Mesenchymal stem cells in human health and diseases

Chapter 11

Mesenchymal stem cells in human health and diseases: general discussion, remarks, and future directions Ahmed H.K. El-Hashash1, 2 1 The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, Zhejiang, PRC; 2Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, Zhejiang, PRC

The stem cell research field has grown fast since the beginning of the 21st century, with many new and astonishing discoveries, representing the most exciting aspects of biological and biomedical and research. Remarkably, stem cell research is growing over twice as fast (7%) as the reported world average growth in research, which is 2.9% (Stem Cell Research report; 2013). The annual growth rate of studies on one rapidly grown type of stem cell, which was awarded the Nobel Prize in Physiology or Medicine in 2012, induced pluripotent stem cells (iPSCs), since 2008 is an astonishing 77% (Stem Cell Research report, 2013). A significant increase in the volume of research output and publications has been reported in all areas of stem cell research. Major advances have been achieved in the stem cell research field, such as the generation of the first functioning whole organ, the thymus, in the laboratory; and the first documented human baby girl conceived through in vitro fertilization now has children of her own. Stem cell and biomedical research is currently under way in different national and international laboratories with ambitious goals of generating several other functioning whole organs, including the kidney and intestine. Mesenchymal stem cells (MSCs) are widely distributed in the body and can therefore be isolated from multiple sources, including the bone marrow, heart, bodily fluids, skin, and perinatal tissues. They can react to microenvironmental changes (stress, pH, oxygen) by releasing immune-modulatory and trophic factors known to regenerate injured cells and tissues. The application of recent discoveries in MSC biology and regenerative medicine to the betterment of Mesenchymal Stem Cells in Human Health and Diseases. https://doi.org/10.1016/B978-0-12-819713-4.00011-6 Copyright © 2020 Elsevier Inc. All rights reserved.

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human diseases has brought forth much hope, but continues to present many challenges. The hope for cures has motivated different states and countries worldwide to invest in stem cell and regenerative medicine research. Experimental findings in neurodegenerative and cardiovascular disease have supported the rapid growth of cell-based research. Research studies have shown evidence of the success of MSC therapy in ameliorating some human diseases or injuries that have been described throughout this book and are discussed in this chapter, in addition to many other cardiovascular diseases, autoimmune diseases, pulmonary diseases, and musculoskeletal diseases. However, adult MSC therapy of human diseases is still challenging, since a limited number of MSCs can be obtained from a single donor and a substantial cell quantity is required for their clinical therapeutic applications. Other challenges include the limited adult MSC proliferation capacity that may significantly constrain their abilities of immunomodulation and secretion of bioactive factors. Remarkable progress has been achieved in the potential application of stem cells in the treatment of different human diseases based on success in obtaining large amounts of iPSC-derived MSCs that are both therapeutically active and patient specific. iPSCs can be induced and converted into many different cell types in a controlled manner that enables obtaining a sufficient cell number and are a major source of induced MSCs (iMSCs). iMSCs are a new stem cell population that can be produced by cellular reprogramming and show the combined advantages of both MSCs and iPSCs and display the characteristics of an MSC population. iPSC-derived iMSCs have a robust potential for both proliferation and differentiation and are a promising cell-based therapy for different immune-mediated human diseases and for tissue repair and regeneration. Many preclinical studies have evaluated the utility of iMSCs that are derived from human iPSCs as a powerful mesenchymal cell source with better proliferation, differentiation, and survival potentials than adult MSCs. In addition, iMSCs derived from human iPSCs have many better properties compared with adult MSCs, such as their cytokine profile, immunomodulation, secretion of paracrine factors, and microenvironment-modulating exosomes. The relatively easy approaches to iMSC derivation and propagation in culture media that are chemically well defined make them an inexhaustible MSC source, which could be used for various clinical applications. However, the current iMSC scope is relatively limited to preclinical studies on tissue repair, regeneration, and engineering for treating various diseases. These preclinical studies have evaluated the utility of iMSCs that are derived from human iPSCs as a powerful mesenchymal cell source with better proliferation, differentiation, and survival potentials than adult MSCs. More preclinical studies and both clinical studies and trials are still needed to scale iMSCs toward routine clinical applications. Nevertheless, iMSCs are a promising cell-based therapy that is remarkably cost efficient and patient specific.

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MSCs are among the most commonly used stem cell types in cell-based therapy in clinical trials because of their regenerative effects. Thus, 695 US clinical trials are testing the utility of MSCs as therapeutic agents for an array of medical conditions as of this writing. Experimental and clinical studies have provided promising results using MSCs to treat diabetes and other diseases, and the results of several clinical trials with MSCs are inspiring (Butler et al., 2017; Staff et al., 2016).The therapeutic potential of MSCs is based on their remarkable ability to differentiate into multiple cell types, ease of isolation, low immunogenicity, and capacity to secrete multiple biologic factors that can restore, repair, and alleviate injured or impaired tissues. Preclinical and clinical evidence has substantiated the therapeutic benefit of MSCs in various medical conditions. However, several factors should be considered before using MSCs in the treatment of human diseases, including the choice of appropriate tissue source and culture conditions for MSCs. In addition, both the safety and the effectiveness should be critical criteria for the clinical application of MSCs. Research studies and clinical applications support the importance of MSCs as the most commonly used stem cell type in the repair, regeneration, and therapy of both soft tissues, including muscles, fascia, ligaments, tendons, nerve and fibrous tissues, synovial membranes, and blood vessels, and hard tissues such as bone and tooth. Research studies and clinical trials show promising results for the application of MSCs in myocardial repair and regeneration after injury. Thus, MSCs have been treated with hypoxic preconditioning for 24 h and tested in a monkey’s infarcted heart. Three months after the first injection, the infarct size and left ventricular (LV) function, as well as cardiomyocyte proliferation, vascular density, and myocardial glucose uptake, were significantly improved compared with the control group (Hu et al., 2016). In addition, when 5% O2etreated human MSCs (hMSCs) were administered intravenously into mice with LV dysfunction, LV ejection fraction and infarct size were significantly improved in the MSC group after 21 days (Luger et al., 2017). Furthermore, a double-blind, placebo-controlled, dose-ranging trial was conducted in 53 patients. The results of this trial were promising and showed that the forced expiratory volume in 1 s and LV ejection fraction were improved in patients who were treated with hMSCs (Hare et al., 2009). MSC therapy has shown promising results in the soft tissues of other body systems, such as the pulmonary and gastrointestinal systems. In pulmonary diseases, for example, a phase I study has shown that in severe chronic obstructive pulmonary disease patients, combined one-way endobronchial valve insertion and MSC treatment can decrease the levels of circulating C-reactive protein at 30 and 90 days, as well as the body mass index, airway obstruction, dyspnea, and exercise index and the modified Medical Research Council score (de Oliveira et al., 2017). In addition, a phase II study showed that in bronchopulmonary dysplasia patients, intratracheal transplantation with

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allogeneic human umbilical cord blood-derived MSCs has lower severity than in the control group (Chang et al., 2014). Notably, when MSC-based therapies were used to treat acute respiratory distress syndrome (ARDS) patients, serious adverse events (SAEs) were subsequently observed in three of nine patients, even though they thought those SAEs were not related to the MSCs. A phase II is already under way to treat ARDS (Wilson et al., 2015). MSC applications in gastrointestinal disease therapy have progressed rapidly. Adipose-derived mesenchymal stem cells (Ad-MSCs), for example, are a promising strategy for reducing intestinal lesions. A 2017 study has evaluated the importance of hydrogel-assisted Ad-MSC therapy and its effects compared with the conventional MSC intravenous injections as used in current clinical applications on both functional and structural damages induced in experimental animal models of colorectal irradiation (Moussa et al., 2017). Interestingly, silanized hydroxypropyl methylcellulose hydrogeleloaded Ad-MSCs improved colonic epithelial structure as well as hyperpermeability in a rat model of radiation-induced severe colonic damage (Moussa et al., 2017). In addition, MSC treatments have shown promising results in Crohn disease patients who were given intravenous infusions of allogeneic MSCs weekly for 4 weeks. Indeed, 42 days after the first MSC administration, 8/15 patients had clinical remission, while 7/15 patients had endoscopic improvement (Forbes et al., 2014). A phase III randomized, double-blind controlled trial used Ad-MSCs for complex perianal fistulas in Crohn disease, in which 212 patients were randomly divided into two groups with MSCs or placebo. Interestingly, after 24 weeks, a significant remission was found in the MSC group compared with the placebo group (Pane´s et al., 2016). Similar progress has been achieved in the application of MSCs in the treatment of liver diseases. Thus, studies have evaluated Ad-MSCs and bone marrowederived MSCs (BM-MSCs) seeded in regenerated silk fibroin (RSF) scaffolds in a CCl4-induced live injury mouse model. The promising results of this study showed that neovascularization, a bile canaliculus-like structure, and some hepatocyte-like cells were observed after transplantation of RSF MSCs (Xu et al., 2017). In addition, in a phase II trial that evaluated the effects of BM-MSCs transplanted into cirrhotic patients, the patients exhibited partial improvement of liver function 3 and 6 months after surgery (El-Ansary et al., 2012). Interestingly, when 30e50 million MSCs were injected into eight patients with end-stage liver disease, serum albumin, bilirubin, and live function score were all improved (Kharaziha et al., 2009). Similarly, injection of BM-MSCs was sufficient to lead to histological improvement in 54.5% of alcoholic cirrhosis patients, and the levels of transforming growth factor-b1, type I collagen, and a-smooth muscle actin were decreased in these patients (Jang et al., 2014). All these data suggest that MSC transplantation can be used as a potential treatment for liver injury. Sound progress has been achieved in many soft tissues that exist in the eye, including those in the cornea and retina. The cornea is the transparent eye front that covers the pupil and iris, and it functions together with the lens to focus

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light on the retina. Corneal epithelial stem cells are placed throughout the entire circumference of the corneoscleral limbus. They have a crucial role in the maintenance of the corneal surface epithelium. Cultivation of corneal limbal cells as limbal stem cell grafts has invited exceptional interest, with their stem cellelike properties considered to rejuvenate and reestablish the ocular surface. A deficiency or lack of corneal epithelium renewal due to limbal epithelial stem cell (LESC) depletion or dysfunction results in the so-called limbal stem cell deficiency (LSCD), which is a difficult and complex disorder to manage when it is complete and severe. The aim of many studies in this area is both to promote cellular expansion and to find variant sources of stem cells. In patients with unilateral LSCD, the contralateral healthy eye could be used as a source to harvest stem cells. Patients with bilateral disease could be treated using allogeneic tissue expanded ex vivo, requiring systemic immunosuppressive therapy. In addition, therapies with stem cells have been considered as a promising therapeutic approach for the degenerative retinal diseases that are among the leading causes of irreparable vision loss (Mead et al., 2015; Sivan et al., 2016; Bobba et al., 2018). MSCs from the bone marrow or adipose tissues, iPSCs, and embryonic stem cells (ESCs) have been tested for the treatment of retinal diseases (Whiting et al., 2015). Indeed, the application of healthy stem cells in the place of degenerated retinal cells promoted the formation of new intercellular connections, cell regeneration, and progression of various visual functions (Whiting et al., 2015). In addition, stem cells are very compatible with retina and can adapt to amacrine, bipolar, horizontal, Mu¨ller, and glial cells as well as photoreceptors (Tucker et al., 2014). MSCs can differentiate to various cell types of the retina. When MSCs were injected into the vitreous chamber in a laser-induced retinal damage model, the utmost of the MSCs had shifted to the ganglion cell layer and both inner and outer nuclear layers (Castanheira et al., 2008). Interestingly, the injected MSCs expressed the markers of photoreceptors, bipolar cells, amacrine cells, and Mu¨ller glial cells, without tumor formation (Castanheira et al., 2008). Moreover, MSCs can differentiate into retinal pigmented epitheliumelike cells with identical morphological features that can replace the damaged cells (Huang et al., 2012). Remarkably, MSCs can survive for almost 3 months in rat vitreous and for 6 months in other retinal tissues and, therefore, MSCs are thought of as an upcoming therapeutic choice for degenerative retinal diseases (Haddad-Mashadrizeh et al., 2013). Furthermore, rat MSCs can activate Mu¨ller cell differentiation and exert a paracrine effect by secreting growth factors in vitro (Konno et al., 2013). Interestingly, the injection of MSCs does not lead to intraocular inflammation, proliferation, or deterioration on electroretinogram (Park et al., 2014). Indeed, the subretinal application of MSCs can reconstruct the disintegrated retina in a rat model of retinal degeneration, and the secreted elements from hMSCs can prevent lightinduced retinal damage (Jian et al., 2015).

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Clinically, MSCs are used for both allogeneic and autologous transplantations. Notably, no constitutional or operative toxicity was detected in the retina in 10 months of follow-up after a single dose of intravitreal analogous BM-MSCs in patients with retinitis pigmentosa or patients with coneerod dystrophy (Siqueira et al., 2011, 2015). Similarly, no indicative basic or functional toxicity in the retina was reported after applying MSCs in the vitreous of degenerative retinal disease patients (Siqueira et al., 2015). Interestingly, a significant improvement in the vision of patients was correlated with quality of life at 3 months. However, the scores had returned back to basic levels at 12 months (Park et al., 2014). The stem cell therapy for different retinal disease types has intriguing potential, but this stem cellebased therapy is currently in its early stages and, therefore, needs more research. There are many studies in progress as of this writing, and the results are highly anticipated, with encouraging and promising reported improvements in visual function. Furthermore, therapeutic approaches to eye diseases using other stem cell types, such as human ESCs and iPSCs, that are specific to patients could be beneficial and efficient and might be promising for conditions currently considered incurable. However, the most important challenges for these therapeutic approaches are precisely diminishing the possibilities of tumorigenicity and targeting differentiation. In addition, it should always be remembered that sight-threatening vitreoretinal complications might develop after intravitreal and subretinal applications. Moreover, the chronic and complex disease process is a major limitation of applying stem cell therapies in such patients. More research with expanded follow-up durations is, therefore, still needed. Before introducing stem cellebased therapy into clinical practice for eye repair and regeneration, certain critical issues should be fixed, including the high cost and the reproducibility of differentiation in different ESC or iPSC clones, as well as the hazards of mutagenesis and tumorigenesis. Nevertheless, it is highly expected that research on stem cellebased therapy will continue to flourish with a promising future for new biological factors implemented to treat vision loss. Indeed, limbal stem cell transplantation therapy can recover both vision and quality of life in many patients who suffer from ocular surface disorders associated with LSCD, and overall, the usage of analogous tissue gives the best consequences (Mead et al., 2015; Sivan et al., 2016; Bobba et al., 2018). However, the expansion of autologous stem cell therapy in corneal diseases associated with LESC damage still requires the optimization of LESC cultures and/or standardization of conducted limbal differentiation of iPSCs. In addition, the therapeutic use of various MSCs may be beneficial due to their autologous nature and capability to reconstruct corneas. However, potential problems that are related to cell support standardization and graft longevity demand further exploration. Clinically, the rejection of limbal allografts is the most critical concern in limbal transplants, and improvement of immunosuppressive regimens should be regularly reviewed.

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Stem cellebased therapies are also important for soft connective tissues such as the cartilages. MSC therapy can be used in cartilage repair and regeneration, as well as in osteoarthritis (OA), which is a common joint disorder caused by cartilage inflammation and breakdown, which may eventually lead to the loss of cartilage in the joints. The articular cartilage damage as seen in OA could be treated in modern medical conditions. Indeed, chondrogenic stem/progenitor cellebased therapy was reported for cartilage regeneration in OA almost a decade ago (Koelling and Miosge, 2009). In addition, recent studies using animal models show that TNF-a inducing LRG1 secretion can promote angiogenesis that is associated with both aberrant bone formation and MSC migration and, therefore, TNF-a could be a potential therapeutic approach for OA treatment (Wang et al., 2017). Notably, MSCs that express recombinant Atsttrin, a novel TNFa blocker, play a chondroprotective role, ameliorating OA development (Xia et al., 2015). In addition, hMSCs injected intraarticularly are activated to express high levels of Hedgehog and trigger type II collagen to enhance rat meniscal regeneration (Horie et al., 2012). Indeed, other studies demonstrated that delayed fracture union can be prevented by MSCs mixed with platelet-rich plasma (Liebergall et al., 2013). Another study has presented a phase I/II study of 12 patients with clinical and objective follow-up coverage for 1 year after intraarticular MSC injection (Orozco et al., 2013). In this study, MSC administration appeared to be safe and the results showed that 65%e78% of patients exhibited rapid and progressive improvement, and most of them emerged with improvement of cartilage quality (Orozco et al., 2013). Interestingly, 2-year follow-up results showed that the quality of cartilage had further improved (Orozco et al., 2014). Moreover, 71% of patients with degenerative disc disease transplanted with MSCs exhibited rapid improvement of pain and disability, but without disc height recovery (Orozco et al., 2011). How to prolong their intraarticular longevity became a focus of attention. Furthermore, MSC pretreatment with inflammatory factors or hypoxia does not influence migration or adhesion to osteoarthritic cartilage and synovium (Leijs et al., 2017). Despite all this progress, alternative approaches should be developed to improve the therapeutic effect. Synovial fluid MSCs (SF MSCs) exist in the synovial fluid of normal human joints and OA patients, and have been proposed to exhibit similar MSC characteristics. SF MSCs were reported to be involved in the hemostasis and reparation of articular cartilage surface. Compared with the increased number of studies using BM-MSCs and iPSCs for OA treatments, not many studies have been carried out on the application of SF MSCs in OA therapy. Early studies by Jay and colleagues reported that human synovial fibroblasts exhibited the same protein, lubricin, as cartilage chondrocytes (Jay et al., 2000, 2001). Lubricin is mainly presented on the surface of the articular cartilage and therefore plays a major role in joint lubrication, protecting joints from load-bearing-induced injuries. Similarly, other studies have identified SF

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MSCs in normal human joints and OA patients (Alsalameh et al., 2004). These SF MSCs exhibited multilineage differentiation potential, and were considerably more clonogenic and less adipogenic compared with BM-MSCs, suggesting that SF MSCs may be more suitable for the repair of articular cartilage (Alsalameh et al., 2004). Interestingly, SF MSCs in early OA increased by sevenfold, suggesting that SF MSCs could be used as a potential treatment for early stage OA (Alsalameh et al., 2004). In addition, other studies show that cells residing on the surface of the articular cartilage exhibit gene expression similar to that of stem cells from synovial fluid (Caldwell and Wang, 2015). Interestingly, in studies that used the knees of pigs that suffered medial meniscal resections as a model, SF MSCs were injected into the knees and led to a significant improvement in the cartilage repair and regeneration, with new synovial tissue filling the area of meniscal resection within 2 weeks (Hatsushika et al., 2014). The general consensus is that SF MSCs could promote the articular cartilage repair process, mainly through their differentiation into chondrocytes and their high expression of type II collagen. Since SF MSCs were identified to be extremely chondrogenic, they can migrate to the cartilage surface to repair the damage. In addition, SF MSCs may help in recruiting MSCs from the bone marrow of the subchondral bone. Some studies on the synovial fluid from healthy donors and OA patients reported similar potential to stimulate MSC migration (Endres et al., 2007), further suggesting the ability of SF MSCs to recruit BM-MSCs for tissue repair. In addition, OA is known to be associated with inflammation. Inflammation was previously proposed to be present in rheumatoid arthritis patients only. However, more recent studies are being shifted toward inflammation in OA, with multiple research groups attempting to find an effective approach to treat OA and repair damaged articular cartilage. Due to their antiinflammatory ability, both MSCs and SF MSCs have gained a continuously increasing interest as a possible therapeutic option for OA (Ozeki et al., 2016). In sum, stem and progenitor cells residing in the cartilage’s superficial zone and the synovial fluid have a promising potential for cartilage repair and regeneration, since both can be easily acquired and applied in cell therapy. With their characteristic antiinflammatory effect and better chondrogenic potential compared with other MSCs, including BM-MSCs, SF MSCs are projected for OA treatment and articular cartilage repair and regeneration. With the pace of advancement on stem cells and regenerative medicine, OA could be cured, along with the improvement of the quality of life. MSC therapy has shown promising results in the hard tissues, including bone and tooth. As of this writing, autologous bone grafting is the standard treatment for the repair of bone fracture. However, there are several drawbacks of this grafting method, including donor-site morbidity (Goulet et al., 1997), the limited availability of autologous bone, and the loss of bone stock, which make scientists turn to other approaches (Rosset et al., 2014). Cell therapy is

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considered as an alternative to autologous bone grafting. To promote bone repair, bone progenitor cells are cultured, alone or on a biomaterial, and supplied to the injury site, combined with a mineral or protein matrix and/or cytokines (Ono and Kronenberg, 2016). MSCs can differentiate into many other cell types, including osteoblasts, chondrocytes, and adipocytes, in vitro (Dominici et al., 2006). In physical bone regeneration, MSCs may play a key role in the mesenchymal and angiogenic activation phase after the early inflammatory phase (Qin et al., 2014). In addition, MSCs characteristically play a key role in bone tissue engineering, since they are reliable multipotent cells for clinical applications (Rosset et al., 2014). Moreover, in physical healing and cell therapy, MSCs can migrate to injury sites and assist in the repair program. However, the underlying mechanisms of these important biological processes are still largely unclear and, therefore, need more research. It is well reported that chemokine receptors with their ligands and adhesion molecules contribute to the tissue-specific homing of leukocytes and transport of hematopoietic precursors into and through tissues. It is very likely that similar mechanisms govern the migration of MSCs. Future research should, therefore, focus on identifying the detailed molecular mechanisms that govern MSC contributions to bone repair and regeneration and bone tissue engineering. In addition, the determination of more internal and external factors affecting MSC homing will definitely make better use of MSCs in bone fractures and improve bone repair and regeneration in the future. Stem cell therapy also has great potential for tendon repair and regeneration. The treatment of tendon injuries has many clinical challenges. The currently available treatment approaches often result in partial healing or repair of the injured tendon, leading to a reduced tendon function. Stem cell therapy is a highly promising intervention for the repair and regeneration of injured or diseased tendon. Another important achievement was the identification of a specific tendon stem/progenitor cell population (TSPCs) by Bi et al. (2007). Since this discovery, researchers in the tendon repair and regeneration field have made great progress in the study of TSPCs. However, there are some difficulties in the study and application of TSPCs. For example, isolated TSPCs show some levels of heterogeneity that are not completely in conformity with the definition of stem cells, indicating that there is an ingredient of progenitor cells in the newly identified cells. Like other stem cells, the behavior of TSPCs is closely connected with the dynamic changes of the cell niche. Thus, to comply with external signal fluctuation, TSPCs could adjust themselves and make corresponding physiological or pathological function changes (Lui, 2015; Liu et al., 2017). Many research laboratories worldwide have characterized TSPCs, including cell isolation and characteristics, and investigated the interactions between TSPCs and the microenvironment, the regulatory mechanisms of TSPC fate, the behavior of TSPCs in tendon disease, and the potential

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applications of TSPCs in clinics (Zhang et al., 2016; Walia and Huang, 2018). However, there are still several challenges to the study and clinical applications of TSPCs. One of these challenges is the need for further and better understanding of the fate of TSPCs during development. In view of that, key transcription factors that control stem cell differentiation have been clearly investigated in other tissue types, such as muscle, bone, and the hematopoietic system, profiting from the current high-throughput technologies. We can, therefore, go a step farther and faster on this road by identifying the critical master genes that work coordinately in the TSPC differentiation process. In addition, a better understanding of the physiological processes involved in the development of TSPCs is necessary to help in our comprehension of the pathology process, and ultimately be beneficial for the treatment of tendonrelated diseases. Another TSPC challenge is their heterogeneity. Like tumor cells, stem cells, including TSPCs, show some levels of heterogeneity, and our previous knowledge about TSPCs was based mostly on the average levels of cell groups. Single-cell analysis is an emerging technology that can be used to avoid this kind of bias error in TSPCs perfectly and analyze gene expression profiles of individual cells with the advantage of high throughput (Guo et al., 2013; Huang et al., 2015). Combining with this the single-cell analysis method, it is possible to uncover the mystery of different gene expression profiles between individual TSPCs, discover subpopulations of cells that have never been detected, and reveal novel signaling regulatory pathways of TSPCs. The interaction between TSPCs and the cell microenvironment still also needs further study. Studies suggest that TSPCs differentiate into the wrong lineage(s) in the pathological microenvironment, but it is still unclear which factors are involved in the pathogenesis of this pathology. In addition, understanding the role of TSPCs in the pathogenesis of tendon disease contributes to our progress in the treatment of disease. Afterward, we need to target those specific signals that regulate the fate of TSPCs. There are many approaches that have been validated, including growth factors, mechanical stimulation, and drugs. Small-molecule drugs, for example, are receiving increasing attention because of their advantages of stability, safety, and high performance. Through high-throughput drug screening, we can acquire effective regulatory methods to maintain the TSPC phenotype and tissue homeostasis in the future. The application of TSPC transplantation in the therapy of tendon diseases is also still a challenge and, therefore, needs more research. TSPC in vitro culture cannot maintain the phenotype. It is, therefore, essential to establish a safe and effective in vitro culture and expansion system. In addition, one view is that the intervening with tendon-endogenous TSPCs may be more reasonable for regenerating tissues, considering the existence of some inherent defects in the cell transplantation (Lee et al., 2015).

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Research has highlighted some of the key issues related to the antiinflammatory effects of MSCs in different pathological conditions, and the potential application of MSCs as an immune-modulatory agent. The major tasks of the immune system include the recognition of foreign antigens and maintenance of self-tolerance. Inadequate and uncontrolled immune response is known to induce tissue damage, which is observed in pathological conditions such as rheumatoid arthritis, atherosclerosis, Crohn disease, and multiple sclerosis. The common strategy to reduce inflammation often includes the administration of antiinflammatory drugs, which show a reduced effect and individual tolerance. Therefore, various approaches for reducing inflammation are needed. Stem cells have an ability to mediate tissue repair and regeneration in many types of tissue injuries. The effect of stem cells in tissue repair and regeneration is largely known to be mediated not only by replacing the damaged cells but at least partially by the cessation of inflammatory processes in the local tissue environment. Stem cells can secrete some critical antiinflammatory products such as TSG6, IL-1ra, NO, IDO, etc., which are able to reduce inflammation and even convert the inflammatory environment toward a reparatory one. Graft-versus-host disease (GVHD) represents one of the most targeted diseases for which the immunomodulatory function of MSCs is utilized. Several clinical trials show the positive effect of MSC infusions in patients with steroid-resistant GVHD. Thus, patients show a response to MSC treatment or have improvement in their conditions, after a few infusions of haploidentical MSCs (Le Blanc et al., 2003, 2004, 2008; Chen et al., 2015). The prospective effect of MSC infusions in cases of acute GVHD was also reported (Kebriaei et al., 2009; Chen et al., 2015). The immunomodulatory effects of MSCs are also tested in other diseases such as Crohn disease and multiple sclerosis. Crohn disease causes inflammation of the digestive system’s lining (Molendijk et al., 2012; Gao et al., 2016). Indeed, MSC infusions showed a promising and partial response without severe adverse effects in patients suffering from Crohn disease, with a decreased disease index in patients (Duijvestein et al., 2010; Forbes et al., 2014; Forbes, 2017). In multiple sclerosis, a chronic immune-mediated and neurodegenerative disease, MSCs are a key therapeutic tool that has the potential use as an immunosuppressive mediator for treating tissue injuries such as central nervous system degenerative and inflammatory injuries, particularly in animal models (Darlington et al., 2011; Dulamea, 2015; Gao et al., 2016). Other studies have demonstrated improvements in multiple sclerosis patients receiving treatments with MSCs (Yamout et al., 2010; Bonab et al., 2012; Llufriu et al., 2014; Harris et al., 2018). Similarly, in diabetes patients, MSC treatments can lead to a significant reduction in the insulin daily dose and in the C-reactive peptide level of type 2 diabetes patients (Jiang et al., 2011). At this writing, there are limited clinical studies on the therapeutic potential of MSCs in the treatment of systemic lupus erythematous, but the

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available studies show promising effects of MSC infusion and no acute adverse effects (Sun et al. 2009, 2010; Liang et al., 2010; Wang et al., 2018). Despite many preclinical and clinical trials showing MSC efficacy in the treatment of inflammatory disorders, the results are sometimes controversial and clinical benefits vary between trials. Differences in experimental design, cell dosages, evaluation of clinical benefits, and cell isolation and cell propagation protocols might influence the results, but overall, it is evident that MSCs may serve as a potent immunomodulatory treatment. Furthermore, the appropriate manufacturing practice protocols and the consensus concerning the definition of MSCs are critical steps in using MSCs as a therapeutic agent for inflammatory disorders. To test MSC efficacy in these disorders, the treatment protocol and ways of cell isolation and expansion of MSCs require high standardization to minimize the off-target effects and safety risks. Another important issue is the long-term safety of using MSCs as a major therapeutic tool for inflammatory disorders. Remarkably, MSCs can act as endogenous regulators of tissue inflammation in culture, but whether this translates to clinical efficacy still needs further investigations. MSCs also play important roles in the therapy of other diseases that are functionally related to inflammation, such as cancer. Indeed, MSCs have elicited a great hope for the treatment of cancer in humans. The characteristic homing abilities make hMSCs a great hope for the treatment of different tumors when administered to the tumor site(s). Importantly, the use of the patient’s MSCs is preferred to avoid the rejection risk of exogenously administered MSCs. MSCs have several other features, including high levels of amphotropic receptors, which can facilitate their transduction using an integrating vector. Moreover, the characteristic hMSC migration and homing abilities and their microvesicle/exosome-mediated regeneration are critical features for treating cancer and other human diseases. Indeed, clinical studies show promising results when using MSC therapy for different human diseases, including cancer. Interestingly, MSC delivery that is mediated by adenoviruses, retroviruses, or lentiviruses was shown to successfully reduce tumor growth and other complications in animal models, suggesting a key role for MSCs in cell-based therapies for cancer. The applications of viral and gene-based therapies for cancer, and a combination of both gene therapy and hMSC therapy, is important for cancer therapy, and progress has been achieved in these applications in recent years. Thus, MSCs have been successfully engineered for cancer therapy with several anticancer genes, and these engineered cells demonstrated anticancer effects in well-established carcinoma models. However, these therapeutic approaches are still risky, since the application of genetically modified cells in cancer therapies can lead to cancer development. Therefore, the application of an MSC-based targeted treatment approach with suicide vehicles in cancer therapy is recommended if it does not lead to the development of a secondary tumor. Despite recent progress, more studies and clinical trials are still needed

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to explore the safe use of MSCs, in general, and genetically modified MSCs in particular, in cancer therapy alone or in combination with conventional cancer therapeutic approaches. Nevertheless, the combination of gene and stem cell therapies, including the use of hMSCs with a suicide gene, is particularly the effective strategy forward. Special attention has been paid to stem cellebased therapies for treatment of neurodegenerative disorders, as well as traumatic events like traumatic brain injury or spinal cord injury since 2000. The nervous system is more susceptible to different types of injury that range from trauma to chronic diseases that cause progressive deterioration. In addition, there is a limited capacity for tissue repair in the central nervous system. A spontaneous neural repair indeed occurs in patients; however, it is not robust enough to promote functional and stable recovery of the nervous system architecture, demanding external intervention. The central nervous system has a characteristic sensitivity to injury and a unique plasticity compared with other organ systems. Intensive studies have been performed to explore both stem cellebased therapies and neural tissue engineering for repair strategies for the central nervous system in different disease contexts. Earlier research studies have primarily attributed the effects behind the success of stem cellebased therapy for the central nervous system and other organ systems to the stem cell differentiation capacity that enables the replacement of damaged tissues. However, more recent studies have paid much attention to the function of the paracrine mechanisms of stem cells in tissue repair and regeneration of the nervous system and other body systems. Remarkably, stem cells can secrete a number of potent combinations of biologically active (bioactive) molecules such as the cytokines, growth factors, lipids, and extracellular vesicles that in turn play key roles in the modulation of many physiological processes. Interestingly, in many instances, the functional benefits of stem cells are due in large part to their secreted bioactive components, including the cytokines, growth factors, and vesicles that are generally referred to as the secretome (Ranganath et al., 2012; Vizoso et al., 2017; Ferreira et al., 2018). The application of the stem cell secretome as a therapeutic option for some disorders of the central nervous system, including namely traumatic brain injury and spinal cord injury, stroke, and Parkinson disease, has attracted the attention of many researchers (Drago et al., 2013; Teixeira et al., 2013; Martins et al., 2017; Vizoso et al., 2017; Ferreira et al., 2018). However, beyond the great enthusiasm for new treatment perspectives using the stem cell secretome, much investigative work is still in progress on the development of robust and customized stem cell secretomeebased therapies for repairing central nervous system injuries. While moving this potential from the “bench to the bedside” is important for human health, it still requires further research studies on several related factors. First, it is necessary to select the source of

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the richest secretome, and pay much attention to possible autologous versus allogeneic donor variability. The second factor is modulation of the secretome and/or extracellular vesicle composition by changing the culture conditions or the overexpression of specific molecules. The last factor is the selection of the regimen (route and dosage) of treatment administration, which can suit each patient’s condition. In addition, although candidate molecules are under investigation, more intensive and detailed research studies are still required to clearly identify and characterize the responsible factors for the stem cell secretomeemediated neuroprotective and regenerative properties. Identifying and understanding the molecular and cellular mechanisms behind these beneficial effects, including the elucidation of activation or inhibition of molecular pathways and their temporal effects, are also important and required. Therefore, rigorous preclinical studies and innovative clinical trials should be designed to better understand the mechanisms of central nervous system diseases and the role of the stem cell secretome that will eventually lead to the development of safe, effective, and controlled therapeutic strategies or approaches based on the use of the stem cell secretome. Despite rapid progress in their research and applications, there are still some controversies and challenges facing the use of MSCs. A controversial basic question is the definition of these cells: what is an MSC? Mesenchymal stromal cells and mesenchymal stem cells are always confused, since there is no clear definition to distinguish between the two terminologies. These terminologies are used to refer to the same cell culture, and were defined in 2006 by the International Society for Cellular Therapy (ISCT; Dominici et al., 2006). According to the ISCT guide, CD73, CD90, and CD105 can be used to identify whether the cells grown in culture are MSCs. CD (cluster of differentiation) antigens are most commonly used as markers for identifying undifferentiated MSCs and excluding other cell types, such as endothelial cells, neural cells, and hematopoietic stem cells. However, there is a limitation when using this conventional panel of CD markers for characterizing MSCs, particularly when large-scale amplification is needed, since they have a limited prediction accuracy for MSC functions. For example, replicative senescence accumulates during MSC expansions (Yang et al., 2017), but the CD markers (CD73, CD90, and CD105) are rarely affected (Kundrotas et al., 2016) and, therefore, CDs are inaccurate markers to predict MSC functions. In addition, many laboratories use different markers to identify MSC subpopulations, including CD146, nestin, and PDGFR-a, but without defining the hierarchical relationship between these cell subpopulations, which is well defined in the hematopoietic system, for example. Other controversial issues regarding MSCs are their existence in different tissues and in in situ form. MSCs exist in almost every tissue of the body. However, to what extent MSCs from different tissues differ is still not clear. The in situ form of MSCs also provokes controversy over these cells. For example, MSCs were first proposed to adopt a perivascular localization, since

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isolated pericytes that are grown in culture show multipotency and express MSC markers (Crisan et al., 2008). This was supported by other studies that concluded that all MSCs are pericytes (Caplan, 2008, 2017). However, lineage tracing studies found that pericytes do not contribute to tissue regeneration or wound healing during either aging or injury (Guimara˜es-Camboa et al., 2017). Therefore, it is still difficult to make a conclusion about the definite relationship between MSCs and pericytes. One possibility is that pericytes partially develop into MSC-like cells during their growth in culture. However, even if these MSC-like cells are derived from pericytes, they are still different in culture.

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