In vivo immunomodulatory effect and safety of MSC-derived secretome

Response to reviewer

Reviewer:1

1. The manuscript investigates important questions however it is not clear what novel information the analyses provide. The abstract should better summarize all the main findings of the manuscript, since it now describes only the results of the in vivo analysis and not the characterization of the in vitro profile.

Thank you for the suggestion to improve our manuscript. We have revised the abstract of the manuscript by providing highlight of our study findings.

Background:  The immune system is a complex network designed to protect the body from harmful pathogen. Phagocytosis process eliminate invaders before they enter the bloodstream. Recent research has highlighted the potential of secreted materials from Mesenchymal Stem Cells (MSCs), called as secretom, as immunomodulatory agent. This study aims to evaluate MSC secretome effect on phagocytosis and its safety.
Methods: Characterization of the secretome content was achieved through nanoparticle tractking analysis (NTA), Transmission Elctron Microscopy (TEM), Enzyme-linked Immunosorbent Assay (ELISA), and nanostring. The assessment of immunomodulatory activity was conducted using carbon clearance assay. Additionally, we measure spleen mRNA CD68, and CD206. Safety evaluation involved a single-dose administration of the secretome, followed by acute toxicity testing using fixed dose method.
Results: The characterization revealed the presence of interleukin 10 (IL-10), exosome-sized particles, and miRNA within the secretome. Mice treated with the secretome displayed a significantly higher phagocytic index compared to untreated controls and higher CD68 mRNA expression in the spleen. Furthermore, acute toxicity testing showed no significant effects on the mice’s body weight, physical examination results, organ weight, or gross anatomy, indicating a favorable safety profile.
Conclusions: This study highlights the novel insights into MSC-derived secretomes, demonstrating their potential as safe and effective immunomodulatory agents.
 

1. Major criticism

1. The rational of the study and the gap of knowledge is not clear and the analyses performed in vitro and the in vivo are not described as part of the same goal. Why has IL-10 been quantified? Is there any relation between the IL-10 amount in the secretome and the immunomodulatory function? What is the link between the top 10 miR found in the secretome with the in vivo findings? Authors should also better contextualize their findings with what already published in terms of miR characterization.

Thank you for the suggestion to improve our manuscript.
Our study integrates in vitro and in vivo analyses to provide a comprehensive understanding of the secretome's immunomodulatory effects. The in vitro characterization identifies the content of the secretome, including IL-10, which is known for its role in phagocytic activity and may influence immunomodulation. The in vivo study evaluates the effect of secretome on phagocytic activity. By measuring IL-10, we aim to correlate its levels with observed immunomodulatory effects. Additionally, analyzing the particles in the secretome, and miRNA content helps identify potential molecular mechanisms underlying these effects. This approach bridges the gap between secretome composition and its functional impact, offering insights into the immunomodulatory mechanisms at play.

We have corrected the introduction of the manuscript:
The immune system is a sophisticated network designed to protect the body from harmful pathogens. It is generally divided into two main categories: the innate immune system and the adaptive immune system [Marshall et al, 2019]. The innate immune system serves as the first line of defence mechanism against pathogens, using processes like phagocytosis to eliminate harmful invaders before they enter the bloodstream and cause further complications [Lim et al, 2017]. Macrophages in the tissue remove pathogens or foreign particles that enter the tissue, while the spleen helps remove pathogens or foreign particles that have entered the bloodstream by filtering the blood and facilitating their destruction [Lewis et al, 2019].
Maintaining homeostasis in the immune system is crucial to ensure that it is prepared to protect the body whenever pathogens or other harmful substances enter. Factors such as daily stress, lifestyle choices, and environmental hazards can weaken the immune system and increase susceptibility to illness. Therefore, it is essential to maintain and enhance immune function to prevent infections and other diseases [Thorbur et al, 2014; Marques et al, 2016]
Researchers are finding that the secreted materials from mesenchymal stem cells (MSCs), known as the "secretome," and the liquid containing these materials, referred to as "conditioned medium," are promising as immunomodulatory agents [Muzes & Sipos, 2022; ]. The secretome includes soluble factors like immunomodulatory molecules, cytokines, and growth factors, as well as vesicular factors such as extracellular vesicles (EVs) including exosome [Rostami et al, 2018Munos-Perez et al, 2021]. Notably, MSC-derived exosomes, which are small vesicles containing proteins, nucleic acids (like mRNA and miRNA), and lipids, have shown significant potential [Asgarpour et al, 2020]. Overexpression of IL-10 in the secretome of human umbilical cord MSCs can enhance macrophage function [Jerkic et al, 2019], while MSC-derived exosomes can improve the anti-inflammatory properties of macrophages and aid in reducing inflammation [song et al, 2017]. Additionally, apoptotic bodies from MSCs can influence macrophage behavior in wound healing models, and MSC exosomes have the potential to inhibit the proliferation, activation, and cytotoxicity of natural killer cells [Tang et al, 2022]. Furthermore, miRNAs present in MSC-derived EVs play a crucial role in regulating macrophage functions during inflammation [Wu et al, 2015; Wu et al, 2017].
Although the immunomodulatory effects of the secretome are well-documented, there is limited information on how secretomes derived from human umbilical cord MSCs (UC-MSCs) support immune function in healthy or normal conditions. This study aims to address this gap by evaluating several key aspects of the UC-MSC secretome in a healthy context. We measured interleukin-10 (IL-10) to understand its role in phagocytosis, profiled the miRNA content to uncover the mechanisms behind the secretome’s effects, characterized the extracellular vesicles (EVs) to explore their therapeutic potential, and assessed the impact of the secretome on phagocytosis in vivo. Additionally, we conducted an acute toxicity assay to ensure the safety of UC-MSC-derived secretomes.
Methods
 

1. In the in vivo analysis, healthy mice have been subjected to intramuscular injection of the secretome or control medium. Then they were subjected to carbon clearance assay after 3 days via intravenous injection of carbon ink suspension. What is the rational of the intramuscular injection? Why not perform the IV secretome infusion (as delivery route mostly used)? What is the rational of studying the phagocytic activity in healthy mice? Having the effect in the context of a pathological condition would increase the translational validity of the findings.

Thank you for the suggestion to improve our manuscript.

Immunomodulatory studies are conducted in healthy animals to establish a baseline understanding of how potential treatments affect the immune system under normal conditions. Healthy animals provide a controlled environment to observe the natural immune responses and assess the impact of interventions without the complications that might arise from pre-existing health conditions. This approach ensures that any observed effects can be attributed to the treatment being studied rather than to underlying health issues.
One common method used in these studies is the carbon clearance assay. This assay measures the efficiency of phagocytosis, the process by which immune cells engulf and eliminate particles such as carbon particles. By introducing carbon particles into the bloodstream and monitoring their clearance rate, researchers can evaluate how well the immune system is functioning, particularly the activity of macrophages, which play a crucial role in the immune response. The carbon clearance assay provides valuable insights into how the treatment modulates immune function and can help in understanding its potential therapeutic benefits.

We appreciate your question regarding the rationale for choosing intramuscular injection over intravenous infusion of the secretome.

While intravenous (IV) delivery is indeed a common method, we opted for intramuscular (IM) administration for several reasons. Firstly, IM delivery allows the secretome to be deposited directly in the muscle tissue, where it can be gradually released into the bloodstream, potentially providing a more sustained effect. Additionally, muscles have a rich blood supply, which facilitates the secretome's absorption into the circulatory system. The large surface area of the muscle also makes intramuscular administration relatively straightforward and practical. Research supports the effectiveness of intramuscular administration for achieving systemic effects. For example, studies have demonstrated that intramuscular delivery of MSCs can result in systemic benefits (Jahromi & Davis, 2019). Furthermore, research has shown that exosomes, a key component of the secretome, can reach systemic circulation even when administered intramuscularly (Rehorova et al., 2019; Fennel et al., 2024). Therefore, while IV infusion is a commonly used route, intramuscular injection offers unique advantages that align with our study's objectives.

Reference:
Hamidian Jahromi S, Davies JE. Concise Review: Skeletal Muscle as a Delivery Route for Mesenchymal Stromal Cells. Stem Cells Transl Med. 2019 May;8(5):456-465. doi: 10.1002/sctm.18-0208. Epub 2019 Feb 5. PMID: 30720934; PMCID: PMC6477141.
Fennel, Z. J., Bourrant, P.-E., Kurian, A. S., Petrocelli, J. J., de Hart, N. M. M. P., Yee, E. M., Boudina, S., Keirstead, H. S., Nistor, G., Greilach, S. A., Berchtold, N. C., Lane, T. E., & Drummond, M. J. (2024). Stem cell secretome treatment improves whole-body metabolism, reduces adiposity, and promotes skeletal muscle function in aged mice. Aging Cell, 23, e14144. https://doi.org/10.1111/acel.14144
Řehořová M, Vargová I, Forostyak S, Vacková I, Turnovcová K, Kupcová Skalníková H, Vodička P, Kubinová Š, Syková E, Jendelová P. A Combination of Intrathecal and Intramuscular Application of Human Mesenchymal Stem Cells Partly Reduces the Activation of Necroptosis in the Spinal Cord of SOD1G93A Rats. Stem Cells Transl Med. 2019 Jun;8(6):535-547. doi: 10.1002/sctm.18-0223. Epub 2019 Feb 25. PMID: 30802001; PMCID: PMC6525562.
We have revised the discussion section to include this important points.

 

1. A characterization of the immune profile of the circulating phagocytic cells after secretome treatment is needed.

We agree that profiling circulating phagocytic cells after secretome treatment would offer valuable insights into the mechanism of the secretome as an immunomodulator. Unfortunately, we do not have data on the phagocytic profile in the circulation. However, we do have mRNA expression data for CD68 and CD206 from the spleen. The spleen is a key organ in the immune system, responsible for filtering the blood and removing foreign materials. To assess macrophage activity, we measured the mRNA levels of CD68, a common marker for macrophages, and CD206, a marker for anti-inflammatory macrophages, in the spleen. We have revised the methods, results, and discussion sections to reflect this update.

Method:
Spleen CD68 mRNA Analysis
Total mRNA was extracted from the spleen using the FavorPrep™ Tissue Total RNA Mini Kit (Favorgen) according to the instructions provided in the kit. The extracted mRNA was then converted into complementary DNA (cDNA) using the ReverTraAce™ qPCR RT Master Mix (Toyobo). The mRNA expression was evaluated using the RT-qPCR SensiFAST™ SYBR® Low-ROX Kit (Bioline), with Beta Actin as the housekeeping gene. The primers used were: F 5'-TCT CCA CCT TCC AGC AGA TGT-3' R 5'-GCT CAG TAA CAG TCC GCC TAGA-3'. The CD68 primers were: F 5'- CGC AGA CAA TCA ACC TA-3' R 5'-GGC ATG GTG AAG TGA GG-3'.

Result:
Spleen CD8 and CD206 mRNA expression
The CD68 mRNA expression of the spleen is higher in dose 3 compare to those on negative control group. Figure 6 present the spleen CD68 mRNA expression.

Figure 6. Spleen CD68 mRNA expression in each group of treatment. The spleen CD68 mRNA expression was significantly higher in dose 3 compare to those on negative control. A negative control is without secretome treatment. * p < 0.05 vs negative control

 

1. Acute toxicity test: no details of the kind of observations and measurements have been provided. In the results, it has been reported only the variance of the body weight of living mice or of post mortem organs. Please provide more details.

Thank you for the suggestion. We conducted the acute toxicity test using the fixed dose method (OECD Acute Toxicity Test No. 420 and BPOM 2014) to determine whether a single injection of the secretome at the highest dose causes toxicity in animals. This test is an internationally accepted protocol for evaluating the toxicity of single exposures to substances. Briefly, after administering the injection, we observed the animals' behavior at specific time points. We also measured their body weight and, finally, sacrificed the animals to examine the macroscopic appearance of their organs and record organ weights.

We agree that our method and results sections may not have clearly described the test conducted. Here is the revised version of the method and results sections related to the acute toxicity test.

Methods (acute toxicity)
Female Swiss albino mice were used in this test. Healthy female 8-10 weeks mice were used because, generally, female animals are more sensitive to toxic substances than male animals [8]. The method for conducting the acute toxicity test was modified fixed dose according to Regulation of the Head of National Agency of Drug and Food Control (BPOM) No. 7 of 2014 [8]. There are two parts to the fixed-dose methods: preliminary and main parts. In this preliminary part, we used two mice divided into two groups, the treatment group, and the control group. The mice received secretome or MEM-α basal medium intramuscularly with the highest possible volume that can be given intramuscularly to mice, which was 0.05 mL in each thigh or 0.1 mL per mouse. The mice were randomly assigned to receive treatment or control. Observations of their behavior and clinical signs were conducted in the first 30 minutes and every 4 hours for 24 hours. After 24 hours, the mice were observed daily until day 15. Since there was no mortality in the preliminary parts of the acute toxicity test, the main test was carried out with the same dose as the dose used in the preliminary part. In the main test, we used four mice in the secretome group and one mouse in the control group. The mice were observed in the first 30 minutes after injection, every 4 hours during 24 hours, and every 24 hours until day 15. On day 15, all mice were killed, and all vital organs were examined.
The behavioral examination includes the following assessments: skin condition, fur piloerection, corneal reflex and corneal opacity, mucous membrane color, type and rate of respiration, palpebral reflexes, digit reflexes, paralysis, seizures, torticollis, somatomotor activity, reverse walking, tremors, salivation, sleep patterns, defecation, and urination.

Result (acute toxicity)
In the preliminary part of the acute toxicity test, the mice received secretome or MEM-α basal medium intramuscularly of the highest possible volume that can be given intramuscularly to mice which is 0.05 mL in each thigh or 0.1 mL per mice. There was no abnormality in the following behavioral assessment: skin condition, fur piloerection, corneal reflex and corneal opacity, mucous membrane color, type and rate of respiration, palpebral reflexes, digit reflexes, paralysis, seizures, torticollis, somatomotor activity, reverse walking, tremors, salivation, sleep patterns, defecation, and urination of the mice in the control and treatment groups.

Reference:
OECD (2002), Test No. 420: Acute Oral Toxicity - Fixed Dose Procedure, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264070943-en.
 

1. Minor criticism

1. In the introduction, authors underline the MSC ability to differentiate into specialized cells as main pharmacological activity. However, the study is focused on the use of MSC-derived secretome, the a-cellular counterpart. Please modify.

Thank you, we have modified the introduction.


Reviewer 2

The number of studies examining the immunomodulatory effects of mesenchymal stem cells through the secretome is increasing, and the safety of the conditioned media used needs to be investigated. In this respect, the authors have drawn attention to a current issue, but essential problems need to be clarified before the article is indexed.

1. First, characterization data of the isolated cells are lacking. Authors must include characterization experiments with immunophenotypic and differentiation data for UCMSCs.

Thank you for your recommendation. We have included the secretome provided by the commercial supplier, PT Tristem Indonesia. We have updated the characterization and modified the methods and results sections based on your suggestions.

Method:
“The characterization of MSCs was performed using flow cytometry at the commercial lab testing facility, PharmaMetricLabs®. Several parameters were measured, including CD105, CD90, CD73, CD45, CD34, CD14, HLA-DR, and CD19. “

Result:
Characterization of MSCs
The characterization of MSCs was performed using flow cytometry (FACS). High percentages of CD105, CD90, and CD73 were observed, while CD45, CD34, CD14, HLA-DR, and CD19 were present in low percentages. The complete results are presented in Table 1.
Table 1. MSC characterization result.
Parameter
Result

CD105
99,98%

CD90
99,97%

CD73
100%

CD45
0,75%

CD34
0,22%

CD14
0,15%

HLA-DR
0%

CD19
0,75%



2. It is not appropriate to store the secretome at -20 °C (especially when miRNA research is involved).

Thank you for your observation. In our study, we stored the secretome at -80°C for long-term preservation. However, for short-term storage, such as when preparing for studies, we stored the secretome at -20°C. Research has shown that storage at -20°C does not significantly affect miRNA levels for up to 4-5 years (Grasedieck et al., 2012). We also update the method section to include this information.

Method:
… The secretome was stored at -80°C for long-term preservation. However, for short-term storage, such as when preparing for studies, we stored the secretome at -20°C…..

Reference
Grasedieck, S., Schöler, N., Bommer, M. et al. Impact of serum storage conditions on microRNA stability. Leukemia 26, 2414–2416 (2012). https://doi.org/10.1038/leu.2012.106

3. In the introduction section, it is stated that there is limited information in the literature about the immunomodulatory effects of the secretome, and it is claimed that the immunomodulatory effects will be evaluated. There are studies in the literature investigating the immunomodulatory effects of MSC-secretome. The authors did not use these studies for their manuscripts.
Additionally, it is unclear how immunomodulatory effects are evaluated. Why has only IL-10 been investigated within the secretome? Why are other cytokines, molecules, and growth factors not mentioned? It should be explained.
protein concentration was very low 0.04 ug/ul and 0.45 ug/ul.

Thank you for your feedback. We have addressed your concerns as follows:
 

1. Introduction: We have now included references to studies on MSC-secretome and its immunomodulatory effects in the introduction. This addition highlights the existing research and aligns our work with the current literature.
2. Evaluation of Immunomodulatory Effects: We have clarified the criteria and methods used to evaluate the immunomodulatory effects. Specifically, we have detailed why IL-10 was selected for measurement.
3. Protein Concentration: We acknowledge that the concentration of IL-10 was indeed very low (0.04 µg/µl and 0.45 µg/µl). We have addressed this issue in the discussion section, providing an explanation and considering its implications for our findings.

Revised sections, including these points, are now incorporated into the introduction and discussion.

4. In vitro experimental setup is not sufficient to examine immunomodulatory effects. It should be investigated in vivo and the results added. The results of acute toxicity testing are unclear.


Why was only IM injection administered? The most common route of administration for conditioned mediums is not IM. Why other routes were not chosen should be stated.
 

1. We conducted the immunomodulatory effect in vivo using carbon clearance assay. We describe the assay in the method section. In the assay the live animal was injected with carbon solution and we calculated the carbon absorbance after 15 minutes.
2. We appreciate your question regarding the rationale for choosing intramuscular injection over intravenous infusion of the secretome. While intravenous (IV) delivery is indeed a common method, we opted for intramuscular (IM) administration for several reasons. Firstly, IM delivery allows the secretome to be deposited directly in the muscle tissue, where it can be gradually released into the bloodstream, potentially providing a more sustained effect. Additionally, muscles have a rich blood supply, which facilitates the secretome's absorption into the circulatory system. The large surface area of the muscle also makes intramuscular administration relatively straightforward and practical. Research supports the effectiveness of intramuscular administration for achieving systemic effects. For example, studies have demonstrated that intramuscular delivery of MSCs can result in systemic benefits (Jahromi & Davis, 2019). Furthermore, research has shown that exosomes, a key component of the secretome, can reach systemic circulation even when administered intramuscularly (Rehorova et al., 2019; Fennel et al., 2024). Therefore, while IV infusion is a commonly used route, intramuscular injection offers unique advantages that align with our study's objectives. We have added this in our discussion section.

5. It should be stated under which conditions diluted exosomes were stored.

Thank you for the sugestion, we have added the information in method section: “Diluted exosomes were stored in -80°C for further characterization.
”
6. The morphology of the TEM images is not compatible with the exosome image. The double-membrane structure is not visible. The authors measured exosome diameters, but there is no scale bar in the TEM images. Details regarding the storage conditions of exosomes and the negative-staining method should be specified in the method section. References should be added for the methods used.

Thank you for the suggestion we have added the reference for the staining protocol that we used in this study. We also have revised the exosome picture based on the reviewer suggestion.

Methods:
For TEM analysis, as much as 200 µL of isolated exosomes were diluted using PBS to reach a volume of 1 mL exosome in PBS. Exosomes were dripped onto the 400 mesh Cu coated grid, allowed to absorb, and dried using filter paper. Negative staining was applied based on protocol by Jung and Mun, 2018, using 1% uranyl acetate. The negative staining was dripped on the grid and allowed to be absorbed, then dried with filter paper. After that, the grid stored in an EM grid box for TEM observation.  TEM visualizations were performed using JEOL 1400 Transmission Electron Microscope at 120 kV, and images were captured using a fully integrated high resolution camera system (8 million pixel CCD camera). Exosome particle size measurements were carried out using ImageJ software version 1.53K.


Figure 3. Size and morphology of isolated exosomes visualization using TEM. The size and morphology of the isolated exosome were varied. The size of the exosome was within the expected size range (30-100 nm). The double membrane appearance is pointed by the black arrow.





7. Authors should indicate the number of animals used in animal experiments. It is inappropriate to perform statistical analysis with parametric tests with the small sample size.

Thank you for the suggestion. We have added the information regarding the number of animals in the Methods section. We have 4 groups, with 5 animals in each group, in total 20 animals used in our study.
The animal number was calculated using the resource equation method, which states that the E value should be between 10 and 20. The formula for E is: total number of animals minus the total number of groups. In our study, we have 4 groups. Therefore, to keep the E value between 10 and 20, the number of animals in each group should be between 4 and 6 (Charan & Kantharia, 2013).
We also conducted statistical analyses using the Kruskal-Wallis and Mann-Whitney tests, and still found a significant difference between the negative control and the dose 3 group.
 
Method:
“Twenty male Swiss albino mice 6-8 weeks old, weighing 15 to 25 g, were used in the study. The animal's room temperature is set to 22° ± 3° C, with a relative humidity of 30–70%, 12 hours of light, and 12 hours of darkness. Animals are fed according to laboratory standards and given indefinitely (ad libitum). The animals were maintained under standard conditions according to Regulation of the Head of National Agency of Drug and Food Control (BPOM) No. 7 of 2014 concerning guidelines for in vivo tests [8]. The person conducting the animal handling was not blinded to the treatment group. However, the person conducting the measurement of the carbon clearance assay was blinded to the treatment group”.

All data were presented as Mean and SEM. The phagocytic index and spleen CD68 mRNA data were analysed using the Kruskal-Wallis and Mann-Whitney tests. The statistical analysis was done using GraphPad Prism version 9.0.0 (86).