Case series of the first three severe COVID-19 patients treated with the secretome of hypoxia-mesenchymal stem cells in Indonesia

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the outbreak of coronavirus disease 2019 (COVID-19), which has been rapidly spreading. Several guideline therapies have been proposed as a possible treatment for SARS-CoV-2, however, these therapies are not sufficient to treat a severe condition of SARS-CoV-2 infection characterised by the increase of D-dimer and C-reactive protein (CRP) levels, and patchy ground-glass opacities (GGOs). Secretome-mesenchymal stem cells (S-MSCs) produced by MSCs under hypoxia could excessively release several anti-inflammatory cytokines and growth factors to control the COVID-19 cytokine storm and accelerate lung injury improvement. This is the first study investigating the clinical outcomes of three severe COVID-19 patients admitted to the intensive care unit of three different hospitals in Indonesia treated with S-MSCs. The decrease of D-dimer and CRP level was reported for all patients treated with S-MSCs. This was in line with improvement of pulmonary radiology, blood gas level, and hematologic assessment. In conclusion, these cases suggest that S-MSCs could effectively control D-dimer, CRP level and GGOs of severe COVID-19 patients associated with recovered pulmonary function.


Introduction
Since December 2019, severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), responsible for the outbreak of coronavirus disease 2019 (COVID- 19), has been rapidly spreading worldwide 1 .The number of infected persons has exceeded 87 million with 2 million deaths globally 2 .Several guideline therapies such as remdesivir and convalescent plasma have been proposed as possible treatment for SARS-CoV-2, however these treatments remain controversial.Moreover, these therapies were not effective to treat severe infection of SARS-CoV-2 due to these treatments potentially inducing the robust cytokine storm 3,4 .A previous study demonstrated that there is a correlation between disease severity and the release of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6, IL-1B, IL-4, IFN-γ, IFN-γ-induced protein 10 (IP10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1a (MIP-1a), and granulocyte-colony stimulating factor (G-CSF) 5 .This finding was confirmed by the high plasma cytokines found in the most severe COVID-19 patients associated with extensive lung damage 6,7 .Therefore, finding an effective therapeutic option to hamper the devastating cytokine storm of COVID-19 and regenerate the damaged lung is crucial.Previous studies recently reported several benefits of mesenchymal stem cells (MSCs) under hypoxia condition to inhibit robust proinflammatory cytokines and repair extensive tissue damage by releasing several anti-inflammatory cytokines and growth factors 8 .
The use of hypoxia-MSCs (H-MSCs) could become an alternative solution to treat the severe cytokine storm of COVID-19.A previous study reported that hypoxia precondition treated on MSCs (H-MSCs) could enhance their survival to reach the damaged area 9 .However, blood clots appearing during the severe phase of COVID-19 could block the H-MSCs trajectory into the damaged area 10 .Other studies reported that H-MSCs could enhance the release of their active soluble molecules, known as secretome-MSCs (S-MSCs) such as IL-10, TGF beta, VEGF and PDGF, which are useful in hampering inflammation and improving tissue healing 11 .Therefore, isolating and concentrating the exact active soluble molecule of S-MSCs is a possible strategy to control the cytokine storm of COVID-19, and, in addition, to accelerate the damaged lung improvement.
In a recent study, we successfully isolate S-MSCs from their culture medium using tangential flow filtration (TFF) strategy with several molecular weight cut-off category 12 .In this Clinical Practice article, we report on three severe COVID-19 patients with several comorbidities who were treated with S-MSCs in three different hospitals in Indonesia.This is the first report to describe the complete monitoring of these three patients.

Ethical considerations
Ethical clearance for the use of S-MSCs in COVID-19 cases and the protocol for administration was obtained from the Health Research Ethics Committee of Bethesda Hospital, Yogyakarta, Indonesia (approval number, No.91/KEPK-RSB/VI/20).Written informed consent for treatment with S-MSCs was obtained from each patient prior to treatment.All patients were treated with standard treatments for severe condition of COVID-19 infection, in addition to novel S-MSCs therapy regarding.Each patient was treated with three, four and six doses of 1 mL S-MSCs every 12 h (with molecular weight cut-off combination of 10-50 kDa 50%, 50-100 kDa 25%, and 100-300 kDa 25%) via deltoid intramuscular injection, respectively.A different S-MSCs dose was utilized in the three patients due to the preliminary nature of this treatment.

Case reports
Case no. 1 A 54-year-old Indonesian male with severe hypertension was diagnosed with COVID-19 on August 28, 2020 and admitted to Dr. Esnawan Antariksa Air Force Hospital, Jakarta, Indonesia intensive care unit (ICU) with cough and dyspnea (Table 1) and was treated with standard treatment (Table 2).The examination showed a temperature of 36°C, a 102/min pulse, a respiratory rate of 32/min, and a blood pressure

Amendments from Version 2
The new version of our article provides 2 additions regarding the clarification of chondrogenic differentiation assessment and the update of isotype staining of MSCs marker analysis using flow cytometry.
The patient was treated with 1 mL S-MSCs three times every 12 h via deltoid intramuscular on August 30 and 31.
On December 24-26, the patient was treated with 1 mL S-MSCs six times every 12 h via deltoid intramuscular injection in addition to other standard treatment (Table 2).The day after the first S-MSC injection, oxygen saturation increased (98%).The examination showed a temperature of 37°C, pulse of 90/min, respiratory rate of 28/min and blood pressure of 120/80 mmHg.On December 28, examination showed a temperature of 37°C, pulse of 80/min, respiratory rate of 24/min and blood pressure of 120/80 mmHg.Oxygen saturation was normal (98%).Laboratory studies showed normal neutrophil count (78.3%) monocyte count (7.2%) and lymphocyte count (25.8%).D-dimer was decreased (86.9 ng/mL).Blood chemistries revealed decreased CRP (8.5 mg/dL (Table 2).Chest X-ray showed decreased bilateral GGOs, cardiomegaly and aortic elongation (Figure 1).On December 30, the patient has no abdominal pain, diarrhea, anosmia, cough and sore throat.The examination showed that SO2 was 99%.He was discharged from ICU and mobilized into rehabilitation room for standard physical activity recovery.On January 6, the patient was negative for COVID-19.

Discussion
Severe pneumonia COVID-19 is characterized by rapid viral infection, excessive inflammatory cell infiltration and robust cytokine storm associated with an increase of D-dimer and CRP levels, resulting in acute respiratory distress syndrome (ARDS) 13 .A previous study reported that the cytokine storm of COVID-19 was associated with an increase of several proinflammatory cytokines, including TNF-α, IL-1, IL-6, IL-17A and granulocyte macrophage colony-stimulating factor (GM-CSF) in the plasma.In line with this phenomenon, lymphocyte count in severe COVID-19 patients' peripheral blood was decreased 14,15 .Another study also revealed that lymphocyte count in severe COVID-19 patients was reduced due to overactivated immune cells, known as macrophage activated syndrome (MAS).The MAS potentially promotes the excessive cytokines storm characterized by the increase of CRP and D-dimer level leading to bilateral GGOs 16 .S-MSCs contain several anti-inflammatory cytokines, including IL-10 and TGF-β, to control the overactivated immunity and hamper the storm's excessive cytokines.In addition, S-MSCs also have several growth factors, such as VEGF and PDGF, that could accelerate lung injury improvement in COVID-19 patient 8,11 .
MSCs were isolated from umbilical cord and exhibited typical monolayers of spindle-shaped fibroblast-like cells with plastic adherent capability under standard culture condition.The cultured MSCs have a high level of surface antigens, including CD90, CD73 and CD105, and lacked the expression CD34, CD45, CD11b, CD19, and HLA-DR, represented as Lin.On the other hand, they also showed the capability to differentiate into osteocyte and adipocyte (see extended data, figure S1 for more details).To induce hypoxia condition, the MSCs were treated under 5% O 2 condition in a hypoxia incubation chamber for 24 h.After incubation, the culture medium of H-MSCs was collected and filtered using TFF strategy with molecular weight cut-off of 10-50 kDa, 50-100 kDa, and 100-300 kDa to produce the S-MSCs (Figure 2).This strategy was employed to obtain the desired cytokines and growth factors in that range, such as IL-10, TGF-β, VEGF and PDGF.In this study, we used S-MSCs with the combination of molecular weight cut-off of 10-50 kDa (50%), 50-100 kDa (25%), and 100-300 kDa (25%).The analysis of cytokines and growth factors level on this S-MSCs combination has been employed using flow cytometry CBA detection and showed a feasible result (see extended data, table S1 for more details).This is the first report that suggests the feasibility of S-MSC therapy of three severe COVID-19 patients in Indonesia.All patients showed bilateral GGOs on chest X-ray before treatment.The hematologic findings showed elevated D-dimer and CRP level in addition to lymphocytopenia and decrease of SO 2 and PO 2 .These patients received several doses of S-MSCs (1 mL/dose) / 12 h which results in favorable outcomes.This study showed that between three and six doses of S-MSCs were well tolerated by the patients.The clinical symptoms were significantly improved with patchy GGO improvement, associated with the decrease of D-dimer and CRP level and increase of SO 2 and PO 2 .From our observation, we would suggest that six doses of S-MSCs performed the most optimal treatment in the COVID-19 patients.These results suggest that the immune system's excessive inflammation and overactivation were alleviated by anti-inflammatory cytokines contained in S-MSCs, while the high level of growth factors in S-MSCs could also accelerate the improvement of GGOs.Based on our preliminary results, S-MSC therapy could be a promising and safe rescue option to treat severe COVID-19 patients.
The first key factor associated with effective S-MSC therapy is the controlled immune system overactivation resulting in the alleviated cytokines storm characterized by the decrease in CRP and D-dimer levels.In our observations, the level of CRP and D-dimer was decreased in all patients treated with S-MSCs.This data suggest that S-MSCs could effectively control the overactivated immune cells.A previous study reported that S-MSCs could control proinflammatory immune cells due to their anti-inflammatory cytokines, such as IL-10 and TGF-β 11,17 .IL-10 could hamper the inflammatory cells' activity by activating tyrosine kinase-2 and Janus tyrosine kinase 1 (JAK1) and inhibiting NF-kB pathways leading to the decreased expression of proinflammatory cytokines such as TNF-α, IL-1, IL-6 and IL-17A 9,18 .On the other hand, TGF-β could activate the regulatory subset of T lymphocyte (Treg) by initiating the FoxP3 expression, resulting in suppressing overactivated immune cells 19 (Figure 2).
Another key factor associated with the efficacy of S-MSCs treatment is the acceleration of lung injury improvement characterized by the improvement pulmonary radiological results, decrease of CRP and D-dimer level resulting in the controlled inflammation leading to normal pulmonary function marked by normal SO 2 .In our patients, decreased CRP and D-dimer was associated with the improvement of patchy GGOs and normal SO 2 in all patients treated with S-MSCs.These data suggest that along with the controlled immune overactivation, the growth factors contained in S-MSCs could rapidly improve lung regeneration post excessive cytokine storm exposure.A previous study revealed that S-MSCs could accelerate wound healing due to their growth factors, particularly VEGF and PDGF 20 .VEGF and PDGF could accelerate the repair of leaky pulmonary blood vessels and accelerate lung injury improvement through the MEK and Akt pathway resulting in the acceleration of angiogenesis and reduced pulmonary infiltrate characterized by decreased bilateral GGOs 21,22 (Figure 2).
In our patients, no severe adverse effects were observed.However, the limitation of our report is that the dynamic changes of cytokine and growth factors during treatment were not investigated.On the other side, we also did not perform the chondrogenic differentiation analysis of MSCs.Nevertheless, the preliminary result seems promising.
In conclusion, we report that S-MSC therapy shows a potential therapeutic effect and low risk in severe COVID-19 patient treatment.We observed that between three and six doses of S-MSCs, with weight cut-off combination of 10-50 kDa (50%), 50-100 kDa (25%) and 100-300 kDa (25%), rapidly controlled the excessive cytokine storm in our patients and improved lung injury.Six doses showed optimum outcome.The treatment time point and the clear clinical advantages of S-MSCs therapy need to be further investigated in randomized clinical studies.characterisation data that include immunophenotyping and mesodermal differentiation would be complete when: chondrogenic differentiation data is included.A complete mesodermal differentiation based on acceptable standards comprises osteo, adipo and chondro differentiations; 1.
it has provided isotype or unstained control for lineage negative staining.As a practice, the negative population is usually located within the first quarter or first half of the antibody axis.In the current supplementary data, the negative population has been depicted in the centre of the antibody axis.

2.
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Mesenchymal Stem Cell and Immunomodulation
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.However, the lack of MSCs differentiation data analysis remains a limitation of our study in which we will improve in further studies."It has provided isotype or unstained control for lineage negative staining.As a practice, the negative population is usually located within the first quarter or first half of the antibody axis.In the current supplementary data, the negative population has been depicted in the center of the antibody axis."The authors indicate their approach as a rescue treatment for patients progressed to severe COVID-19 stage (often presenting with pronounced coagulopathy).Here, the application of cellular secretome instead of the actual cellular MSC products may be somewhat superior/safer given the expression of varying levels of highly procoagulant Tissue Factor (TF/CD142) on MSCs (See PMID's 32574263 3 and 30711482 4 .In this context, another key advantage of this study may be that the S-MSC was applied via deltoid intramuscular injection.

Guido Moll
Future studies should also put more emphasis on well-controlled dose escalation, pharmacokinetics and pharmacodynamics, as well as biodistribution of the product with tight monitoring of coagulation/complement markers and other safety parameters, adjunct with deeper monitoring of a broader panel of biomarkers of response (e.g.systemic and potentially tissue cytokine profiling in blood and BAL as well as longitudinal monitoring of immune cell subsets in the periphery and local tissue BAL).Another important point is the timing of the treatment relative to the diseases stage (e.g.newly diagnosed vs. progressed patients, identify optimal treatment window).Presumably, S-MSC is an allogeneic off-the-shelf product, thus aspects of alloimunization should be studied in more detailed (humoral and cellular alloimunization).Another important issue is the GMP-compliant production of S-MSC for use in human patients.
Second maybe just as efficient as cellular MSCs in treating COVID-19.Future studies should also put more emphasis on well-controlled dose-escalation, pharmacokinetics, and pharmacodynamics, as well as biodistribution of the product with tight monitoring of coagulation/complement markers and other safety parameters, adjunct with deeper monitoring of a broader panel of biomarkers of response (e.g.systemic and potentially tissue cytokine profiling in blood and BAL as well as longitudinal monitoring of immune cell subsets in the periphery and local tissue BAL).Another important point is the timing of the treatment relative to the disease's stage (e.g.newly diagnosed vs. progressed patients, identify optimal treatment window).

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Author response: We agree that this is a potential limitation of the study.In our future study, we will analyze the pharmacokinetics, pharmacodynamics, and biodistribution of the secretome.Furthermore, in the prospective study, we will also evaluate the parameter related to cytokine profiling in blood and BAL and monitoring of immune cell subsets in the periphery and local tissue BAL.We also agree that another potential limitation is no information about the disease stage of patients receiving secretome MSCs treatment.In the future study, it is planned to make several groups of patients who describe the different disease stages to determine the optimal treatment window.
S-MSC is an allogeneic off-the-shelf product, thus aspects of alloimmunization should be studied in more detail (humoral and cellular alloimmunization).Another important issue is the GMP-compliant production of S-MSC for use in human patients ○ Author response: Thank you for pointing this out.We agree that this is an important consideration, we have added the MSCs characterization in extended data, figure S1.Based on this data, the expression CD34, CD45, CD11b, CD19, and HLA-DR are represented as Lin <2%.It can be concluded that the possibility of an allergic reaction is quite limited.Although the potential of allergic reactions is limited, further studies need to be carried out.
secretome MSCs administered is consistent on all three patients (See extended data, table S1 for more details).
The discussion should be made with achieved results rather than the anticipated immunosuppressive action of MSCs.There is no standardisation of MSC secretome.It could be possible that the exosome and microRNA in the secretome mediate the reparative function of MSCs through variable actions that include tissue repair.We also agree with the reviewer's assessment that there is no standardization of secretome MSCs.Secretome MSCs contain exosome and soluble molecule active.This is a potential limitation of the study because we have not standardized the secretome MSCs.Therefore, further studies of secretome MSCs standardization need to be carried out.
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Figure 2 .
Figure 2. Schematic of cytokine storm on severe COVID-19 treated by S-MSCs.MSCs were incubated in 5% O 2 hypoxia condition and S-MSCs was isolated from the culture medium using TFF technique 12 .S-MSCs contain soluble molecules, including IL-10, TGF-β, PDGF and VEGF.IL-10 and TGF-β inhibit NF-kB pathways activation of overactivated immune cells leading to cytokine storm inhibition, characterized by the decreased level of CRP and D-dimer.Under controlled inflammatory milieu, VEGF and PDGF promote the improvement of bilateral GGOs marked by pulmonary recovery.
Center for Regenerative Therapies (BCRT) and Berlin-Brandenburg School of Regenerative Therapies (BSRT), Berlin Institute of Health, Charité -University Medicine Berlin, Berlin, Germany In this Case Study, Putra et al. report the experimental treatment of three patients suffering from severe COVID-19 with hypoxia-conditioned mesenchymal stromal cell (MSC)-derived secretome (S-MSC) prepared by tangential flow filtration (TFF), to prepare the highly concentrated/enriched MSC secretome for clinical application.In their introduction and discussion, the authors identify among others the COVID-19 cytokine storm and concomitant severe lung and tissue damage, as targets of MSC therapy, anticipating that MSC therapy may exert pleiotropic beneficial effects.

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Author Response: Regarding the efficacy of S-MSCs to treat the severe condition of COVID-19, we suggest that the S-MSCs is more efficient than cellular MSCs.This is due to the pulmonary intravascular coagulopathy (PIC) that present in severe COVID-19 patients could blockage the direction of cellular MSCs to homing to injured pulmonary.We use S-MSCs molecules under 300 kDA which makes it possible to get past the blockages due to PIC to modulate the inflammatory condition and enhance pulmonary regeneration.Interestingly, not only inflammation and lung parameters improved, but also D-dimer levels, as typically observed in the follow-up of successfully treated patients ○ Author response: We suggest that the possibility of S-MSCs to improve D-dimer levels, besides lung parameters is the controlled immune system's excessive inflammation and overactivation and the improvement of the lung caused by anti-inflammatory cytokines and growth factors contained in S-MSCs which possible to pass the PIC blockages.

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Author response: Thank you for your suggestion.Accordingly, throughout the manuscript, we have revised and discussed the results in the discussion section such as: The clinical symptoms were significantly improved with patchy GGO improvement, associated with the decrease of D-dimer and CRP level and increase of SO 2 and PO 2. From our observation, we would suggest that six doses of S-MSCs performed the most optimal treatment in the COVID-19 patients.These results suggest that the immune system's excessive inflammation and overactivation were alleviated by antiinflammatory cytokines contained in S-MSCs, while the high level of growth factors in S-MSCs could also accelerate the improvement of GGOs." "In our observations, the level of CRP and D-dimer was decreased in all patients treated with S-MSCs.This data suggest that S-MSCs could effectively control the overactivated immune cells.A previous study reported that S-MSCs could control proinflammatory immune cells due to their anti-inflammatory cytokines, such as IL-10 and TGF-β11,17 ."

Table 2 . Standard treatment received by the three patients receiving S-MSCs. Patient no. Drugs Administrated Oxygen support Antiviral treatment Antibiotic or antifungal treatment Corticosteroids treatment Before S-MSCs Therapy After S-MSCs therapy
po., per os; i.v., i.v.injection; q12h, every 12 h; q24h, every 24 h.

Table 3 . Comparison of laboratory parameters before and after S-MSCs treatment. Clinical Factors Before S-MSCs treatment After S-MSCs treatment Patient 1 Patient 2 Patient 3 Patient 1 Patient 2 Patient 3
CRP (mg/L, normal reference: 0-8.1 mg/L) 2021;12:621744.Published 2021 Mar 10. doi:10.3389/fimmu.2021.621744 3. Qu X, Liu X, Cheng K, Yang R, Zhao RC.Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion.Exp Hematol.2012;40(9):761-770.doi:10.1016/j.exphem.2012.05.006 The Case Study was conducted at Bethesda Hospital in Yogyakarta, Indonesia (With Local Health Research Committee approval number No.91/KEPK-RSB/V1/20), with written informed consent for the treatment with S-MSCs obtained from each patient prior to treatment.All patients received standard treatment for severe COVID-19, with on-top adjunct treatment with S-MSCs.This is a Case Report of three patients, who were all treated in exploratory fashion with different doses of earlier in "MSC Therapies for COVID19: Importance of Patient Coagulopathy, Thromboprophylaxis, Cell Product Quality and Mode of Delivery for Treatment Safety and Efficacy" 3 published in Frontiers in Immunology 2020 and "Intravascular MSC Therapy Product Diversification: Time for New Clinical Guidelines" 4 published in Trends in Molecular Medicine 2019).Another crucial point is the appropriate timing and dosing and mode of application of MSC therapy in COVID-19, "Mesenchymal Stromal Cell Therapy for Coronavirus Disease 2019: Which?When?And How Much?" 5 published in Cytotherapy 2021.