Extracellular vesicles rejuvenate the microenvironmental modulating function of recipient tissue-specific mesenchymal stem cells in osteopenia treatment

Systemic transplantation of mesenchymal stem cells (MSCs), such as bone marrow MSCs (BMMSCs) and stem cells from human exfoliated deciduous teeth (SHED), is considered a prominent treatment for osteopenia. However, the mechanism of action of the transplanted MSCs has been poorly elucidated. In the recipient target tissue, including bone and bone marrow, only a few donor MSCs can be detected, suggesting that the direct contribution of donor MSCs may not be expected for osteopenia treatment. Meanwhile, secretomes, especially contents within extracellular vesicles (EVs) released from donor MSCs (MSC-EVs), play key roles in the treatment of several diseases. In this context, administrated donor MSC-EVs may affect bone-forming function of recipient cells. In this review, we discuss how MSC-EVs contribute to bone recovery recipient tissue in osteopenia. We also summarize a novel mechanism of action of systemic administration of SHED-derived EVs (SHED-EVs) in osteopenia. We found that reduced telomerase activity in recipient BMMSCs caused the deficiency of microenvironmental modulating function, including bone and bone marrow-like niche formation and immunomodulation in estrogen-deficient osteopenia model mice. Systemic administration of SHED-EVs could exert therapeutic effects on bone reduction via recovering the telomerase activity, leading to the rejuvenation of the microenvironmental modulating function in recipient BMMSCs, as seen in systemic transplantation of SHED. RNase-preconditioned donor SHED-EVs diminished the therapeutic benefits of administrated SHED-EVs in the recipient osteopenia model mice. These facts suggest that MSC-EV therapy targets the recipient BMMSCs to rejuvenate the microenvironmental modulating function via telomerase activity, recovering bone density. We then introduce future challenges to develop the reproducible MSC-EV therapy in osteopenia.


Introduction
Owing to the osteoblast differentiation capacity, mesenchymal stem cells (MSCs) have been considered a promising source for degenerative skeletal disease, such as osteopenia. Recent studies show that systemic transplantation of MSCs rescues the bone mineral reduction in both Fas-mutated MRL/lpr and ovariectomy-induced estrogen-deficient (OVX) mice (1,2). However, despite the persisting therapeutic effects of systemic MSC transplantation, infrequent engraftments of donor MSCs are recognized in the recipient target tissues, including bone and bone marrow, suggesting that the direct contribution of donor MSCs does not fully explain the solid mechanism of systemic MSC transplantation in osteopenia.
Increasing reports suggest that donor MSCs exert therapeutic effects via releasing secretomes, such as cytokines, chemokines, and growth factors (3). Notably, recent advances in the biology of extracellular vesicles (EVs) lead to an increased understanding of intercellular communication between donor cells and recipient cells in MSC therapy (4). EV-containing bioactive molecules, such as RNAs, DNA, lipid, and proteins, are transferred to affect the functions of recipient cells (5,6). In addition, current advances in the procedure of EV isolation promote the number of studies on EV therapy as a novel promising option (4).
As of February 2022, according to ClinicalTraials.gov (https:// clinicaltrials.gov) as a reference, there is no clinical trial using MSCderived EVs (MSC-EVs) for treating osteopenia/osteoporosis, even though less than ten clinical studies of MSC therapy are conducted in osteopenia/osteoporosis.
Previous preclinical studies nominate the various mechanisms of MSC-EV therapy. On the other hand, the precise criteria to clarify the quality of MSC-EVs in osteopenia treatment remains unclear because the definitive molecules and their targets are not identified. Here, we review the current understanding of molecular actions to recover bone reduction by systemic administration of MSC-EVs in osteopenia. We further introduce a novel mechanism of MSC-EV therapy for osteopenia treatment; the administrated MSC-EVs are involved in rejuvenizing the tissue-specific microenvironmental modulating function of recipient bone marrow MSCs (BMMSCs) through promoting Tert gene expression and telomerase activity, resulting in exerting the bone mineral recovery in osteopenia model animals.
Based on current preclinical knowledge, as a future perspective, we propose that focusing on the microenvironmental modulating function of recipient BMMSCs as a target of MSC-EV therapy for osteopenia enables to establish a criterion for the quality control of MSC-EVs to achieve MSC-EV therapy a standard option for osteopenia.

Discovery and property of MSCs
In the 1970s, a Russian biologist Alexander J. Friedenstein first discovers a clonogenic cell population in bone marrow stromal cells, colony-forming units-fibroblast (CFU-F) (7). The later studies report that CFU-F-forming stromal cells exhibit spindle-shaped and plastic-adherent features and show the properties of in vitro and in vivo osteoblast differentiation (8). Sequential transplantation assay also reveals the self-renewal capacity of the bone marrow stromal cells (9, 10). Further investigations identify that the bone marrow stromal cells exhibit the multipotency into mesenchymal tissue-specific cells, such as adipocytes and chondrocytes, as well as osteoblasts (8,11). Thus, in 1991, Arnold I. Caplan refers to the stromal cells as stem cells and named them MSCs (12). MSCs are now identified in a variety of postnatal tissues, such as adipose tissue, umbilical cord blood, umbilical cord, dental pulp, periodontal tissue, and liver (13). However, the rapidly increasing knowledge of MSCs and their clinical trials makes it challenging to compare the study outcomes.
In 2006, the International Society for Cellular Therapy addresses minimal criteria to define human MSCs (14); MSCs exhibit plastic adherent and express surface antigens positive to CD105, CD90, and CD73 and negative to CD45, CD34, CD14 or CD11b, CD79a or CD19, and human leukocyte antigen DR. MSCs are capable of differentiating into mesenchymal cells including adipocytes, chondrocytes, and osteoblasts. Currently, this criterion has been well-recognized and helpful for the characterization and application of MSCs in pre-clinical and clinical studies.

Mechanisms of actions of MSC-EV therapy in osteopenia
Clinical applications of systemic MSC transplantation have been explored in various diseases (15). The mechanisms of MSC therapy were explained by the in vitro and in vivo properties of multipotency or immune modulation of donor MSCs at the recipient disease sites. Especially the osteogenesis of MSCs promotes several investigations of MSC therapy in osteopenia model animals and demonstrates the bone regenerative outcomes (16).
However, a sufficient number of systemically transplanted MSCs fail to engraft in recipient target tissues (17,18), a critical question of why single transplanted MSCs exert long-term effects without the direct potency of donor MSCs.
EVs contain a variety of bioactive molecules, including nucleic acids, such as messenger RNAs, micro RNA (miRNA), and long noncoding RNA (lncRNA), lipids, and proteins, indicating that EVs perform as cargo for transferring their intercellular signals to recipient cells and control the functions of recipient cells (5,6).
Recent several studies revealed the mechanism of actions of systemic MSC therapy for osteopenia; the transplanted donor MSCs can release paracrine factors within MSC-EVs and functionally activate recipient cells by transferring therapeutical signal(s) via donor MSC-EVs, leading to ameliorate the reduced bone mineral density (22).
Apoptotic bodies (ApBs) of donor MSCs play crucial roles in MSC-based therapy through the intercellular communications between transplanted donor MSCs and recipient cells (23). Donor MSC-mediated T lymphocyte apoptosis via FasL/Fas pathway is proposed as a mechanism of MSC therapy (24). Systemic infusion of exogenous ApBs of BMMSCs ameliorates bone mineral reduction in several osteopenia models, including MRL/lpr, Caspase 3 -/-, and OVX mice (25). The infused ApBs activate the Wnt/b-catenin pathway in the recipient BMMCs by transferring ubiquitin ligase RNF146 and miR-328-3p within ApBs and regulate the differentiation of recipient osteoblasts and osteoclasts. Thus, the BMMSC ApB-mediated action is focused on the potential control of recipient bone metabolism to treat osteoporosis. However, there is no information on whether MSC-derived ApBs exhibit similar therapeutic benefits to osteopenia treatment of MSC-EVs. Further studies may be necessary to evaluate the effects and mechanism of MSC-derived ApBs in osteopenia treatment.

Target cells and therapeutic molecules in MSC-EV therapy for osteopenia
Numerous studies of MSC-EV therapy have been performed in osteopenia model animals (22,26) (Table 1).

MSC-EVs target recipient osteoblasts and osteoclasts
MSC-EV-containing RNAs enhance the proliferation and bone formation of recipient osteoblasts; lncRNA of metastasis associated lung adenocarcinoma transcript 1 in MSC-EVs acts as a sponge of miR-34c to promote the alkaline phosphatase activity and calcified nodule formation of osteoblasts associated with the increased gene expression of special AT-rich sequence-binding protein 2, runtrelated transcription factor 2, and activating transcription factor 4 and decreased gene expression of homeobox A2 (27). MiR-935contained MSC-EVs promote the proliferation and differentiation of osteoblasts by inhibiting signal transducer and activator of transcription 1 expression (28). MiRNAs within MSC-EVs enhance the proliferation and osteogenesis of osteoblasts, while the osteogenic MSC-EVs do not affect the proliferation of osteoblasts (29).
A single administration of MSC-EVs exerts bone recovery; MSC-EVs provide Fas to rescue Fas-deficient osteogenesis of recipient BMMSCs via regulating miR-29b-Notch pathway (38). Moreover, MSC-EVs can inhibit the apoptosis of recipient BMMSCs by transferring miR-1263 to inhibit the Hippomediated signaling pathway (39).
Taken together, the action of singly administrated MSC-EVs may cause the epigenetic memorization of stemness, including self-renew and cellular functions, in recipient tissue-specific MSCs and exert the long-term recovery and maintenance of bone metabolism. Meanwhile, multiple injections of MSC-EVs for osteopenia treatment may exert undesirable inconvenience to the patients of osteopenia in the clinical application of MSC-EV therapy, suggesting that single MSC-EV administration, targeting recipient tissue-specific MSCs, may be clinically safe and beneficial application for osteopenia.

A novel mechanism of MSC-EV therapy through microenvironmental modulating function of recipient BMMSCs
In the following sections, we will focus on the microenvironmental modulating function of recipient BMMSCs, which include bone metabolism interplayed with osteoblasts and osteoclasts, as an action of MSC-EV therapy.

Microenvironmental modulating function of MSCs
MSCs have been known to interact with neighboring cells directly (cell-to-cell contact) and indirectly (paracrine factors, EVs, and apoptosis) and modulate the microenvironment for immune cells and other tissue-specific cells, suggesting that tissuespecific MSCs potentially act as tissue-specific microenvironmental modulators in recipients after transplantation (23): in bone and bone marrow, resident BMMSCs can communicate with microenvironmental cells directly and indirectly and participate in stem cell niche formation, bone marrow hematopoiesis (42), and microenvironmental reorganization regulated by osteoblasts and osteoclasts (43,44).
Here, we focus on two properties of immune modulation and niche-organization as the microenvironmental modulating function of MSCs (Figure 1).

Immunomodulatory properties of MSCs
MSCs exhibit an immunomodulatory property to a variety of immune cells, such as T lymphocytes, B lymphocytes, and macrophages (45,46). The immunosuppressive functions of MSCs are explained by the following three mechanisms: 1) indirect intercellular communications with innate and adaptive immune cells by paracrine factors, including growth factors, cytokines, and container within EVs, such as miRNA; 2) direct intercellular communications with T lymphocytes through cell surface molecules, such as FasL and program death ligand 1; 3) efferocytosis of T lymphocyte-induced ApBs of MSCs, followed by secreting immunosuppressive factors from phagocytes (23, 47).

Niche-organizing properties of MSCs
When BMMSCs are subcutaneously implanted with hydroxyapatite/beta-tricalcium phosphate (HA/TCP) as a carrier into immunocompromised mice, donor BMMSC-derived osteoblasts deposit de novo lamellar-bone-like matrix on the carriers (48). Interestingly, in a space surrounded by the de novo bone-like matrix, mononuclear cells exhibit hematopoietic properties of hematopoietic colony formation and expression of hematopoietic cell markers, including Sca-1 and c-Kit stem cell markers and CD45 lymphoid progenitor cell marker (49, 50). The mononuclear cells can also systemically circulate in the recipient body, indicating that the de novo microenvironment is implicated in a hematopoietic niche, likely in the bone marrow. These findings indicate that BMMSCs potentially serve as hematopoietic nicheorganizing cells, as well as bone-forming cells, and contribute to the regulation of lymphocyte production and differentiation. mitotic divisions and become two daughter cells. One daughter cell maintains the properties of MSCs, while the others undergo further symmetric division to produce more progenitor cells of tissuespecific cells. Thus, MSCs can provide an internal repair system in the human body by recruiting tissue-specific cells into damaged tissues and organs. MSCs must acquire the sustained self-renewal and proliferative capacity to maintain homeostasis in the human body (13).

MSCs are defined by two important properties of self-renewal and multipotency into various types of functional cells, as likely to embryonic stem cells (ESCs) (51, 52). MSCs undergo asymmetric
Telomerase reverse transcriptase (TERT) is a catalytic subunit of telomerase and is essential to acquire telomerase activity in cells (53). Suppression of TERT inhibits the pluripotency and differentiation of human ESCs; meanwhile, ectopic TERT expression enhances the colony-forming ability, proliferation, and differentiation of ESCs (54) and BMMSCs (55).
Telomerase plays critical roles in stemness of stem cells (54). Telomerase reactivation in adult stem cells can help to repaired of tissue degeneration (56). Telomere attrition is considered one of the hallmarks of epigenetic changes in aging stem cells (57). Furthermore, in nuclear reprogramming process of induced pluripotent stem cells, telomere rejuvenation is occurred by regulating epigenetic modification on chromatin and DNA (58).
Telomerase activity is detectable in HSCs and MSCs, but the levels are lower than those in ESCs. The moderate levels of telomerase activity in HSCs are essential to support the rapid turnover of differentiated blood cells (59). Furthermore, telomerase knockout mice express several disorders (60,61). Telomerase dysfunction impairs the function and engraftment of HSCs (62). Moreover, TERT-deficient mice exhibit bone loss by suppressing osteoblasts and accelerating osteoclasts (63).
Thus, TERT is a critical rate-limiting component to control telomerase activity for maintaining and regulating the sustained self-renewal, proliferation, and functions of MSCs. In other words, TERT is considered a significant key enzyme to control the microenvironmental modulating function of MSCs.

Impaired microenvironmental modulating function of recipient BMMSCs in osteopenia model mice
Recipient BMMSCs isolated from non-treated osteopenia model mice (recipient OPe-BMMSCs) display deficient MSC stemness of self-renewal and osteogenic capacity. In addition, the recipient OPe-BMMSCs exhibit a suppressed immunomodulatory function to interleukin 17 helper T lymphocytes (Th17) and suppressed regulatory T lymphocytes (Tregs). Moreover, the recipient OPe-BMMSCs display damaged hematopoietic functions, including the de novo formation of bone marrow-like hematopoietic niche and in vitro induction of hematopoietic colony formation (40). Thus, the recipient OPe-BMMSCs may be epigenetically damaged in the stemness, microenvironmental modulatory function, and osteogenic ability ( Figure 2). Therefore, the dysfunctions of recipient OPe-BMMSCs are considered one of the critical pathogenesis in osteopenia.

Systemic administration of stem cells from human exfoliated deciduous teethderived EVs target the recipient BMMSCs
to rejuvenate the microenvironmental modulating function via their telomerase reverse transcriptase/telomerase activity in osteopenia treatment 4
Increasing studies report SHED-EVs carry in vitro and in vivo biological actions of SHED (47,85). SHED-EV-containing various bioactive molecules, such as nucleic acids and protein, are transported to target cells selectively by an unknown mechanism, resulting in implicating cell-cell communication, signal t r a n s d u c t i o n , i m m u n e m o d u l a t i o n , a n d e p i g e n e t i c reprogramming of recipient cells. Recently, the benefits of SHED-EV administration are evaluated in bone regeneration (86), neuroprotective effects (87,88), and anti-inflammatory function (89, 90) in disease model mice.

SHED-EVs are mechanistically involved in systemic transplantation of SHED for osteopenia treatment
The systemic transplantations of SHED rescued the osteopenia phenotype as indicated by the significant increase in bone mineral density and trabecular bone structure (2,72,76). The single SHED transplantation improved the reduced osteogenic capacity of recipient BMMSCs and suppressed the differentiation and bone resorptive activity of recipient osteoclasts. The systemic transplantations of SHED also recovered the abnormal immune reactions of enhanced Th17 cells and suppressed Tregs in the osteopenia model mice. Interestingly, the single systemic transplantations rescued the microenvironmental modulating function of forming de novo bone and bone marrow by recipient BMMSCs, indicating that SHED therapy can exert therapeutic effects on osteopenia phenotype.
Meanwhile, the very low frequency of donor SHED are engrafted in the recipient bone and bone marrow tissues in osteopenia model animals (2,72,76). Further systemic infusion of SHED-derived conditioned medium exert the bone regeneration in OVX mice (91), suggesting that the indirect manner of transplanted donor SHED is involved in the improvement of the reduced bone mineral density (25,31).
Systemic SHED-EV administration ameliorates the recovery of bone loss associated with osteoclast activation and hyperactivation of Th17 cells in the osteopenia model mice (40,92), indicating that SHED-EV therapy is an alternative option for treating osteopenia and may target the recipient BMMSC function(s).
In the following literature, we discuss a unique mechanism of action of SHED-EV therapy for osteopenia treatment, which rejuvenate the bone marrow microenvironment through recipient BMMSCs (40,47,92).
When SHED-EVs are systemically administrated into osteopenia model mice, SHED-EVs are up-taken in the recipient OPe-BMMSCs and the microenvironmental modulating dysfunction of recipient OPe-BMMSCs are improved. The SHED-EV administration rescues the reduced Tert expression and reduced telomerase activity of recipient OPe-BMMSCs. Systemic administration of RNase-preconditioned SHED-EVs mostly attenuated the phenotypical and cellular effects of SHED-EV administration.
Small RNA, such as microRNAs, is well-known to epigenetically regulate gene expression (93, 94). MIR346 can bind to a region in the 3'UTR of TERT mRNA, leading to upregulating TERT expression (95, 96). In fact, SHED-EVs contain MIR346, and when SHED-EVs are incubated with human BMMSCs, MIR346 is increased in human BMMSCs. Therefore, SHED-EV-derived MIR346 may act as a candidate to regulate Tert expression and telomerase activity epigenetically, leading to participation in SHED-EV therapy for osteopenia ( Figure 2).
Semaphorin 3A (SEMA3A) plays an osteoprotective factor produced by osteoblasts (97,98) and inhibits osteoclast differentiation via receptor activator of nuclear factor-kB ligand by binding to neuropilin-1. The recipient osteogenic OPe-BMMSCs show the suppressed expression of SEMA3A and exhibit the decreased capacity for mineralized tissue deposition. Systemic SHED-EV administration increases the mineralized tissue deposition associated with the increased expression of SEMA3A in the recipient OPe-BMMSCs. The hyperactivated osteoclasts in osteopenia model mice are suppressed by the SHED-EV administration. Meanwhile, RNase-preconditioned SHED-EV administration attenuates the SHED-EV efficacy to the expression o f S E M A 3 A i n t h e r e c i p i e n t O P e -B M M S C s a n d osteoclast suppression.
Taken together, systemic administration of SHED-EVs exerts a therapeutic effect in osteopenia by improving the microenvironmental modulating function of recipient BMMSCs through enhancement of Tert expression and telomerase activity by SHED-EV-transferred miRNA. Thus, the rejuvenated recipient BMMSCs mainly contribute to bone reconstruction by regulating the function of osteoblasts and osteoclasts through SEMA3A (Figure 2).

Summary and future challenge
On regenerative treatment for osteopenia, MSC therapy is a promising option to recover bone mineral reduction by regulating the balance between osteoclast and osteoblast activity and immunomodulation. MSC-EV-mediated intercellular signal communications play an important role in MSC therapy. On the other hand, MSC-EV therapy is considered the alternative to MSC therapy for treating osteopenia. The common mechanism in MSCbased and MSC-free therapies is that MSC-EVs transfer a signal(s) to rejuvenate the impaired functions of recipient BMMSCs; this process is accompanied by epigenetic changes of telomerase activity in the recipient BMMSCs. Following that, the impaired recipient BMMSCs can acquire normalized self-renewal, stemness, and function to reconstruct the destructed bone matrix and maintain bone homeostasis. Once the self-renewal properties are epigenetically rescued in recipient BMMSCs, especially through TERT-telomerase pathway, the therapeutic effects in the recipient tissue may last in the long term. Thus, the epigenetic regulation in recipient BMMSCs is a promising target for systemic degenerative diseases, such as osteopenia. Further investigations, which are focused on epigenetic regulations in recipient tissue-specific MSCs, will open the novel door for establishing a safe and highly effective therapy not only for osteopenia but also for systemic degenerative disorders.
Generally, EVs cannot proliferate themselves (4, 21) and can circulate systemically without tissue trapping (18,99,100). Meanwhile, tumorigenesis and pulmonary embolism are major risks in general MSC therapy. MSC-EVs exhibit stable components under cryopreserved and freshly thawed conditions without losing their functional properties, while cryopreserved MSCs significantly lose their immunomodulatory properties and must be cultured to recover their full properties (101,102). Thus, due to the safety and practical and functional off-the-shelf option, MSC-EV therapy may have a promising potential compared to MSC therapy.
Despite the advantages of MSC-EV therapy, there are several challenges to achieve MSC-EV therapy as a standard option for osteopenia. Clinically, the large-scale manufacturing of MSC-EVs is a considerable matter for delivering to many patients. As the quantity of MSC-EVs affects the quality, the consistent reproducibility of therapeutic effects on osteopenia relies on the quality of MSC-EVs. Therefore, to make MSC-EV therapy successful, the quality control of MSC-EVs must be primally overcome. However, the quality control of MSC-EVs is much more challenging due to the heterogeneity of MSC-EV, source difference, and donor difference of the parent MSCs (22). Although further evaluation of the mechanisms of action by MSC-EVs for osteopenia treatment will be required, once the definitive molecular mechanism is identified, the bioactive factor (s) within MSC-EVs will enable us to use it as the key molecule for quality control. Further engineering may produce artificial nanoparticles that contain enough key molecules for osteoporosis treatment in the future.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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