Comparing the Therapeutic Potential of Stem Cells and their Secretory Products in Regenerative Medicine.

1. Introduction

Cellular therapy, also known as “cell-based therapy,” involves the transplantation of human cells to stimulate the regeneration of damaged tissues and modulate the mechanisms underlying disease initiation and progression. Multiple types of human cells, including stem cells and progenitor cells, have been used to treat different diseases. Stem cell therapies using induced pluripotent stem cells (iPSCs) [ 1 ] , embryonic stem cells (ESCs) [ 2 ], and adult stem cells such as mesenchymal stem cells (MSCs) [ 3 ] have been tested preclinically and clinically for years. Nowadays, MSC is widely used in the field of tissue engineering and regenerative medicine. In general, stem cell therapy has grown to become an attractive option to reduce the overall need for tissue transplantation and minimize the waiting time for patients [ 4 ]. Numerous clinical studies have indicated that stem cell administration is a safe and promising therapeutic approach. The transplanted cells can differentiate to restore the structure and function of injured tissues [ 5 , 6 ]. However, more and more evidence suggested that the transplanted cells promote tissue regeneration mainly through paracrine secretion.

Recent studies have shown that the transplanted cells secrete paracrine factors that directed the proliferation and differentiation of surrounding cells as well as produce chemoattractants that attracted the migration of effector cells to the injured sites. The “cell-free therapy” that utilizes the therapeutic molecules, i.e., secretome, secreted by stem cells has become more popular as it offers many advantages and avoids many limitations bothering the cell-based therapy. The composition of secretome is very dynamic, depending on the cell type and stimulus from their surrounding microenvironment [ 7 ]. Generally, stem cell-secreted secretome comprises (i) a complex mixture of soluble components such as growth factor and cytokines (obtained as the conditioned medium), (ii) a vesicular portion composed of extracellular vehicles (EVs), and (iii) cell organelles (e.g., mitochondria). It has been suggested that secretome can promote cell-cell communication, interact with other cells in their immediate environment, and transfer functional biomolecules to initiate tissue repair or regeneration. Generally, secretome has been found to possess proangiogenic, antiapoptotic, antifibrotic, anti-inflammatory, immunomodulatory, and proproliferative properties [ 8 – 10 ]. Nevertheless, extensive investigations are still required to better understand the therapeutic mechanism of secretome transplantation, its safety issues, and the clinical efficacy, mainly through clinical trials. In this review, the focus is on the values of stem cells and secretome in regenerative medicine, as well as discussing the latest insights on the safety, efficacy, advantages, and disadvantages of using these two modes of treatment.

2. Classification of Cell-Based Therapy

2.1. Stem Cell Therapy

Stem cell therapy can be categorized into autologous and allogeneic based on the tissue donor. To date, autologous stem cell transplantation has been performed for a broad range of purposes, such as to promote cardiac and cartilage regeneration, expedite wound healing, and improve aesthetic appearance. Autologous stem cells are used as they are readily available from many tissue sources and have a lower risk of life-threatening complications such as graft-versus-host disease (GVHD), free of ethical issues, and nonimmunogenic. The adverse events reported in the transplantation of stem cells are most likely unrelated to the treatment but to the underlying disease instead [ 11 ].

Allogeneic stem cell transplantation is gaining more attention in the past decade due to its advantages, such as reduction of functional variability through the pooling of cell products from multiple donors in a master bank, and it is readily available off-the-shelf for clinical applications. Allogeneic bone marrow-derived MSCs (BMSCs) mixed with autologous chondrocytes have been transplanted into the knee joint of patients with symptomatic cartilage defect, and the results showed the regeneration of hyaline cartilage with a high concentration of proteoglycans and type II collagen at 12 months [ 12 ]. A clinical trial on end-stage liver cirrhosis also revealed that allogeneic stem cell transplantation positively affects the patients' condition by improving the serum albumin levels and model for end-stage liver disease (MELD) scores after six months [ 13 ]. Paul et al. reported the immunomodulatory benefit of allogeneic MSC infusion by reducing the rejection of transplanted corneal during the immediate posttransplant period [ 14 ].

2.2. Stem Cell Derivatives/Secretome

In the field of regenerative medicine, the therapeutic effects of stem cells are not constricted to cell-cell interactions. A broad range of bioactive molecules is found in stem cell secretion, including growth factors, cytokines, chemokines, enzymes, extracellular matrix (ECM), and extracellular vesicles (EVs), collectively known as the secretome [ 15 ]. The secretome is a crucial component in exhibiting the therapeutic effect of stem cells ( Figure 1 ).

3. Stem Cells

3.1. Source of Stem Cells

The collection of pluripotent stem cells (PSCs) such as ESCs is ethically controversial as it involves the destruction of possible human life. Furthermore, ESC is also considered an allogeneic source of cells which may cause immune incompatibility. However, there is an immediate solution to avoid ethical repercussions; the adult somatic cells can be reprogrammed into iPSCs which essentially functionally behaved as ESCs [ 16 ]. Genetically modified PSC is utilized in disease modeling to overcome the species-specific differences as observed in an animal model. It also serves as a potential cure to a currently permanent condition such as thyroid disease [ 17 – 19 ], cardiovascular disease [ 20 ], macular degeneration [ 21 , 22 ], or Parkinson's disease that only can be managed with lifelong medications. Unfortunately, PSC has raised safety concerns as some research reported tumorigenicity [ 23 ] or epigenetic aberrations posttreatment [ 24 – 26 ]. A fail-safe suicide gene known as inducible caspase-9 (iCasp9) has been tested extensively in vitro and in vivo as a potentially viable solution to remove the residual pluripotent cell that may cause teratoma formation [ 27 , 28 ].

On the other hand, multipotent cells have a narrower spectrum of differentiation than pluripotent stem cells and can differentiate into discrete cells of specific lineages. Examples of multipotent cells are hematopoietic stem cells (HSCs) and MSCs. HSCs can be isolated from peripheral blood, bone marrow, and umbilical cord blood, whereas MSCs can be found in bone marrow, umbilical cord, cord blood, placental, peripheral blood, adipose tissue, dental tissues, skin, salivary gland, and synovial fluid [ 29 – 32 ]. Although there are variations in molecular composition, surface antigen expression, differentiation capacity, and immunomodulatory property in MSCs isolated from different tissue sources [ 23 , 33 ], however, functional analyses showed that all the secretome have similar functionality, i.e., to promote cell migration and inhibit cell apoptosis [ 34 ].

3.2. Mechanism of Therapeutics

MSCs were thought to promote tissue regeneration via transdifferentiation to replace the damaged cells and cell fusion to save the dying cells. However, many studies have found that these mechanisms are insufficient, and MSCs seem to secrete a myriad of paracrine factors, e.g., growth factors, chemokines, and cytokines, to promote tissue regeneration and modulate the immune response. This notion is supported by the low engraftment of the transplanted cells at the target site and rapid loss of the transplanted cells in vivo. The mechanisms employed by MSCs in tissue repair and immunomodulation have been excellently reviewed in previous publications [ 35 – 39 ]. In this section, we will only provide a glimpse at their mechanism of action in a short summary. In brief, MSCs secrete anti-inflammation, antiapoptosis, antioxidative, antifibrosis, proangiogenesis, promitosis, and chemotactic factors to stimulate tissue regeneration ( Figure 2 ).

3.3. Multifactorial Crosstalk

3.3.1. Direct Signaling

Cell-cell signaling by direct contact allows stem cells to communicate and respond to other cells. It is not always necessary as stem cells have other soluble-dependent crosstalk as well. In a mixed immune cell culture such as peripheral blood mononuclear cells (PBMCs), cell contact is not required for MSCs to exert their anti-inflammatory effect. Contrastingly, when MSCs were introduced to a lymphocyte-only culture and cell-cell contact was prohibited, MSCs failed to induce FoxP3 and CD25 expressions in CD4 + T cells [ 40 ]. The adhesion molecules ICAM-1 and VCAM-1 will not form in the lack of direct MSC-lymphocyte contact [ 41 ]. Moreover, MSCs require direct contact with immune cells to upregulate cell-surface proteins such as programmed death-ligand 1 (PD-L1) and Fas ligand to suppress inflammation [ 42 , 43 ]. As a result, the immunoregulatory properties of MSC will not be exerted to their full potential. The modulation of dendritic cell maturation by MSCs also requires both direct cellular contact and the soluble factor, interleukin (IL)-6. Loibl et al. reported better results when endothelial progenitor cells were cocultured with MSCs as it significantly upregulated the mature endothelial cell marker, PECAM-1, relative to the transwell setup [ 44 ]. The immunosuppression of B cells was more efficient in direct cocultured with MSCs [ 45 ].

3.3.2. Secondary Crosstalk

Paracrine signaling is the main mechanism of MSC therapy. It was initially thought that MSC would migrate and engraft at the site of injury. Nonetheless, most of the MSC administrated intravenously are sequestered in the vasculature of the lungs, with only a few MSCs homed to the tissue of interest. Studies have also noted that exogenous MSCs unable to retain their population long enough to completely replace the affected tissue. Hence, the lasting reparative effect of MSCs is largely attributed to its ability to secrete trophic factors to ameliorate the inflammation in other parts of the body [ 35 , 46 – 48 ]. Chin et al. claimed that the anti-inflammatory cytokine levels remained elevated from baseline up until six months post-MSC transfusion [ 46 ]. MSCs are known to secrete immunosuppressive factors such as transforming growth factor-beta (TGF- β ), vascular endothelial growth factor (VEGF), IL-10, prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and galectin-1 into the circulatory system [ 47 – 51 ]. These molecules interact with the immune cells such as T and B cells to suppress their proliferation and differentiation, causing the polarization of macrophage to an anti-inflammatory phenotype and reduction of the pro-inflammatory milieu consists of cytokines such as tumor necrosis factor-alpha (TNF- α ), interferon-gamma (IFN- γ ), and IL-6 [ 46 , 47 , 49 , 52 – 55 ].

3.3.3. Necrobiology

Necrobiology is a term used to describe the life processes associated with morphological, biochemical, and molecular changes related to cell death and the consequences and tissue response to cell death [ 56 ]. It encompasses four mechanisms by which derivatives of MSCs can retain significant clinical efficacy, including apoptosis, autophagy, mitochondrial transfer, and extracellular vesicle production [ 57 ]. The bioactive parts of dead or dying MSCs can trigger immunomodulatory properties in the host without the concern over cell survival and the formation of large aggregates [ 58 – 60 ].

(1) Apoptosis . Apoptosis of cultured MSCs can be induced via nutrient deprivation. In addition, some studies found that IFN- γ and TNF- α also can trigger MSC apoptosis through the nitric oxide (NO) [ 61 ] and Fas [ 62 ] pathways, respectively. One can consider inhibiting NO to prolong MSC survival, noting that it also will restrict the immunosuppression capacity of MSCs on the lymphocytes. Interestingly, Mancuso et al.'s study of knee osteoarthritis using an in vitro model revealed that apoptotic MSCs were more immunosuppressive than healthy MSCs [ 63 ]. Moreover, Chang et al. found that apoptotic MSCs were more effective in attenuating organ damage in rat sepsis models compared to the healthy MSCs [ 64 ]. Cheung et al. found that monocytes that efferocytosed the apoptotic MSCs have higher expression of IDO, PD-L1, and cyclooxygenase 2 (COX2), and these cells secrete more PGE2 and IL-10 as well as lower TNF- α to subside the inflammation. Then, they proceeded to monitor the serum PGE2 levels of eight patients with severe steroid-resistant GVHD who received MSC therapy. It was found that the responders demonstrated increment in PGE2 levels while the nonresponders showed not changes in PGE2 levels. Albeit, in a small sample size of eight GVHD patients, this study teased the possibility of apoptotic MSCs in translational medicine [ 65 ].

(2) Autophagy . In terms of stem cells, autophagy plays a pivotal role in maintaining genomic stability and retain its potency and differentiation capacity [ 66 ]. Gao et al. discovered that autophagy may regulate MSC immunoregulation through TGF- β 1 signaling. The proliferation of CD4 + T helper cells was inhibited when cocultured with autophagic MSCs. However, when autophagy is inhibited, MSCs failed to suppress the proliferation of T cells [ 67 ]. Additionally, autophagy activation in MSC transplantation has protective effects on the damaged tissues. These protective effects of autophagy can be reversed using autophagy inhibitors such as 3-methyladenine and chloroquine. Autophagy can be induced by hypoxia and nutrient depletion, and it has been shown to protect MSCs in vitro [ 68 ]. Furthermore, Zhang et al. showed that hypoxic preconditioning on MSCs can enhance its functional survival to restore cardiac function in ischemia models [ 69 ]. Using rapamycin to induce the autophagy mechanism, Wang et al. showed that MSC-derived exosomes prevented acute kidney injury caused by cisplatin [ 70 ]. Similarly, Hou et al. induced autophagy by pretreating MSCs with starvation and rapamycin. In their study, it was shown that autophagy prevented the autophagic MSCs from irradiation injury and maintained the stemness after exposure to reactive oxygen species- (ROS-) induced damage [ 71 ]. Park et al. attributed the neuroprotective effects of MSCs to the higher levels of autophagy in a parkinsonian mice model and MPP + treated neuronal cell culture [ 72 ].

(3) Mitochondrial Transfer . MSCs are known to reprogram the host cells by the transfer of mitochondria. It is a process that requires direct cell-cell contact through tunneling nanotubes (TNT) or gap junctions [ 73 ]. Mitochondria also can be transferred via secreted EVs [ 56 ]. The mitochondrial transfer has a prominent role in protecting the recipient cells from oxidative stress, radiation injury, and hypoxic injury as well as recovering the mitochondrial membrane potential and aerobic respiration and modulating the host immune response [ 74 , 75 ]. Upregulation of Miro 1, a mitochondrial Rho-GTPase, has been reported to enhance mitochondrial transfer, subsequently improve the MSC therapeutic efficacy [ 76 ].

(4) Extracellular Vesicles . According to MISEV 2018, EVs are nonreplicating particles of size 100-200 nm and encapsulated by a lipid bilayer [ 77 ]. MSC-derived EVs contain bioactive molecules including genetic materials, microRNAs, enzymes, signaling proteins, immunomodulatory factors, and growth factors [ 78 ]. EVs have the potential to be developed into cell-free therapy with the benefits of MSC immunomodulation but without the concerns of maintaining the cell viability or risk of immune rejection in allogeneic transplantation. Many studies showed that MSC-derived EVs are as effective as MSCs in treating diseases [ 79 – 81 ]. Apoptotic cells are known to produce different types of EVs and apoptotic bodies that can influence the surrounding cells. Apoptotic cell-derived EVs are rich in spliceosomes that alter the RNA splicing in recipient cells [ 82 ]. More data are showing that apoptotic cell-derived EVs play a significant role in immune modulation in autoimmunity, infection, and cancer, implicating that they are not just cell debris [ 83 ]. All these findings indicating that apoptotic cell-derived EVs could be an important medium of communication between the dead and living cells [ 84 ]. Nonetheless, to date, apoptotic cell-derived EVs are not well studies. Thus, what we know is still very limited.

4. Secretome

Secretome is often referred as a group of biologically active molecules or factors that are released by cells into their extracellular environment [ 85 ]. Although MSCs derived from various anatomical sites may exhibit similar morphological and immunophenotypic characteristics, numerous evidence showed that they secrete a distinct set of secretome that is normally associated with the host age and specific microenvironment that the cells were grown. The secretome may even fluctuate in response to various physiological changes and pathological circumstances. In general, MSC secretome is made up of a variety of growth factors, cytokines, and EVs that conferred its tissue repair and regenerative potential, mainly attributed to their capability to stimulate cell proliferation, formation of new blood vessels, and their immunomodulatory effects ( Figure 3 ) [ 85 , 86 ].

4.1. Growth Factor

Different investigations have shown that the growth factors present in MSC secretome may either work synergistically to exert their tissue regenerative potential or the presence of individual growth factors could be sufficient to achieve the desired therapeutic objective. For instance, brain injury such as stroke usually involves brain tissue damage due to a lack of blood supply. Hence, stroke therapy usually requires the promotion of new blood vessel formation and brain cell production, along with suppression of further cell death and inflammatory processes [ 85 ]. These have been successfully achieved via administration of BMSC and adipose tissue-derived MSC (AT-MSC) secretome that contain a mixture of hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), fibroblast growth factors (FGF), and platelet-derived growth factor (PDGF) [ 7 , 87 ]. Meanwhile, another study by Ding et al. has suggested a direct involvement of increased insulin-like growth factor-1 (IGF-1) levels that exhibited neuroprotective effect in a mouse model of brain stroke through regulating its ischemic and inflammatory condition to reduce the volume of brain infarct while improving the function of brain cells [ 88 ]. Similar observations also have been reported in Huntington's disease mouse model whereby transplantation of BMSCs led to elevated expression of stromal cell-derived factor-1 (SDF-1) to improve blood supply to the damaged brain striatum tissue via stimulation of angiogenesis [ 89 ]. The neuroprotective role of SDF-1 had also been verified in another rat model of Parkinson's disease whereby the grafted BMSCs inhibited apoptotic activities in the affected dopaminergic neuronal tissue, which significantly recovered the behavior of the diseased rats [ 90 ]. Besides brain injuries, therapeutic effects of growth factors present in MSCs have also been investigated for other pathologic conditions such as cutaneous injury, whereby the use of AT-MSC secretome that contains VEGF, HGF, transforming growth factor β (TGF- β ), and keratinocyte growth factor (KGF) was able to induce greater cellular proliferation, trigger cell migration, and decrease the wound size at a faster rate [ 91 – 95 ]. The positive impact of these diverse growth factors in promoting angiogenesis [ 96 ], regenerating muscle tissue [ 97 ], and reducing incidences of premature infant diseases such as periventricular leukomalacia, retinopathy of prematurity, bronchopulmonary dysplasia, and necrotizing enterocolitis [ 98 ] also has been implicated.

4.2. Cytokines

Whilst growth factors are more frequently associated with induction of cellular proliferation or prevention of cell death for tissue regeneration, cytokines present in the MSC secretome play a more important role in regulating inflammatory activities in pathologic conditions to attain the therapeutic effect. In the MSC secretome, both anti-inflammatory cytokines (such as tumor necrosis factor β 1 (TNF- β 1), IL-10, IL-12 p70, IL-13, IL-18 binding protein, IL-25, and IL-27) and proinflammatory cytokines (such as TNF- α , interferon γ (IFN- γ ), IL-1b, IL-6, IL-8, and IL-9) could be present. The impact of MSC secretome on the inflammatory process is usually governed by the balance of these anti- and proinflammatory cytokines [ 86 ]. For example, the destruction of pancreatic cells in autoimmune diabetes mellitus type 1 disease by proinflammatory cytokines, including TNF- α , IFN- γ , and IL-1b, could be reversed by treating the primary islet cells with MSC secretome containing significantly elevated levels of anti-inflammatory cytokines (IL-4 and IL-10), resulted in the prevention of cell apoptosis and improvement in insulin secretion [ 99 ]. The importance of anti-inflammatory cytokines produced by MSCs was further demonstrated by Hsu et al. who utilized MSCs to suppress inflammation-associated transplant arteriosclerosis through the secretion of IL-10 [ 100 ]. In another study by Ogata et al., MSC secretome was shown to stimulate bone healing in a rat bone defect model by increasing the migration of endogenous stem cells into the defect area. Subsequent analysis revealed the presence of various important cytokines in the MSC secretome which are essential to suppress inflammation as well as induce cell proliferation, angiogenesis, recruitment, and osteogenesis. These cytokines include chemokine ligand 2, chemokine ligand 5, chemokine ligand 7, and TNF- β [ 101 ].

4.3. Extracellular Vesicles

Other than growth factors and cytokines, EVs are another important subset of MSC secretome that play a crucial role in both normal and pathological processes through maintenance of homeostasis as well as regulation of immune function, tissue regeneration processes, and tumorigenesis. These EVs that carry therapeutic cargo, including nucleic acids, proteins, and lipids, are originally a method of communication between neighboring and distant cells. They can be divided into two types depending on their sizes, that is either exosomes (40-200 nm) or microvesicles (50-1000 nm) [ 4 ]. Example of the therapeutic proteins abundantly present in EVs secreted by MSCs includes osteoprotegerin and angiogenin that were found to be the key players for bone regeneration in a rat model of bone defect [ 101 ]. Besides proteins, microRNAs (miRNAs) inside the EVs secreted by BMSCs, such as miR27a, miR196a, and miR206, were also found to be crucial in triggering the expression of osteogenic genes for acceleration of bone regeneration in a rat model of calvarial bone defect [ 102 ]. On the other hand, miR133 is an important miRNA produced by MSCs to stimulate neuronal tissue remodeling in a rat model of stroke disease [ 103 , 104 ]. Meanwhile, miR125b-5p is an example of miRNA that could exert antiapoptotic effect as demonstrated by its ability to suppress expression of proapoptotic BAK1 and p53 genes in a myocardial infarction model, hence preventing the death of cardiomyocytes and subsequently allowing the repair of the ischemic tissue [ 105 ]. Apart from modulation of tissue regeneration, some miRNAs are also able to regulate the immune system to suppress the extent of tissue injury. For instance, miR15a, miR15b, and miR16 could inhibit the expression of CX3CL1 to prevent recruitment of macrophages to the ischemic kidney, therefore reducing the inflammatory process in the injured kidney [ 106 ].

5. Delivery and Homing of Stem Cells and Secretome

Stem cells and secretome can be delivered via various routes of administration to elicit their therapeutic actions. Thus far, direct injection to the target site and intravenous injection is most widely used as they deliver the biologics to the target tissue more effective compared to other routes of administration.

Compared to exosomes, homing of stem cells to the target tissue is critical for the cells to exert their therapeutic effects. The efficacy of stem cell homing to the target tissue upon transplantation is very much dependent on the route of administration. There is an intrinsic relationship between different chemical factors and MSCs that influence its homing and reparative effects. Stromal-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis is imperative in the recruitment of MSCs to the injured tissue [ 107 , 108 ] and inadvertently promotes neovascularization [ 109 , 110 ]. The increased expression of SDF-1 after tissue injury stimulates the expression of CXCR4 on MSCs which improve stem cell homing and engraftment to the injured site [ 111 ]. Besides, Qin et al. described that SDF-1 regulates the MSC immunomodulatory effects through CXC chemokine receptor 7 (CXCR7). In low concentration, the proliferation of MSCs is induced, and the regulatory B cells produce various cytokines including (IL-6, IL-10, IL-4, IFN- γ , TNF- α ) [ 112 ]. Zheng et al. suspected enhanced homing of CXCR4-overexpressing MSCs to the site of colitis resulted in the significant reduction of tumor formation when compared to the untreated group [ 113 ]. Similarly, Wang et al. observed improvement in cell migration using CXCR4-overexpressing MSCs, and the progression of diabetic retinopathy was hampered [ 47 ]. Without CXCR4 gene transfection, MSC is considerably less effective in repairing cardiovascular damage as the necessary vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) cannot be stimulated solely with SDF-1 [ 110 , 114 ].

5.1. Direct Injection to the Target Tissue

Direct injection is a straightforward approach to deliver stem cells to the target tissue. For example, stem cells can be injected through the intraspinal and intrathecal route to treat spinal cord injury and intraarticularly to treat osteoarthritis [ 115 , 116 ]. Direct injection can increase the homing of stem cells to the target tissue, and this is crucial as stem cells can self-renew and differentiate into the desired cells to repopulate and regenerate the injured or lost tissue. In addition, local delivery of stem cells to the target tissue is necessary as the secreted bioactive factors act in a paracrine manner and may be degraded in the bloodstream before reaching the target tissue when administered distantly. In the context of secretome such as exosomes, direct injection is applicable as well. MSC exosomes were effective in repairing critical size osteochondral defects in immunocompetent rats, as evidenced by the increased cellular proliferation and infiltration, enhanced matrix synthesis, and presence of regenerative immune phenotype [ 117 ]. These results are achieved via intraarticular injection of exosomes.

Even though many researchers have analyzed the therapeutic efficacy of different routes of delivery for stem cell therapy, however, there is still no congruous consensus on the optimal delivery method among the different reports [ 118 ]. Although direct injection of cell-based treatment (either stem cell or secretome) to the affected tissue is appealing and has long been documented, this approach may accompany problematic complications if it is not carefully planned, performed, or managed. The migration of stem cell or lymphatic drainage is the physiological process that would reduce the number of injected cells or quantity of secretome initially present in the target tissue vicinity. In addition, the hostile wound environment with intense inflammation is not ideal to support the survival of the transplanted cells [ 119 ].

5.2. Intravenous Administration

Delivery of cells through vein has been suggested in numerous preclinical studies and clinical trials [ 120 ]. Intravenous administration is advantageous because of its systemic distribution and ability to reach deeper tissues. However, intravenous administration also carries the risk of cell entrapment in the lung vasculature, and the retention time for the cells and their effects are short. The main concern of intravenous administration is to get enough cells to the target tissues. Harting et al. managed to infuse rats with MSCs intravenously to treat traumatic brain injury [ 121 ]. The group did not find cell homing to the target tissue. However, the rats still showed improvement in motor and cognitive functions. In terms of secretome, delivery via the intravenous route is safe due to the lower risk of embolism compared to the delivery of stem cells. In the case of neurological disorders, emboli of administered cells in the cerebral microvascular can exacerbate the disease and can be life-threatening. Intravenous administration of whole secretome or its components, i.e., exosomes, has been reported to be safe and capable to ameliorate several diseases ( Table 1 ).

5.3. Scaffold

On top of that, new delivery strategies utilizing the biomaterials such as polymeric scaffolds and cell sheets can increase cell retention on top of providing a supporting matrix to enhance cell survival and functionality [ 131 ]. The polymeric scaffold stabilizes the stem cells and their soluble factors as well as permits sustained delivery of these bioactive factors. The structure also supports cell growth. The architecture of the scaffolds including stiffness and pore arrangement is an important regulator of stem cell differentiation. The microarchitecture of the scaffold has an impact on the differentiation of MSCs into cells of interest. Phadke et al. found the randomly oriented pores were better suited for osteogenic differentiation of MSCs when compared to the lamellar column-arranged pore network [ 132 ]. Multiple studies have focused on the development of insulin-producing cells to treat diabetes. Enderami et al. noted a significantly higher expression of glucose-regulating genes including Pdx1, insulin, glucagon, and Ngn3 genes in poly-L-lactic acid and polyvinyl alcohol (PLLA/PVA) 3D scaffold than in the regular 2D culture [ 133 ]. The 3D scaffold provides a supporting structure to maintain the cell-cell and cell-matrix interactions. The stem cells cultured in nanofibrous scaffolds generate pancreatic organoids which are morphologically and functionally similar to the mature pancreatic β -cells [ 134 – 136 ].

The main advantage of using cell sheets is the fabrication techniques that will not disrupt the cell-cell and cell-matrix contact [ 137 ]. Usage of cell sheets fabrication techniques such as temperature-responsive culture surface, photoresponsive polymer, and ultrasound irradiation enables the detached cells to maintain their cell surface proteins, cellular junctions, and extracellular matrix [ 138 ]. Cell sheets may be developed into an advanced cell delivery method for the treatment of many tissue injuries, including cardiovascular diseases, cutaneous wound healing, and tendon/ligament injuries. The combination of multiple cell sources in the fabrication of cell sheets may mimic the natural state of tissue to allow better grafting of cells and better tissue regeneration.

6. Stem Cells and Secretome Clinical Trials

6.1. Stem Cell Clinical Trials

Thus far, many clinical trials using MSCs have been completed, and some of the therapies, e.g., Cupistem®, Queencell®, Cartistem®, Cellgram®, Neurorata-R®, Prochymal®, Stempeucel®, and MesestroCell, have received market authorization in Korea, Canada, India, and Iran [ 139 ]. A list of worldwide clinical studies using stem cells in different phases can be found in National Institutes of Health Clinical Trials.gov website ( https://www.clinicaltrials.gov/ ) ( Table 2 ).

6.1.1. Pluripotent Stem Cells

There are eight completed clinical trials on ESC transplantation, and seven of them are associated with eye disease and one for ischemic heart disease. The PSC clinical trials mainly focus on eye disease as the tissue is easily accessible for transplantation, and serious adverse events (SAEs) on the eye are less likely to be life-threatening. Furthermore, PSC transplantation is associated with higher risks of tumor formation. Since the eyeball is a confined space that has few vasculature connections with the rest of the body, the tumor is less likely to metastasize. However, none of the studies listed is presented with results. All these studies are either phase I or phase I/II, indicating that the translational research of PSC therapy is still in the early phase.

6.1.2. Multipotent Stem Cells

There are more completed MSC clinical trials compared to the PSC clinical trials as the cells are safer and have fewer ethical concerns. Many MSC clinical trials have published their results. Generally, MSC therapy is found to be safe and well-tolerated by the patients. In addition, some studies also reported the efficacy of MSC therapy to treat a battery of diseases. MSC therapy has received market authorization in several countries for the treatment of diseases such as Crohn's fistula, cartilage defects, osteoarthritis, major adverse cardiac events, amyotrophic lateral sclerosis, aGvHD, and critical limb ischemia [ 139 ]. Very recently, a parallel assigned controlled, nonrandomized phase I clinical trial has been conducted to evaluate the safety of human umbilical cord-derived mesenchymal stem cell (UC-MSC) infusion to treat patients with moderate and severe COVID-19 pulmonary disease [ 140 ]. Eighteen hospitalized COVID-19 patients were enrolled on the study, and nine of them received three cycles of intravenous infusion of UC-MSCs (3 × 10 7 cells/infusion). Twenty-eight days after the first infusion, no UC-MSC infusion associated SAEs were observed except for one patient in the treatment group that required mechanical ventilation compared to four patients in the control group. All patients recovered following the treatment and were discharged. These data showed that intravenous UC-MSC infusion is safe and well-tolerated in patients with moderate and severe COVID-19.

6.2. Secretome Clinical Trials

Clinical trials of cell-free therapy are taking the emerging field from basic science to clinical application. Numerous trials are/have been conducted for a huge variety of conditions. While there are reviews that have summarized previous clinical trials pertaining to the use of cell-free therapy, we intend to highlight several clinical studies that are recently published at the time of this writing ( Table 3 ). Unfortunately, to date, the results from many of these clinical trials have yet to be published.

Overall, stem cell therapy has a longer history compared to cell-free therapy. A review on the stem cell clinical trials was published in the year 2011 [ 156 ]. One decade has passed since then, and a significant change in the current trend of stem cell clinical trials has been observed, most noticeably, the quantity ( Figure 4 ). In 2011, 123 clinical trials using MSCs were recorded. Although some of the studies are in the combination of phase I/II, the majority are in phase II. The quantity of MSC clinical trials has grown tremendously, circa 25 times since the past decade. Notwithstanding, a total of 152 clinical trials using exosomes have also been recorded in the last 10 years. Although there is a huge surge in the number of clinical trials on MSCs and exosomes, the disease treated has not varied significantly and most of which are chronic diseases and disorders. While it is too hasty to draw a conclusion of the efficacy of cell-based therapy, the early observations of these trial results demonstrated that it is safe and feasible.

However, the clinical applications of MSCs or secretome are not without risks. Several pertaining concerns are promoting the growth of cancerous cells and nonspecific and undesirable differentiation of the transplanted cells at the target tissue. Perhaps, the most relevant risk of stem cell therapy is the malignant transformation of the administered cells. Many researchers have reported genomic instability in MSCs at higher passage [ 157 , 158 ]. Thus, genotyping might be relevant to ensure the safety of the cells before transplantation.

7. Stem Cell vs. Secretome

7.1. Manufacturing

As a cellular product, the cell source poses the first major challenge to reproducibly manufacture clinically effective stem cells and secretome products. Stem cell manufacturing has been critically reviewed and discussed in the previous publication [ 159 ]. The production of stem cells is indeed a quite straightforward process. The stem cells can be grown on a large scale using bioreactors or large cell culture flasks under specific culture conditions [ 160 ]. Large-scale expansion is crucial to produce enough cells for downstream clinical application. Human platelet lysate (HPL) is often recommended as an alternative to fetal bovine serum (FBS) for good manufacturing practize- (GMP-) compliant stem cell expansion. Generally, stem cells cultured with HPL are smaller in size, display a tighter spindle-shaped morphology, and increased cell growth [ 161 ]. In addition, a chemically defined serum-free medium also can be used to replace the serum-based medium to avoid the batch-to-batch variation bothering the serum-based medium.

The manufacturing of clinically effective secretome is not an easy process. Notably, the quality and quantity of secretome are greatly influenced by the cell source and culture condition. Although secretome has been proven to work as effective as stem cells, nevertheless, it does not guarantee that the secretome harvested could work in the same way or as effective as the cultured cells. In vivo, transplanted stem cells produce secretome that could regenerate/repair the tissue or modulate the immune function in response to the signalling from the surrounding tissue. In contrast, this does not happen when the cells are grown in the laboratory. Therefore, it might be necessary to customize the culture condition that mimics the pathophysiological environment to produce clinically effective secretome [ 162 ].

When an appropriate cell source of clinically effective secretome is identified, the consistency of the cell source for all subsequent batches of secretome production must be addressed. This could be achieved by pooling the cells from the same tissue source of different healthy donors to produce multiple batches of secretome, mitigating the challenges of biological variation in tissue sources and donors [ 163 ]. Due to the finite replication of the primary cells, a more practical approach is to use “immortalized” cell lines or PSC-derived stem cells. Although studies have reported that the regenerative properties of secretome [ 164 ] and sEVs [ 165 ] harvested from immortalized stem cells are not compromised. Nevertheless, all these must be carefully investigated to ensure that the immortalized cells are stable and continue to produce secretome products that are bioequivalent to those from nonimmortalized parental cells. In addition, secretome enrichment protocols could also be employed to enhance the production of secretome in the laboratory [ 166 ]. It should be taken into consideration that the type of media used to harvest secretome may also affect the quality and efficacy of secretome. To avoid interference from HPL, basal media is often employed to harvest the secretome to determine the efficacy of stem cell secretome. Nevertheless, the sudden switch from nutrient-rich to basal media may change the cell's behavior and subsequently modifying the secretome profile. Would the basal media harvested secretome work as effective as the stem cells in the host and whether the secretome harvested from cells cultured in complete growth media might have better clinical efficacy compared to the secretome harvested from basal media are some of the questions that remain to be answered. Based on the abovementioned arguments, the quality of secretome preparation is dependent on the source, culture condition, and secretome enrichment protocol. Therefore, secretome manufactured using different protocols may have different disease-relevant potency.

7.2. Quality Control

Quality control is crucial to ensure the safety and efficacy of cell-based products. Adherence to the GMP regulations assures the identity, strength, quality, and purity of the products. Strict adherence to the quality management system helps to prevent contamination, mix-up, deviation, failure, and error during production [ 167 ]. It is important to note that a lot of efforts have been given to establish GMP facilities to produce cellular products for clinical applications [ 168 ]. Apart from the GMP facility, guidelines are in place to characterize the stem cells. For instance, MSCs should be characterized according to the guideline recommended by the International Society of Cellular Therapy (ISCT) [ 169 ].

The quality control for secretome is way more complex compared to the stem cells. As the secretome is a mixture of EVs and soluble proteins, it is challenging to identify the active components from this mixture and hence, more efforts are required to characterize the secretome. For instance, proteomic analysis is needed to identify the type of proteins and their concentration in the preparation [ 96 , 170 ]. For EVs, particularly small EVs (sEVs), it needs to be characterized according to the guidelines published by the International Society of Extracellular Vesicles (ISEV). The identification of size and number would require either zetasizer or nanoparticle tracking analysis (NTA). Notably, quantifiable metrics defining the identity of sEV preparations should reflect the cellular origin of the sEVs in preparation, the presence of lipid membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. The combination of these metrics could quantify the identity of sEVs and facilitate stratification and comparison of different secretome preparations [ 77 , 171 ]. As EVs contain miRNAs, the molecular technique is also required to characterize the miRNA profile [ 172 , 173 ].

7.3. Cost of Production and Treatment

There is an argument whether the cost of production and treatment is lower in secretome therapy in comparison to stem cell therapy. For stem cell therapy, the number of stem cells that could be isolated from patients/donors is low; therefore, the harvested cells are usually expanded in the laboratory to attain enough cells for clinical applications. The process can take weeks to months. During the cell expansion, media change is typically done every 3-4 days. The high volume of spent media means to be discarded is a potential source of secretome that can be used clinically after proper processing. The preparation of cell-free therapies from the spent media can greatly reduce the cost of production. However, need to bear in mind that the secretome or exosomes collected form the spent media could have different biological components when the cells are cultured in different conditions. Thus, it is imperative to determine safety and efficacy as well as to characterize the secretome or exosomes secreted by cells cultured in different conditions. In another words, not all spent media can be processed to produce safe and effective secretome and exosomes.

Furthermore, as the secretome cannot self-replicate and have a short half-life in vivo, thus, the secretome might need to be given more frequently to exert its therapeutic effect. In contrast, stem cells can self-renew and survive in the body for a longer period. Stem cells can respond to the signaling molecules released by the injured cells by secreting the appropriate paracrine factors to stimulate tissue regeneration. On the other hand, the contents of secretome are already defined in vitro. Thus, preconditioning of the cells at the culture condition that mimic the disease pathophysiological condition might be needed to produce clinically relevant secretome. The used of specific instruments or biochemicals to replicate the disease pathophysiological condition in vitro will incur extra costs. Finally, the cost of secretome production is still likely to be higher than the cells as it requires extra concentration and purification steps [ 174 ].

7.4. Advantages and Disadvantages

The use of stem cells as regenerative medicine for various diseases has been progressing well since the past decade. However, the type of stem cell suitable for different diseases is still under vigorous debate since each stem cell subtype possesses its advantages and limitations. For instance, ESCs can differentiate into various types of tissue but its limitations, i.e., ethical dilemma, genetic instability, and teratocarcinoma, might overweight the benefits [ 175 ]. MSCs show several superior properties for therapeutic use compared to other types of stem cells, including easy to harvest and expand, both autologous and allogeneic cells can be used with minimal risk of rejection, free from ethical issues and have limited replicative lifespan, and hence have lower risks of malignant formation. However, MSCs are only capable to differentiate into certain lineages thus limiting their usage to only certain diseases [ 176 ]. Notably, stem cells could be differentiated into specific tissue as cell replacement therapy [ 177 , 178 ]. This is the main advantage of stem cells over secretome. Another prominent property of stem cells is their ability to migrate to the site of injury (homing effect). Surprisingly, sEVs also possessed the homing ability. Studies showed that MSC-EVs were mainly accumulated in the inflamed kidneys [ 179 ] and injured brains [ 180 , 181 ] in the acute kidney injury model and intracerebral haemorrhage models, respectively. The accumulation of systemically injected sEVs in the intracerebral hemorrhage model also showed that sEVs can cross the blood-brain barrier (BBB). Research also has indicated that stem cells could cross BBB. MSCs integrated into the endothelium through the adhesion molecules VCAM-1/VLA-4 and β 1 integrin. After crossing the endothelial barrier, MSCs invade the host tissue via plasmic podia [ 182 ]. MSCs were also found to cross BBB through paracellular pathways that are normally inhibited by the presence of tight junctions [ 183 ]. These showed that both stem cells and secretome could be intravenously injected and reach the brain. Nevertheless, the bioavailability of these two subjects in the brain remains to be elucidated.

Accumulating evidence suggests that secretome possesses many advantages over stem cell transplantation. Cell degeneration or senescence in the host after transplantation is not a concern for secretome therapy. It was also reported that secretome has lower cell surface proteins compared to stem cells, which makes allogeneic secretome safer than allogeneic stem cells because of the lower risk of immunogenicity [ 184 ]. Irreplicable property and absence of DNAs in secretome greatly reduce the risk of DNA mutation and tumor formation in the host. The use of secretome also reduces the possibility of vascular obstruction compared to larger stem cells. The bioactive components of secretome can be easily modulated by culturing the cells in different conditions. Secretome is also easier to store compared to stem cells, i.e., stem cells need to be stored in liquid nitrogen to maintain their viability while secretome can be stored in -20°C. Finally, the requirement to evaluate the safety and dosage of secretome is less stringent in comparison to the stem cells, making the journey to the clinical setting smoother and faster. This is because stem cells are living cells, and the fate of the transplanted cells is more difficult to predict. Table 4 and Figure 5 summarize the comparison between stem cells and secretome from the perspective of manufacturing, quality control, cost of production, and treatment, as well as their advantages and disadvantages in clinical applications.

8. Future Perspective

Currently, cell-based therapies face two great challenges; how to anticipate decreased cell viability and biological functions during in vitro culture and how to prolong survival of transplanted cells. Consequently, several strategies can be envisaged to increase survival, immunomodulatory potential, and regenerative functions of cell-based therapy. Preconditioning, genetic modification, and tissue engineering are the dominant strategies. Furthermore, combinatorial approaches using nanotechnology could also improve the therapeutic performance of stem cells and secretome.

8.1. Stem Cells

8.1.1. Genetic Modification

The combination of stem cell biology and genetic engineering has great potential in regenerative medicine. Through genetic modification, the researcher could induce or determine the cell's specific differentiation pathway after injection or enhance the adhesion potential of the stem cell to specific target. After transplantation, the fate of MSCs would be stochastically determined based on the microenvironment and biochemical stimulation of the host body; therefore, not all transplanted cells would contribute to the regeneration of damaged organs. As recently demonstrated in mice, transplanted MSCs could differentiate into osteoblasts in the heart [ 185 ]. Although cell differentiation can be achieved using the biochemical or biophysical stimulus in vitro, however, reverse differentiation may occur after transplantation or withdrawal of stimulants [ 186 ]. Therefore, genetic modification of the transplanted stem cells would be the key to achieve a directed and irreversible differentiation into the desired lineage. Several studies have been conducted on the therapeutic applicability of genetically modified MSCs in animal models of diabetes, myeloma bone disease, GvHD, and myocardial infarction. Table 5 summarizes the modifiers, cell source, genetic engineering method, and applications from various studies.

In addition to the ability to differentiate, MSCs can be genetically engineered to home to the target tissue. For example, MSCs transduced with CXCR4 demonstrated higher homing in the mice model of myocardial infarction after intravenous administration [ 110 , 235 ]. The overexpression of CXCR4 facilitated MSC aggregation and etching of collagenous tissue of the infarcted area [ 236 ]. Such strategies will help in the development of noninvasive cell therapies, since the route of administration is also important to avoid the formation of heterotopic tissue, especially in the case of genetically modified MSCs. On the other hand, poor cell survival after transplantation is a yet to resolved hurdle in MSC-based therapies. Studies show that genetic modification of MSCs with hypoxia-regulated heme oxygenase-1 (HO-1), Akt1, and Bcl-2 increased cell survival after transplantation in animal models by inhibiting cell apoptosis [ 236 – 238 ]. Thus, these strategies might be the possible solutions to increase the survival of MSCs after transplantation.

8.1.2. Tissue Engineering Using MSCs

Another area of regenerative medicine is to combine cells and scaffolds to create a 3D implant. Tissue engineering seeks to recreate the in vivo environment to promote the development of tissues needed for transplantation. Various approaches have been studied, including protein-impregnated scaffolds [ 239 ], gene vector-incorporated templates [ 240 ], and cell-scaffold combinations ( Table 6 ). Scaffolding alone has been shown to help repair certain types of damage [ 239 ]. However, incorporating MSCs into the scaffold improves the in situ repair process by acting as the precursors and stimulators [ 241 ].

Over the decades, much effort has been devoted to study the physical and chemical properties of various biomaterials, as these properties affect the differentiation pathway and adhesion capacity of MSCs. For example, the elasticity of a polyacrylamide matrix seeded with MSCs determines their differentiation pathway into neuronal, muscle, or bone lineage based on crosslink density [ 247 ]. Furthermore, studies indicated that the presence of carboxyl or hydroxyl groups on the scaffold surface prioritizes chondrogenic differentiation, while amino and sulfhydryl groups promote bone formation [ 248 ]. In addition to biometric properties, graft angiogenesis is another important factor in ensuring cell survival and therapeutic efficiency. The host's blood vessels can invade the transplant, but the process is very slow, and it takes weeks to vascularize just a few millimeters. Therefore, researchers incorporate angiogenesis-promoting factors such as endothelial progenitor cells (EPCs) and VEGF to hasten graft angiogenesis [ 249 ]. Unfortunately, no perfusion was observed upon implantation [ 250 ]. Currently, there are no established angiogenesis strategies available to support transplantation of large tissue due to delayed angiogenesis which resulted in cell apoptosis and necrosis. The approaches mentioned above only could increase the likelihood of angiogenesis.

8.2. Exosomes

8.2.1. Preconditioning

Both 3D culturing and pretreatment of MSCs with cytokines, hypoxia, or chemicals are reliable methods to increase exosome secretion ( Figure 6 ) [ 251 ]. In addition, MSC gene and cell surface modifications may be used to improve the therapeutic effect of exosomes.

(1) Increasing Exosome Production . Increasing the secretion of exosomes is an important but unmet process. Studies have shown that 3D culturing methods such as bioreactors and microcarriers could significantly increase the production of exosomes by cultured MSCs [ 252 ]. Generally, MSCs are processed on 2D surfaces in plastic dishes that do not reflect the physiological niches of MSCs. Therefore, the use of a 3D porous scaffold structure, such as beads, microfiber, or any other type of carrier is an attractive method to increase exosome production. One study showed that antifungal agents, i.e., imidazole and nitrefazole, significantly increase the production of exosomes in prostate cancer cells [ 253 ]. In this case, nitreprazole increased the level of the protein Rab27a, which regulates MVB exocytosis. Other chemicals, such as azole and pentetrazole, have also been shown to activate exosome biogenesis-related molecules, i.e., Alix and NSmas2. The techniques may be employed to increase the production of exosomes from MSCs by modulating the biogenesis and release of exosomes [ 253 ]. On the other hand, gene editing is another effective way to increase the production of exosomes. There are several important genes, such as phospholipase D2, that are important for the biosynthesis and secretion of exosomes, and the overexpression or dysfunction of these genes promotes exosome secretion. For example, the overexpression of phospholipase D2 led to a twofold increase in the number of secreted exosomes [ 254 ].

(2) Hypoxia Preconditioning . Hypoxia culture is commonly used to prime MSCs. Several studies found that exosomes derived from MSCs cultured in hypoxic condition showed greater angiogenic potential compared to exosomes secreted by MSCs cultured in normoxic condition [ 255 ]. The exosomes secreted by hypoxia primed MSCs were uptaken more effectively by the target cells compared to exosomes derived from MSCs cultured in normoxic condition. The uptaken exosomes promoted the VEGF expression and protein kinase A signaling pathway activation in the target cells, which resulted in improved angiogenesis [ 256 , 257 ]. However, the reason for these phenomena is still unclear, and how different culture conditions influence the uptake of exosomes needs to be further investigated.

(3) Chemical Preconditioning . In contrast to hypoxic priming, the effects and mechanisms of biomolecule priming in exosomes are better studied. Various studies have compared the therapeutic effect between lipopolysaccharides (LPS) preconditioned and unconditioned exosomes. LPS conditioned exosome showed higher regeneration potential for liver disease preclinically by reducing the expression of IL-6 and TNF- β [ 258 ] and upregulated the expression of THP-1, which in turn stimulate the synthesis of more anti-inflammatory cytokines and contributed to the polarization of M2 macrophages [ 259 ]. A recent study has also shown that macrophages cultivated with exosomes from LPS-primed MSCs expressed higher levels of STAT3 gene, secretion of cytokines (IL-10 and IL-15), and growth factors (FLT-3 L) which play vital roles in cell regeneration and antiapoptosis [ 260 ]. Several other molecules have been tested as preconditioners, including thrombin to improve fibroblast proliferation, enhance anti-inflammatory effects, accelerate wound healing [ 176 ] and melatonin to increase BCL2, HO1, IL-10, and VEGF expression, and suppress the expression of various apoptosis-related genes such as ICAM1, HIF1, NFkB, and IL-1 β in a rat model [ 261 ]. Exosomes derived from deferoxamine-primed MSCs contained higher levels of miR-126a that support angiogenesis [ 262 ].

8.2.2. Genetic Modification

In 2010, a study reported that the paracrine factor secreted by MSC-overexpressed GATA-4 increased blood vessel formation and cell survival [ 263 ]. Next, in a mouse model of myocardial infarction, exosomes secreted by the genetically modified stem cells with GATA-4 were more effective in increasing angiogenesis and reducing the number of apoptotic cardiac cells compared to the exosomes secreted by native stem cells [ 264 ]. MSC-derived exosomes that overexpress GATA-4 and CXCR4 have been shown to contain cardioprotective antiapoptotic miR-19a that activates Akt and ERK signaling pathways [ 265 , 266 ]. Similarly, exosomes from MSCs that overexpress SDF-1 have been shown to prevent apoptosis of cardiomyocytes and exhibit improved cardiac regeneration properties [ 267 ]. Genetic modification methods have also been investigated to improve the therapeutic potential of exosomes for musculoskeletal disorders, liver and lung disorders, and inflammation-related disorders.

8.2.3. Combining Nanoparticles, Exosomes, and Stem Cells

Nanotechnology is the term used to cover the design, construction, and utilization of functional structures with at least one characteristic dimension measured in nanometers. In recent years, the application of nanotechnology in stem cells has made great advances. Currently, nanotechnology is utilized to control the proliferation and differentiation of the transplanted stem cells.

Carbon nanotubes (CNTs) are widely used in various fields, including medicinal chemistry, molecular electronics, and tissue engineering, due to their unique mechanical, physical, and chemical properties. CNTs can be designed and filled with DNA or peptide molecules to improve their properties and functions, such as biocompatibility and recognition capability in the molecular treatment of diseases [ 268 – 270 ]. In a study that examined the effect of CNTs on the proliferation and differentiation of human stem cells, the result showed that CNTs inhibit the proliferation of cells of the embryonic kidney cell line HK293 and reduce the adhesion efficiency of cells in a dose- and time-dependent manner, but similar CNTs can stimulate the formation of bumps on the surface of human osteoblasts and fibroblasts, which are one of the active cells in the immune response [ 271 ]. Nanomaterials such as CNTs have enormous potential in the field of regenerative medicine in several areas, including (1) the development of nanovehicles to deliver biomolecule-based products to MSCs and (2) the creation of new biomedical applications for electroactive CNTs in combination with MSCs. However, despite the immense potential of nanoparticles, the method of delivering nanoparticles to the target cells was still a major problem. The maximum size of particles entering cells is 25 nm to 700 nm; so, it is difficult for nanosized particles to penetrate cells due to the tension and adhesion strength of the cell surface. As an alternative, the nanoparticles can be bonded to the external cell membrane.

9. Conclusion

Regenerative medicine holds an immense potential for a variety of diseases in which there is a high unmet clinical need. Regenerative medicine covered a wide range of subbranches including cell and gene therapies and tissue engineering applications. Stem cells have been the focus for years because of their biological potential, and paracrine effect is the pivotal mechanism in stem cell-based tissue regeneration. Thus, cell secretome has attracted great attention as therapeutics in recent years and has been suggested as alternative to stem cell therapy as cell-free agents. The high degree of confidence in cell-based therapy is vividly indicated by the significant increase in the number of ongoing and planned clinical trials worldwide. Despite the relatively slow rate of translational success from laboratory to clinics, expectations, optimism, and excitement surrounding this field remain great.

Acknowledgments

This work was funded by the Fundamental Research Grant Scheme (FRGS/1/2020/SKK0/UKM/02/5) provided by the Ministry of Higher Education Malaysia.

Data Availability

No data were used to support this study.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

1 Yang J. Li S. He X. B. Cheng C. Le W. Induced pluripotent stem cells in Alzheimer's disease: applications for disease modeling and cell-replacement therapy Molecular Neurodegeneration 2016 11 1 p. 39 10.1186/s13024-016-0106-3 2-s2.0-84969233464 27184028 PMC4869261

2 Wang H. Yan X. Jiang Y. Wang Z. Li Y. Shao Q. The human umbilical cord stem cells improve the viability of OA degenerated chondrocytes Molecular Medicine Reports 2018 17 3 4474 4482 10.3892/mmr.2018.8413 2-s2.0-85041180149 29328479 PMC5802223

3 Jin H. J. Bae Y. K. Kim M. Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy International Journal of Molecular Sciences 2013 14 9 17986 18001 10.3390/ijms140917986 2-s2.0-84883628131 24005862 PMC3794764

4 Daneshmandi L. Shah S. Jafari T. Emergence of the stem cell secretome in regenerative engineering Trends in Biotechnology 2020 38 12 1373 1384 10.1016/j.tibtech.2020.04.013 32622558 PMC7666064

5 Guthrie K. Bruce A. Sangha N. Rivera E. Basu J. Potency evaluation of tissue engineered and regenerative medicine products Trends in Biotechnology 2013 31 9 505 514 10.1016/j.tibtech.2013.05.007 2-s2.0-84882660585 23932143

6 Basu J. Ludlow J. W. Cell-based therapeutic products: potency assay development and application Regenerative Medicine 2014 9 4 497 512 10.2217/rme.14.25 2-s2.0-84906770525 25159066

7 Drago D. Cossetti C. Iraci N. The stem cell secretome and its role in brain repair Biochimie 2013 95 12 2271 2285 10.1016/j.biochi.2013.06.020 2-s2.0-84887820815 23827856 PMC4061727

8 Geiger A. Walker A. Nissen E. Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice Biochemical and Biophysical Research Communications 2015 467 2 303 309 10.1016/j.bbrc.2015.09.166 2-s2.0-84944387386 26454169

9 Tran C. Damaser M. S. Stem cells as drug delivery methods: application of stem cell secretome for regeneration Advanced Drug Delivery Reviews 2015 82-83 1 11 10.1016/j.addr.2014.10.007 2-s2.0-84927137683 25451858 PMC4398586

10 Kusuma G. D. Carthew J. Lim R. Frith J. E. Effect of the microenvironment on mesenchymal stem cell paracrine signaling: opportunities to engineer the therapeutic effect Stem Cells and Development 2017 26 9 617 631 10.1089/scd.2016.0349 2-s2.0-85018267276 28186467

11 Comella K. Parlo M. Daly R. Safety analysis of autologous stem cell therapy in a variety of degenerative diseases and injuries using the stromal vascular fraction Journal of Clinical Medicine Research 2017 9 11 935 942 10.14740/jocmr3187w 29038672 PMC5633095

12 de Windt T. S. Vonk L. A. Slaper-Cortenbach I. C. M. Allogeneic mesenchymal stem cells stimulate cartilage regeneration and are safe for single-stage cartilage repair in humans upon mixture with recycled autologous chondrons Stem Cells 2017 35 1 256 264 10.1002/stem.2475 2-s2.0-84983667051 27507787

13 Liang J. Zhang H. Zhao C. Effects of allogeneic mesenchymal stem cell transplantation in the treatment of liver cirrhosis caused by autoimmune diseases International Journal of Rheumatic Diseases 2017 20 9 1219 1226 10.1111/1756-185X.13015 2-s2.0-85013393867 28217916

14 Lohan P. Murphy N. Treacy O. Third-party allogeneic mesenchymal stromal cells prevent rejection in a pre-sensitized high-risk model of corneal transplantation Frontiers in Immunology 2018 9, article 2666 10.3389/fimmu.2018.02666 2-s2.0-85057401818 PMC6255848 30515159

15 Nawaz M. Fatima F. Vallabhaneni K. C. Extracellular vesicles: evolving factors in stem cell biology Stem Cells International 2016 2016 17 1073140 10.1155/2016/1073140 2-s2.0-84948799678 26649044 PMC4663346

16 Bacakova L. Zarubova J. Travnickova M. Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - a review Biotechnology Advances 2018 36 4 1111 1126 10.1016/j.biotechadv.2018.03.011 2-s2.0-85044303109 29563048

17 Posabella A. Alber A. B. Undeutsch H. J. Derivation of thyroid follicular cells from pluripotent stem cells: insights from development and implications for regenerative medicine Frontiers in Endocrinology 2021 12 1 16 10.3389/fendo.2021.666565 PMC8095374 33959101

18 Arauchi A. Matsuura K. Shimizu T. Okano T. Functional thyroid follicular cells differentiation from human-induced pluripotent stem cells in suspension culture Frontiers in Endocrinology 2017 8 p. 103 10.3389/fendo.2017.00103 2-s2.0-85020033584 PMC5439004 28588551

19 Kurmann A. A. Serra M. Hawkins F. Regeneration of thyroid function by transplantation of differentiated pluripotent stem cells Cell Stem Cell 2015 17 5 527 542 10.1016/j.stem.2015.09.004 2-s2.0-84947706099 26593959 PMC4666682

20 Ito E. Miyagawa S. Takeda M. Tumorigenicity assay essential for facilitating safety studies of hi PSC-derived cardiomyocytes for clinical application Scientific Reports 2019 9 1 1 10 30760836 10.1038/s41598-018-38325-5 PMC6374479

21 Mandai M. Watanabe A. Kurimoto Y. Autologous induced stem-cell-derived retinal cells for macular degeneration New England Journal of Medicine 2017 376 11 1038 1046 10.1056/NEJMoa1608368 2-s2.0-85015803917 28296613

22 Kanemura H. Go M. J. Shikamura M. Tumorigenicity studies of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration PLoS One 2014 9 1 1 11 10.1371/journal.pone.0085336 PMC3891869 24454843

23 Dayem A. A. Lee S. B. Kim K. Production of mesenchymal stem cells through stem cell reprogramming International Journal of Molecular Sciences 2019 20 8 1 42 10.3390/ijms20081922 PMC6514654 31003536

24 Quinlan A. R. Boland M. J. Leibowitz M. L. Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangementduring reprogramming Cell Stem Cell 2011 9 4 366 373 21982236 10.1016/j.stem.2011.07.018 PMC3975295

25 Laurent L. C. Ulitsky I. Slavin I. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ES and iPS cells during reprogramming and time in culture Cell 2012 8 1 106 118 10.1016/j.stem.2010.12.003 PMC3043464 21211785

26 Johannesson B. Sagi I. Gore A. Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors Cell Stem Cell 2014 15 5 634 642 10.1016/j.stem.2014.10.002 2-s2.0-84922637049 25517467

27 Itakura G. Kawabata S. Ando M. Fail-safe system against potential tumorigenicity after transplantation of iPSC derivatives Stem Cell Reports 2017 8 3 673 684 10.1016/j.stemcr.2017.02.003 2-s2.0-85014119461 28262544 PMC5355810

28 Lipus A. Janosz E. Ackermann M. Targeted integration of inducible caspase-9 in human iPSCs allows efficient in vitro clearance of iPSCs and iPSC-macrophages International Journal of Molecular Sciences 2020 21 7, article 2481 10.3390/ijms21072481 32260086 PMC7177583

29 Mushahary D. Spittler A. Kasper C. Weber V. Charwat V. Isolation, cultivation, and characterization of human mesenchymal stem cells Cytometry A 2018 93 1 19 31 10.1002/cyto.a.23242 2-s2.0-85032300260 29072818

30 Hassan M. N. F. B. Yazid M. D. Yunus M. H. M. Large-scale expansion of human mesenchymal stem cells Stem Cells International 2020 2020 17 9529465 10.1155/2020/9529465 32733574 PMC7378617

31 Liau L. L. Al-Masawa M. E. Koh B. The potential of mesenchymal stromal cell as therapy in neonatal diseases Frontiers in Pediatrics 2020 8, article 591693 10.3389/fped.2020.591693 PMC7672022 33251167

32 Looi Q. H. Eng S. P. Liau L. L. Mesenchymal stem cell therapy for sports Injuries - From research to clinical practice Sains Malaysiana 2020 49 4 825 838 10.17576/jsm-2020-4904-12

33 Liau L. L. Ruszymah B. H. I. Ng M. H. Law J. X. Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells Current Research in Translational Medicine 2020 68 1 5 16 10.1016/j.retram.2019.09.001 2-s2.0-85072268987 31543433

34 Shin S. Lee J. Kwon Y. Comparative proteomic analysis of the mesenchymal stem cells secretome from adipose, bone marrow, placenta and wharton’s jelly International Journal of Molecular Sciences 2021 22 2 1 17 10.3390/ijms22020845 PMC7829982 33467726

35 Spees J. L. Lee R. H. Gregory C. A. Mechanisms of mesenchymal stem/stromal cell function Stem Cell Research & Therapy 2016 7 1 125 125 10.1186/s13287-016-0363-7 2-s2.0-84984889454 27581859 PMC5007684

36 Gnecchi M. Danieli P. Malpasso G. Ciuffreda M. C. Paracrine mechanisms of mesenchymal stem cells in tissue repair 2016 Springer 10.1007/978-1-4939-3584-0_7 27236669

37 da Silva Meirelles L. Fontes A. M. Covas D. T. Caplan A. I. Mechanisms involved in the therapeutic properties of mesenchymal stem cells Cytokine & Growth Factor Reviews 2009 20 5-6 419 427 10.1016/j.cytogfr.2009.10.002 2-s2.0-70849103924 19926330

38 Fatima F. Ekstrom K. Nazarenko I. Non-coding RNAs in mesenchymal stem cell-derived extracellular vesicles: deciphering regulatory roles in stem cell potency, inflammatory resolve, and tissue regeneration Frontiers in Genetics 2017 8 p. 161 10.3389/fgene.2017.00161 2-s2.0-85032186711 PMC5662888 29123544

39 Zhao Q. Ren H. Han Z. Mesenchymal stem cells: Immunomodulatory capability and clinical potential in immune diseases Journal of Cellular Immunotherapy 2016 2 1 3 20 10.1016/j.jocit.2014.12.001 2-s2.0-85038124867

40 English K. Ryan J. M. Tobin L. Murphy M. J. Barry F. P. Mahon B. P. Cell contact, prostaglandin E2and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25Highforkhead box P3+regulatory T cells Clinical and Experimental Immunology 2009 156 1 149 160 10.1111/j.1365-2249.2009.03874.x 2-s2.0-61849123247 19210524 PMC2673753

41 Pourgholaminejad A. Aghdami N. Baharvand H. Moazzeni S. M. The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells Cytokine 2016 85 51 60 10.1016/j.cyto.2016.06.003 2-s2.0-84978924301 27288632

42 Augello A. Tasso R. Negrini S. M. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway European Journal of Immunology 2005 35 5 1482 1490 10.1002/eji.200425405 2-s2.0-18844404360 15827960

43 Nasef A. Chapel A. Mazurier C. Identification of IL-10 and TGF-beta transcripts involved in the inhibition of T-lymphocyte proliferation during cell contact with human mesenchymal stem cells Gene Expression 2007 13 4-5 217 226 10.3727/000000006780666957 17605296 PMC6032462

44 Loibl M. Binder A. Herrmann M. Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro BioMed Research International 2014 2014 10 395781 10.1155/2014/395781 2-s2.0-84893791551 24563864 PMC3915932

45 Lee H. K. Kim E. Y. Kim H. S. Effect of Human Mesenchymal Stem Cells on Xenogeneic T and B Cells Isolated from Lupus-Prone MRL.Faslpr Mice Stem Cells International 2020 2020 10 5617192 10.1155/2020/5617192 32215018 PMC7077055

46 Chin S. P. Mohd-Shahrizal M. Y. Liyana M. Z. Then K. Y. Cheong S. K. High dose of intravenous allogeneic umbilical cord-derived mesenchymal stem cells (CLV-100) infusion displays better immunomodulatory effect among healthy volunteers: a phase 1 clinical study Stem Cells International 2020 2020 13 10.1155/2020/8877003 8877003 33061992 PMC7539086

47 Wang J. Zhang W. He G. H. Wu B. Chen S. Transfection with CXCR4 potentiates homing of mesenchymal stem cells in vitro and therapy of diabetic retinopathy in vivo International Journal of Ophthalmology 2018 11 5 766 772 10.18240/ijo.2018.05.08 2-s2.0-85050383383 29862173 PMC5957026

48 Fan J. Tang X. Wang Q. Mesenchymal stem cells alleviate experimental autoimmune cholangitis through immunosuppression and cytoprotective function mediated by galectin-9 Stem Cell Research and Therapy 2018 9 1 1 12 30223894 10.1186/s13287-018-0979-x PMC6142687

49 Liu F. Qiu H. Xue M. MSC-secreted TGF- β regulates lipopolysaccharide-stimulated macrophage M2-like polarization via the Akt/Fox O1 pathway Stem Cell Research and Therapy 2019 87 1 14 10.1186/s13287-019-1447-y PMC6878630 31771622

50 Haghighitalab A. Matin M. M. Amin A. Investigating the effects of IDO1, PTGS2, and TGF- β 1 overexpression on immunomodulatory properties of hTERT-MSCs and their extracellular vesicles Scientific Reports 2021 11 1 1 19 10.1038/s41598-021-87153-7 33837229 PMC8035148

51 Maertens L. Erpicum C. Detry B. Bone marrow-derived mesenchymal stem cells drive lymphangiogenesis PLoS One 2014 9 9, article e106976 10.1371/journal.pone.0106976 2-s2.0-84907155405 25222747 PMC4164522

52 El-Gendy H. Hawass S. E. D. Awad M. Comparative study between human mesenchymal stem cells and etanercept as immunomodulatory agents in rat model of rheumatoid arthritis Immunologic Research 2020 68 5 255 268 10.1007/s12026-020-09132-w 32734446

53 Shin S. Kim Y. Jeong S. The therapeutic effect of human adult stem cells derived from adipose tissue in endotoxemic rat model International Journal of Medical Sciences 2013 10 1 8 18 10.7150/ijms.5385 2-s2.0-84871355986 23289000 PMC3534872

54 Tinoco R. Alcalde V. Yang Y. Sauer K. Zuniga E. I. Cell-intrinsic transforming growth factor- β signaling mediates virus-specific CD8+ T cell deletion and viral persistence in vivo Immunity 2009 31 1 145 157 10.1016/j.immuni.2009.06.015 2-s2.0-68249152608 19604493 PMC3039716

55 Rosado M. M. Bernardo M. E. Scarsella M. Inhibition of B-cell proliferation and antibody production by mesenchymal stromal cells is mediated by T cells Stem Cells and Development 2015 24 1 93 103 10.1089/scd.2014.0155 2-s2.0-84919782445 25036865 PMC4273193

56 Weiss D. J. English K. Krasnodembskaya A. Isaza-Correa J. M. Hawthorne I. J. Mahon B. P. The necrobiology of mesenchymal stromal cells affects therapeutic efficacy Frontiers in Immunology 2019 10 1 12 31214185 10.3389/fimmu.2019.01228 PMC6557974

57 Goodman R. R. Davies J. E. Mesenchymal Stromal Cells and Their derivatives – putative Therapeutics in the Management of Autoimmune Pancreatitis FEBS Open Bio 2020 10 6 969 978 10.1002/2211-5463.12866 32323467 PMC7262915

58 Galipeau J. Krampera M. Leblanc K. Mesenchymal stromal cell variables influencing clinical potency: the impact of viability, fitness, route of Administration and Host Predisposition Cytotherapy 2021 23 5 368 372 10.1016/j.jcyt.2020.11.007 33714704 PMC11708105

59 Bazhanov N. Ylostalo J. H. Bartosh T. J. Intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs Stem Cell Research and Therapy 2016 7 1 1 14 10.1186/s13287-016-0284-5 2-s2.0-84957588494 26864573 PMC4748482

60 Eggenhofer E. Benseler V. Kroemer A. Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion Frontiers in Immunology 2012 3 p. 297 10.3389/fimmu.2012.00297 2-s2.0-84872914245 PMC3458305 23056000

61 Li X. Shang B. Li Y.-n. Shi Y. Shao C. IFN γ and TNF α synergistically induce apoptosis of mesenchymal stem/stromal cells via the induction of nitric oxide Stem Cell Research & Therapy 2019 10 18 1 11 10.1186/s13287-018-1102-z 2-s2.0-85059870305 30635041 PMC6330503

62 Liu Y. Wang L. Kikuiri T. Mesenchymal stem cell-based tissue regeneration is gverned by recipient T lymphocyte via IFN- γ and TNF- α Nature Medicine 2012 17 12 1594 1601 10.1038/nm.2542 PMC3233650 22101767

63 Mancuso P. Burke A. J. Raman S. Mesenchymal stem cell therapy for osteoarthritis: how apoptotic cells modulate inflammation Osteoarthritis and Cartilage 2018 26 S297 S297 10.1016/j.joca.2018.02.596

64 Chang C. L. Leu S. Sung H. C. Impact of apoptotic adipose-derived mesenchymal stem cells on attenuating organ damage and reducing mortality in rat sepsis syndrome induced by cecal puncture and ligation Journal of Translational Medicine 2012 10 1 p. 1 10.1186/1479-5876-10-244 PMC3543276 23217183

65 Cheung T. S. Galleu A. von Bonin M. Bornhäuser M. Dazzi F. Apoptotic mesenchymal stromal cells induce prostaglandin E2 in monocytes: implications for the monitoring of mesenchymal stromal cell activity Haematologica 2019 104 438 441 10.3324/haematol.2018.214767 PMC6886441 30846505

66 Amin N. Tan X. Ren Q. Recent advances of induced pluripotent stem cells application in neurodegenerative diseases Progress in Neuro-Psychopharmacology and Biological Psychiatry 2019 95, article 109674 10.1016/j.pnpbp.2019.109674 2-s2.0-85068193274 31255650

67 Gao L. Cen S. Wang P. Autophagy improves the immunosuppression of CD4+T cells by mesenchymal stem cells through transforming growth Factor- β 1 Stem Cells Translational Medicine 2016 5 11 1496 1505 10.5966/sctm.2015-0420 2-s2.0-84992116203 27400793 PMC5070508

68 Lv B. Hua T. Li F. Hypoxia-inducible factor 1 α protects mesenchymal stem cells against oxygen-glucose deprivation-induced injury via autophagy induction and PI3K/AKT/mTOR signaling pathway American Journal of Translational Research 2017 9 5 2492 2499 28559999 PMC5446531

69 Zhang Z. Yang C. Shen M. Autophagy mediates the beneficial effect of hypoxic preconditioning on bone marrow mesenchymal stem cells for the therapy of myocardial infarction Stem Cell Research and Therapy 2017 8 1 1 17 28420436 10.1186/s13287-017-0543-0 PMC5395756

70 Wang B. Jia H. Zhang B. Pre-incubation with huc MSC-exosomes prevents cisplatin-induced nephrotoxicity by activating autophagy Stem Cell Research and Therapy 2017 8 1 1 14 10.1186/s13287-016-0463-4 2-s2.0-85018466363 28388958 PMC5385032

71 Hou J. Han Z. P. Jing Y. Y. Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells Cell Death and Disease 2013 4 10 1 10 10.1038/cddis.2013.338 2-s2.0-84887445881 PMC3824648 24113178

72 Park H. J. Shin J. Y. Kim H. N. Oh S. H. Lee P. H. Neuroprotective effects of mesenchymal stem cells through autophagy modulation in a Parkinsonian model Neurobiology of Aging 2014 35 8 1920 1928 10.1016/j.neurobiolaging.2014.01.028 2-s2.0-84899915023 24629674

73 Tseng N. Lambie S. C. Huynh C. Q. Mitochondrial transfer from mesenchymal stem cells improves neuronal metabolism after oxidant injury in vitro: the role of miro 1 Journal of Cerebral Blood Flow and Metabolism 2021 41 4 761 770 10.1177/0271678X20928147 32501156 PMC7983509

74 Li C. Cheung M. K. H. Han S. Mesenchymal stem cells and their mitochondrial transfer: a double-edged sword Bioscience Reports 2019 39 5, article BSR20182417 10.1042/BSR20182417 2-s2.0-85065599515 30979829 PMC6500894

75 Liu D. Gao Y. Liu J. Intercellular mitochondrial transfer as a means of tissue revitalization Signal Transduction and Targeted Therapy 2021 6 1 p. 65 10.1038/s41392-020-00440-z 33589598 PMC7884415

76 Ahmad T. Mukherjee S. Pattnaik B. Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy The EMBO Journal 2014 33 9 994 1010 10.1002/embj.201386030 2-s2.0-84899805263 24431222 PMC4193933

77 Théry C. Witwer K. W. Aikawa E. Minimal information for studies of extracellular vesicles 2018 (MISEV 2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines Journal of Extracellular Vesicles 2018 7 1, article 1535750 10.1080/20013078.2018.1535750 PMC6322352 30637094

78 Lin W. Huang L. Li Y. Mesenchymal stem cells and cancer: clinical challenges and opportunities BioMed Research International 2019 2019 12 2820853 10.1155/2019/2820853 2-s2.0-85066047248 31205939 PMC6530243

79 Ni H. Yang S. Siaw-Debrah F. Exosomes derived from bone mesenchymal stem cells ameliorate early inflammatory responses following traumatic brain injury Frontiers in Neuroscience 2019 13 p. 14 10.3389/fnins.2019.00014 2-s2.0-85065481503 PMC6354067 30733666

80 Li Z. Liu F. He X. Yang X. Shan F. Feng J. Exosomes derived from mesenchymal stem cells attenuate inflammation and demyelination of the central nervous system in EAE rats by regulating the polarization of microglia International Immunopharmacology 2019 67 268 280 10.1016/j.intimp.2018.12.001 2-s2.0-85058446393 30572251

81 Mao F. Wu Y. Tang X. Exosomes derived from human umbilical cord mesenchymal stem cells relieve inflammatory bowel disease in mice BioMed Research International 2017 2017 12 5356760 10.1155/2017/5356760 2-s2.0-85020045834 28589143 PMC5447283

82 Pavlyukov M. S. Yu H. Bastola S. Apoptotic cell-derived extracellular vesicles promote malignancy of glioblastoma via intercellular transfer of splicing factors Cancer Cell 2018 34 1 119 135.e10 10.1016/j.ccell.2018.05.012 2-s2.0-85048162598 29937354 PMC6048596

83 Caruso S. Poon I. K. H. Apoptotic cell-derived extracellular vesicles: More than just debris Frontiers in Immunology 2018 9 1486 1486 10.3389/fimmu.2018.01486 2-s2.0-85049240185 30002658 PMC6031707

84 Kakarla R. Hur J. Kim Y. J. Kim J. Chwae Y.-J. Apoptotic cell-derived exosomes: messages from dying cells Experimental & Molecular Medicine 2020 52 1 1 6 10.1038/s12276-019-0362-8 31915368 PMC7000698

85 Meiliana A. Dewi N. M. Wijaya A. Mesenchymal stem cell secretome: Cell-free therapeutic strategy in regenerative medicine The Indonesian Biomedical Journal 2019 11 2 113 124 10.18585/inabj.v11i2.839 2-s2.0-85070738571

86 Pokrovskaya L. A. Zubareva E. V. Nadezhdin S. V. Lysenko A. S. Litovkina T. L. Biological activity of mesenchymal stem cells secretome as a basis for cell-free therapeutic approach Research Results in Pharmacology 2020 6 1 57 68 10.3897/rrpharmacology.6.49413

87 Cunningham C. J. Redondo-Castro E. Allan S. M. The therapeutic potential of the mesenchymal stem cell secretome in ischaemic stroke Journal of Cerebral Blood Flow & Metabolism 2018 38 8 1276 1292 10.1177/0271678X18776802 2-s2.0-85047414403 29768965 PMC6077926

88 Ding D.-C. Shyu W.-C. Chiang M.-F. Enhancement of neuroplasticity through upregulation of β 1-integrin in human umbilical cord-derived stromal cell implanted stroke model Neurobiology of Disease 2007 27 3 339 353 10.1016/j.nbd.2007.06.010 2-s2.0-34548324336 17651977

89 Lin Y.-T. Chern Y. Shen C.-K. J. Human mesenchymal stem cells prolong survival and ameliorate motor deficit through trophic support in Huntington's disease mouse models PloS One 2011 6 8, article e22924 10.1371/journal.pone.0022924 2-s2.0-79961147771 21850243 PMC3151281

90 Wang F. Yasuhara T. Shingo T. Intravenous administration of mesenchymal stem cells exerts therapeutic effects on parkinsonian model of rats: focusing on neuroprotective effects of stromal cell-derived factor-1 α BMC Neuroscience 2010 11 1 1 9 10.1186/1471-2202-11-52 2-s2.0-77951248824 PMC2873592 20420688

91 Collawn S. S. Patel S. Adipose-derived stem cells, their secretome, and wound healing Journal of Cell Science & Therapy 2014 5 3 p. 1 10.4172/2157-7013.1000165

92 Ebrahimian T. G. Pouzoulet F. Squiban C. Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing Arteriosclerosis, Thrombosis and Vascular Biology 2009 29 4 503 510 10.1161/ATVBAHA.108.178962 2-s2.0-63449142051 19201690

93 Huang S.-P. Huang C.-H. Shyu J.-F. Promotion of wound healing using adipose-derived stem cells in radiation ulcer of a rat model Journal of Biomedical Science 2013 20 1 1 10 10.1186/1423-0127-20-51 2-s2.0-84880334562 23876213 PMC3724586

94 Zografou A. Papadopoulos O. Tsigris C. Autologous transplantation of adipose-derived stem cells enhances skin graft survival and wound healing in diabetic rats Annals of Plastic Surgery 2013 71 2 225 232 10.1097/SAP.0b013e31826af01a 2-s2.0-84880573343 23636118

95 Ahangar P. Mills S. J. Cowin A. J. Mesenchymal stem cell secretome as an emerging cell-free alternative for improving wound repair International Journal of Molecular Sciences 2020 21 19, article 7038 10.3390/ijms21197038 32987830 PMC7583030

96 Kehl D. Generali M. Mallone A. Proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential NPJ Regenerative Medicine 2019 4 1 1 13 31016031 10.1038/s41536-019-0070-y PMC6467904

97 Mitchell R. Mellows B. Sheard J. Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins Stem Cell Research & Therapy 2019 10 1 p. 116 10.1186/s13287-019-1213-1 2-s2.0-85063996688 30953537 PMC6451311

98 Wang Y. Long W. Cao Y. Li J. You L. Fan Y. Mesenchymal stem cell-derived secretomes for therapeutic potential of premature infant diseases Bioscience Reports 2020 40 5, article BSR20200241 10.1042/BSR20200241 32320046 PMC7953482

99 Al-Azzawi B. McGuigan D. H. Koivula F. N. M. The secretome of mesenchymal stem cells prevents islet beta cell apoptosis via an IL-10-dependent mechanism The Open Stem Cell Journal 2020 6 1 1 12 10.2174/1876893802006010001

100 Hsu W.-T. Lin C.-H. Chiang B.-L. Jui H.-Y. Wu K. K.-Y. Lee C.-M. Prostaglandin E2Potentiates Mesenchymal Stem Cell–Induced IL-10+IFN- γ +CD4+Regulatory T Cells To Control Transplant Arteriosclerosis The Journal of Immunology 2013 190 5 2372 2380 10.4049/jimmunol.1202996 2-s2.0-84874239821 23359497

101 Ogata K. Osugi M. Kawai T. Secretomes of mesenchymal stem cells induce early bone regeneration by accelerating migration of stem cells Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 2018 30 5 445 451 10.1016/j.ajoms.2018.04.002 2-s2.0-85046351715

102 Qin Y. Wang L. Gao Z. Chen G. Zhang C. Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo Scientific Reports 2016 6 1 1 11 10.1038/srep21961 2-s2.0-84959421655 26911789 PMC4766421

103 Eleuteri S. Fierabracci A. Insights into the secretome of mesenchymal stem cells and its potential applications International Journal of Molecular Sciences 2019 20 18, article 4597 10.3390/ijms20184597 2-s2.0-85072377863 31533317 PMC6770239

104 Xin H. Li Y. Cui Y. Yang J. J. Zhang Z. G. Chopp M. Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats Journal of Cerebral Blood Flow & Metabolism 2013 33 11 1711 1715 10.1038/jcbfm.2013.152 2-s2.0-84887117028 23963371 PMC3824189

105 Zhu L.-P. Tian T. Wang J.-Y. Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction Theranostics 2018 8 22 6163 6177 10.7150/thno.28021 2-s2.0-85059327416 30613290 PMC6299684

106 Zou X. Zhang G. Cheng Z. Microvesicles derived from human Wharton’s Jelly mesenchymal stromal cells ameliorate renal ischemia-reperfusion injury in rats by suppressing CX3CL1 Stem Cell Research & Therapy 2014 5 2 1 13 10.1186/scrt428 2-s2.0-84919772481 24646750 PMC4055103

107 Mazini L. Rochette L. Admou B. Amal S. Malka G. Hopes and limits of adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) in wound healing International Journal of Molecular Sciences 2020 21 4, article 1306 10.3390/ijms21041306 32075181 PMC7072889

108 Wu Q. Ji F. K. Wang J. H. Nan H. Liu D. L. Stromal cell-derived factor 1 promoted migration of adipose-derived stem cells to the wounded area in traumatic rats Biochemical and Biophysical Research Communications 2015 467 1 140 145 10.1016/j.bbrc.2015.09.097 2-s2.0-84944880782 26392311

109 Yu X. Chen D. Zhang Y. Overexpression of CXCR4 in mesenchymal stem cells promotes migration, neuroprotection and angiogenesis in a rat model of stroke Journal of the Neurological Sciences 2012 316 1-2 141 149 10.1016/j.jns.2012.01.001 2-s2.0-84862832130 22280945

110 Zhang D. Fan G. C. Zhou X. Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium Journal of Molecular and Cellular Cardiology 2008 44 2 281 292 10.1016/j.yjmcc.2007.11.010 2-s2.0-39149089319 18201717 PMC2601571

111 Fu L. Liu G. Halim A. Ju Y. Luo Q. Song A. G. Mesenchymal Stem Cell Migration and Tissue Repair Cells 2019 8 8 p. 784 10.3390/cells8080784 31357692 PMC6721499

112 Qin Y. Zhou Z. Zhang F. Induction of regulatory B-cells by mesenchymal stem cells is affected by SDF-1 α -CXCR7 Cellular Physiology and Biochemistry 2015 37 1 117 130 10.1159/000430338 2-s2.0-84940665386 26303308

113 Zheng X. B. He X. W. Zhang L. J. Bone marrow-derived CXCR4-overexpressing MSCs display increased homing to intestine and ameliorate colitis-associated tumorigenesis in mice Gastroenterology Report 2019 7 2 127 138 10.1093/gastro/goy017 2-s2.0-85065746081 30976426 PMC6454852

114 Lim M. Wang W. Liang L. Intravenous injection of allogeneic umbilical cord-derived multipotent mesenchymal stromal cells reduces the infarct area and ameliorates cardiac function in a porcine model of acute myocardial infarction Stem Cell Research and Therapy 2018 9 1 1 17 29751831 10.1186/s13287-018-0888-z PMC5948807

115 Liau L. L. Looi Q. H. Chia W. C. Subramaniam T. Ng M. H. Law J. X. Treatment of spinal cord injury with mesenchymal stem cells Cell & Bioscience 2020 10 1 p. ??? 10.1186/s13578-020-00475-3 PMC7510077 32983406

116 Al Faqeh H. Nor Hamdan B. M. Y. Chen H. C. Aminuddin B. S. Ruszymah B. H. I. The potential of intra-articular injection of chondrogenic-induced bone marrow stem cells to retard the progression of osteoarthritis in a sheep model Experimental Gerontology 2012 47 6 458 464 10.1016/j.exger.2012.03.018 2-s2.0-84861196480 22759409

117 Zhang S. Chuah S. J. Lai R. C. Hui J. H. P. Lim S. K. Toh W. S. MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity Biomaterials 2018 156 16 27 10.1016/j.biomaterials.2017.11.028 2-s2.0-85034834386 29182933

118 Kurtz A. Mesenchymal stem cell delivery routes and fate International Journal of Stem Cells 2008 1 1 1 7 10.15283/ijsc.2008.1.1.1 2-s2.0-80051578440 24855503 PMC4021770

119 Kanelidis A. J. Premer C. Lopez J. Balkan W. Hare J. M. Route of delivery modulates the efficacy of mesenchymal stem cell therapy for myocardial infarction: a meta-analysis of preclinical studies and clinical trials Circulation Research 2017 120 7 1139 1150 10.1161/CIRCRESAHA.116.309819 2-s2.0-85008350885 28031416 PMC5656247

120 Klass M. Gavrikov V. Drury D. Intravenous mononuclear marrow cells reverse neuropathic pain from experimental mononeuropathy Anesthesia & Analgesia 2007 104 4 944 948 10.1213/01.ane.0000258021.03211.d0 2-s2.0-34047254693 17377111

121 Harting M. T. Jimenez F. Xue H. Intravenous mesenchymal stem cell therapy for traumatic brain injury Journal of Neurosurgery 2009 110 6 1189 1197 10.3171/2008.9.JNS08158 2-s2.0-67650845362 19301973 PMC2889620

122 Vandergriff A. C. de Andrade J. B. M. Tang J. Intravenous cardiac stem cell-derived exosomes ameliorate cardiac dysfunction in doxorubicin induced dilated cardiomyopathy Stem Cells International 2015 2015 8 960926 10.1155/2015/960926 2-s2.0-84940746206 26351465 PMC4553317

123 Chen J.-y. An R. Liu Z.-j. Therapeutic effects of mesenchymal stem cell-derived microvesicles on pulmonary arterial hypertension in rats Acta Pharmacologica Sinica 2014 35 9 1121 1128 10.1038/aps.2014.61 2-s2.0-84906913674 25088001 PMC4155533

124 Keyhanmanesh R. Rahbarghazi R. Aslani M. R. Hassanpour M. Ahmadi M. Systemic delivery of mesenchymal stem cells condition media in repeated doses acts as magic bullets in restoring IFN- γ /IL-4 balance in asthmatic rats Life Sciences 2018 212 30 36 10.1016/j.lfs.2018.09.049 2-s2.0-85054526366 30268855

125 Tan J. L. Lau S. N. Leaw B. Amnion epithelial cell-derived exosomes restrict lung injury and enhance endogenous lung repair Stem Cells Translational Medicine 2018 7 2 180 196 10.1002/sctm.17-0185 2-s2.0-85041114330 29297621 PMC5788876

126 Sun X. Shan A. Wei Z. Xu B. Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy Biochemical and Biophysical Research Communications 2018 503 4 2611 2618 10.1016/j.bbrc.2018.08.012 2-s2.0-85051656322 30126637

127 Hu G.-w. Li Q. Niu X. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice Stem Cell Research & Therapy 2015 6 1 1 15 10.1186/scrt546 2-s2.0-84938929476 26268554 PMC4533800

128 Melzer C. Ohe J. V. D. Hass R. Anti-tumor effects of exosomes derived from drug-incubated permanently growing human MSC International Journal of Molecular Sciences 2020 21 19, article 7311 10.3390/ijms21197311 33023058 PMC7582671

129 Wang L. Chopp M. Szalad A. Exosomes derived from schwann cells ameliorate peripheral neuropathy in type 2 diabetic mice Diabetes 2020 69 4 749 759 10.2337/db19-0432 31915154 PMC7085247

130 Sengupta V. Sengupta S. Lazo A. Woods P. Nolan A. Bremer N. Exosomes derived from bone marrow mesenchymal stem cells as treatment for severe COVID-19 Stem Cells and Development 2020 29 12 747 754 10.1089/scd.2020.0080 32380908 PMC7310206

131 Rashidbenam Z. Jasman M. H. Hafez P. Overview of urethral reconstruction by tissue engineering: current strategies, clinical status and future direction Tissue Engineering and Regenerative Medicine 2019 16 4 365 384 10.1007/s13770-019-00193-z 2-s2.0-85069996108 31413941 PMC6675804

132 Phadke A. Hwang Y. S. Hee Kim S. Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells European Cells and Materials 2012 25 114 129 10.22203/ecm.v025a08 PMC4387005 23329467

133 Enderami S. E. Kehtari M. Abazari M. F. Generation of insulin-producing cells from human induced pluripotent stem cells on PLLA/PVA nanofiber scaffold Artificial Cells, Nanomedicine and Biotechnology 2018 46 Supplement 1 1062 1069 10.1080/21691401.2018.1443466 2-s2.0-85042920706 29486602

134 Abazari M. F. Soleimanifar F. Nouri Aleagha M. PCL/PVA nanofibrous scaffold improve insulin-producing cells generation from human induced pluripotent stem cells Gene 2018 671 50 57 10.1016/j.gene.2018.05.115 2-s2.0-85047854832 29860065

135 Mansour R. N. Barati G. Soleimani M. Generation of high-yield insulin producing cells from human-induced pluripotent stem cells on polyethersulfone nanofibrous scaffold Artificial Cells, Nanomedicine and Biotechnology 2018 46 Supplement 1 733 739 10.1080/21691401.2018.1434663 2-s2.0-85041920450 29433332

136 Nadri S. Barati G. Mostafavi H. Esmaeilzadeh A. Enderami S. E. Differentiation of conjunctiva mesenchymal stem cells into secreting islet beta cells on plasma treated electrospun nanofibrous scaffold Artificial Cells, Nanomedicine and Biotechnology 2018 46 Supplement 1 178 187 10.1080/21691401.2017.1416391 2-s2.0-85038091199 29241367

137 Guo R. Morimatsu M. Feng T. Stem cell-derived cell sheet transplantation for heart tissue repair in myocardial infarction Stem Cell Research & Therapy 2020 11 1 p. 19 10.1186/s13287-019-1536-y 31915074 PMC6950817

138 Imashiro C. Shimizu T. Fundamental technologies and recent advances of cell-sheet-based tissue engineering International Journal of Molecular Sciences 2021 22 1 p. 425 10.3390/ijms22010425 33401626 PMC7795487

139 Ramezankhani R. Torabi S. Minaei N. Two decades of global progress in authorized advanced therapy medicinal products: an emerging revolution in therapeutic strategies Frontiers in Cell and Developmental Biology 2020 8 547653 547653 10.3389/fcell.2020.547653 33392179 PMC7773756

140 Meng F. Xu R. Wang S. Human umbilical cord-derived mesenchymal stem cell therapy in patients with COVID-19: a phase 1 clinical trial Signal Transduction and Targeted Therapy 2020 5 1 p. 172 10.1038/s41392-020-00286-5 32855385 PMC7450163

141 Chambers D. C. Enever D. Ilic N. A phase 1b study of mesenchymal stromal cell therapy for idiopathic pulmonary fibrosis Cytotherapy 2014 16 4 p. S12 10.1016/j.jcyt.2014.01.029 25039426

142 Wilson J. G. Liu K. D. Zhuo H. Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial The Lancet Respiratory Medicine 2015 3 1 24 32 10.1016/S2213-2600(14)70291-7 2-s2.0-84920500515 25529339 PMC4297579

143 Brizuela C. Meza G. Urrejola D. Cell-based regenerative endodontics for treatment of periapical lesions: a randomized, controlled phase I/II clinical trial Journal of Dental Research 2020 99 5 523 529 10.1177/0022034520913242 32202965

144 Tanikawa D. Y. S. Pinheiro C. C. G. Almeida M. C. A. Deciduous dental pulp stem cells for maxillary alveolar reconstruction in cleft lip and palate patients Stem Cells International 2020 2020 9 6234167 10.1155/2020/6234167 32256610 PMC7091546

145 Vega A. Martín-Ferrero M. A. Canto F. D. Treatment of knee osteoarthritis with allogeneic bone marrow mesenchymal stem cells: a randomized controlled trial Transplantation 2015 99 8 1681 1690 10.1097/TP.0000000000000678 2-s2.0-84939518963 25822648

146 Tompkins B. A. Difede D. L. Khan A. Allogeneic mesenchymal stem cells ameliorate aging Frailty: a phase II randomized, double-blind, placebo-controlled clinical trial Journal of gerontology 2017 72 11 1513 1522 10.1093/gerona/glx137 2-s2.0-85032646300 28977399 PMC5861900

147 Bolli R. Hare J. M. March K. L. Rationale and design of the CONCERT-HF trial (combination of mesenchymal and c-kit+Cardiac stem cells as regenerative therapy for heart failure) Circulation Research 2018 122 12 1703 1715 10.1161/CIRCRESAHA.118.312978 2-s2.0-85051711213 29703749 PMC5993622

148 Bolli R. Hare J. M. Henry T. D. Rationale and design of the SENECA (StEm cell iNjECtion in cAncer survivors) trial American Heart Journal 2018 201 54 62 10.1016/j.ahj.2018.02.009 2-s2.0-85046798158 29910056 PMC7282462

149 Hare J. M. Fishman J. E. Gerstenblith G. Comparison of allogeneic vs autologous bone marrow–derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy Journal of Autism and Developmental Disorders 2012 47 3 549 562

150 Hare J. M. DL D. F. Rieger A. C. Randomized Comparison of Allogeneic Versus Autologous Mesenchymal Stem Cells for Nonischemic Dilated Cardiomyopathy: POSEIDON-DCM Trial Journal of the American College of Cardiology 2017 69 5 526 537 10.1016/j.jacc.2016.11.009 2-s2.0-85011650223 27856208 PMC5291766

151 Florea V. Rieger A. C. DiFede D. L. Dose comparison study of allogeneic mesenchymal stem cells in patients with ischemic cardiomyopathy (The TRIDENT Study) Circulation Research 2017 121 11 1279 1290 10.1161/CIRCRESAHA.117.311827 2-s2.0-85037708130 28923793 PMC8742223

152 Heldman A. W. Difede D. L. Fishman J. E. Transendocardial mesenchymal stem cells and mononuclear bone marrow cells for ischemic cardiomyopathy: the TAC-HFT randomized trial JAMA 2014 311 1 62 73 10.1001/jama.2013.282909 2-s2.0-84891941987 24247587 PMC4111133

153 Kharaziha P. Hellström P. M. Noorinayer B. Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial European Journal of Gastroenterology and Hepatology 2009 21 10 1199 1205 10.1097/MEG.0b013e32832a1f6c 2-s2.0-70349733505 19455046

154 Lee W. S. Kim H. J. Kim K. I. Kim G. B. Jin W. Intra-articular injection of autologous adipose tissue-derived mesenchymal stem cells for the treatment of knee osteoarthritis: a phase IIb, randomized, placebo-controlled clinical trial Stem Cells Translational Medicine 2019 8 6 504 511 10.1002/sctm.18-0122 2-s2.0-85062590697 30835956 PMC6525553

155 Bartolucci J. Verdugo F. J. González P. L. Safety and efficacy of the intravenous infusion of umbilical cord mesenchymal stem cells in patients with heart failure Circulation Research 2017 121 10 1192 1204 10.1161/CIRCRESAHA.117.310712 2-s2.0-85032618937 28974553 PMC6372053

156 Trounson A. Thakar R. G. Lomax G. Gibbons D. Clinical trials for stem cell therapies BMC Medicine 2011 9 1 1 7 21569277 10.1186/1741-7015-9-52 PMC3098796

157 Kim M. Rhee J.-K. Choi H. Passage-dependent accumulation of somatic mutations in mesenchymal stromal cells during in vitro culture revealed by whole genome sequencing Scientific Reports 2017 7 1 14508 14510 10.1038/s41598-017-15155-5 2-s2.0-85032982297 29109420 PMC5674020

158 Nikitina V. Astrelina T. Nugis V. Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture PLoS One 2018 13 2, article e0192445 10.1371/journal.pone.0192445 2-s2.0-85042070805 29432491 PMC5809118

159 Cabral J. M. S. Stem Cell Manufacturing 2017 Elsevier

160 Kwon S. Y. Chun S. Y. Ha Y.-S. Hypoxia enhances cell properties of human mesenchymal stem cells Tissue Engineering and Regenerative Medicine 2017 14 5 595 604 10.1007/s13770-017-0068-8 2-s2.0-85029953634 30603513 PMC6171625

161 Dessels C. Durandt C. Pepper M. S. Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion Platelets 2019 30 3 356 367 10.1080/09537104.2018.1445840 2-s2.0-85044214149 29553865

162 Wangler S. Kamali A. Wapp C. Uncovering the secretome of mesenchymal stromal cells exposed to healthy, traumatic, and degenerative intervertebral discs: a proteomic analysis Stem Cell Research & Therapy 2021 12 1 p. 11 10.1186/s13287-020-02062-2 33413584 PMC7789679

163 Gimona M. Brizzi M. F. Choo A. B. H. Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles Cytotherapy 2021 23 5 373 380 10.1016/j.jcyt.2021.01.001 33934807

164 Dou L. Wu Y. Yan Q. Wang J. Zhang Y. Ji P. Secretome profiles of immortalized dental follicle cells using iTRAQ-based proteomic analysis Scientific Reports 2017 7 1, article ??? 10.1038/s41598-017-07467-3 2-s2.0-85026808227 28779163 PMC5544752

165 Chen T. S. Arslan F. Yin Y. Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs Journal of Translational Medicine 2011 9 1 p. 47 10.1186/1479-5876-9-47 2-s2.0-79955031226 PMC3100248 21513579

166 Waters R. Subham S. Pacelli S. Modaresi S. Chakravarti A. R. Paul A. Development of MicroRNA-146a-Enriched stem cell secretome for wound-healing applications Molecular Pharmaceutics 2019 16 10 4302 4312 10.1021/acs.molpharmaceut.9b00639 2-s2.0-85072331637 31398053 PMC7260687

167 Giancola R. Bonfini T. Iacone A. Cell therapy: cGMP facilities and manufacturing Muscles, Ligaments and Tendons Journal 2012 2 3 243 247 PMC3666518 23738304

168 Viganò M. Giordano R. Lazzari L. Challenges of running a GMP facility for regenerative medicine in a public hospital Regenerative Medicine 2017 12 7 803 813 10.2217/rme-2017-0051 2-s2.0-85033552062 29115906

169 Dominici M. Le Blanc K. Mueller I. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement Cytotherapy 2006 8 4 315 317 10.1080/14653240600855905 2-s2.0-33747713246 16923606

170 Kalinina N. Kharlampieva D. Loguinova M. Characterization of secretomes provides evidence for adipose-derived mesenchymal stromal cells subtypes Stem Cell Research & Therapy 2015 6 1 p. ??? 10.1186/s13287-015-0209-8 2-s2.0-84947867463 PMC4642680 26560317

171 Witwer K. W. Van Balkom B. W. M. Bruno S. Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications Journal of Extracellular Vesicles 2019 8 1, article 1609206 10.1080/20013078.2019.1609206 2-s2.0-85065065615 31069028 PMC6493293

172 Qiu G. Zheng G. Ge M. Mesenchymal stem cell-derived extracellular vesicles affect disease outcomes via transfer of microRNAs Stem Cell Research & Therapy 2018 9 1 p. 320 10.1186/s13287-018-1069-9 2-s2.0-85056982817 30463593 PMC6249826

173 Asgarpour K. Shojaei Z. Amiri F. Exosomal microRNAs derived from mesenchymal stem cells: cell-to-cell messages Cell Communication and Signaling 2020 18 1 p. 149 10.1186/s12964-020-00650-6 32917227 PMC7488404

174 Wang K. X. Xu L. L. Rui Y. F. The effects of secretion factors from umbilical cord derived mesenchymal stem cells on osteogenic differentiation of mesenchymal stem cells PLoS One 2015 10 3, article e0120593 10.1371/journal.pone.0120593 2-s2.0-84925847497 25799169 PMC4370627

175 Sun Q. Zhang Z. Sun Z. The potential and challenges of using stem cells for cardiovascular repair and regeneration Genes and Diseases 2014 1 1 113 119 10.1016/j.gendis.2014.07.003 2-s2.0-84931826011 25642448 PMC4307803

176 Kim H. J. Park J.-S. Usage of human mesenchymal stem cells in Cell-based therapy: advantages and disadvantages Development and Reproduction 2017 21 1 1 10 10.12717/DR.2017.21.1.001 28484739 PMC5409204

177 Katolikova N. V. Malashicheva A. B. Gainetdinov R. R. Cell replacement therapy in Parkinson’s disease—history of development and prospects for use in clinical practice Molecular Biology 2020 54 6 827 839 10.1134/S0026893320060060 33276357

178 Schuetz C. Anazawa T. Cross S. E. β cell replacement therapy: the next 10 years Transplantation 2018 102 2 215 229 10.1097/TP.0000000000001937 2-s2.0-85041572027 28885496

179 Grange C. Tapparo M. Bruno S. Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging International Journal of Molecular Medicine 2014 33 5 1055 1063 10.3892/ijmm.2014.1663 2-s2.0-84899577244 24573178 PMC4020482

180 Han Y. Seyfried D. Meng Y. Multipotent mesenchymal stromal cell-derived exosomes improve functional recovery after experimental intracerebral hemorrhage in the rat Journal of Neurosurgery 2018 131 1 290 300 10.3171/2018.2.JNS171475 2-s2.0-85068498755 30028267

181 Otero-Ortega L. Laso-García F. Gómez-de Frutos M. D. White matter repair after extracellular vesicles administration in an experimental animal model of subcortical stroke Scientific Reports 2017 7 1, article 44433 10.1038/srep44433 2-s2.0-85015433259 PMC5353554 28300134

182 Steingen C. Brenig F. Baumgartner L. Schmidt J. Schmidt A. Bloch W. Characterization of key mechanisms in transmigration and invasion of mesenchymal stem cells Journal of Molecular and Cellular Cardiology 2008 44 6 1072 1084 10.1016/j.yjmcc.2008.03.010 2-s2.0-44649178243 18462748

183 Matsushita T. Kibayashi T. Katayama T. Mesenchymal stem cells transmigrate across brain microvascular endothelial cell monolayers through transiently formed inter-endothelial gaps Neuroscience Letters 2011 502 1 41 45 10.1016/j.neulet.2011.07.021 2-s2.0-80051791998 21798315

184 Vizoso F. J. Eiro N. Cid S. Schneider J. Perez-Fernandez R. Mesenchymal stem cell secretome: toward cell-free therapeutic strategies in regenerative medicine International Journal of Molecular Sciences 2017 18 9, article 1852 10.3390/ijms18091852 2-s2.0-85028305639 28841158 PMC5618501

185 Breitbach M. Bostani T. Roell W. Potential risks of bone marrow cell transplantation into infarcted hearts Blood 2007 110 4 1362 1369 10.1182/blood-2006-12-063412 2-s2.0-34548044258 17483296

186 Song L. Webb N. E. Song Y. Tuan R. S. Identification and functional analysis of candidate genes regulating mesenchymal stem cell self-renewal and multipotency Stem Cells 2006 24 7 1707 1718 10.1634/stemcells.2005-0604 2-s2.0-33749060495 16574750

187 Ma J. Zhao Y. Sun L. Exosomes derived from AKt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D Stem Cells Translational Medicine 2017 6 1 51 59 10.5966/sctm.2016-0038 2-s2.0-85017344289 28170176 PMC5442756

188 Xu X. P. Huang L. L. Hu S. L. Genetic modification of mesenchymal stem cells overexpressing angiotensin II type 2 receptor increases cell migration to injured lung in LPS-induced acute lung injury mice Stem Cells Translational Medicine 2018 7 10 721 730 10.1002/sctm.17-0279 2-s2.0-85053180647 30133167 PMC6186265

189 Park J. S. Suryaprakash S. Lao Y.-H. Leong K. W. Engineering mesenchymal stem cells for regenerative medicine and drug delivery Methods 2015 84 3 16 10.1016/j.ymeth.2015.03.002 2-s2.0-84935851134 25770356 PMC4526354

190 Fierro F. A. Kalomoiris S. Sondergaard C. S. Nolta J. A. Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy Stem Cells 2011 29 11 1727 1737 10.1002/stem.720 2-s2.0-80054885259 21898687 PMC3784258

191 Lim J. Y. Park S. I. Kim S. M. Neural differentiation of brain-derived neurotrophic factor-expressing human umbilical cord blood-derived mesenchymal stem cells in culture via TrkB-Mediated ERK and β -catenin phosphorylation and following transplantation into the developing brain Cell Transplantation 2011 20 11-12 1855 1866 10.3727/096368910X557236 2-s2.0-84863012475 21375801

192 Scheper V. Schwieger J. Hamm A. Lenarz T. Hoffmann A. BDNF-overexpressing human mesenchymal stem cells mediate increased neuronal protection in vitro Journal of Neuroscience Research 2019 97 11 1414 1429 10.1002/jnr.24488 2-s2.0-85072508885 31257632 PMC6772136

193 Zhang Y. Li R. Rong W. Therapeutic effect of hepatocyte growth factor-overexpressing bone marrow-derived mesenchymal stem cells on CCl 4-induced hepatocirrhosis Cell Death & Disease 2018 9 12 1 12 30538216 10.1038/s41419-018-1239-9 PMC6290007

194 Zhang S. Jiang W. Ma L. Liu Y. Zhang X. Wang S. Nrf 2 transfection enhances the efficacy of human amniotic mesenchymal stem cells to repair lung injury induced by lipopolysaccharide Journal of Cellular Biochemistry 2018 119 2 1627 1636 10.1002/jcb.26322 2-s2.0-85038882777 28905450

195 Jiang J. Wei D. Sun L. A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells Oncology Reports 2014 31 2 781 787 10.3892/or.2013.2898 2-s2.0-84892409893 24317390

196 Duffy G. P. D'Arcy S. Ahsan T. Nerem R. M. O'Brien T. Barry F. Mesenchymal stem cells overexpressing ephrin-b2 rapidly adopt an early endothelial phenotype with simultaneous reduction of osteogenic potential Tissue Engineering Part A 2010 16 9 2755 2768 10.1089/ten.tea.2009.0623 2-s2.0-77956074155 20491587

197 Chae Y. J. Jun D. W. Lee J. S. The use of Foxa2-Overexpressing adipose tissue-derived stem cells in a scaffold system attenuates acute liver injury Gut and Liver 2019 13 4 450 460 10.5009/gnl18235 2-s2.0-85069295336 30602218 PMC6622567

198 Wang W. Zhang Y. Yang C. Transplantation of neuregulin 4-overexpressing adipose-derived mesenchymal stem cells ameliorates insulin resistance by attenuating hepatic steatosis Experimental Biology and Medicine 2019 244 7 565 578 10.1177/1535370219839643 2-s2.0-85063954393 30935234 PMC6545697

199 Huang W. Wang T. Zhang D. Mesenchymal stem cells overexpressing CXCR4 attenuate remodeling of postmyocardial infarction by releasing matrix metalloproteinase-9 Stem Cells and Development 2012 21 5 778 789 10.1089/scd.2011.0126 2-s2.0-84859464219 21671800 PMC3295858

200 Kopru C. Z. Cagnan I. Akar I. Esendagli G. Korkusuz P. Gunel-Ozcan A. Dual Effect of Glucocorticoid-Induced Tumor Necrosis Factor-Related Receptor Ligand Carrying Mesenchymal Stromal Cells on Small Cell Lung Cancer: A Preliminary in vitro Study Cytotherapy 2018 20 7 930 940 10.1016/j.jcyt.2018.05.002 2-s2.0-85049111882 30180943

201 Kim S.-W. Lee D.-W. Yu L.-H. Mesenchymal stem cells overexpressing GCP-2 improve heart function through enhanced angiogenic properties in a myocardial infarction model Cardiovascular Research 2012 95 4 495 506 10.1093/cvr/cvs224 2-s2.0-84865212543 22886775

202 Kearns-Jonker M. Dai W. Gunthart M. Genetically engineered mesenchymal stem cells influence gene expression in donor cardiomyocytes and the recipient heart Journal of Stem Cell Research & Therapy 2012 2012 Supplement 1 p. 5 10.4172/2157-7633.s1-005 PMC3487477 23125947

203 Song Y. S. Lee H. J. Doo S. H. Mesenchymal stem cells overexpressing hepatocyte growth factor (HGF) inhibit collagen deposit and improve bladder function in rat model of bladder outlet obstruction Cell Transplantation 2012 21 8 1641 1650 10.3727/096368912X637488 2-s2.0-84868146978 22506988

204 Zhao L. Liu X. Zhang Y. Enhanced cell survival and paracrine effects of mesenchymal stem cells overexpressing hepatocyte growth factor promote cardioprotection in myocardial infarction Experimental Cell Research 2016 344 1 30 39 10.1016/j.yexcr.2016.03.024 2-s2.0-84962106729 27025401

205 Lai L. Chen J. Wei X. Transplantation of MSCs overexpressing HGF into a rat model of liver fibrosis Molecular Imaging and Biology 2016 18 1 43 51 10.1007/s11307-015-0869-x 2-s2.0-84955757026 26194009

206 Li J. Zheng C.-Q. Li Y. Yang C. Lin H. Duan H.-G. Hepatocyte growth factor gene-modified mesenchymal stem cells augment sinonasal wound healing Stem Cells and Development 2015 24 15 1817 1830 10.1089/scd.2014.0521 2-s2.0-84937714739 25835956 PMC4507357

207 Liu X.-S. Li J.-F. Wang S.-S. Human umbilical cord mesenchymal stem cells infected with adenovirus expressing HGF promote regeneration of damaged neuron cells in a Parkinson’s disease model BioMed Research International 2014 2014 7 909657 10.1155/2014/909657 2-s2.0-84930989487 25276829 PMC4167956

208 Seo K. W. Sohn S. Y. Bhang D. H. Nam M. J. Lee H. W. Youn H. Y. Therapeutic effects of hepatocyte growth factor-overexpressing human umbilical cord blood-derived mesenchymal stem cells on liver fibrosis in rats Cell Biology International 2014 38 1 106 116 10.1002/cbin.10186 2-s2.0-84892998885 24115681

209 Jeong S. R. Kwon M. J. Lee H. G. Hepatocyte growth factor reduces astrocytic scar formation and promotes axonal growth beyond glial scars after spinal cord injury Experimental Neurology 2012 233 1 312 322 10.1016/j.expneurol.2011.10.021 2-s2.0-84856231980 22079829

210 Wu N. Zhang Y.-L. Wang H.-T. Overexpression of hepatocyte nuclear factor 4 α in human mesenchymal stem cells suppresses hepatocellular carcinoma development through Wnt/ β -catenin signaling pathway downregulation Cancer Biology & Therapy 2016 17 5 558 565 10.1080/15384047.2016.1177675 2-s2.0-84969245584 27124543 PMC4910927

211 Lee E. J. Choi E.-K. Kang S. K. N-cadherin determines individual variations in the therapeutic efficacy of human umbilical cord blood-derived mesenchymal stem cells in a rat model of myocardial infarction Molecular Therapy 2012 20 1 155 167 10.1038/mt.2011.202 2-s2.0-84856953479 22068423 PMC3255601

212 Razban V. Lotfi A. S. Soleimani M. HIF-1 α overexpression induces angiogenesis in mesenchymal stem cells BioResearch Open Access 2012 1 4 174 183 10.1089/biores.2012.9905 2-s2.0-84886751351 23514846 PMC3559201

213 Payne N. L. Dantanarayana A. Sun G. Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination Cell Adhesion & Migration 2012 6 3 179 189 10.4161/cam.20341 2-s2.0-84864917325 22568986 PMC3427232

214 Choi J. S. Jeong I. S. Han J. H. Cheon S. H. Kim S.-W. IL-10-secreting human MSCs generated by TALEN gene editing ameliorate liver fibrosis through enhanced anti-fibrotic activity Biomaterials Science 2019 7 3 1078 1087 10.1039/C8BM01347K 2-s2.0-85062258492 30631870

215 Nakajima M. Nito C. Sowa K. Mesenchymal stem cells overexpressing interleukin-10 promote neuroprotection in experimental acute ischemic stroke Molecular Therapy-Methods & Clinical Development 2017 6 102 111 10.1016/j.omtm.2017.06.005 2-s2.0-85029291200 28725658 PMC5502709

216 Yang F. Wu R. Jiang Z. Leptin increases mitochondrial OPA1 via GSK3-mediated OMA1 ubiquitination to enhance therapeutic effects of mesenchymal stem cell transplantation Cell Death & Disease 2018 9 5 1 17 29748581 10.1038/s41419-018-0579-9 PMC5945599

217 Xie C. Du L.-Y. Guo F. Li X. Cheng B. Exosomes derived from microRNA-101-3p-overexpressing human bone marrow mesenchymal stem cells suppress oral cancer cell proliferation, invasion, and migration Molecular and Cellular Biochemistry 2019 458 1-2 11 26 10.1007/s11010-019-03526-7 2-s2.0-85067077008 31165315

218 Yu L. Gui S. Liu Y. Exosomes derived from microRNA-199a-overexpressing mesenchymal stem cells inhibit glioma progression by down-regulating AGAP2 Aging 2019 11 15 5300 5318 10.18632/aging.102092 2-s2.0-85071039901 31386624 PMC6710058

219 Zhu G. Pei L. Lin F. Exosomes from human-bone-marrow-derived mesenchymal stem cells protect against renal ischemia/reperfusion injury via transferring miR‐199a‐3p Journal of Cellular Physiology 2019 234 12 23736 23749 10.1002/jcp.28941 2-s2.0-85071092347 31180587

220 Huleihel L. Sellares J. Cardenes N. Modified mesenchymal stem cells using mi RNA transduction alter lung injury in a bleomycin model American Journal of Physiology-Lung Cellular and Molecular Physiology 2017 313 1 L92 L103 28385811 10.1152/ajplung.00323.2016 PMC5538868

221 Wei Z. Qiao S. Zhao J. miRNA-181a over-expression in mesenchymal stem cell-derived exosomes influenced inflammatory response after myocardial ischemia-reperfusion injury Life Sciences 2019 232, article 116632 10.1016/j.lfs.2019.116632 2-s2.0-85068523367 31278944

222 Ryu S. Lee J.-M. Bae C. A. Moon C.-E. Cho K.-O. Therapeutic efficacy of neuregulin 1-expressing human adipose-derived mesenchymal stem cells for ischemic stroke PloS One 2019 14 9, article e0222587 10.1371/journal.pone.0222587 2-s2.0-85072700020 31560696 PMC6764745

223 Han S.-M. Han S.-H. Coh Y.-R. Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells Experimental & Molecular Medicine 2014 46 6, article e101 10.1038/emm.2014.28 2-s2.0-84902126864 24946789 PMC4081551

224 Bonilla-Porras A. Arevalo-Arbelaez A. Alzate-Restrepo J. Velez-Pardo C. Jimenez-Del-Rio M. PARKIN overexpression in human mesenchymal stromal cells from Wharton's jelly suppresses 6-hydroxydopamine-induced apoptosis: Potential therapeutic strategy in Parkinson's disease Cytotherapy 2018 20 1 45 61 10.1016/j.jcyt.2017.09.011 2-s2.0-85032175828 29079356

225 Gao Y. Yao A. Zhang W. Human mesenchymal stem cells overexpressing pigment epithelium-derived factor inhibit hepatocellular carcinoma in nude mice Oncogene 2010 29 19 2784 2794 10.1038/onc.2010.38 2-s2.0-77952426237 20190814

226 Ji M. Wang W. Li S. Hu W. Implantation of bone mesenchymal stem cells overexpressing miRNA-705 mitigated ischemic brain injury Molecular Medicine Reports 2017 16 6 8323 8328 10.3892/mmr.2017.7626 2-s2.0-85032721758 28983620

227 Martínez-González I. Cruz M.-J. Moreno R. Morell F. Muñoz X. Aran J. M. Human mesenchymal stem cells resolve airway inflammation, hyperreactivity, and histopathology in a mouse model of occupational asthma Stem Cells and Development 2014 23 19 2352 2363 10.1089/scd.2013.0616 2-s2.0-84907448675 24798370 PMC4172465

228 Martínez-González I. Roca O. Masclans J. R. Human mesenchymal stem cells overexpressing the IL-33 antagonist soluble IL-1 receptor–like–1 attenuate endotoxin-induced acute lung injury American Journal of Respiratory Cell and Molecular Biology 2013 49 4 552 562 23656573 10.1165/rcmb.2012-0406OC

229 Hu J. Yang Z. Wang J. Infusion of Trx-1-overexpressing huc MSC prolongs the survival of acutely irradiated NOD/SCID mice by decreasing excessive inflammatory injury PLoS One 2013 8 11, article e78227 10.1371/journal.pone.0078227 PMC3817237 24223778

230 Ni J. Liu X. Yin Y. Zhang P. Xu Y.-W. Liu Z. Exosomes Derived from TIMP2-Modified Human Umbilical Cord Mesenchymal Stem Cells Enhance the Repair Effect in Rat Model with Myocardial Infarction Possibly by the Akt/Sfrp2 Pathway Oxidative Medicine and Cellular Longevity 2019 2019 19 1958941 10.1155/2019/1958941 2-s2.0-85067785594 31182988 PMC6512021

231 Xia P. Wang W. Bai Y. Claudin-7 suppresses the cytotoxicity of TRAIL-expressing mesenchymal stem cells in H460 human non-small cell lung cancer cells Apoptosis 2014 19 3 491 505 24242915 10.1007/s10495-013-0938-z

232 Jiang X. Fitch S. Wang C. Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery Proceedings of the National Academy of Sciences 2016 113 48 13857 13862 10.1073/pnas.1615396113 PMC5137687 27849590

233 Spano C. Grisendi G. Golinelli G. Soluble TRAIL armed human MSC as gene therapy for pancreatic cancer Scientific Reports 2019 9 1 1 14 30742129 10.1038/s41598-018-37433-6 PMC6370785

234 Man A. J. Kujawski G. Burns T. S. Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF Cellular and Molecular Bioengineering 2016 9 1 96 106 27087859 10.1007/s12195-015-0425-4 PMC4830493

235 Cheng Z. Ou L. Zhou X. Targeted migration of mesenchymal stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac performance Molecular Therapy 2008 16 3 571 579 18253156 10.1038/sj.mt.6300374

236 Tang Y. L. Tang Y. Zhang Y. C. Qian K. Shen L. Phillips M. I. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector Journal of the American College of Cardiology 2005 46 7 1339 1350 16198853 10.1016/j.jacc.2005.05.079

237 Li W. Ma N. Ong L. L. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function Stem Cells 2007 25 8 2118 2127 17478584 10.1634/stemcells.2006-0771

238 Mangi A. A. Noiseux N. Kong D. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts Nature Medicine 2003 9 9 1195 1201 10.1038/nm912 12910262

239 Burkus J. K. Transfeldt E. E. Kitchel S. H. Watkins R. G. Balderston R. A. Clinical and radiographic outcomes of anterior lumbar interbody fusion using recombinant human bone morphogenetic protein-2 Spine 2002 27 21 2396 2408 12438990 10.1097/00007632-200211010-00015

240 Fang J. Zhu Y.-Y. Smiley E. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes Proceedings of the National Academy of Sciences 1996 93 12 5753 5758 10.1073/pnas.93.12.5753 PMC39133 8650165

241 Franceschi R. Biological approaches to bone regeneration by gene therapy Journal of Dental Research 2005 84 12 1093 1103 16304438 10.1177/154405910508401204

242 Liu Y. Shu X. Z. Prestwich G. D. Osteochondral defect repair with autologous bone marrow–derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix Tissue Engineering 2006 12 12 3405 3416 17518677 10.1089/ten.2006.12.3405

243 Syková E. Jendelová P. Urdzíková L. Lesný P. Hejčl A. Bone marrow stem cells and polymer hydrogels—two strategies for spinal cord injury repair Cellular and Molecular Neurobiology 2006 26 7-8 1111 1127 10.1007/s10571-006-9007-2 PMC11520705 16633897

244 Tu Q. Valverde P. Li S. Zhang J. Yang P. Chen J. Osterix overexpression in mesenchymal stem cells stimulates healing of critical-sized defects in murine calvarial bone Tissue Engineering 2007 13 10 2431 2440 17630878 10.1089/ten.2006.0406 PMC2835465

245 Hoffmann A. Pelled G. Turgeman G. Neotendon formation induced by manipulation of the Smad 8 signalling pathway in mesenchymal stem cells The Journal of Clinical Investigation 2006 116 4 940 952 16585960 10.1172/JCI22689 PMC1421340

246 Guo C.-A. Liu X.-G. Huo J.-Z. Jiang C. Wen X.-J. Chen Z.-R. Novel gene-modified-tissue engineering of cartilage using stable transforming growth factor- β 1-transfected mesenchymal stem cells grown on chitosan scaffolds Journal of Bioscience and Bioengineering 2007 103 6 547 556 17630127 10.1263/jbb.103.547

247 Engler A. J. Sen S. Sweeney H. L. Discher D. E. Matrix elasticity directs stem cell lineage specification Cell 2006 126 4 677 689 16923388 10.1016/j.cell.2006.06.044

248 Curran J. M. Chen R. Hunt J. A. The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate Biomaterials 2006 27 27 4783 4793 16735063 10.1016/j.biomaterials.2006.05.001

249 Cassell C. O. Hofer O. S. Morrison W. A. Knight K. R. Vascularisation of tissue-engineered grafts: the regulation of angiogenesis in reconstructive surgery and in disease states British Journal of Plastic Surgery 2002 55 8 603 610 12550111 10.1054/bjps.2002.3950

250 Rouwkema J. Boer J. D. Blitterswijk C. A. V. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct Tissue Engineering 2006 12 9 2685 2693 16995802 10.1089/ten.2006.12.2685

251 de Cássia Noronha N. Mizukami A. Caliári-Oliveira C. Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies Stem Cell Research & Therapy 2019 10 1 1 21 31046833 10.1186/s13287-019-1224-y PMC6498654

252 Mendt M. Kamerkar S. Sugimoto H. Generation and testing of clinical-grade exosomes for pancreatic cancer JCI Insight 2018 3 8, article e99263 10.1172/jci.insight.99263 PMC5931131 29669940

253 Datta A. Kim H. McGee L. High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: a drug repurposing strategy for advanced cancer Scientific Reports 2018 8 1 1 13 29802284 10.1038/s41598-018-26411-7 PMC5970137

254 Laulagnier K. Grand D. Dujardin A. PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes FEBS Letters 2004 572 1-3 11 14 15304316 10.1016/j.febslet.2004.06.082

255 Zhang H.-C. Liu X.-B. Huang S. Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo Stem Cells and Development 2012 21 18 3289 3297 22839741 10.1089/scd.2012.0095 PMC3516422

256 Almeria C. Weiss R. Roy M. Hypoxia conditioned mesenchymal stem cell-derived extracellular vesicles induce increased vascular tube formation in vitro Frontiers In Bioengineering and Biotechnology 2019 7 p. 292 10.3389/fbioe.2019.00292 PMC6819375 31709251

257 Xue C. Shen Y. Li X. Exosomes derived from hypoxia-treated human adipose mesenchymal stem cells enhance angiogenesis through the PKA signaling pathway Stem Cells and Development 2018 27 7 456 465 29415626 10.1089/scd.2017.0296

258 Lee S. C. Jeong H. J. Lee S. K. Kim S.-J. Lipopolysaccharide preconditioning of adipose-derived stem cells improves liver-regenerating activity of the secretome Stem Cell Research & Therapy 2015 6 1 1 11 25890074 10.1186/s13287-015-0072-7 PMC4416308

259 Ti D. Hao H. Tong C. LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b Journal of Translational Medicine 2015 13 1 1 14 26386558 10.1186/s12967-015-0642-6 PMC4575470

260 Kink J. A. Forsberg M. H. Reshetylo S. Macrophages educated with exosomes from primed mesenchymal stem cells treat acute radiation syndrome by promoting hematopoietic recovery Biology of Blood and Marrow Transplantation 2019 25 11 2124 2133 31394269 10.1016/j.bbmt.2019.07.026 PMC6861683

261 Alzahrani F. A. Melatonin improves therapeutic potential of mesenchymal stem cells-derived exosomes against renal ischemia-reperfusion injury in rats American Journal of Translational Research 2019 11 5, article 2887 PMC6556638 31217862

262 Ding J. Wang X. Chen B. Zhang J. Xu J. Exosomes derived from human bone marrow mesenchymal stem cells stimulated by deferoxamine accelerate cutaneous wound healing by promoting angiogenesis BioMed Research International 2019 2019 12 9742765 10.1155/2019/9742765 2-s2.0-85066036330 31192260 PMC6525840

263 Li H. Zuo S. He Z. Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survival American Journal of Physiology-Heart and Circulatory Physiology 2010 299 6, article H1772 10.1152/ajpheart.00557.2010 PMC3006287 20870802

264 He J.-G. Li H.-R. Han J.-X. GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes Scientific Reports 2018 8 1 1 11 29899566 10.1038/s41598-018-27435-9 PMC5998064

265 Yu B. Kim H. W. Gong M. Exosomes secreted from GATA-4 overexpressing mesenchymal stem cells serve as a reservoir of anti-apoptotic microRNAs for cardioprotection International Journal of Cardiology 2015 182 349 360 10.1016/j.ijcard.2014.12.043 2-s2.0-84947201437 25590961 PMC4382384

266 Kang K. Ma R. Cai W. Exosomes secreted from CXCR4 overexpressing mesenchymal stem cells promote cardioprotection via Akt signaling pathway following myocardial infarction Stem Cells International 2015 2015 14 659890 10.1155/2015/659890 2-s2.0-84929649938 26074976 PMC4436515

267 Gong X. H. Liu H. Wang S. J. Liang S. W. Wang G. G. Exosomes derived from SDF1-overexpressing mesenchymal stem cells inhibit ischemic myocardial cell apoptosis and promote cardiac endothelial microvascular regeneration in mice with myocardial infarction Journal of Cellular Physiology 2019 234 8 13878 13893 10.1002/jcp.28070 2-s2.0-85061067210 30720220 PMC13483023

268 Cui D. Advances and prospects on biomolecules functionalized carbon nanotubes Journal of Nanoscience and Nanotechnology 2007 7 4 1298 1314 10.1166/jnn.2007.654 2-s2.0-34447544574 17450892

269 Pantarotto D. Singh R. McCarthy D. Functionalized carbon nanotubes for plasmid DNA gene delivery Angewandte Chemie 2004 116 39 5354 5358 10.1002/ange.200460437 15455428

270 Lu Q. Moore J. M. Huang G. RNA polymer translocation with single-walled carbon nanotubes Nano Letters 2004 4 12 2473 2477 10.1021/nl048326j 2-s2.0-10844277454

271 Cui D. Tian F. Ozkan C. S. Wang M. Gao H. Effect of single wall carbon nanotubes on human HEK293 cells Toxicology Letters 2005 155 1 73 85 15585362 10.1016/j.toxlet.2004.08.015