J. Pineal Res. 2014; 56:382–397

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Doi:10.1111/jpi.12133

Journal of Pineal Research

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

REVIEW ARTICLE

Melatonin regulates mesenchymal stem cell differentiation: a review Abstract: Among the numerous functions of melatonin, the control of survival and differentiation of mesenchymal stem cells (MSCs) has been recently proposed. MSCs are a heterogeneous population of multipotent elements resident in tissues such as bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, gaining thus increasing interest for tissue repair and restoration therapeutic protocols. Receptor-dependent and receptor-independent responses to melatonin are suggested to occur in these cells. These involve antioxidant or redox-dependent functions of this indolamine as well as secondary effects resulting from autocrine and paracrine responses. Inflammatory cytokines and adipokines, proangiogenic/mitogenic stimuli, and other mediators that influence the differentiation processes may affect the survival and functional integrity of these mesenchymal precursor cells. In this scenario, melatonin seems to regulate signaling pathways that drive commitment and differentiation of MSC into osteogenic, chondrogenic, adipogenic, or myogenic lineages. Common pathways suggested to be involved as master regulators of these processes are the Wnt/b-catenin pathway, the MAPKs and the, TGF-b signaling. In this respect melatonin emerges a novel and potential modulator of MSC lineage commitment and adipogenic differentiation. These and other aspects of the physiological and pharmacological effects of melatonin as regulator of MSC are discussed in this review.

Francesca Luchetti1, Barbara
e Bartolini3, Canonico2, Desire Marcella Arcangeletti1, Silvia Ciffolilli3, Giuseppe Murdolo3, Marta Piroddi3, Stefano Papa1, Russel J. Reiter4 and Francesco Galli3 1

Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy; Department of Earth, Life and Environmental Sciences, University of Urbino “Carlo Bo”, Urbino, Italy; 3Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy; 4 Department of Cellular and Structural Biology, UT Health Science Center, San Antonio, TX, USA 2

Key words: adipogenesis, differentiation, melatonin, mesenchymal stem cells, osteogenesis, oxidative stress, PPRAc, proliferation, Wnt Address reprint requests to Francesco Galli, Nutrition and Clinical Biochemistry Lab, Department of Pharmaceutical Sciences, University of Perugia, Istituti biologici di Via del Giochetto, 06126 Perugia, Italy. E-mail: [email protected] Received February 25, 2014; Accepted March 14, 2014.

Introduction Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine originally isolated from bovine pineal tissue [1]. Thereafter, it was recognized that in mammals the nighttime increase in blood levels of melatonin is entirely a result of its synthesis and secretion from the pineal gland [2]. However, this pineal secretory product is also produced in multiple, perhaps all, cells and organs including the retina, Harderian gland, gut, ovary, and testes [3, 4]. Moreover, melatonin is produced in all organisms of the plant [5–7] and animal kingdoms [8, 9]. Many biological functions of melatonin have been identified so far and take place throughout organisms. Thus, melatonin influences circadian rhythms, sleep-wake cycle, tumor growth inhibition, immune function [10–14] and provides antioxidant protection and redox homeostasis in tissues [15–17]. Ubiquitously distributed, melatonin receptors, MT1 and MT2, belong to the family of G-protein-coupled receptors [18, 19] and binding sites (putative receptors) have also been found in the nuclei of many cell types [20, 21]. Melatonin also binds to calmodulin, which assists in regu382

lating intracellular events such as nitric oxide synthase (NOS) activity [22]. Subsequently, a third, nonmammalian melatonin receptor subtype MT3 [23] has been cloned and its binding site has been identified and characterized as the hamster homologue of the human enzyme quinone reductase [24]. Melatonin and its metabolites also have multiple receptor-independent effects [25–29]. Some of these receptorindependet actions rely on its potential as free radical scavenger [30, 31], and on the transcriptional regulation of antioxidant and detoxification genes [32, 33]. The antioxidant actions of melatonin have been documented during therapeutic interventions in animal models of ischemia [34] and reperfusion of hepatic [35], renal [36] and brain tissues [37, 38], as well as in brain trauma [39, 40], extra-hepatic bile duct ligation [41] and infection with Schistosoma mansoni [42]. Moreover, beneficial effects on the control of immune–inflammatory and oxidative stress reactions have been reported in some human conditions such as pain in neonatal intensive care [26], Duchenne muscular dystrophy [43], in respiratory distress syndrome of preterm newborns [44] as well as in the para-physiological settings of a strenuous exercise in healthy human males [45, 46].

Melatonin in mesenchymal stem cell biology There is increasing evidence that antioxidant effects of melatonin are just a component of the homeostatic and cell protective effects [47, 48], the impact of which could be particularly relevant in the case of immune and inflammatory components. These are at the same time players/ promoters and victims of oxidative stress events, and the balance between the two roles is key to preserve the integrity of the same immune system and thus of the entire organism along the life cycle [49] as this system with its different cell elements is ultimately responsible for the control of the host response and cancer cell surveillance, as well as of tissue repair and regeneration. Recent studies suggest that the biological and multifaceted effects of melatonin may also include a regulatory function on mesenchymal stem cell (MSC) differentiation, a process primarily involved in the development and regeneration of several tissues as bone, muscle and the adipose tissue. In bone marrow-derived MSC, for instance, melatonin enhances osteogenesis and inhibits adipogenesis; as a result, melatonin can shift bone marrow precursor cells from an adipocytic to osteoblastic differentiation that be of importance in bone repair technologies [50, 51]. Such early evidence, however, suggests implications in the physiological control of bone components in which melatonin receptor-dependent and receptor-independent effects promote tissue formation while preventing deterioration [52]. Further inferences for a regulatory role of melatonin in MSC differentiation relate to the pathophysiology of adipose tissue [53]. Here, the control of oligopotent elements and then the terminal differentiation of pre-adipocytes to mature adipocytes remain poorly understood processes, which may have profound effects on the endocrine and paracrine functions as well as on metabolic control of this organ [51]. This review recognizes and critically examines the available information on the effect of melatonin as a regulator of MSC differentiation and protection in different organs and tissues.

Mesenchymal stem cells: biology and classification Stem cells are undifferentiated elements endowed with the property of indefinitely proliferating. Two main types of such self-regenerating elements are identified: the former is present in embryonic organisms (thus, their definition as embryonic stem cells or ESC), and the latter group is present in adult organisms and is defined as somatic stem cells (SSC). Both cell types show the potential of differentiating into various specialized cells and may eventually organize into tissues. Depending on the extent of this potency, stem cells are divided in totipotent or omnipotent and pluripotent cells; these have the capability to give origin to any type of mature cell, and multipotent and oligopotent elements have a progressively narrower potential of differentiation. Totipotent SC are the first elements formed from the fusion of gametes and form embryonic cells; pluripotent cells are embryonic cells derived from the blastocyst or the inner cell mass of the embryo. These differentiate into tissue from all three germ layers (endoderm, mesoderm, and

ectoderm). Alternatively, pluripotent elements can be formed following reprogramming of somatic cells (induced pluripotent SC). Multipotent SC that differentiate into tissue components and derive from a single germ layer are present as resident populations of cells in almost all tissues of an adult organism and play a major role in regeneration processes, as well as in immunoregulatory and homeostatic mechanisms [54]. MSC that show the potential to form several lineages such as adipose, bone, and cartilage appertain to this group of SC. Other tissue-resident SC are oligopotent because they can form terminally differentiated cells of a specific tissue. A putative form of MSC originally defined as colonyforming unit fibroblasts (CFU-Fs) was first described in the early 1970s by Friedenstein et al. [55] as nonphagocytic, nonhemapoietic, fibroblast-like cells that after ex vivo isolation in small numbers by plastic adherence from rat whole bone marrow cultures, showed clonogenic properties, formed colonies in culture conditions, differentiated in vitro to various tissues as bone, cartilage, adipose tissue, tendon, muscle, and fibrous tissue. With such biological properties, it was thought that these SC may be responsible for the normal turnover and maintenance of adult tissue, at least that of mesenchymal origin. These prototypal MSC were thus investigated in a series of studies describing their origin and isolation methods, phenotypes, and biological properties [56]. In these studies, MSCs have been described with various terms in an attempt to highlight differences in the developmental origin and differentiation capacity of cellular subsets [57], such as marrow stromal cells, multipotent stromal cells, mesenchymal stromal cells, CFU-Fs, bone marrow stromal stem cells (BMSSCs), stromal precursor cells (SPCs), skeletal stem cells (SSCs), and multipotent adult progenitor cells (MAPCs). The definition of ‘mesenchymal stem cells’ remains, however, the most often employed, and this will be used in this review paper. Despite a limited differentiating potential, MSCs from various sources have been employed in the treatment of age-dependent and age-independent degenerative disorders and traumatic injuries [58]. Clinical applications in the repair of skeletal muscle lesions and in regenerative and esthetic medicine have been proposed [59], and the therapeutic efficacy of MSC has been confirmed in animal models of meniscus injury, neurological disorder, myocardial infarction, lung injury, ischemic acute renal failure, and others [60]. Recent evidence also suggests the potential for therapeutic applications in the treatment of morbid obesity and insulin resistance (IR) that are associated with type 2 diabetes (T2D) and metabolic syndrome [61]. The origin of the MSC lineage during embryonic development remains debated, and this has hindered the process of delineating an unambiguous taxonomy and the molecular mechanisms that regulate MSC self-renewal and fate determination. The widespread tissue distribution of MSC or MSC-like cells has been interpreted as the demonstration that the cells reside in the vascular pericyte population in vivo. This concept is consistent with studies showing that MSCs express antigens detected on pericytes, endothelial and perivascular cells and that postcapillary venule pericytes from bone marrow and peri-vascular cells 383

Luchetti et al. from most tissues exhibit MSC-like characteristics [62, 63]. Alternatively, several studies [64–66] indicated that MSCs are derived from neuro-epithelium via a neural crest intermediate during the development. Takashima et al. [64, 67] demonstrated that cultured expansion of embryonic stem cells (ESCs) under conditions that drive mesodermal specification yielded a PDGFR alpha lineage restricted to the adipogenic lineage. Based on this and other findings reported in the literature, it is possible to assume that specification of the MSC lineage from a neuro-epithelial intermediate occurs via an epithelial-to-mesenchymal transition (EMT), a process that plays a well-established role in cellular diversification during development. Other studies, however, have focused on human adipose-derived stem cells (ASC) [53, 68] and demonstrate how these are intimately associated with perivascular cells. Using a set of antibodies focused on the cell surface markers, a stromal cell precursor antigen (STRO-1), 3G5, and CD146, [68] in fact, isolated ASC which showed characteristics of MSC and were clonogenic and multipotent. Taken together, these findings do not exclude the pericytic origin of MSC in adipose tissue, but rather suggest that once pericytic cells are released from the vascular wall and become MSC they do not necessarily retain the entire phenotype and localization of the ancestors. Moreover, the transcription factor TWIST1 has also been implicated in mesoderm specification, and it is known to function as a potent inducer of the EMT program. Thus, one may assume that TWIST also plays an important role in lineage specification of MSC, and, indeed, several lines of evidence support such a hypothesis. A recent paper [69] demonstrated in postmigratory neural crest cells the role of TWIST1 as a repressor of pro-neural factors and thereby a regulator of cell fate determination between ectodermal and mesodermal lineages. As stated above, the tissue source is another and more straightforward criterion of taxonomy for MSC. Previously, adult MSCs were commonly isolated from bone marrow. Bone marrow-derived stem cells form only 0.001–0.01% of total nucleated cells in the aspirate [70]; therefore, their production in large scale requires a considerable amount of primary cell material and a time-consuming expansion period. It has now been recognized that MSCs are found in nearly all adult tissues, for example adipose, dermis, periosteum, peripheral and menstrual blood, and in solid organs like liver, spleen, and lung [71]. ASC qualify as an excellent source of MSC alternative to BMSC. The adipose tissue in fact shows a 2500-fold higher frequency of stem cells compared to BM, and the abundance and accessibility of adipose tissue in the body is obviously advantageous [72]. Moreover, this tissue is often a surgical waste, and the method for obtaining a lipo-aspirate is less invasive than that of obtaining a BM aspirate. The International Society for Cellular Therapy has provided three minimal criteria to define MSC independent of their source: (i) plastic adherence in standard culture conditions (ii) expression of nonspecific markers CD105, CD90, and CD73 along with the lack of expression of CD34, CD45, CD14, or CD11b, CD79a or CD19, and class II major histocompatibility complex (MHC-II) mole384

cules, mainly HLA-DR, and (iii) differentiation into osteoblast, adipocytes, and chondroblasts under specific in vitro stimulation. However, it should be emphasized that the immunophenotype of MSC is dynamic and changes over the course of culturing; some of these changes may represent alterations in the biological features of MSC [73]. It has been reported that MSCs from different sources, for example BM and adipose tissue, share a similar immunophenotype and capacity for in vitro multilineage differentiation, although some differences are present. For example, after expansion in culture, bone marrow-derived MSCs express the surface marker CD29, CD73, CD90, CD105, CD106, CD140b, and CD166, but not CD31, CD45, CD34, CD133, or MHC class II. Cultured MSC from other sources, such as chorion- and amnion-derived cells of placenta, adipose tissue, peripheral blood, umbilical cord blood, amniotic fluid, fetal hepatic and pulmonary tissue, skin, and prostate [73] also seems to be negative for CD31, CD45, CD80, but uniformly express CD9, CD10, CD13, CD29, CD73, CD90, CD105, and CD106, and additional tissue-specific expression of other surface antigens has also been reported. Moreover, only adipose tissue-derived MSCs express high levels of CD34, while amnion-derived MSCs are positive for stage-specific embryonic antigen (SSEA)-4 and tumor rejection antigen (TRA). In contrast, bone marrow-derived MSCs, but not placenta-derived MSC, express CD271 as well as tissuenonspecific alkaline phosphatase (TNAP). It is important to highlight, however, that MSC populations exhibit donor-to-donor heterogeneity. In fact, an analysis of BM-derived MSC from 17 healthy human donors revealed marked disparities in growth rate, alkaline phosphatase levels, and osteogenic potential [74]. Other studies have confirmed these findings and attributed this heterogeneity to several factors including sampling procedure during BM aspiration [75], age of the donor [76], and postharvesting methods used to expand MSC populations [77]. Regardless of the origin and tissue source, MSCs are easily isolated, cultured, and manipulated for terminal differentiation thus providing an excellent tool for research with a huge potential for clinical applications. As far as homeostatic and regeneration mechanisms of culturedexpanded MSC are concerned, it was originally thought that after the delivery of these SCs into the injured host, they would migrate to the site of injury and directly differentiate into the cells of an appropriate phenotype and function, thus contributing to repair of injured tissue. Furthermore, several studies demonstrated that MSC-conditioned media alone have therapeutic effects. Collectively, these data suggest that MSC could exert their reparative and physiological functions also, or possibly exclusively, through paracrine effects [78]. Paracrine factors are expected to regulate the physiological role that MCSs have in healthy tissues and, besides such a regulatory component, the differentiating potential of MCSs from various origins is under the influence of several other factors that include a series of metabolic and endocrine effectors that, in turn, integrate to influence developmental origin, maintenance, and differentiation potential of MSCs. The available knowledge on these

Melatonin in mesenchymal stem cell biology aspects, along with the potential role of melatonin on the biological properties of MSC, will be discussed below.

MSC differentiation: signaling and biological effects by melatonin Mesenchymal stem cells are multipotent progenitor cells that can be differentiated under appropriate conditions into several cell types such as osteoblasts, chondrocytes and adipocytes [56, 74, 79, 80]. MSC differentiation is finely regulated by the action of mechanical and molecular signals from the extracellular environment. Emerging evidence suggests that melatonin may also be an important regulator of precursor cell commitment and differentiation. Therefore, the signaling pathways involved in these melatonin-dependent responses are discussed in the sections below (Fig. 1). Wnt/b-catenin signaling The effects of melatonin on the Wnt pathway have been investigated under various experimental conditions [81] that have strengthen the evidence on the role for the indolamine in the control of MSC survival and differentiation. Wnt proteins constitute a large family of cysteine-rich secreted ligands that control development in organisms ranging from nematode worms to mammals. These proteins are the leading element of a signaling pathway and the component and its biological roles of which have been exhaustively reviewed elsewhere [51, 82, 83]. In adult mammals, Wnt/b-catenin signaling is crucial for regulating cell proliferation and fate determination, apoptosis, and axis polarity induction [84]. The human genome harbors almost 20 Wnt genes. A growing body of evidence demonstrates the role of Wnt/b-catenin signaling in the control of MSC differentiation [85]. Signaling is initiated when Wnt ligands engage their cognate receptor complex, consisting of a serpentine

receptor of the Frizzeled family and a member of the LDL receptor family, Lrp5/6. The central player is a cytoplasmic protein termed b-catenin, the stability of which is regulated by the destruction complex b-catenin-independent, noncanonical, signaling induced by Wnt has been also described, and its mechanism of action has been summarized elsewhere [84]. When Wnt receptors are not engaged, two scaffolding proteins in the destruction complex bind newly synthesized-b-catenin, that is, APC and axin. CK1 and GSK3, two kinases residing in the destruction complex, sequentially phosphorylate a set of conserved Ser and Thr residues in the amino terminus of b-catenin. The resulting phosphorylated footprint recruits a b-TrCP containing E3 ubiquitin ligase, which targets b-catenin for proteosomal degradation. Receptor occupancy inhibits the kinase activity of the destruction complex by an incompletely understood mechanism involving the direct interaction of axin with Lrp5/6 and/or the actions of an axin-binding molecule. As a consequence, b-catenin accumulates and travels into the nucleus where it engages the N-terminus of DNA-binding proteins of the Tcf/Lef family. The canonical Wnt/b-catenin signaling is a key regulator of bone formation and MSC differentiation to either the osteogenic or chondrogenic lineage through rather high or low Wnt canonical activity, respectively [86]. On the other hand, in the absence of Wnt signaling, adipogenic differentiation is enhanced. Furthermore, Taipaleenmak et al. [86] showed that an appropriate level of b-catenin signaling is required for commitment to the chondrogenic lineage. Zhang et al. [87] demonstrated for the first time that the Wnt/b-catenin signaling plays an important role in stem cell aging. Although the mechanisms of cell senescence induced by Wnt/b-catenin signaling are still poorly understood, the authors showed that the activated Wnt/b-catenin pathway can induce MSC aging possibly by the activation of ROS-generating pathways or lower control of their reactivity by antioxidant genes. Actually, ROS generation was increased in MSCs when high levels of

MELATONIN (putative targets of the Wnt signaling)

MSC Fig. 1. (Main panel) differentiation of MSCs toward the three main lineages in which melatonin (MLT) has been proposed to produce a regulatory effect (upward and downward arrows), namely osteocytes, chondrocytes, and adipocytes. Other components with main regulatory function in the three lineages are also described. (Insert) Scheme of Wnt signaling with the proposed targets of melatonin (MLT) (highlighted by the dashed box).

+Wnt

–Wnt MLT

Proliferation and differentiation

+Wnt

PPARγ MLT

COL2A1, Sox9, BMP

Adipocytes

MLT

Runx2 BMP

Osteocytes Chondrocytes

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Luchetti et al. Wnt3a were expressed, whereas lower levels of this Wnt form were unable to induce ROS generation. The data of Zhang’s group are corroborated by other studies showing that the Wnt/b-catenin signaling is increased in aged tissue and in a mouse model of accelerated aging [88]. Recent data reported by Wang [89] showed that the Wnt signaling strongly activates mitochondrial biogenesis, which in turn produces elevated levels of ROS implying the need for an improved antioxidant protection to hamper oxidative damage effects at the cellular level. The antioxidant and cellular protective roles of melatonin could thus represent an important support for the control of ROS-generating and metabolic effects of Wnt signaling in the aging MSC. Also, the melatonin-dependent activation of MT2 receptors expressed on MSCs influences Wnt signaling. As further described below, the results in osteoblast formation via G1-protein/b-arrestin/MEK involve stimulation of MAPK subfamily ERK1/2 [81]. This signaling by melatonin regulates bone morphogenic protein (BMP) activity and links to the canonical signaling of Wnt because b-arrestin 1 is an essential component and positive modulator of the Wnt/b-catenin pathway, and b-arrestin 2 is a mediator for the agonist-induced internalization of FZD4 [90]. Further support for a role of melatonin as a regulator of b-catenin/Wnt signaling of SC comes from the evidence obtained in murine embryonic SC of the inhibition of GSK-3 a/b, a component of this pathway (Fig. 1, right top panel) that together with PI3K/Akt signaling [91] mediates the melatonin-dependent promotion of stem cell proliferation. Specific signals acting on the Wnt pathway of MSCs have been found to inhibit adipogenesis [92–94] while promoting osteogenesis [95–98], two melatoninresponsive processes described in detail in the next sections. Wnt inhibition of adipogenesis is mediated via b-catenin, which interferes with PPARc transcriptional activation of downstream targets [98, 99], and the b-catenin function can be modulated by the inhibition of GSK3b activity. Together these two components, namely b-catenin and GSK3b, represent putative targets of the regulatory role of melatonin in the Wnt signaling pathway (Fig. 1, right top panel) that warrant further investigation to elucidate their response mechanism upon melatonin treatment. Melatonin and the signaling of MSC differentiation Several lines of evidence suggest that melatonin may play an important role in the regulation of MSC commitment and differentiation. The differentiation into osteoblasts or adipocytes of MSC provides an example of this. This dichotomy is based on the activation of several lineagespecific signaling pathways and transcription factors that act as ‘switches’ of the differentiation process to control the fate of mesenchymal progenitor cells as they enter the bone or fat lineage, thus demonstrating that adipogenesis and osteogenesis are the phenotypic result of the multipotent nature of MSCs. Studies by Gimble et al. [100] strongly support an inverse relationship between the commitment of bone marrow-derived MSCs or stromal cells to the adipocyte and osteoblast lineage pathways. Likewise, agents that induce osteoblast differentiation inhibit adipogenesis [101]. These findings are consistent with pathologi386

cal and epidemiological studies [102] linking increased marrow adiposity with aging, bone loss, and osteoporosis. More components of the transcriptional complex may influence MSC differentiation in the different settings. A shared co-activator protein, known as the transcriptional co-activator with PDZ binding motif (TAZ), accounts for a link between functional components in the cell’s lineage commitment as Runx2 and PPARc [103] that are described in detail below. In murine cell lines, the TAZ protein co-activates Runx2 and osteogenesis while suppressing PPARc and adipogenesis. Some aspects of the cell signaling studied in bone marrow may help to discriminate the routes of MSC differentiation in these two cell lineages also providing hints on the possible effect of melatonin. The CCAAT/enhancerbinding protein (C/EPB) family and PPARc regulate adipocyte differentiation, while the transcription factor Runx2 and osterix regulate osteoblast differentiation. Activation of PPARc down-regulates the expression of Runx2, whereas insufficient PPARc leads to a decrease in marrow adipogenesis and an increase in Runx2-mediated osteogenesis. Melatonin enhances osteogenesis and inhibits adipogenesis by suppressing PPARc expression and enhancing Runx-2 [52]. Botolin and McCabe [104] demonstrated that the PPARc antagonist bisphenol-A-diglycidyl ether (BADGE) prevents BM adiposity in type 1 diabetic mice, but at the same time, this effect was not associated with an improvement in bone density. Furthermore, melatonin was unable to prevent triglyceride accumulation in rat osteoblast-like ROS17/2.8 cell line in presence of forskolin, a stimulator of cAMP accumulation [105], and adipocyte differentiation of 3T3L1 murine pre-adipocytes through inhibiting the activity of critical transcriptional factor, C/EBPb [106]. As mentioned above, the Wnt signaling (Fig. 1) plays a key role in MSC differentiation encompassing multiple ligands, antagonists, receptors, co-receptors, and transcriptional mediators [85]. The Wnt signaling in MSCs has opposite effects on adipogenesis [92–94] and osteogenesis, leading to inhibition or activation, respectively [95–98]. bcatenin and GSK3b mediate Wnt inhibition of adipogenesis interfering with PPARc transcriptional activation [98, 99]. The PPAR signaling pathway has been intensively studied in adipocyte differentiation (described in Fig. 2 and in the next section). Peroxisome proliferator-activated receptors are a group of ligand-dependent nuclear receptors responsible for gene expression regulation [107] that generally function as transcriptional regulators of adipogenic differentiation and lipid metabolism [108]. So far, three members of the PPAR subfamily have been identified: PPARa, PPARb (also called PPARd), and PPARc [109, 110] each have different ligands, target genes, and biological functions. PPARc is expressed in humans and rodents as two protein isoforms produced from a single gene, where PPAR1c is expressed in many different cell types, while the expression of PPARc 2, which contains an additional 28–30 amino acids at the N-terminus, is limited to adipocytes and bone marrow stromal cells. PPARc 2 is recognized as an essential transcriptional regulator of both adipocyte differentiation and lipid storage in mature

Melatonin in mesenchymal stem cell biology adipocytes. Furthermore, PPARc 2 is highly expressed in mature adipocytes, and it is absent in na€ıve undifferentiated precursor cells isolated from adipose tissue, indicating that these cells are not committed to an adipocytic phenotype. Expression of PPARc 2 during the early stages of adipocyte differentiation is directly induced by members of the C/EPB family of transcription factors, which are themselves induced by adipogenic hormones. Four members of the C/EBP family, a, b, d, and CHOP-10, are expressed at specific times during adipogenesis in a manner that is consistent with a distinct regulatory role for each protein [111]. C/EBPb and d are induced very early and have been shown to play a crucial role in initiating the differentiation of pre-adipocytes by activating the expression of PPARc2. PPARc2 activity, however, can also be regulated at the post-translational level, for example by MAPK pathway or noncanonical Wnt signaling. Wu et al. [112] demonstrated the activation of MEK/ERK signaling by higher serum-promoted PPARc expression and phosphorylation, which subsequently enhanced adipogenic differentiation of MSCs. As reviewed elsewhere [48] and further discussed below, melatonin is known to regulate ERK1/2 activity in several cell model systems. In human leukocytes, for instance, a positive effect on the activation of this kinase has been observed [113]. PPARc appears, therefore, a confluent transcription element in the transregulatory activity that melatonin exerts on Wnt and MAPK-ERK routes of MSCs and even of pre-adipocytes; moreover, melatonin is expected to follow its regulatory function on this nuclear receptor also in mature adipocytes that provide a molecular framework for the proposed beneficial effects of this hormone and its analogues on comorbidity of obese patients reviewed in [114, 115]. In the human adipose, PPARc operates to control both inflammatory and metabolic aspects reviewed elsewhere [61, 116, 117], and different roles in adipogenesis and insulin resistance are reported for PPARc 1 and 2, but the possible effect of melatonin on selective regulation of these two isoforms is still

undefined. Importantly, besides the effect on adipogenesis, a specific function for form 2 in maintaining insulin sensitivity independently of its effects on adipogenesis has been described in a knockout mice model also pointing to a role of this element in the development of adverse effects of a high-fat diet on glucose homeostasis [117]. Altogether, these data suggest that melatonin may be a novel regulator of adipose organ homeostasis. The identification of functional MT2 receptors in human adipocytes increases the interest on these aspects providing further support to the hypothesis of a physiological role of melatonin in the development and functional regulation of the adipose tissue. To address this point, Brydon et al. [118] investigated if the treatment of PAZ6 adipocytes with melatonin could alter the expression of key genes involved in metabolic and functional aspects of these cells. When administered through the differentiation process of PAZ6 adipocytes, melatonin affects Glut4 mRNA levels in a way that Glut4 protein levels and glucose uptake were reduced, and it is important to highlight that Glut4 overexpression in the adipose can produce an increase in fat cell number and up to a 2- to 3-fold of increase in total body lipids. Thus, the in vitro experiments on PAZ6 adipocytes suggest that melatonin has a prominent effect on the increase in Glut4 mRNA levels. Adipogenetic differentiation of MSC: is there a role for melatonin? In the adipose tissue, MSCs are present in a limited number as a main component of the stromal vascular cell fraction (see above in the section MSC: biology and classification). Signaling and transcriptional interpretation of multiple pathways involved in MSC differentiation to pre-adipocytes have disclosed the complexity of this process [51, 119], which it is believed to actively contribute to metabolic compensation and insulin function in the

MSC

Commitment and differenaon differentiation

Anoxidant and Antioxidant paracrine effects effects

PREADIPOCYTE

MELATONIN

Signaling and transcription regulaon transcripon regulation

MELATONIN

Survival and proliferation proliferaon

MATURE ADIPOCYTE Insulin resistance (IR)

Adipocytokines

Fig. 2. Proposed effects of melatonin (MLT) on homeostatic processes and differentiation of MSC in the adipose.

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Luchetti et al. adipose as well as at the systemic level [61]. Pre-adipocyte differentiation from MSCs and their maturation to functionally and metabolically competent adipocytes is key to prevent dysfunctional fatty acid storage in the adipose and thus to avoid systemic lipotoxicity of released free fatty acids (FFA). The latter produce inflammatory and degenerative effects in insulin-sensitive organs, one of the proposed underling events in the onset of insulin resistance (IR). A role of melatonin in the control of adipogenesis may thus have important consequences in the pathophysiology of IR and type 2 diabetes (T2D). This hypothesis is further supported by Jimenez-Aranda et al. [120] who investigated the role of melatonin in Zucker diabetic fatty (ZDF) rats, a model of obesity-related type 2 diabetes. In this study, the findings clearly demonstrated how melatonin reduces obesity and improves the metabolic profile of the diabetic animals [121]. Several types of stimuli appear to drive adipogenic commitment as well as the other steps that accompany the developmental process by which MSCs differentiate into pre-adipocyte and thus become to mature adipocyte. Melatonin has been shown to regulate many aspects of adipocyte biology. In pinealectomized rats, a reduced lipolysis and increased lipogenesis were observed [122]. In vitro, MT1 and MT2 melatonin receptor mRNA are detected in the adipose tissue, and long-term melatonin treatment decreased the expression of the glucose transporter Glut4 and glucose uptake in adipocytes [118], which clearly demonstrate a role of this hormone in the metabolic control of these cells and thereby of the entire organ. Such a metabolic responsiveness to melatonin may suggest effects on MSC differentiation. Indeed, this process in adipose tissue is known to be under the influence of endocrine and paracrine factors (Fig. 2) such as insulin, adipokines, and inflammatory cytokines, which drive the metabolic control of the organ and thereby that of the entire organism [61]. The presence of melatonin receptors in MSCs and preadipocytes of the human adipose tissue, however, has not been conclusively demonstrated, and thus, further investigation is needed to elucidate this aspect. The actual contribution of melatonin to the control of adipogenesis and more in general of the trophism of the human adipose is thus a matter of speculation. However, recent evidence has emerged to suggest that alterations in circadian systems and sleep participate in the pathogenesis of IS and metabolic syndrome, through the onset of obesity [123]. A relationship nocturnal work between a serious alteration of melatonin secretion suggests an increased risk for health problems such as obesity and diabetes [124]. Melatonin is also proposed to influence the positive metabolic effects of the brown adipose tissue [125]. The differentiation of pre-adipocytes to adipocytes is a well-regulated process performed by the sequential activation of signals and transcription factors. This signaling described in detail in the next section and Fig. 2 controls the expression of proteins responsible for establishing the mature fat cell phenotype, a process that has been extensively studied in mouse pre-adipocyte cell lines as 3T3-L1 and 3T3-F442A [126, 127], but not in human cell homologues. 388

Melatonin and osteoblast and chondroblast differentiation Early studies [124, 125] demonstrated the ability of melatonin to promote in vitro osteoblast differentiation and mineralization of matrix, thereby suggesting that this indolamine may play an essential role in the regulation of bone growth. In the study of Roth et al. [128], MC3T3 cells grown in the presence of 50 nM melatonin underwent cell differentiation and mineralization by day 12 instead of day 21, the span usually required for cell growth in conventional media not supplemented with melatonin. Although not performed on MSC cells, this study identified the capability of melatonin of promoting bone growth through differentiation and functional competence of osteoblasts that was also supported by the analysis of the dose-dependent response of molecular players such as bone-secreted protein (BSP) and the other bone marrow proteins, including alkaline phosphatases (ALP) and osteopontin. Nakade et al. [129] demonstrated that melatonin at concentrations between 50 and 100 lM acts directly on normal human bone cells (HOB-M cells) and human osteoblastic cell line (SV-HFO) to affect osteogenic action in vitro. This study provided new evidence that melatonin stimulates the proliferation and type I collagen synthesis in human bone cells. Subsequent studies by Radio et al. [130] detailed for the first time the involvement of melatonin in MSC regulation, demonstrating that this neurohormone at a physiological concentrations (50 nM) in combination with an osteogenic medium, significantly increased ALP, a biomarker of the enhanced proliferation and differentiation of osteoblasts. This was accompanied by gene expression of type I collagen, osteopontin, bone sialoprotein, and osteocalcin. The melatonin-mediated increase in these biomarkers was blocked by the presence of the melatonin receptor inhibitor pertussis toxin and the antagonists luzindole and 4PPDOT. The results showed that MT2 receptor is the most probable receptor form involved in that osteogenic response. To further elucidate the mechanisms underlying a MT2 receptor-mediated effect on ALP activity, the signaling occurring through the receptor tyrosine kinase- and mitogen-activated pathways was assessed. Pharmacologic inhibitors of MEK and EGF activity blocked the melatonin-induced increase in ALP levels, thus pointing to an involvement of MEK/ERK (1/2) signaling. MEK activation by G-protein-coupled receptors (GPCRs) can also be modulated by clathrin-mediated endocytosis. Indeed, the clathrin-coated pit inhibitor monodansyl-cadaverine prevents the melatonin-induced increase in ALP activity [131]. Therefore, EFG receptors, the MEK signal transduction cascade, as well as clathrin-mediated endocytosis, all have the potential to represent signaling components of the melatonin induce control of osteoblastic differentiation of MSC. Sethi et al. [132] further confirmed the role of MT2 receptor in the bone formation by the selective MT2 antagonist 4P-DOT, first highlighting the importance of both timing and duration of melatonin treatment in inducing MSC differentiation to osteoblast lineage. In this study, an increase in differentiation rate was observed when the cells were exposed to melatonin either before the differentiation

Melatonin in mesenchymal stem cell biology is initiated or at later stages of the differentiation induced by 19-day pre-exposure to osteogenic medium. On the contrary, the intermediate stage of differentiation that is featured by a sharp increase in proliferation was not under the influence of melatonin. These findings were similar to those of the study by Roth et al. [128], which suggested that MC3T3 cells must first undergo differentiation before they became responsive to melatonin. Interestingly, the increase in ALP activity induced by melatonin was coupled to cytoplasmic localization of ERK1/2, which further supports a specific involvement of melatonin in cellular differentiation rather than in proliferation [132]. Other components in the ostogenic and chondrogenic function of Wnt signaling are BMPs, a unique group of proteins within the TGFb superfamily of genes, with a pivotal role in the regulation of heart, neural, cartilage, and bone development [133]. The main biological function is, however, the promotion of bone mineralization. Indeed, these proteins were originally identified in bone-inductive extracts of demineralized bone. The role that these proteins play in MSC differentiation to osteoblasts via Wnt signaling activation has been demonstrated [134]. In fact, the impairment of Wnt signaling results in a significant reduction in the capacity of BMP-2 to induce ALP activity in MSC. BMP-2 and BMP-6 strongly promote osteogenesis in MSC. Downstream propagators of BMP signals involve the Smad family of proteins (Smad-1, -5, and -8) which induce Runx2 gene expression, and Smads interact physically with the Runx2 protein to induce osteoblast differentiation [135]. Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes, and it is a major target of the BMP pathway. Runx2 is known to represent a key regulator of chondroblast and osteoblast differentiation, and of bone development in vivo, influencing the expression of major extracellular matrix genes of chondroblasts and osteoblasts such as ALP, osteopontin, osteocalcin, type I collagen, and type X collagen [136]. Several observations [137] reported that Runx2 identified a possible target of Wnt signaling for early specification of the osteoblast lineage. With respect to promoting chondrogenesis, the most potent inducers are the TGF-b family of mediators, including TGF-b1, TGF-b2, and TGF-b3, as well as BMPs. For human, MSC, TGF-b2, and TGF-b3 were shown to be more active than TGF-b1 in promoting chondrogenesis, and although cellular content is similar after culture, significantly more proteoglycans and collagen type II can be produced after stimulation with the former two inducers [138]. BMPs, known for their involvement in cartilage formation, can act alone or in concert with other growth factors to induce or enhance MSC chondrogenic differentiation. For example, BMP-2, BMP-4, or BMP-6, combined with TGF-b3, induced chondrogenic phenotype in cultured human bone marrow-derived MSC pellets, with BMP-2 having the most pronounced effect [139]. The role of melatonin in the osteogenesis was also investigated in the differentiation of mouse osteoblatic MC3T3-E1 cells by Park et al. [50]. Melatonin promotes cell differentiation in these cells through the BMP/ERK/ Wnt signaling pathway. At the same time, an interruption

of BMP/SMAD signaling is reported to allow neural induction [140], and further studies have shown that such an inductive effect of melatonin on neural progenitor phenotype also involves an increase in nestin gene expression and the stimulation of AKT1. These findings point to a role of a melatonin-sensitive pathway as a sort of switch in MSC differentiation at the crossroads between bone and neural compartments [141, 142]. The role of melatonin in the chondrogenic differentiation of human MSC was investigate by Go et al.[143]. Cells were induced during chondrogenic differentiation via high-density micromass culture in chondrogenic medium containing vehicle or 50 nM melatonin. Histological examination and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen isotypes in the melatonin group than in the untreated controls. Besides the collagen type II (COL2A1) and X (COL10A1), the genes involved in chondrogenic differentiation up-regulated by the melatonin treatment included aggrecan (ACAN), SRY (Sex determining Region Y), Sox 9 (SOX9), runt-related transcription factor 2 (RUNX2), and the potent inducer of chondrogenic differentiation BMP2.

Melatonin and the paracrine control of MSC differentiation Besides a multipotent differentiation potential, a strong paracrine capacity has been proposed for MSC which may contribute a major role in tissue repair. This capability of MSC was first reported by Gnecchi et al. [144] in experimental models of ventricular remodeling during myocardial infarction (MI). These cells, in fact, secrete a wide array of cytokines, chemokine, and growth factors that can suppress the immune system, inhibit fibrosis and apoptosis, enhance angiogenesis, and stimulate differentiation of tissue-specific stem cells. These unique properties of MSC make them an invaluable regulator of repair and regeneration of lesions as well as of tissue and organ adaptation during their lifetime. Besides a role in the post-MI myogenic response, a release of growth factors by MSC was also described in experimental models of CNS injury, including traumatic brain injury (TBI). Indeed, MSC transplantation in rats exposed to TBI resulted in a restoration of the balance between pro-and anti-inflammatory cytokines and reduced the infiltration of microglia, macrophages, and peripheral leukocytes [145]. Amniotic fluid-derived MSCs also release a number of chemokines and cytokines such as IL-8, IL-6, TGF-b, TNFRI, VEGF, and EGF [146]. MSCs obtained from rat bone marrow release soluble immunosuppressive factors, including indoleamine 2,3-dioxygenase (IDO), IL-6, and prostaglandin E2 (PGE2). The IL-6 secreted by MSC is believed to be a pro-inflammatory cytokine. Recently, however, IL-6 has been reported to be involved in the suppression of T-cell proliferation and local inflammation, and the MSC-derived IL-6 was reported to contribute to immunoregulatory activities [146]. Paracrine mediators can be produced, as a consequence of selected metabolic events that include the response to energy and nutrient intake [61]. This is observed in the 389

Luchetti et al. different cell types such as in differentiated hepatocytes, adipocytes, and endothelial cells as well as in bone marrow-derived MSC. These stem cells obtained from high-fat diet animals, indeed, showed increased production of IL-1, IL-6, and TNF-a, and increased NF-kB and reduced PPARc expression, suggesting that obesity can affect MSC differentiation through such a paracrine mechanism [147]. The role of MSC and pre-adiopcytes as active players in the paracrine activity of the human adipose remains so far unexplored. Melatonin is now considered a regulator of the paracrine function in a range of immune and nonimmune cell subsets [48]. In vitro and in vivo studies have convincingly revealed that melatonin, while protecting leukocytes from the damaging effects of environmental and endogenous stressors [113], promotes immunoregulatory effects and the host response activating T helper cell-induced IL-2 production and the monocyte-dependent production of IL-1, IL-6, TNFa, and ROS/RNS [148]. Melatonin also enhances IL-12 production by monocytes driving T-cell differentiation toward the Th1 phenotype also causing an increase in the production of IFN-c [149]. Early evidence suggested that both the cell protection and regulatory effect of melatonin may extend to MSC. In fact, the study of Mias et al. [150] showed that a pretreatment of MSC with 5 lM melatonin prevented apoptosis while promoting the secretion of proangiogenic/mitogenic factors such as b-FGF and HGF, which suggests an advantage in the use of melatonin in the strategies of handling MSCs ex vivo. Melatonin improves at the same time cell survival and the ability of MSCs to produce a paracrine effect with a positive function on surrounding cell components, which is important in the physiological or therapeutic role that MSCs have on regeneration and repair processes of solid organs. To further support this view, functional tests carried out with the nonselective melatonin receptor antagonist, luzindole, have revealed that MSCs express melatonin receptors on their surface [130, 132, 150, 151], pointing to a role for this neurohormone as an important preconditioning and protective factor for MSC. These findings could have important implications in cellular therapy protocols. In fact, MSCs are the most promising stem cells to be employed in this type of therapy [152] because their expansion and differentiation potentials are particularly advantageous; MSC, however, lost this potential after several passages, which suggests the need for protection and conditioning treatments along the cell therapy procedures. At the same time, the effect of melatonin on endogenous MSC is expected to provide a key contribution to protection of healthy tissue, possibly representing a cell reservoir for tissue rejuvenation and immunohomeostasis. In this respect, melatonin may act as a positive stimulus on this reservoir of repair cells and immunomodulatory sentinels that may control inflammatory responses of tissues [153]. The immunomodulatory effect of MSC is an emerging aspect with expected implications in vascular biology and cardiovascular prevention. Luu et al. [154] have recently shown that MSCs co-cultured with endothelial cells downregulate adhesion of flowing neutrophils or lymphocytes, 390

also decreasing their subsequent transendothelial migration. The supernatant of the co-culture had much higher levels of IL-6 than the supernatants of cultures from the individual cell types. Carrero et al. [155] showed that IL-1b increases migration and adhesion of MSC promoting leukocyte chemotaxis through the secretion of soluble factors. As described in other cell types, IL-1b activates NF-jB resulting in transcriptional activation of a wide variety of genes such as inflammatory cytokines and chemokines, adhesion molecules, growth factor, and enzymes involved in the production of metabolites and arachidonic acid-derived inflammatory mediators. Besides soluble mediators, the inflammatory cells recruited into the site of a lesion cause the so-called ‘respiratory burst’ that is the result of an increased flux of oxygen and metabolites with consequent generation of ROS [156]. These mediators activate signal transduction pathways and transcription factors, such as NF-jB and STAT3, hypoxia-inducible factor-1a (HIFa), AP-1, Nrf2, and others, which further sustain inflammation while mediating the cell stress response. An understanding the paracrine signaling of MSC may represent, therefore, an important advancement in tissue repair and regeneration strategies; Chang et al. [157] clearly demonstrated that the MSC-mediated repair is affected by the inflammatory burden of the lesion. In this context, the pro-inflammatory cytokines TNF and IL-17 were shown to stimulate IjB kinase (IKK)/NF-jB activity thus impairing osteogenic differentiation of MSC. On the contrary, the inhibition of IKK-NF-jB significantly enhanced MSC-mediated bone formation. In this scenario, Liu et al. [158] demonstrated a positive effect of melatonin on MSC proliferation and osteogenic differentiation that was observed either with or without the exposure to an inflammatory challenge operated by the cytokine IL-1b. In these experiments, melatonin improved in a dose-dependent manner cell viability and reduced ROS generation of MSC also up-regulating the expression of CuZnSOD and MnSOD and lowering the expression of Bax [158].

Melatonin as redox regulator and cellular protective factor of MSC Reactive oxygen species are products of a normal cellular metabolism and play vital roles in the stimulation of signaling pathways in plant and animal cells in response to changes in intra- and extracellular environmental conditions. However, ROS have the potential of behaving as dangerous toxicants that under selected circumstances can lead to damaging effects to biomolecules and cells, with a potential impact on the entire organism [156, 159]. Most ROS are generated in cells by the leakage from mitochondrial components of the respiratory chain [156, 160] which can assume an abnormal proportion during cellular stress and mitochondrial dysfunction [113]. Alternatively, high fluxes of ROS are generated in mononuclear and polymorphonuclear phagocytic cells by NADPH oxidase isoenzymes, inducible NO synthase, and myeloperoxidase [156]. Most relevant ROS produced during such endogenous metabolic reactions include superoxide anion (˙O2
) and

Melatonin in mesenchymal stem cell biology hydrogen peroxide (H2O2), as well as the highly reactive hydroxyl radical (˙OH), organic peroxides, and NOderived species such as peroxynitrite. Protein thiols are the main target and sensing system of ROS fluxes at the cellular level providing the molecular backbone for the redoxsensitive component of cellular signaling. Several lines of evidence suggest that redox changes can affect MSC integrity and biological functions. Hypoxia seems to reduce MSC adipogenic differentiation through the hypoxia-inducible factor-1a transcription [161]. At the same time, superoxide dismutase (SOD)-deficient mice show spontaneous adipogenesis [162]. These changes suggest that the redox balance in bone marrow as well as in other tissues may induce differentiation of MSC cells toward osteogenesis or adipogenesis, suggesting a role for ROS in these regulatory pathways. High levels of melatonin have been demonstrated in bone marrow, about two orders of magnitude higher than that in the circulation, and further studies performed in mouse bone marrow suggested that these levels exhibit a circadian rhythm [163, 164]. Such a high level of melatonin in the bone marrow may be required for maintaining optimal activity of immunocompetent cells thus preventing the onset of immunodeficiencies, and, at the same time, these could provide a protective environment for MSC within such a metabolically active tissue. Furthermore, Gao et al. [165] in agreement with previous findings demonstrated that melatonin is involved in the reprogramming of iPSCs. These cells have the capacity to differentiate in MSC, and this represents an attractive property in view of developing high-throughput technologies to produce large quantities of stem cells for the application in regenerative medicine. Intriguingly, the reprogramming involves epigenetic changes that can result in cell death, especially in the early stages, suggesting that cell death is one of the barriers limiting the efficiency of the procedure. Moreover, the increased cell death observed during reprogramming is associated with a higher generation of ROS offering a mechanistic explanation to the effect of melatonin on iPSC generation [165]. Actually, melatonin may act as a ROS scavenger at the cellular level, and this is believed to represent a major underlying event in apoptosis inhibition and cellular protection function of this hormonal substance [48].

than 80%–90% of grafted cells die within 72 hr after injection, highlighting the extensive and early sufferance of transplanted cells that may undergo premature apoptotic cell death [170] by the stressors generated in the microenvironment of a tissue lesion. Different mechanisms have been involved in the early death of grafted cells, including oxidative stress, hypoxia, and inflammation [171, 172]. The role of melatonin as a potent free radical scavenger and stimulator of cellular antioxidants in different cell types may thus provide crucial mechanisms for MSC protection during the grafting into damaged tissues. Besides its well-known physiological activities [48, 173, 174], melatonin also lowers tissue injury through the reduction of oxidative damage and regulation of cytokine secretion in immune and bone marrow cells [148, 175]. In the study of Mias et al. [150], cited in the previous section, the anti-apoptotic/pro-survival effect of melatonin on injected MSCs was also verified after exposure to H2O2, a pro-oxidant that stimulates the expression of antioxidant genes such as catalase and SOD-1, and a challenge commonly used to trigger oxidative stress-induced apoptotic cell death. Melatonin produced the anti-apoptotic activity and the regulatory effect on antioxidant genes of MSC through the engagement of its receptors. Other authors [151, 158] have recently confirmed that melatonin protects transplanted MSCs from the damaging effects of oxidative stress in the microenvironment of a injured tissue. Early studies by Newsome et al. [176] suggested that the cellular protective function of melatonin in MSC may be the consequence of an increased expression of antioxidant enzymes in the cytoplasm and mitochondria, also modulating the ROS-generating enzyme NADPH oxidase which, depending on its activity rate, can play alternatively a physiological role in self-renewal of MSC or a cytotoxic effect. Collectively, these pieces of evidence highlight the role that melatonin may have in the control of ROS-generating and detoxification/antioxidant genes as upstream events in cellular protection and anti-apoptotic mechanisms of MSC. This interpretation encourages further studies aimed at establishing the therapeutic relevance of this indolamine in stem cell therapy of various injuries and in tissue regeneration.

Melatonin and MSC protection

Concluding remarks

Different studies have shown that MSC administration improves structural and functional recovery of injured organs. Morigi et al. [166] have partially associated the beneficial effects of injected MSC to their transdifferentiation in the cell phenotype of host organs. Recently, it was demonstrated that MSC get better tissue regeneration by secretion of mitogenic and vasculotropic factors [167–169]. Consequently, approaches that enhance MSC survival and secreted cytokine concentrations within the damaged area would significantly improve the beneficial effects of cell therapy. However, the efficacy of direct administration of MSC is restricted because the transplanted cells do not survive efficiently within the damaged organ. Indeed, several studies performed in solid organs showed that more

Over the last decades a growing number of studies have addressed physiological roles and therapeutic potential of MSCs. These cells transdifferentiate into various lineages. There is sparse data in the literature concerning the effects that melatonin may have on MSC from different tissues, but the available evidence is supportive of its roles in the physiological control of proliferation and cellular protection mechanisms. Signaling and transcriptional responses elicited by the exposure of MSC to melatonin are now partially disclosed (Fig. 1), but obviously further work is needed to provide a more precise picture of regulatory and differentiation mechanisms. Main lessons have come from studies carried out on bone marrow MSCs that unanimously suggest a 391

Luchetti et al. role of melatonin in the inhibition of adipogenesis to leave space for osteoblastic differentiation and, thus, bone formation. This level of knowledge has been implemented by the investigation of MSC from other tissues in which melatonin was confirmed to differentially control the signaling that drives these multipotent cells along the steps of osteogenesis, chondrogenesis, and myogenesis processes, gaining great relevance in tissue repair science. Therefore, the proposed effects of melatonin on MSC pave the way for a role of this hormone in the ex vivo handling of MSC as well as in the pharmacological support to grafting and preservation, and in the active interplay between MSC and the immune and nonimmune components involved in tissue regeneration. In this context, radical scavenging and transcriptional properties of melatonin are expected to contribute significant homeostatic effects starting from a support to feedback mechanisms that control prooxidant and inflammatory pathways. These properties and the aforementioned osteogenic potential of melatonin have suggested intriguing applications of this hormone in the prevention and therapy for bone and articular diseases associated with aging and metabolic defects. Another and so far poorly investigated option for the function that melatonin may have on MSC regulation is that of modulating the adipogenetic potential in the adipose. Early evidence obtained from investigations carried out in bone marrow MSC and from other sparse reports, indicates that melatonin may have important metabolic consequences in both physiological or diabetogenic conditions. In this respect, the adipogenetic process and its possible role in the development of pathologic obesity and IR mechanisms associated with T2D, are potential targets of therapeutic interventions with melatonin. These clinical inferences clearly demonstrate the importance of further exploring this area.

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Acknowledgements This overview of the literature has been compiled within the scientific activities of the research project ‘PROS.IT’, of the ‘Cluster Tecnologico Nazionale Agrifood’ and sponsored by the Italian Ministry of University and Research (grant # CTN01_00230_413096). We are indebted with Miss Cristina Tortoioli for the assistance provided in the phases of literature search and interpretation and for the invaluable support that she is providing to research activities carried out on MSC by this team of scientists.

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