Effects of cell culture conditions on Mesenchymal Stem Cells and strategies to improve their therapeutic application

Effects of cell culture conditions on Mesenchymal Stem Cells and strategies to improve their therapeutic application Máster Universitario de Biología Avanzada: Investigación y Aplicación. Trabajo Fin de Máster Laura Olmedo Moreno CURSO ACADÉMICO 2019/2020 Universidad de Sevilla Departamento de Biología Celular Centro Andaluz de Biología Molecular (CABIMER) Departamento de Terapia Celular y Regeneración 1 INDEX 1. Introduction………………………………………………………………………2 2. Changes induced in MSCs during cell culture……..…….………………..…….6 2.1. Morphological alterations…………………………………………………..7 2.2. Modifications in the markers profile………………..……………………....7 2.3. Physiological perturbations………………...………………………………..8 3. Consequences of the in vitro MSCs modifications for their use in cell therapy….8 4. Strategies to potentiate the therapeutic properties of MSCs……………..…...…..10 4.1. Biomaterials…………………………………………………………………13 4.2. Secretome…..………………………………………………………………...14 5. Conclusions and future prospects..……………………………………………..…15 6. Acknowledgment…..…………………………………………………………….…16 7. Bibliography……..………………………………………………….……………...17 2 Effects of cell culture conditions on Mesenchymal Stem Cells and strategies to improve their therapeutic application. 1. Introduction. Mesenchymal Stem Cells (MSCs) were discovered in 1974 by Friedenstein who isolated them from bone marrow and described their morphology in vitro as fibroblast-like spindle shaped 1. MSCs are multipotent cells that can be obtained from various tissues, including placenta 2, umbilical cord 3, amniotic fluid 4, bone marrow 5, muscle 6, compact bone 7 , synovial fluid 8, fat 9, dental pulp 10, hair follicles 11 and blood 12. MSCs have two principal characteristics: self-renewal and multilineage differentiation 13,14. Self-renewal concerns to the MSCs ability to generate identical copies of themselves, while multilineage differentiation refers to their capacity to give rise to cells into the mesodermal, ectodermal and endodermal lineages 15. Given that MSCs show heterogeneous qualities depending on their tissue source, the International Society for Cellular Therapy (ISCT) has established three minimal standards to define MSCs: they adhere to plastic in standard conditions, they express specific markers (positive in antigens like CD73, CD105 and CD90 while negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR) and they have the ability to differentiate into adipocytes, chondrocytes, and osteoblast in specific culture conditions 16. As mentioned above, the self-renewal and multilineage differentiation properties of MSCs are interesting points for basic and translational investigation, but also for clinical studies on several pathologies, such as cardiology, neurology, The beneficial characteristics of Mesenchymal Stem Cells (MSCs) allow us to use them in translational and clinical research. Recent studies have shown beneficial effects of MSCs for the treatment of several pathologies, such as retinal degenerative disorders, neurodegenerative diseases, diabetes, myocardial infarction, skin problems, bone, and liver disorders, among others. These cells are found in various tissues, but they appear in low quantities, which makes necessary to expand MSCs in vitro before application. However, in vitro manipulation has noticeable consequences on MSCs morphology, physiology and function. The expression profile of molecules and receptors of MSCs undergoes drastic changes during cell culture. These alterations give rise to different results when MSCs are used in cell-based therapies, such as different immune response in the host. In this overview, our main aim will be to analyze the different modifications of MSCs during cell culture, and how these changes alter their therapeutic properties after transplantation. In addition, we will discuss potential strategies to improve the therapeutic effects of MSCs. 3 orthopaedics, among others areas 17–20, as they promote tissue repair and regeneration (Figure 1 and Table 1) 21–23. For instance, MSC-based therapies are generating increasing interest in the current pandemic situation with the Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. Several research groups have reported the beneficial effects of MSC application for pulmonary complications of COVID patients. Shetty and collaboration have demonstrated that intravenous application of human MSCs (hMSCs) produced improvements in 7 patients with COVID19 pneumonia for 14 days compared to 3 placebotreated patients. It was suggested that this could be due to reduced hyperactivation of the immune system and increased endogenous repair due to the paracrine effects of MSCs 24. They observed that the administration of hMSCs originated changes in inflammatory markers, such as a significant increase in IL-10 and a decrease in TNF-α. In addition, computed tomography images showed that MSCs reduced the lesion area in the lungs at the end of the treatment in a critically ill patient with COVID-19 24,25. Furthermore, MSCs possess intrinsic tropism toward damaged tissues that is mediated by chemotaxis signalling pathways 13, being the C-XC motif chemokine ligand 12 (CXCL12) – C-X-C chemokine receptor type 4 (CXCR4) axis one of the key players 26. The CXCL12 is found in different tissues and is released in high concentrations during injury 27. Importantly, it has been demonstrated that MSCs express the CXCR4, one of the receptors to which CXCL12 binds to mediate migration towards injury tissues 28,29. The low quantity of MSCs in their multiple sources creates the need to expand them in vitro to obtain sufficient cells for therapeutic application 30. MSCs cultures are not subject to standardized protocols. There are unequal culture media and different methods to isolate the cells, such as the FIGURA 1. Representation of some MSCs therapeutic applications. MSCs as treatment in pathological conditions: liver disorders (e.g. cirrhosis), autoimmune diseases (e.g. Crohn's disease), skin problems (e.g. skin ulcers), bone disorders (e.g. imperfect osteogenesis and osteonecrosis), heart diseases (e.g. myocardial infarction and cardiac ischemia), pulmonary pathologies (e.g. pulmonary COVID-19 infection) and neurological damage (e.g. Parkinson’s disease and spinal cord injury). explant culture method 31,32 or the enzymatic method 32,33. In addition, MSCs can be expanded 4 on different plastic surfaces, which have peculiar hydrophobicity characteristics affecting cell growth 14,34. The lack of common rules generates a huge variety of results when MSCs are used in preclinical therapies 35. It is suggested that alterations in therapies could be associated with modifications during MSC culture, such as distinct morphologies, different membrane receptors and modifications in their secretome 30,36. The major goal of this review is to provide a general overview about the modifications occurring in cultured MSCs that may lead to interlaboratory variability observed when working with this cell type. In addition, we discuss alternatives in vitro conditions that may help to obtain more effective MSCs for cell-based therapies. The significance of this review lies in the importance of MSCs as therapeutic tool for the treatment of a wide range of pathologies due to their benefits on tissue repair and regeneration. 5 CLINICALTRIALS IDENTIFIER PATHOLOGY DISEASE NAME TIME N MSC TYPE ADMINISTRATION PHASE COUNTRY NCT0042013437 Liver Cirrhosis 24 wk 30 BMhMSC Intravenous I/II Iran NCT0122049238 Liver Cirrhosis 48 wk 45 UChMSC Intravenous I/II China NCT0145433639 Liver Liver fibrosis 48 wk 3 BMhMCS Intravenous I Iran NCT0159120040 Liver Alcoholic Cirrhosis 96 wk 40 BMhMCS Intraarterial II India NCT0115765041 Autoimmune Crohn's disease 144 wk 15 AdhMSC Unknown I/II Spain NCT0165976242 Autoimmune Crohn’s disease 48 wk 16 BMhMSC Intravenous I EE. UU. NCT0377833343 Autoimmune Multiple Sclerosis 48 wk 7 BMhMSC Intravenous I Sweden NCT0187362544 Autoimmune Rheumatoid Arthritis 48 wk 60 BMhMSC Intraarticular II/III Iran NCT0282439345 Skin Chronic autoimmune urticaria 48 wk 10 AdhMSC Intravenous I Turkey NCT0388720846 Skin Cutis laxa senile and scars 27 wk 100 AdhMSC Subcutaneus I/II Poland NCT0268572247 Skin Skin ulcers 24 wk 20 UChMSC Topic I China NCT0249165848 Skin Vulgar Psoriasis 48 wk 30 UChMSC Intravenous I/II China NCT0151369449 Bone Intervertebral Degenerative Disc disease 24 wk 15 BMhMSC Implantation I/II Spain NCT0160538350 Bone Osteonecrosis of the Femoral Head 48 wk 23 BMhMSC Implantation I/II Spain NCT0217288551 Bone Osteogenesis imperfecta 96 wk 2 MSC Intravenous I Spain NCT0018691452 Bone Osteodysplasia Unkno wn 8 BMhMCS Intravenous I EE. UU. NCT0173977753 Heart Cardiopathy 48 wk 30 UChMSC Intravenous I/II Chile NCT0144903254 Heart Chronic myocardial ischemia 24 wk 60 AdhMSC Intramyocardial II Denmark NCT0238772355 Heart Severe Heart Failure 24 wk 10 AdhMSC Intramyocardial I Denmark NCT0246738756 Heart Non-Ischemic Heart Failure 64 wk 23 BMhMSC Intravenous II EE. UU. NCT0436632357 Pulmonary COVID-19 48 wk 26 AdhMSC Intravenous I/II Spain NCT0428810258 Pulmonary COVID-19 12 wk 90 UChMSC Intravenous II China NCT0259483959 Pulmonary Progressive Interstitial Lung Disease 48 wk 20 BMhMSC Intravenous I/II Russia NCT0191982760 Pulmonary Idiopathic pulmonary fibrosis 48 wk 17 BMhMSC Endobronchial I Spain NCT0266806861 Pulmonary Pneumoconiosis 24 wk 80 UChMSC Lavage I China NCT0105647162 Neurological Secondary Progressive Multiple Sclerosis 48 wk 30 AdhMSC Intravenous I/II Spain NCT0261116763 Neurological damage Idiopathic Parkinson’s Disease 52 wk 20 BMhMSC Intravenous I/II EE. UU. NCT0132510364 Neurological Spinal Cord Injury 24 wk 14 BMhMSC Intralesional I Brazil NCT0224967665 Neurological Neuromyelitis Optica 48 wk 15 BMhMSC Intravenous II China *Abreviations: Mesenchymal Stem Cells (MSC), Bone Marrow human Mesenchymal Stem Cells (BM-hMSC), Umbilical Cord human Mesenchymal Stem Cells (UC-hMSC), Adipose derived human Mesenchymal Stem Cells (Ad-hMSC). TABLE 1. Clinical trials of Mesenchymal Stem Cells therapy for different types of pathologies. 6 2. Changes induced in MSCs during cell culture. Each step in cell culture is parallel to MSCs morphological disorders, changes in their markers profile and physiological perturbations. Furthermore, the alterations are determined by many variable conditions such as donor age 66, tissue source 67, passages number 68, oxygen levels 69 or medium composition 70 (Figure 2). Zaim et al. demonstrated that donor age affects differentiation of bone marrow hMSCs (BM-hMSCs). BMhMSCs from children between 0-12 years old showed more adipogenic, neurogenic and osteogenic differentiation potential and more proliferation than BM-hMSCs from adults between 25-50 years old or from elderly over 60 years old in the same passage 66 . Another study demonstrated that the source of MSCs affects later differentiation. The use of BM-hMSCs presented a greater differentiation potential to osteogenic cells, while MSCs derived from adipose tissue (AdhMSCs) revealed a greater differentiation potential to adipogenic cells 67. Furthermore, the passage number of the cell culture is another significant point. Tan and co-workers showed that surface markers of bovine synovial membrane-derived MSCs (SD-MSCs) change in passages (P) 4 68. There was an increase in the expression of CD73 between P1 and P2, whereas CD73 levels had a significant reduction in P3. In this report, they suggested that the decrease on CD73 expression could be responsible for the changes in migration. When they applied a direct current electric field (DC-EF), 85% of cells moved towards the positive pole in P1, while a 75% of cells migrated towards the negative pole in P4. This variations in the direction of SD-MSCs migration correlated with the changes in CD73 expression 68. All these investigations evidence how the cell culture conditions influence SD-MSCs. Throughout this section we will focus on describing morphological alterations, changes in the markers profile and physiological perturbations that MSCs undergo during culture. FIGURE 2. Representation of influencing factors on morphological and physiological characteristics, in addition to surface markers. Factors responsible for changes in cultured MSCs include tissue origin, donor age, medium and supplements, passage number, and incubation conditions such as oxygen levels. All these parameters cause morphological alterations in MSCs, which change from a spindle form to a flattened form. Moreover, there are changes in surface markers such as higher expression of CD146, CD105 and CD271 and a lower expression of CD34 and CD90. Physiological changes include the generation of free radicals that directly affect the amino acid profile and lipid peroxidation. Abbreviations: Cluster of Differentiation 146 (CD146), Cluster of Differentiation 105 (CD105), Cluster of Differentiation 271 (CD271), Cluster of Differentiation 34 (CD34), Cluster of Differentiation 90 (CD90), Reactive oxygen species (ROS). 7 2.1 Morphological alterations. The MSCs normally have a spherical form in vivo 71. When MSCs are seeded as adherent cells, they usually acquire spindle form 72. However, the MSCs morphology can change in response to medium supplements 73,74, passage number 75 and/or oxygen conditions 76. One of the most common supplements used in cell culture is fetal calf serum (FCS) as a nutrition source, protein and growth factors 74. Chase et al., demonstrated that the use of FCS affects MSCs morphology. Using light microscopy, they observed that BM-hMSCs cultured in a medium with FCS had a flattened shape, while BM-hMSCs cultured in a serum-free medium had their characteristic spindle morphology 73. Another influential factor affecting cell morphology is the passage number, which refers to MSC aging. A study grew BM-hMSCs in two different media, Minimum Essential Medium Eagle - Alpha Modification (α-MEM) and Dulbecco's Modified Eagle Medium (DMEM), and observed that MSCs acquired atypical and flat shapes by P6 75. In addition to medium supplements and aging, another factor to consider is the oxygen concentration. Holzwarth's team demonstrated how low oxygen levels and donor affect cell morphology. They cultured BM-hMSCs from 10 donors under two conditions 21% and 1% oxygen. When examining the cells under the light microscope, they observed that BM-hMSCs from most donors showed the same spindle morphology and all the cells appeared as a monolayer at 21% and 1% oxygen after one or three weeks. Conversely, BM-hMSCs from 7 donors did not adopt the typical spindle shape and they did not create monolayers at 1% oxygen in the two measures of time 76. Examples such as these demonstrate that MSCs undergo dynamic changes. The question is, which are the molecular mechanisms underlying the morphological alterations? 2.2. Modifications in the markers profile. There is not a set of definitive markers which define a unique phenotype in MSCs due to their variability (origin, conditions, age, isolation method). However, there are common receptors for growth factors, chemokines, cytokines, matrix proteins, cell-cell receptors and inmunomodulating receptors 26. Most membrane markers have been determined in vitro, hence there is a limited knowledge of their properties in vivo 77. These antigens are not exclusive of MSCs as Schrage et al. showed in their study by demonstrating that CD46 is also an endothelial marker (antibody ME-9f1) 78. Moreover, MSCs can have a different expression receptor fraction according to their source, for example Stro-1 appears in BM-hMSCs but there is a lack of this antigen in Ad-hMSCs 79. As previously discussed, the ISCT indicated that the positive MSCs surface markers are CD73, CD90 and CD105. Furthermore, Maleki et al. compared hMSCs from ovary, testis, hWJ-MSCs and hair follicle, and they found that all these hMSCs also shared Stro-1, CD44, CD166 and CD106 80. On the other hand, Ly and co-workers in their review added three repetitive membrane markers, e.g. Stage-specific 8 Embryonic Antigen-4 (SSEA4), CD271 and CD46 81. Several investigations have demonstrated that the markers profile changes when MSCs are cultured in vitro. For example, Braun and collaboration have shown that clusters of differentiation, such as CD146, CD105 and CD271 of adventitial stromal cells (AST), were overexpressed after four days in vitro. However, CD34 had less expression between the fourth and sixth day 72. Another example of marker that changes in vitro was identified by Yu et al., who demonstrated high expression of Stro-1 in dental pulp stem cells (DPSCs) of rat and human at P9, as compared to P1 82. Moreover, it has been shown that BMhMSCs seeded in a 3D alginate culture or under mechanical stimulation present low CD90 expression 83,84. This protein is involved in the regulation of cell-cell contact and cell-matrix junctions. The lack of this marker has consequences such as impaired cell migration, affected actin filaments and loss of cell-cell and cell-matrix junctions, which could alter cell morphology 85. 2.3. Physiological perturbations. Cell culture conditions, such as oxygen concentration, passages, supplements, contribute to maintain cellular homeostasis. The oxygen levels applied in culture are usually those that we have in the atmosphere (20%), but this point is questionable since cells in the body are indeed exposed to 2-7% oxygen. This is an important issue because high oxygen levels generate metabolism perturbations and oxidative stress, generating one of the most notorious consequences that is the increase of the radical oxidative species (ROS) concentration, one of the typical senescence marks in cultures cells. In addition to the activation of the senescence process, high oxygen levels reduces cell survival and proliferation, which is a bottleneck to use MSC in therapy 86. ROS are small molecules usually generated in mitochondria and they can react easily due to their free electron (superoxide anions [O2-], hydrogen peroxide [H2O2] and hydroxyl radicals [OH-]). ROS can coordinate different cell levels because of its ability of regulating redox state of proteins and lipids, among others, generating cellular perturbations87. Shin et al. demonstrated that increased ROS levels relate to changes in amino acid profile and lipid peroxidation. BM-hMSCs with high ROS levels due to serum starvation exhibited alteration of amino acids like lysine, tyrosine, and γ-aminobutyric acid (GABA) accumulation. At the same time, there is a gain of lipid peroxidation giving rise to a decreased membrane permeability and fluency 88. 3. Consequences of the in vitro MSCs modifications for their use in cell therapy. The changes occurring in cultured MSCs, such as morphological alterations, the different profile of surface markers and the physiological modifications, limit their widely use in clinical application. For example, the increased size of MSCs in vitro influences their therapeutic effect 15 secretome induced behaviour improvements in vivo through Rotarod and Staircase tests (Figure 5) 146. 5. Conclusions and future prospects. Throughout this review, three main points have been addressed: 1) changes induced in MSCs during cell culture, 2) how these changes affect the use of MSC-based therapies and 3) strategies to improve the therapeutic effects of MSCs. MSCs have great potential for cell therapy and regenerative medicine due to their easy extraction from multiple sources 147, their ability to migrate to damaged tissues 148,149, their multilineage differentiation 150,151, their self-renewal 152, and the lack of ethical concerns 153. All these advantages reinforce the use of MSCs to treat several diseases, such as myocardial infarction 154,155, cardiac ischemia 156,157, neurological disorders 100,158,159, imperfect osteogenesis 160, or more recently the COVID-19 24,25,126,161,162. Latest advances in this research area hold promises for the applications of MSCs in regenerative medicine. However, MSC-based therapies still present barriers that need to be overcome. In conclusion, all the procedures that are carried out during cell culture generate morphological and physiological modifications in MSCs that directly affect their clinical application. Moreover, the lack of a unanimous protocol originates discrepancies in the results obtained by researchers, and, in many cases, this makes difficult to reproduce the experiments. Therefore, it is important that the investigators make the effort to unify protocols to obtain conclusions that are more robust. On the other hand, strategies are being implemented to enhance the therapeutic properties of MSCs, but there is still much potential to be improved. The scientific community should further investigate how to develop strategies that significantly increase the efficacy of MSC-based therapies in a safety way, allowing the translation to humans. FIGURE 5. Motor coordination and balance, and spatial memory and learning some behavioural tests. On the left, it is represented two behavioral tests related to motor coordination and balance. Staircase test. In this test, two stairs are used with feed in each step on both sides of the transparent container, and between them a platform where the animal is placed. It can be measured the effectiveness and the distance reached by forelimbs depending on the feed consumed and observations. Rotarod test. This methodology consists of a rotating cylinder with modifiable speed. Rodents are placers on the rod, and it can be measured their resistance, strength, balance, and coordination. On the right, there is a test connected with spatial memory and learning. Maze Morris test. In this test, rodents are placed in a container of water in which there is a platform that can be viewed by individuals. After several repetitions, the platform is hidden to check the memory and learning of rodents. The length of the route, the time spent, the analysis of different quadrants can be measured. 16 As a critical analysis, the communication between the different research teams is required to the unification of protocols. The main obstacle to achieve this point is the lack of transparency in the publications, which often do not specify all the information in a detailed manner or even the obtained results. A universal protocol to culture MSCs should include an effective isolation method, the use of a specific cell culture vessel, the detailed media composition (including reagent references) and the standardized culture conditions, such as oxygen levels. Furthermore, the type of MSCs must also be taken into account since it has been shown that MSC properties may vary between source tissues. Apart from that, the experimental strategies to improve the therapeutic properties of MSCs are based on three main points: the minimization of the changes that MSCs undergo in vitro, the use of biomaterials to favor MSCs application, and the utilization of MSC secretome. The choice of the most appropriated strategy will depend on the specific aim of the therapy. For instance, the use of MSCs combined with biomaterials to facilitate their engraftment and survival, would be an appropriated option when doing local cell administration (e.g., intracranial injections). However, when a systemic administration is used in allogenic therapies, the MSC-derived secretome could be an interesting choice since it has a low risk of rejection, as well as a reduced tumorigenic potential, unlike live cells. In conclusion, the scientific community should make efforts in synergy to seek solutions to the aforementioned issues, bringing new perspectives for a personalized medicine that will allow us to successfully treat the specific pathological condition of each patient. 6. Acknowledgment. We would like to acknowledge the ‘Master's Degree in Advanced Biology: Research and Application’ of the University of Seville. The research group of Stem Cells and Translational Neurology of CABIMER receives the support of the Andalusian Regional Ministry of Health (PI-0272-2017), the Institute of Health Carlos III co-funded by Fondos FEDER (CP19/00046), and the crowdfunding platform PRECIPITA of the Spanish Foundation for Science and Technology (2018-000237). 17 7. Bibliography. 1. Friedenstein, A., AJ, F. & AF, P. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method (1974). 2. Fukuchi, Y., Nakajima, H., Sugiyama, D., Hirose, I., Kitamura, T., & Tsuji, K. Human placenta‐derived cells have mesenchymal stem/progenitor cell potential. Stem cells, 22, 649-658 (2004). 3. Wang, H. S., Hung, S. C., Peng, S. T., Huang, C. C., Wei, H. M., Guo, Y. J., ... & Chen, C. C. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem cells, 22, 13301337 (2004). 4. int Anker, P. S., Scherjon, S. A., Kleijburg-Van der Keur, C., Noort, W. A., Claas, F. H., Willemze, R., ... & Kanhai, H. H. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood, 102, 1548-1549 (2003). 5. Rojas, M., Xu, J., Woods, C. R., Mora, A. L., Spears, W., Roman, J., & Brigham, K. L. Bone marrow–derived mesenchymal stem cells in repair of the injured lung. American journal of respiratory cell and molecular biology, 33, 145-152 (2005). 6. Čamernik, K., Mihelič, A., Mihalič, R., Presen, D. M., Janež, A., Trebše, R., ... & Zupan, J. Skeletal-muscle-derived mesenchymal stem/stromal cells from patients with osteoarthritis show superior biological properties compared to bone-derived cells. Stem cell research, 38, 101465 (2019). 7. Short, B. J., Brouard, N., & Simmons, P. J. Prospective isolation of mesenchymal stem cells from mouse compact bone. Stem Cells in Regenerative Medicine, 259-268 (2009). 8. Kim, Y. S., Lee, H. J., Yeo, J. E., Kim, Y. I., Choi, Y. J., & Koh, Y. G. Isolation and characterization of human mesenchymal stem cells derived from synovial fluid in patients with osteochondral lesion of the talus. The American journal of sports medicine, 43, 399-406 (2015). 9. Yoshimura, H., Muneta, T., Nimura, A., Yokoyama, A., Koga, H., & Sekiya, I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell and tissue research, 327, 449-462 (2007). 10. Tamaki, Y., Nakahara, T., Ishikawa, H. & Sato, S. In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow. Odontology, 101, 121–132 (2013). 11. Hoogduijn, M. J., Gorjup, E., & Genever, P. G. Comparative characterization of hair follicle dermal stem cells and bone marrow mesenchymal stem cells. Stem cells and development, 15, 49-60 (2006). 12. Lee, O. K., Kuo, T. K., Chen, W. M., Lee, K. D., Hsieh, S. L., & Chen, T. H. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood, 103, 1669-1675 (2004). 13. Hmadcha, A., Martin-Montalvo, A., Gauthier, B. R., Soria, B. & Capilla-Gonzalez, V. Therapeutic Potential of Mesenchymal Stem Cells for Cancer Therapy. Frontiers in Bioengineering and Biotechnology, 8, 43 (2020). 14. Ringe, J., Kaps, C., Burmester, G. R., & Sittinger, M. Stem cells for regenerative medicine: advances in the engineering of tissues and organs. Naturwissenschaften, 89, 338-351 (2002). 15. Flores-Figueroa, E., Montesinos, J. J., & Mayani, H. Células troncales mesenquimales: historia, biología y aplicación clínica. Revista de investigación clínica, 58, 498-511 (2006). 16. Dominici, M. L. B. K., Le Blanc, K., Mueller, I., SlaperCortenbach, I., Marini, F. C., Krause, D. S., ... & Horwitz, E. M. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8, 315-317 (2006). 17. Byrne, S. N., Knox, M. C., & Halliday, G. M. TGFβ is responsible for skin tumour infiltration by macrophages enabling the tumours to escape immune destruction. Immunology and cell biology, 86, 92-97 (2008). 18. Mukai, T., Tojo, A., & Nagamura-Inoue, T.. Mesenchymal stromal cells as a potential therapeutic for neurological disorders. Regenerative Therapy, 9, 32-37 (2018). 19. Oliveira, M. S., & Barreto-Filho, J. B. Placental-derived stem cells: culture, differentiation and challenges. World journal of stem cells, 7, 769 (2015). 20. Wang, S., Qu, X. & Zhao, R. C. Clinical applications of mesenchymal stem cells. Journal of Hematology and Oncology, 5, 19 (2012). 21. Alsaeedi, H. A., Lam, C., Koh, A. E. H., Teh, S. W., Mok, P. L., Higuchi, A., ... & Muthuvenkatachalam, B. S. Looking into dental pulp stem cells in the therapy of photoreceptors and retinal degenerative disorders. Journal of Photochemistry and Photobiology B: Biology, 203, 111727 (2020). 22. Hsiao, C. Y., Chen, T. H., Huang, B. S., Chen, P. H., Su, C. H., Shyu, J. F., & Tsai, P. J. Comparison between the therapeutic effects of differentiated and undifferentiated Wharton's jelly mesenchymal stem cells in rats with streptozotocin-induced diabetes. World Journal of Stem Cells, 12, 139. (2020). 23. Tatullo, M., Codispoti, B., Spagnuolo, G., & Zavan, B. Human periapical cyst-derived stem cells can be a smart “lab-on-a-cell” to investigate neurodegenerative diseases and the related alteration of the exosomes’ content. Brain Sciences, 9, 358 (2019). 24. Shetty, A. K. Mesenchymal stem cell infusion shows promise for combating coronavirus (COVID-19)-induced pneumonia. Aging and Disease, 11, 462–464 (2020). 25. Leng, Z., Zhu, R., Hou, W., Feng, Y., Yang, Y., Han, Q., ... & Fan, J. Transplantation of ACE2-mesenchymal stem cells improves the outcome of patients with COVID-19 pneumonia. Aging and disease, 11, 216 (2020). 26. Docheva, D., Haasters, F., & Schieker, M. Mesenchymal stem cells and their cell surface receptors. Current Rheumatology Reviews, 4, 155-160 (2008). 27. Blanco, B. V. Aplicacion de la resonancia de plasmon superficial al estudio de la interacción cxcl12/cxcr4. Doctoral dissertation, Universidad Autónoma de Madrid, 2012. 28. Yu, X., Chen, D., Zhang, Y., Wu, X., Huang, Z., Zhou, H., ... & Zhang, Z. Overexpression of CXCR4 in mesenchymal stem cells promotes migration, neuroprotection and angiogenesis in a rat model of stroke. Journal of the neurological sciences, 316, 141149 (2012). 29. Zhang, D., Fan, G. C., Zhou, X., Zhao, T., Pasha, Z., Xu, M., ... & Wang, Y. Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium. Journal of molecular and cellular cardiology, 44, 281-292 (2008). 30. Drela, K., Stanaszek, L., Nowakowski, A., Kuczynska, Z., & Lukomska, B. Experimental strategies of mesenchymal stem cell propagation: adverse events and potential risk of functional changes. Stem cells international, 2019 (2019). 18 31. Hendijani, F.. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues. Cell proliferation, 50, e12334 (2017). 32. Lee, D. H., Joo, S. D., Han, S. B., Im, J., Lee, S. H., Sonn, C. H., & Lee, K. M. Isolation and expansion of synovial CD34− CD44+ CD90+ mesenchymal stem cells: comparison of an enzymatic method and a direct explant technique. Connective tissue research, 52, 226-234 (2011). 33. Zhang, H., Zhang, B., Tao, Y., Cheng, M., Hu, J., Xu, M., & Chen, H. Isolation and characterization of mesenchymal stem cells from whole human umbilical cord applying a single enzyme approach. Cell biochemistry and function, 30, 643-649 (2012). 34. Sotiropoulou, P. A., Perez, S. A., Salagianni, M., Baxevanis, C. N. & Papamichail, M. Characterization of the Optimal Culture Conditions for Clinical Scale Production of Human Mesenchymal Stem Cells. Stem Cells, 24, 462–471 (2006). 35. Parekkadan, B., & Milwid, J. M. Mesenchymal stem cells as therapeutics. Annual review of biomedical engineering, 12, 87-11 (2010). 36. Eggenhofer, E., Luk, F., Dahlke, M. H., & Hoogduijn, M. J. The life and fate of mesenchymal stem cells. Frontiers in immunology, 5, 148 (2014). 37. Kharaziha. P. Improvement of Liver Function in Liver Cirrhoses Patients After Autologous Mesenchymal Stem Cell Injection: a Phase I-II Clinical Trial. National Library of Medicine, (20072009). Identifier: NCT00420134. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT00420134. 38. Chen, C. Human Umbilical Cord Mesenchymal Stem Cells Transplantation for Patients With Decompensated Liver Cirrhosis. National Library of Medicine, 2011. Identifier: NCT01342250. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01342250. 39. Vosough, M. Transplantation of Autologous Mesenchymal Stem Cell in Decompensate Cirrhotic Patients With Pioglitazone. National Library of Medicine, (2011-2014). Identifier: NCT01454336. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01454336. 40. Tantry, B. V, Samir, S., Kini, D., Deepak, N., Saraswat, V. A., Habeeb, M. A., ... & Nai, P. V. Dose Finding Study to Assess Safety and Efficacy of Stem Cells in Liver Cirrhosis. National Library of Medicine, (2012-2016). Identifier: NCT01591200. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01591200. 41. Prosper, F. Treatment of Fistulous Crohn’s Disease by Implant ofAutologous Mesenchymal Stem Cells Derived From Adipose Tissue. National Library of Medicine, (2010-2016). Identifier: NCT01157650. Retrieved from: https://www.clinicaltrials.gov/ct2/show/study/NCT01157650. 42. Kugathasan,S. & Dhere, T. A Phase I Study Evaluating Autologous Bone Marrow Derived Mesenchymal Stromal for Crohn’s Disease. National Library of Medicine, (2012-2016). Identifier: NCT01659762. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01659762. 43. Iacobaeus, E. Mesenchymal Stem Cells for Progressive Multiple Sclerosis_Sweden. National Library of Medicine, (2018). Identifier: NCT03778333. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT03778333. 44. Shadmanfar, S. Transplantation of Bone Marrow Derived Mesenchymal Stem Cells in Affected Knee Osteoarthritis by Rheumatoid Arthritis. National Library of Medicine, (2013). Identifier: NCT01873625. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01873625. 45. Özdemir, A. T. & Ovalı, E. Experimental Autologous Mesenchymal Stem Cell Therapy in Treatment of Chronic Autoimmune Urticaria. National Library of Medicine, (20162018). Identifier: NCT02824393. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02824393. 46. Jaworowski, J. Therapy of Scars and Cutis Laxa With Autologous Adipose Derived Mesenchymal Stem Cells. National Library of Medicine, (2019-2020). Identifier: NCT03887208. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT03887208. 47. Fu, X. UC-MSCs Gel Treatment Difficult Healing of Skin Ulcers. National Library of Medicine, (2016). Identifier: NCT02685722. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02685722. 48. Hu, C. Safety and Efficacy of UC-MSCs in Patients With Psoriasis. National Library of Medicine, (2015). Identifier: NCT02491658. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02491658. 49. del Cañizo, R. D. M. C. Clinical Trial Based on the Use of Mesenchymal Stem Cells From Autologous Bone Marrow in Patients With Lumbar Intervertebral Degenerative Disc Disease. National Library of Medicine, (2012-2017). Identifier: NCT01513694. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01513694. 50. Aguirre, M. Mesenchymal Stem Cells in Osteonecrosis of the Femoral Head. National Library of Medicine, (2012-2020). Identifier: NCT01605383. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01605383. 51. Rodriguez, C. E. Mesenchymal Stem Cell Based Therapy for the Treatment of Osteogenesis Imperfecta National Library of Medicine, (2014-2019). Identifier: NCT02172885. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02172885. 52. Kasow, K. A. Stromal Therapy of Osteodysplasia After Allogeneic Bone Marrow Transplantation. National Library of Medicine, (2005-2015). Identifier: NCT00186914. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT00186914. 53. Bartolucci, J. Randomized Clinical Trial of Intravenous Infusion Umbilical Cord Mesenchymal Stem Cells on Cardiopathy. National Library of Medicine, (2012-2015). Identifier: NCT01739777. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01739777. 54. Kastrup, J. MesenchYmal STROMAL CELL Therapy in Patients With Chronic Myocardial Ischemia (MyStromalCell Trial). National Library of Medicine, (2011-2014). Identifier: NCT01449032. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01449032. 55. Kastrup, J. CSCC_ASC Therapy in Patients With Severe Heart Failure. National Library of Medicine, (2015-2016). Identifier: NCT02387723. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02387723. 56. Stardal, K. A Study to Assess the Effect of Intravenous Dose of (aMBMC) to Subjects With Non-ischemic Heart Failure. National Library of Medicine, (2015-2020). Identifier: NCT02467387. Retrieved from:https://clinicaltrials.gov/ct2/show/NCT02467387. 57. Cardesa, A. G. Clinical Trial to Assess the Safety and Efficacy of Intravenous Administration of Allogeneic Adult Mesenchymal Stem Cells of Expanded Adipose Tissue in Patients With Severe Pneumonia Due to COVID-19. National Library of Medicine, (2020). Identifier: NCT04366323. Retrieved from:https://clinicaltrials.gov/ct2/show/NCT04366323 58. Wang, F. S. Treatment With Human Umbilical Cord-derived Mesenchymal Stem Cells for Severe Corona Virus Disease 2019 (COVID-19) National Library of Medicine, (2020). Identifier: NCT04288102. Retrieved from: 19 https://clinicaltrials.gov/ct2/show/NCT04288102. 59. Federal Research Clinical Center of Federal Medical & Biological Agency of Russia. Safety and Efficacy of Allogeneic Mesenchymal Stem Cells in Patients With Rapidly Progressive Interstitial Lung Disease. National Library of Medicine, (20152018). Identifier: NCT02594839. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02594839. 60. Universidad de Navarra. Study of Autologous Mesenchymal Stem Cells to Treat Idiopathic Pulmonary Fibrosis. National Library of Medicine, (2013-2018). Identifier: NCT01919827. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01919827. 61. Dai, J., Xiong, W., Dai, X., Zhang, Y. & Fang S. A Study on Pneumoconiosis Treated With Whole-lung Lavage Combined With Mesenchymal Stem Cells. National Library of Medicine, (2016-2019). Identifier: NCT02668068. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02668068. 62. Fernández, O. F. & Ayuso G. I. Autologous Mesenchymal Stem Cells From Adipose Tissue in Patients With Secondary Progressive Multiple Sclerosis. National Library of Medicine, (2010-2015). Identifier: NCT01056471. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT01056471. .63. Schiess, M. C. Allogeneic Bone Marrow-Derived Mesenchymal Stem Cell Therapy for Idiopathic Parkinson’s Disease. National Library of Medicine, (2015-2019). Identifier: NCT02611167. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02611167. 64. Dos Santo, R. R., Soares, M.B. P., Mendonça, M. V. P., Pinheiro, P. C., Ferreira, T. X., Villarreal, C. F., … & Ubirajara, B. J. Autologous Bone Marrow Stem Cell Transplantation in Patients With Spinal Cord Injury. National Library of Medicine, (20112017). Identifier: NCT01325103. Retrieved from:. https://clinicaltrials.gov/ct2/show/NCT01325103. 65. Shi, F. D. Autologous Mesenchymal Stem Cells for the Treatment of Neuromyelitis Optica Spectrum Disorders. National Library of Medicine, (2014-2018). Identifier: NCT02249676. Retrieved from: https://clinicaltrials.gov/ct2/show/NCT02249676. 66. Zaim, M., Karaman, S., Cetin, G. & Isik, S. Donor age and longterm culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells. Annals of Hematology. 91, 1175–1186 (2012). 67. Xu, L., Liu, Y., Sun, Y., Wang, B., Xiong, Y., Lin, W., ... & Li, G. Tissue source determines the differentiation potentials of mesenchymal stem cells: a comparative study of human mesenchymal stem cells from bone marrow and adipose tissue. Stem cell research & therapy, 8, 1-11 (2017). 68. Tan, A. R., Alegre-Aguarón, E., O'Connell, G. D., VandenBerg, C. D., Aaron, R. K., Vunjak-Novakovic, G., ... & Hung, C. T. Passage-dependent relationship between mesenchymal stem cell mobilization and chondrogenic potential. Osteoarthritis and cartilage, 23, 319-327 (2015). 69. Sheehy, E. J., Buckley, C. T., & Kelly, D. J. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells. Biochemical and biophysical research communications, 417, 305-310 (2012). 70. Adamzyk, C., Emonds, T., Falkenstein, J., Tolba, R., JahnenDechent, W., Lethaus, B., & Neuss, S. Different culture media affect proliferation, surface epitope expression, and differentiation of ovine MSC. Stem cells international, 2013 (2013). 71. Beijer, N. R., Nauryzgaliyeva, Z. M., Arteaga, E. M., Pieuchot, L., Anselme, K., van de Peppel, J., ... & de Boer, J. Dynamic adaptation of mesenchymal stem cell physiology upon exposure to surface micropatterns. Scientific reports, 9, 1-14 (2019). 72. Braun, J., Kurtz, A., Barutcu, N., Bodo, J., Thiel, A., & Dong, J. Concerted regulation of CD34 and CD105 accompanies mesenchymal stromal cell derivation from human adventitial stromal cell. Stem cells and development, 22, 815-827 (2013). 73. Chase, L. G., Lakshmipathy, U., Solchaga, L. A., Rao, M. S., & Vemuri, M. C. A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem cell research & therapy, 1, 8 (2010). 74. Popov, A., Scotchford, C., Grant, D., & Sottile, V. Impact of Serum Source on Human Mesenchymal Stem Cell Osteogenic Differentiation in Culture. International Journal of Molecular Sciences, 20, 5051 (2019). 75. Yang, Y. H. K., Ogando, C. R., See, C. W., Chang, T. Y., & Barabino, G. A. Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem cell research & therapy, 9, 1-14 (2018). 76. Holzwarth, C., Vaegler, M., Gieseke, F., Pfister, S. M., Handgretinger, R., Kerst, G., & Müller, I. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells. BMC cell biology, 11, 11 (2010). 77. Niehage, C., Steenblock, C., Pursche, T., Bornhäuser, M., Corbeil, D., & Hoflack, B. The cell surface proteome of human mesenchymal stromal cells. PloS one, 6, e20399 (2011). 78. Schrage, A., Loddenkemper, C., Erben, U., Lauer, U., Hausdorf, G., Jungblut, P. R., ... & Klugewitz, K. Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1. Histochemistry and cell biology, 129, 441-451 (2008). 79. Kern, S., Eichler, H., Stoeve, J., Klüter, H., & Bieback, K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem cells, 24, 1294-1301 (2006). 80. Maleki, M., Ghanbarvand, F., Behvarz, M. R., Ejtemaei, M., & Ghadirkhomi, E. Comparison of mesenchymal stem cell markers in multiple human adult stem cells. International journal of stem cells, 7, 118 (2014). 81. Lv, F. J., Tuan, R. S., Cheung, K. M. C. & Leung, V. Y. L. Concise review: The surface markers and identity of human mesenchymal stem cells. Stem Cells, 32, 1408–1419 (2014). 82. Yu, J., He, H., Tang, C., Zhang, G., Li, Y., Wang, R., ... & Jin, Y. Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging. BMC cell biology, 11, 32 (2010). 83. Lee, H. J., Choi, B. H., Min, B. H. & Park, S. R. Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitro. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology, 60, 2325–2332 (2009). 84. Wiesmann, A., Bühring, H.-J., Mentrup, C. & Wiesmann, H.-P. Decreased CD90 expression in human mesenchymal stem cells by applying mechanical stimulation. Head Face Medicine, 2 (2006). 85. Rege, T. A. & Hagood, J. S. Thy‐1 as a regulator of cell‐cell and cell‐matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis. FASEB Journal,. 20, 1045–1054 (2006). 86. Gu, Y., Li, T., Ding, Y., Sun, L., Tu, T., Zhu, W., ... & Sun, X. Changes in mesenchymal stem cells following long-term culture in 20 vitro. Molecular medicine reports, 13, 5207-5215 (2016). 87. Bigarella, C. L., Liang, R., & Ghaffari, S. Stem cells and the impact of ROS signaling. Development, 141, 4206-4218 (2014). 88. Shin, T. H., Lee, S., Choi, K. R., Kim, Y., Paik, M. J., Seo, C., ... & Lee, G. Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buffered saline. Scientific reports, 7, 1-8 (2017). 89. Eggenhofer, E., Benseler, V., Kroemer, A., Popp, F., Geissler, E., Schlitt, H., ... & Hoogduijn, M. J. Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion. Frontiers in immunology, 3, 297 (2012). 90. Schrepfer, S., Deuse, T., Reichenspurner, H., Robbins, R. & Pelletier, M. Stem cell transplantation: The lung barrier. The Thoracic and Cardiovascular Surgeon, 56, 24 (2008). 91. Ge, J., Guo, L., Wang, S., Zhang, Y., Cai, T., Zhao, R. C., & Wu, Y. The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke. Stem Cell Reviews and Reports, 10, 295-303 (2014). 92. Janowski, M., Lyczek, A., Engels, C., Xu, J., Lukomska, B., Bulte, J. W., & Walczak, P. Cell size and velocity of injection are major determinants of the safety of intracarotid stem cell transplantation. Journal of Cerebral Blood Flow & Metabolism, 33, 921-927 (2013). 93. Wang, Y., Huang, J., Gong, L., Yu, D., An, C., Bunpetch, V., ... & Liu, H. The plasticity of mesenchymal stem cells in regulating surface HLA-I. Iscience, 15, 66-78 (2019). 94. Capilla-González, V., López-Beas, J., Escacena, N., Aguilera, Y., de la Cuesta, A., Ruiz-Salmerón, R., ... & Soria, B. PDGF restores the defective phenotype of adipose-derived mesenchymal stromal cells from diabetic patients. Molecular Therapy, 26, 2696-2709 (2018). 95. Djouad, F., Plence, P., Bony, C., Tropel, P., Apparailly, F., Sany, J., ... & Jorgensen, C.. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood, 102, 3837-3844 (2003). 96. Nagaya, N., Fujii, T., Iwase, T., Ohgushi, H., Itoh, T., Uematsu, M., ... & Kitamura, S. Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis. American Journal of Physiology-Heart and circulatory physiology, 287, H2670-H2676 (2004). 97. Osaka, M., Honmou, O., Murakami, T., Nonaka, T., Houkin, K., Hamada, H., & Kocsis, J. D. Intravenous administration of mesenchymal stem cells derived from bone marrow after contusive spinal cord injury improves functional outcome. Brain research, 1343, 226-235 (2010). 98. Danielyan, L., Schäfer, R., von Ameln-Mayerhofer, A., Bernhard, F., Verleysdonk, S., Buadze, M., ... & Koehle, C. Therapeutic efficacy of intranasally delivered mesenchymal stem cells in a rat model of Parkinson disease. Rejuvenation research, 14, 3-16 (2011). 99. Donega, V., Nijboer, C. H., van Tilborg, G., Dijkhuizen, R. M., Kavelaars, A., & Heijnen, C. J. Intranasally administered mesenchymal stem cells promote a regenerative niche for repair of neonatal ischemic brain injury. Experimental neurology, 261, 5364 (2014). 100. Soria, B., Martin-Montalvo, A., Aguilera, Y., Mellado-Damas, N., López-Beas, J., Herrera-Herrera, I., ... & Capilla-González, V. Human mesenchymal stem cells prevent neurological complications of radiotherapy. Frontiers in cellular neuroscience, 13, 204 (2019). 101. Amado, L. C., Saliaris, A. P., Schuleri, K. H., John, M. S., Xie, J. S., Cattaneo, S., ... & Lehrke, S. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proceedings of the National Academy of Sciences, 102, 11474-11479 (2005). 102. Makkar, R. R., Price, M. J., Lill, M., Frantzen, M., Takizawa, K., Kleisli, T., ... & Bick-Forrester, J. Intramyocardial injection of allogenic bone marrow-derived mesenchymal stem cells without immunosuppression preserves cardiac function in a porcine model of myocardial infarction. Journal of cardiovascular pharmacology and therapeutics, 10, 225-233 (2005). 103. Braid, L. R., Wood, C. A., Wiese, D. M., & Ford, B. N. Intramuscular administration potentiates extended dwell time of mesenchymal stromal cells compared to other routes. Cytotherapy, 20, 232-244 (2018). 104. Chen, C. H., Chang, Y., Wang, C. C., Huang, C. H., Huang, C. C., Yeh, Y. C., ... & Sung, H. W.. Construction and characterization of fragmented mesenchymal-stem-cell sheets for intramuscular injection. Biomaterials, 28, 4643-4651 (2007). 105. Gao, L. R., Chen, Y., Zhang, N. K., Yang, X. L., Liu, H. L., Wang, Z. G., ... & Wang, L. H. Intracoronary infusion of Wharton’s jellyderived mesenchymal stem cells in acute myocardial infarction: double-blind, randomized controlled trial. BMC medicine, 13, 162 (2015). 106. Kang, W. J., Kang, H. J., Kim, H. S., Chung, J. K., Lee, M. C., & Lee, D. S. Tissue distribution of 18F-FDG-labeled peripheral hematopoietic stem cells after intracoronary administration in patients with myocardial infarction. Journal of Nuclear Medicine, 47, 1295-1301 (2006). 107. Zeinaloo, A., Zanjani, K. S., Bagheri, M. M., Mohyeddin‐Bonab, M., Monajemzadeh, M., & Arjmandnia, M. H. Intracoronary administration of autologous mesenchymal stem cells in a critically ill patient with dilated cardiomyopathy. Pediatric transplantation, 15++ E183-E186 (2011). 108. Singer, W., Dietz, A. B., Zeller, A. D., Gehrking, T. L., Schmelzer, J. D., Schmeichel, A. M., ... & Coon, E. A. Intrathecal administration of autologous mesenchymal stem cells in multiple system atrophy. Neurology, 93, e77-e87 (2019). 109. Zhang, T., Lee, Y. W., Rui, Y. F., Cheng, T. Y., Jiang, X. H., & Li, G. Bone marrow-derived mesenchymal stem cells promote growth and angiogenesis of breast and prostate tumors. Stem cell research & therapy, 4, 1-15 (2013). 110. Cui, L. L., Kerkelä, E., Bakreen, A., Nitzsche, F., Andrzejewska, A., Nowakowski, A., ... & Jolkkonen, J. The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity. Stem Cell Research & Therapy, 6, 11(2015). 111. Watanabe, M., & Yavagal, D. R. Intra-arterial delivery of mesenchymal stem cells. Brain circulation, 2, 114 (2016). 112. Ge, J., Guo, L., Wang, S., Zhang, Y., Cai, T., Zhao, R. C., & Wu, Y. The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke. Stem Cell Reviews and Reports, 10, 295-303 (2014). 113. Almeida, S. O., Skelton, R. J., Adigopula, S., & Ardehali, R. Arrhythmia in stem cell transplantation. Cardiac electrophysiology clinics, 7, 357-370 (2015). 21 114. Chang, M. G., Tung, L., Sekar, R. B., Chang, C. Y., Cysyk, J., Dong, P., ... & Abraham, M. R. Proarrhythmic potential of mesenchymal stem cell transplantation revealed in an in vitro coculture model. Circulation, 113, 1832-1841 (2006). 115. Zhang, T., Lee, Y. W., Rui, Y. F., Cheng, T. Y., Jiang, X. H., & Li, G. Bone marrow-derived mesenchymal stem cells promote growth and angiogenesis of breast and prostate tumors. Stem cell research & therapy, 4, 1-15 (2013). 116. Lukomska, B., Stanaszek, L., Zuba-Surma, E., Legosz, P., Sarzynska, S., & Drela, K. Challenges and controversies in human mesenchymal stem cell therapy. Stem Cells International, 2019 (2019). 117. Lund, P., Pilgaard, L., Duroux, M., Fink, T., & Zachar, V. Effect of growth media and serum replacements on the proliferation and differentiation of adipose-derived stem cells. Cytotherapy, 11, 189-197 (2009). 118. Neuhuber, B., Swanger, S. A., Howard, L., Mackay, A., & Fischer, I. Effects of plating density and culture time on bone marrow stromal cell characteristics. Experimental hematology, 36, 11761185 (2008). 119. Anderson, D. G., Levenberg, S., & Langer, R. Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells. Nature biotechnology, 22, 863-866 (2004). 120. Gharibi, B., & Hughes, F. J. Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells. Stem cells translational medicine, 1, 771782 (2012). 121. Bianchi, G., Banfi, A., Mastrogiacomo, M., Notaro, R., Luzzatto, L., Cancedda, R., & Quarto, R. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Experimental cell research, 287, 98-105 (2003). 122. Hagmann, S., Moradi, B., Frank, S., Dreher, T., Kämmerer, P. W., Richter, W., & Gotterbarm, T. FGF‐2 addition during expansion of human bone marrow‐derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential. Cell Proliferation, 46, 396-407 (2013). 123. Hu, C., Zhao, L., Peng, C., & Li, L. Regulation of the mitochondrial reactive oxygen species: Strategies to control mesenchymal stem cell fates ex vivo and in vivo. Journal of cellular and molecular medicine, 22, 5196-5207 (2018). 124. Nawrocka, D., Kornicka, K., Szydlarska, J., & Marycz, K. Basic fibroblast growth factor inhibits apoptosis and promotes proliferation of adipose-derived mesenchymal stromal cells isolated from patients with type 2 diabetes by reducing cellular oxidative stress. Oxidative Medicine and Cellular Longevity, 2017 (2017). 125. Giuliani, M., Poggi, A., Griscelli, B. A., & Lataillade, J. J. IFNGamma priming protects fetal and embryonic MSC from NK cell-mediated killing and improves their immunosuppressive properties: role of activating and inhibitory receptors. Journal of Cell Science & Therapy, 5, 1 (2014). 126. Yip, H. K., Fang, W. F., Li, Y. C., Lee, F. Y., Lee, C. H., Pei, S. N., ... & Lee, M. S. Human umbilical cord-derived mesenchymal stem cells for acute respiratory distress syndrome. Read Online: Critical Care Medicine Society of Critical Care Medicine, 48, e391-e399 (2020). 127. Zheng, G., Huang, L., Tong, H., Shu, Q., Hu, Y., Ge, M., ... & Xu, J. Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebocontrolled pilot study. Respiratory research, 15, 39 (2014). 128. Kim, H., Na, D. L., Lee, N. K., Kim, A. R., Lee, S., & Jang, H. Intrathecal Injection in a Rat Model: A Potential Route to Deliver Human Wharton’s Jelly-Derived Mesenchymal Stem Cells into the Brain. International Journal of Molecular Sciences, 21, 1272 (2020). 129. Xu, W., Xu, R., Li, Z., Wang, Y., & Hu, R. Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF‐1α signalling. Journal of cellular and molecular medicine, 23, 18991907 (2019). 130. Dolatshahi-Pirouz, A., Nikkhah, M., Kolind, K., Dokmeci, M. R., & Khademhosseini, A. Micro-and nanoengineering approaches to control stem cell-biomaterial interactions. Journal of functional biomaterials, 2, 88-106 (2011). 131. Kobolak, J., Dinnyes, A., Memic, A., Khademhosseini, A., & Mobasheri, A. Mesenchymal stem cells: Identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods, 99, 62-68 (2016). 132. Amer, M. H., Rose, F. R., Shakesheff, K. M., & White, L. J. A biomaterials approach to influence stem cell fate in injectable cellbased therapies. Stem cell research & therapy, 9, 1-15 (2018). 133. Ozsvar, J., Mithieux, S. M., Wang, R., & Weiss, A. S. Elastinbased biomaterials and mesenchymal stem cells. Biomaterials science, 3, 800-809 (2015). 134. Nomura, H., Zahir, T., Kim, H., Katayama, Y., Kulbatski, I., Morshead, C. M., ... & Tator, C. H. Extramedullary chitosan channels promote survival of transplanted neural stem and progenitor cells and create a tissue bridge after complete spinal cord transection. Tissue Engineering, 14, 649-665 (2008). 135. Jin, K., Mao, X., Xie, L., Galvan, V., Lai, B., Wang, Y., ... & Greenberg, D. A. Transplantation of human neural precursor cells in Matrigel scaffolding improves outcome from focal cerebral ischemia after delayed postischemic treatment in rats. Journal of Cerebral Blood Flow & Metabolism, 30, 534-544 (2010). 136. King, V. R., Alovskaya, A., Wei, D. Y., Brown, R. A., & Priestley, J. V. The use of injectable forms of fibrin and fibronectin to support axonal ingrowth after spinal cord injury. Biomaterials, 31, 44474456 (2010). 137. Kim, H. S., Mandakhbayar, N. E., Kim, H. W., Leong, K. W., & Yoo, H. S. Protein-reactive nanofibrils decorated with cartilagederived decellularized extracellular matrix for osteochondral defects. Biomaterials, 120214 (2020). 138. Roche, E. T., Hastings, C. L., Lewin, S. A., Shvartsman, D. E., Brudno, Y., Vasilyev, N. V., ... & Mooney, D. J. Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart. Biomaterials, 35, 6850-6858 (2014). 139. Lu, D., Mahmood, A., Qu, C., Hong, X., Kaplan, D., & Chopp, M. Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury. Neurosurgery, 61, 596-603 (2007). 140. Zhang, M., Methot, D., Poppa, V., Fujio, Y., Walsh, K., & Murry, C. E. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. Journal of molecular and cellular cardiology, 33, 907-921 (2001). 141. Teixeira, F. G., Carvalho, M. M., Sousa, N., & Salgado, A. J. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration?. Cellular and Molecular Life Sciences, 70, 3871-3882 (2013). 142. Kumar, P., Kandoi, S., Misra, R., Vijayalakshmi, S., Rajagopal, K., 22 & Verma, R. S. The mesenchymal stem cell secretome: a new paradigm towards cell-free therapeutic mode in regenerative medicine. Cytokine & Growth Factor Reviews, 46, 1-9 (2019). 143. Eleuteri, S., & Fierabracci, A. Insights into the secretome of mesenchymal stem cells and its potential applications. International journal of molecular sciences, 20, 4597 (2019). 144. Eleuteri, S., & Fierabracci, A. Insights into the secretome of mesenchymal stem cells and its potential applications. International journal of molecular sciences, 20, 4597 (2019). 145. Teixeira, F. G., Carvalho, M. M., Neves-Carvalho, A., Panchalingam, K. M., Behie, L. A., Pinto, L., ... & Salgado, A. J. Secretome of mesenchymal progenitors from the umbilical cord acts as modulator of neural/glial proliferation and differentiation. Stem Cell Reviews and Reports, 11, 288-297 (2015). 146. Teixeira, F. G., Carvalho, M. M., Panchalingam, K. M., Rodrigues, A. J., Mendes‐Pinheiro, B., Anjo, S., ... & Salgado, A. J. Impact of the secretome of human mesenchymal stem cells on brain structure and animal behavior in a rat model of Parkinson's disease. Stem cells translational medicine, 6, 634-646 (2017). 147. Secunda, R., Vennila, R., Mohanashankar, A. M., Rajasundari, M., Jeswanth, S., & Surendran, R. Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study. Cytotechnology, 67, 793-807 (2015). 148. Satake, K., Lou, J., & Lenke, L. G. Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue. Spine, 29, 1971-1979 (2004). 149. Wu, G. D., Nolta, J. A., Jin, Y. S., Barr, M. L., Yu, H., Starnes, V. A., & Cramer, D. V. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 75, 679-685 (2003). 150. Song, B. Q., Chi, Y., Li, X., Du, W. J., Han, Z. B., Tian, J. J., ... & Lu, S. H. Inhibition of Notch signaling promotes the adipogenic differentiation of mesenchymal stem cells through autophagy activation and PTEN-PI3K/AKT/mTOR pathway. Cellular Physiology and Biochemistry, 36, 1991-2002 (2015). 151. Takahashi, Y., Yamamoto, M., & Tabata, Y. Osteogenic differentiation of mesenchymal stem cells in biodegradable sponges composed of gelatin and β-tricalcium phosphate. Biomaterials, 26, 3587-3596 (2005). 152. Bruder, S. P., Jaiswal, N., & Haynesworth, S. E. Growth kinetics, self‐renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. Journal of cellular biochemistry, 64, 278-294 (1997). 153. Li, W., Ren, G., Huang, Y., Su, J., Han, Y., Li, J., ... & Zhang, L. Mesenchymal stem cells: a double-edged sword in regulating immune responses. Cell Death & Differentiation, 19, 1505-1513 (2012). 154. Miyahara, Y., Nagaya, N., Kataoka, M., Yanagawa, B., Tanaka, K., Hao, H., ... & Sano, S. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nature medicine, 12, 459-465 (2006). 155. Wen, Z., Zheng, S., Zhou, C., Wang, J., & Wang, T. Repair mechanisms of bone marrow mesenchymal stem cells in myocardial infarction. Journal of cellular and molecular medicine, 15, 1032-1043 (2011). 156. Jiang, W., Ma, A., Wang, T., Han, K., Liu, Y., Zhang, Y., ... & Wang, J. Intravenous transplantation of mesenchymal stem cells improves cardiac performance after acute myocardial ischemia in female rats. Transplant international, 19, 570-580 (2006). 157. Quevedo, H. C., Hatzistergos, K. E., Oskouei, B. N., Feigenbaum, G. S., Rodriguez, J. E., Valdes, D., ... & Heldman, A. W. Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. Proceedings of the National Academy of Sciences, 106, 14022-14027 (2009). 158. Park, H. J., Lee, P. H., Bang, O. Y., Lee, G., & Ahn, Y. H. Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson’s disease. Journal of neurochemistry, 107, 141-151 (2008). 159. Weiss, M. L., Medicetty, S., Bledsoe, A. R., Rachakatla, R. S., Choi, M., Merchav, S., ... & Troyer, D. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem cells, 24, 781-792 (2006). 160. Ali, T. F., & Hasan, T. Phlorotannin-incorporated mesenchymal stem cells and their promising role in osteogenesis imperfecta. Journal of Medical Hypotheses and Ideas, 6, 85-89 (2012). 161. Zheng, G., Huang, L., Tong, H., Shu, Q., Hu, Y., Ge, M., ... & Xu, J. Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebocontrolled pilot study. Respiratory research, 15, 39 (2014). 162. Simonson, O. E., Mougiakakos, D., Heldring, N., Bassi, G., Johansson, H. J., Dalén, M., ... & Wiklander, O. P. In vivo effects of mesenchymal stromal cells in two patients with severe acute respiratory distress syndrome. Stem cells translational medicine, 4, 1199-1213 (2015).