Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration

Background: Human umbilical cord blood-mesenchymal stem cell (hUCB-MSC)-derived secretome is known to be able to promote neovascularization and angiogenesis, so it is also thought to have a capability to modulate endothelial progenitor cell (EPC) functions. Atorvastatin is the cornerstone of coronary artery disease (CAD) treatment which can enhance EPCs proliferation and migration. This study aims to analyze the effect of the hUCB-MSC-derived secretome and its combination with atorvastatin toward EPCs proliferation and migration. Methods: EPCs were isolated from a CAD patient’s peripheral blood. Cultured EPCs were divided into a control group and treatment group of 2.5 µM atorvastatin, hUCB-MSC-derived secretome (2%, 10%, and 20% concentration) and its combination. EPCs proliferation was evaluated using an MTT cell proliferation assay, and EPC migration was evaluated using a Transwell migration assay kit. Results: This research showed that hUCB-MSC-derived secretomes significantly increase EPC proliferation and migration in a dose-dependent manner. The high concentration of hUCB-MSC-derived secretome were shown to be superior to atorvastatin in inducing EPC proliferation and migration (p<0.001). A combination of the hUCB-MSC-derived secretome and atorvastatin shown to improve EPCs proliferation and migration compared to hUCB-MSC-derived secretome treatment or atorvastatin alone (p<0.001). Conclusions: This study concluded that the hUCB-MSC-derived secretome work synergistically with atorvastatin treatment in improving EPCs proliferation and migration.


Introduction
Coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide 1 . It is responsible for the deaths of 7.2 million people or 12.2% of total deaths per year worldwide. Despite advancement in CAD management (e.g. novel antiplatelet therapy, coronary stents, percutaneous coronary intervention techniques and devices, and coronary artery bypass surgery), there are some clinical subsets of CAD which remain untreatable such as ischemic cardiomyopathy, refractory angina, and patients who cannot undergo revascularization due to clinical and anatomical complexity 2,3 .
It is already known that CAD is caused by atherosclerosis, which is followed by reduced levels of circulating endothelial progenitor cells (EPCs) 4 . EPCs can differentiate into mature endothelial cells and also promote endothelial repair. Hence, increasing circulating EPC levels is proven to improve endothelial function 5 . EPCs also had a critical role in the stimulation of angiogenesis and vasculogenesis. Hence, increasing EPC proliferation and migration may reduce ischemia and improve myocardial performance 4,6 .
Regenerative treatment for CAD using stem cells has been extensively studied in the last decade 7 . However, these treatments faced challenges of low engraftment, poor survival, and low differentiation of the transplanted cells. Despite regenerative treatment shown to be promising in vitro, clinical studies showed unsatisfying results 8 . Hence, the researcher started to shift regenerative treatment from cell based-treatment into cell-free treatment using paracrine stimulation 9 . Nowadays, the usage of cell-free therapeutics as a regenerative therapy in cardiovascular diseases also started to be emerged 9 .
The secretome is the wide array variety of paracrine factors produced by mesenchymal stem cells (MSCs). Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) derived secretome was proven could promote neovascularization, angiogenesis 10-13 and improved cardiac systolic function by protecting myocardial cells from apoptosis 14 . However, using this approach to improve neovascularization is yet to be investigated. Hence, it is hypothesized that increasing EPCs proliferation and migration by the hUCB-MSC-derived secretome may be responsible for this effect. Statins, through their pleiotropic effect, are the cornerstone of CAD treatment. Atorvastatin is one of the most prescribed statins, whose ability to modulate EPCs proliferation and migration has already been well studied in both laboratory and clinical settings [15][16][17] . Furthermore, this study aims to compare the effect of the hUCB-MSC-derived secretome, atorvastatin and the two in combination in modulating EPC proliferation and migration.

Methods
This is an experimental laboratory study of atorvastatin and hUCB-MSC-derived secretome and also their combinations on EPCs proliferation and migration. We conducted a controlled, posttest-only group design.

Sample collection
A 50-100 mL peripheral blood sample was obtained from a patient with CAD. The patient was recruited from the outpatient cardiovascular clinic at Pusat Pelayanan Jantung Terpadu, Dr. Soetomo General Hospital, Surabaya, in March 2020. The inclusion criteria were as follows: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries 18 . The exclusion criteria were as follows: a history of percutaneous coronary intervention procedures or coronary artery bypass grafting surgery, acute coronary syndromes, and anemia.
This study protocol has an ethical clearance from the Health Research Ethics Committee of Dr. Soetomo General Hospital, Surabaya (No.1567/KEPK/X/2019, approved on 8 October 2019). The included subjects provided written informed consent before subject recruitment. All details which include personal information were omitted.

HUCB-MSCs-derived secretome preparation
The HUCB-MSCs-derived secretome was prepared according to the previous study 19 . HUCB-MSCs (3H Biomedical AB, Uppsala, Sweden) was cultured in MesenCult TM MSC Basal medium, supplemented with MesenCult TM Stimulatory supplement (StemCell Technologies Inc., Vancouver, Canada), and also added with penicillin and streptomycin. Upon reaching 80% confluency, the media was replaced with MesenCult TM MSC Basal medium (supplement-free media) and incubated for 24 hours. The media was collected and centrifuged. The supernatant was used as a conditioned medium that contained hUCB-MSCs-derived secretome 19 .
Isolation and culture of EPCs Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation of CAD patient's peripheral blood using Histopaque-1077 (Sigma-Aldrich, USA). After centrifugation of peripheral blood, PBMCs then cultured with STEMLINE-II hematopoietic stem cell expansion medium (Sigma-Aldrich, USA)

Amendments from Version 1
The revised version was improved with feedback from both reviewers which are: -Clear diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries.
-We stated the design of the study which is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration. -Reference has been added for the statement: HUCB-MSCsderived secretome preparation -All abbreviations has been spelt out in the beginning or first time used Any further responses from the reviewers can be found at the end of the article REVISED supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6. A total of 5×10 6 mononuclear cells/ml were seeded into fibronectincoated 6-well plate dish and cultured at 37°C and 5% CO 2 levels for 5 days. Non-adherent cells were then transferred for the proliferation and migration assay. After five days of culture, EPCs were confirmed using FITC-labeled anti-human CD34 antibody (animal source was mouse, 5 μL antibody was diluted at 500 μL per 1 × 10 6 cells; catalog number 60013FI, Gene ID: 947, by StemCell Technologies Inc., Vancouver, Canada) staining and examined with immunofluorescence microscopy.

Treatment of EPCs
Cultured EPCs were divided into eight treatment groups for each proliferation and migration assays. Those treatment include control group, 2.5 μM atorvastatin, low (2%), medium (10%) and high (20%) doses of hUCB-MSC-derived secretome, and combination of 2.5 μM atorvastatin with each dose of the hUCB-MSC-derived secretome. There were n=5 replications made from each treatment. To determine the volume of hUCB-MSC-derived secretome given, the concentration was multiplied with total solution given at each treatment.

EPCs proliferation assay
The MTT cell proliferation assay kit (Sigma-Aldrich, St Louis, MO, USA) was used to measure EPCs proliferation as described previously 20 . Treated EPCs were added with MTT reagent and incubated in a 37°C incubator with 5% CO 2 for 4 hours. Proliferation was determined from the reduction of tetrazolium (MTT) into insoluble formazan product by viable EPCs mitochondria. Absorbance was measured with a microplate reader at 595 nm wavelength. EPCs proliferation was measured as optical density (OD). MTT assay was measured at day 3 after reagent addition.

EPCs migration assay
EPCs migration was evaluated using the 24-mm diameter insert, 3-μm pore size, 6-well Transwell migration assay kit (Corning, USA). A total of 5×10 5 cultured EPCs were placed in the upper part of the Transwell migration assay kit. Next, 2 mL of EPC media and each treatment were added in the lower chamber compartment and then incubated for 24 hours at 37°C. Nonmigratory cells were removed manually. On the receiver plate, the new basal medium was placed and added 500 μL of trypsin + EDTA solution 0.5%, followed by 10 minutes incubation. Then, cells on the bottom surface of the membrane were stained with Giemsa and cell images were obtained on a light microscope and counted manually in n=5 random fields/sample 21 .

Statistical analysis
Statistical analyses were conducted using SPSS Statistics 23.0 to detect significance level at p<0.05. One-way ANOVA was used to compare groups, with Fisher's least significant difference (LSD) post hoc test. Kruskal-Wallis test was used if there are violations to the assumption of normality and the assumption of homogeneity of variance. Correlation between variables was obtained using Spearman's correlation followed by a linear regression test.

Results
Baseline characteristics and demography of CAD patient Clinical examination, blood sampling, electrocardiography, echocardiography and coronary angiography was conducted and evaluated in order to examine the inclusion and exclusion criteria. Our sample had a 1-year history of coronary artery disease, he suffered from refractory chest pain despite the optimum medical therapy. The coronary angiography showed complex lesion (three-vessel disease with chronic total occlusion) which was not amenable to undergo revascularization. The baseline characteristics of the patient are presented in Table 1.

EPC characteristics
EPCs were successfully isolated and cultured from the CAD patient's peripheral blood. It was confirmed through light microscopy that displayed a spindle-shape morphology, which is typical for early EPCs and an immunofluorescence assay that showed FITC CD 34+ expression ( Figure 1). Raw images are available as Underlying data 22 . In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research 20, [23][24][25] . There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs 26,27 HUCB-MSCs-derived secretome and atorvastatin increase EPCs proliferation EPCs were evaluated using the MTT proliferation assay. As shown in Figure 2, both atorvastatin and hUCB-MSCsderived secretome treatment groups at all doses increase EPCs proliferation compared to the control (p<0.05, ANOVA). hUCB-MSC-derived secretome treatment showed a dosedependent relationship with EPCs proliferation. At medium (10%) and high (20%) doses, hUCB-MSC-derived secretome was shown to elicit superior EPC proliferation than atorvastatin (OD 1.252±0.104 and 1.585±0.029, respectively, vs 0.738±0.025; p<0.01). Raw absorbance data for MTT assays are available as Underlying data 22 .
Pearson's correlation showed a significant and strong correlation between hUCB-MSCs-derived secretome treatment with EPC proliferation (r=0.954; p<0.001). The linear regression test showed an R 2 of 0.910.

Discussion
This research showed that treatment with hUCB-MSC-derived secretome, atorvastatin and a combination of the two increased the proliferation and migration of EPCs (isolated from CAD patient's peripheral blood). HUCB-MSC-derived secretome enhances EPCs proliferation and migration in a dose-dependent manner. The combination of hUCB-MSC-derived secretome and atorvastatin was shown to be superior to atorvastatin or hUCB-MSC-derived secretome alone.
In this research, hUCB-MSC-derived secretome treatment increased EPC proliferation in a dose-dependent manner, with the concentrations of 10% and 20% shown to be superior to atorvastatin. Previous studies showed that atorvastatin treatment is superior to other statins at improving EPC proliferation 23,24,28 . The HUCB-MSC-derived secretome is also composed of cytokines, chemokines, growth factors, proteins, and extracellular vesicles which may be involved in EPCs proliferation and migration 13,29 . Vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), insulin-like growth factor (IGF-1) are contained in the hUCB-MSC-derived secretome which may be involved in increasing EPC proliferation 30 . VEGF has been shown to improve the proliferation and differentiation of EPCs through activation of Ras signaling, and the MAPK/ERK pathway 31-33 . SDF-1 and IGF 1 also been shown to increase the EPCs proliferation in response to the PI3K/protein kinase B signaling pathway and promote angiogenesis 34-36 . Hence, it is suggested that hUCB-MSC-derived secretome treatment is beneficial to improve EPC proliferation which may involve MAPK/ERK and PI3K/protein kinase B pathway.
HUCB-MSC-derived secretome treatment shown to increase EPCs migration in a dose-dependent manner, with a concentration of 20% shown to be superior to atorvastatin. Similarly, the previous study showed secretome-derived from placental-MSCs is able to significantly increase EPCs migration 37 . The HUCB-MSC-derived secretome contains pro-angiogenic factors, such as human angiopoietin-1 (Ang-1), hepatocyte growth  factor (HGF), insulin-like growth factor I (IGF-I), prostaglandin E2 (PGE2), transforming growth factor-beta 1 (TGF-ß1), vascular cell adhesion protein 1 (VCAM-1) and vascular endothelial growth factor (VEGF) 38 . MSCs also have immunomodulatory and anti-inflammatory properties, as it contributes to the maintenance of self-renewal capacity through E-Prostanoid 2 (EP2) 39 and immune cell activation and maturation, including CD4 + helper T cells, B cells, dendritic cells, natural killer cells, monocytes and macrophages 40 . The HUCB-MSC-derived secretome also has a higher anti-inflammatory effect than other MSCs 41 and antioxidant properties, as proven in previous studies conducted in renal injury 42 and ischemic stroke 43 . Inflammatory stimuli and oxidative stress are also known to impair EPCs migration 44 . Chemoattractant gradient was an important driving factor to induce EPCs mobilization. Hence, a high concentration of growth factors in the HUCB-MSC-derived secretome increase the gradient between the top and lower parts of the Transwell migration assay may lead into an increase of EPCs migration. taken together, wide array molecules and multiple possible pathways involved in HUCB-MSCs secretome treatment seem to be responsible for its superiority against atorvastatin.
The synergistic effect of the HUCB-MSC-derived secretome with atorvastatin in enhancing EPCs proliferation and migration was demonstrated in this study. These combinations significantly increase EPCs proliferation and migratory activity by up to two-fold. Previously, The combination of MSCs with another compound, including statins, was shown to have beneficial effects in angiogenesis and neovascularization 45-47 . Co-culture of MSCs and EPCs have been shown to demonstrate improved EPC proliferation and migration, and enhance their angiogenic capacity 48,49 . However, the exact mechanism of these combinations to improve EPCs proliferation and migration is yet to be investigated. It is speculated that the involvement of multiple pathways may be responsible for its superiority against HUCB-MSCs-derived secretome or atorvastatin alone.
A mitogen-activated protein kinase (MAPK) pathway has been known to play a role in increasing EPCs proliferation 25 . Cell cycle progression through increased Cyclin D1 expression mediated by PI3K/Akt and MAPK pathway also involved in EPCs proliferation 50 . While increasing microRNA 221/222 expression shown to reduce EPCs proliferation capabilities 51 . HUCB-MSC-derived secretome treatment was speculated to improve EPCs proliferation through MAPK/ERK and PI3K/protein kinase B pathway. While atorvastatin improves EPCs proliferation through downregulation of microRNA 221/222 expression 51 . The involvement of these multiple pathways may result in higher EPCs proliferation in the combination group.
Enhanced growth factor levels through hUCB-MSC-derived secretome treatment will augment the chemoattractant gradient 42 , thus leading to enhanced migration of EPCs. The antiinflammatory and antioxidant properties of hUCB-MSC-derived secretome also speculated to improve EPCs migration 40,42 . While atorvastatin can increase the production of endothelial nitric oxide synthase and nitric oxide, which reduces the oxidative stress that impairs EPCs migration 47 . Statin also could prevent EPCs senescence by upregulating TRF2 of EPCs, hence enhance migratory capacity 52 . Those facts suggested that the combination of hUCB-MSC-derived secretome and atorvastatin will have superior EPCs migration through the involvement of multiple pathways.
In summary, hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. Through those mechanisms, secretome could be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy. However, this research did not measure the exact composition of the hUCB-MSC-derived secretome. The previous study showed that the secretome from another type of MSC can increase EPCs migration but not EPCs proliferation 53 . Hence, further research should be directed to identify the substance within the hUCB-MSC-derived secretome which is responsible for increasing EPC proliferation and migration, and compare it with other MSC secretomes. Further research should also verify the multiple pathways which may be responsible for the improvement of EPCs proliferation and migration in the combination group.

Conclusions
High dose hUCB-MSC-derived secretome outperforms atorvastatin to improve EPC proliferation and migration. A combination of hUCB-MSC-derived secretome with atorvastatin seems to be beneficial in promoting neovascularization through improved EPCs proliferation and migration effect compared to hUCB-MSCderived secretome or atorvastatin alone.

Open Peer Review
The exact composition of the hUCB-MSc derived secretome should be described. 9.
In summary, this research needs more diligent work especially with the number of samples from more robust patients population.

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Author Response 30 Nov 2020
Yudi Her Oktaviono, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia Dear Dr Anwar Tandar Thank you very much for your valuable feedback I am truly honoured with your feedback In this opportunity, please let me answer the question Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria. A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.
Q: Typographical error in the Introduction... refractory angina, and patients A: Thank you for the feedback, we will revise accordingly Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3). A: We have added the reference, thank you Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5 A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.
Q: All abbreviations need to be spelt out in the beginning or first time used A: Thank you for the feedback, we will revise accordingly Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field. A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.
Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.
Q: Other statins should also be investigated as they are being used widely in real world. A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia.
Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.
Q: The exact composition of the hUCB-MSc derived secretome should be described. A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.
Again, many thanks for the review,, In this opportunity, please let me answer the question Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria. A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Best Regards Yudi Her
Q: Typographical error in the Introduction... refractory angina, and patients A: Thank you for the feedback, we will revise accordingly Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3). A: We have added the reference, thank you Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5 A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.
Q: All abbreviations need to be spelt out in the beginning or first time used A: Thank you for the feedback, we will revise accordingly Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field. A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.
Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically. Q: Other statins should also be investigated as they are being used widely in real world. A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.
Q: The exact composition of the hUCB-MSc derived secretome should be described. A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.
In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Yordinate)? ○ How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement? ○ In the #Table 1 -Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or enddiastolic dimension"? ○ In #summary, the authors should open the opportunity on the horizon, whether the cellbased therapy or cell-free measures that win the future game? 3 . ○ Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine. and diabetes mellitus 3. Hypertension 4. Stem cell and regenerative medicine 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.
controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.
Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.