MSC infusion to sample collection. All cytokines were plotted in the log scale. To estimate the proportion of samples stable or decreasing after MSC infusions, we created a binary response variable based on the post-MSC sample being less than or equal to the baseline cytokine level and fit a repeated measures logistic model with an independent correlation structure. For each cytokine, we present the proportion of samples less than or equal to baseline levels with corresponding valid 95% confidence intervals. The null hypothesis of this single sample test is that this proportion is equal to 50%. Significantly different estimates at the p= 0.05 level were shown by whether the 95% confidence intervals contained the null. To compare risk of death (i.e., cumulative incidence) in the absence of censoring since all were known to have either died or survived to discharge, we present the proportion who died with exact 95% confidence intervals using the Klopper-Pearson method among those who supported with ECMO for the 9 who received MSC infusions and the 31 who did not receive MSC infusions. Fisher's exact test were used to compare survival among those who received MSC infusions to those who did not locally, and then in comparison with the ELSO registry. All analyses were conducted in SAS 9.4 software (Cary, NC) and graphs were constructed in R 4.0.0 (Vienna, Austria). From April 1,2020 to September 1, 2020, we administered MSCs under FDA eIND to patients (n=12; age range, 39-72 years; 5 women) who met the inclusion criteria (Table1). All MSC administered patients had pre-existing complex medical conditions and one was a former . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. . 9 smoker (Supplement Table 1 ,2). All had received hydroxychloroquine (100%), none had received antivirals and most received corticosteroids (83.33%). Nine patients (75%) were supported with venous-venous ECMO. MSC infusions were administered between 15 and 28 days after hospital admission. During this period, 31 contemporaneous ECMO patients with COVID 19 and similar baseline conditions (table 2) did not receive MSCs and served as a control group (Table 2) . No acute-infusion related or allergic reactions were observed within two hours after MSC infusion, Six patients were successfully decannulated from ECMO on post-MSC infusion day 4, 15, 16, 18, 26, and 28 days, respectively, and of these, 2 patients required a tracheostomy and 5 patients were discharged home. The two tracheostomy patients had their tracheostomy decannulated. One patient remained on ECMO at the end of the follow-up period, and two patients died on ECMO after MSC infusion. Two of 3 MSC administered patients not on ECMO were extubated and discharged from the hospital with length of stay 34 and 67 days. A third MSC administered patient not on ECMO suffered a myocardial infarction that resulted in a left ventricle ejection fraction of 10%, cardiogenic shock and died on hospital day 29 which was 9 days after MSC infusion. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. . https://doi.org/10.1101/2020.10.15.20122523 doi: medRxiv preprint 10 The SOFA score ranged from 4 to 19 prior to MSC infusion, and decreased to a range of 2 to 15 at 14 days following infusion (Supplement Table 1 and Figure 1 ). The PaO2/FiO2 ranged from 78.33 to 371.43 prior to MSC infusion, and increased for most patients (83.33%), ranging from 109 to 503.57 14 days after MSC infusion (Supplement Table 1 We next examined the levels of 11 pro-inflammatory and anti-inflammatory cytokines from plasma samples taken at baseline and approximately three days after each MSC infusion by examining their ratio of change from baseline and after each MSC infusion ( Figure 2, Supplement Figure4 ). These samples were grouped as 3 days after first infusion (black; n= 6), 3 or 4 days after the second MSC infusion (blue, n= 5), and 2 days after the third infusion (red, n= 2). VEGF-A in 25% of post-MSC infusion samples was less than or equal to baseline levels (95% CI: 11%, 48%) indicating that VEGF-A was increased in 75% of post-infusion. This was significantly different from the null of 50% (p= 0.035). For the remaining cytokines, 61% to 77% of samples decreased, apart from IL-8 in which 46% of the samples decreased. These were not significantly different from the null, except for IL-12-p70 in which 77% were decreased (95% CI: 51%, 91%; p= 0.043). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In this case-control study, 12 critically ill COVID-19 patients were administered intravenous infusions of culture-expanded allogeneic MSCs. In response, inflammatory cytokines declined within days of MSC infusion, patients improved clinical status, SOFA scores, and PaO2/FiO2 ratios, and exhibited resolution of COVID-19 pneumonia on chest radiographs. Among the sickest cohort requiring ECMO support, mortality was numerically less than that of the global world-wide experienced as tabulated by the registry. Of 7 surviving ECMO patients, 6 have been successfully decannulated with one subject remaining on ECMO at study endpoint. These findings, which are preliminary, nevertheless add to other early stage reports, substantiate the safety of these infusions, and strongly support efforts to rigorously test this strategy in placebocontrolled trials. The novel SARS-CoV2 virus can induce ARDS as a serious manifestation of COVID-19 which can rapidly progress to refractory pulmonary failure. In the most advanced cases, ECMO . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. . 12 support may be considered as a rescue therapy, with limited prognosis for viral infections in general. For instance, ECMO support for ARDS in patients with Middle East Respiratory Syndrome Coronavirus (MERS-CoV) reduced the in-hospital mortality rate to 65% and decreased length of intensive care unit stay when compared to conventional therapy (16) . However, a pooled analysis of early ECMO support in COVID-19 patients reported a 94% in-hospital mortality rate as compared to 70.9% with conventional therapy (11) . Query of the Extracorporeal Life Support Even so, the major limitation of the latter study is that the 7 COVID-19 patients were not categorized as having severe ARDS, rather they had at most mild ARDS. In our study, we administered MSCs to 12 patients admitted to hospital with severe ARDS, In this preliminary series of 12 critically ill patients with COVID-19 pneumonia, most requiring ECMO support, clinical status improved after MSC infusion. While this study only reports results from relatively few patients, this experience suggests that a patient population with great unmet need, like ECMO patients, who are often excluded from current clinical trial design should be studied in randomized, placebo-controlled trials. MSCs may represent a potentially safe antiinflammatory therapy that can contribute to patient recovery in the most severe forms of COVID-19 pneumonia. Drs Kaushal and Hare had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. Infusions 3 3 3 2 2 2 3 3 3 3 3 3 Table 1 . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. -Intubation to ECMO hours, Positive end expiratory pressure, stroke, renal failure, renal insufficiency, intracranial hemorrhage, and intubation to ECMO hours was not included for one patient with MSC infusion from Miami -Any patient that was remained in the hospital were not accounted for in the length of hospital admission. -Two patients remained on ECMO and was not accommodated in ECMO run time hours -ECMO run time was calculated by the number of days on ECMO times 24 hours . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. Table 2 for more complete definition). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. Figure 3 . Chest radiograph images of the critically severe COVID-19 patients at baseline and interval times after MSC treatment. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 20, 2020. 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