Electromagnetic field exposure as a plausible approach to enhance the proliferation and differentiation of mesenchymal stem cells in clinically relevant scenarios

Electromagnetic field exposure as a plausible approach to enhance the proliferation and differentiation of mesenchymal stem cells in clinically relevant scenarios 电磁场暴露作为在临床相关情况下增强间充质干细胞增殖和分化的一种可行方法 1 first-author 2 3 4 4 1 corresp Azizi MISKON, azizimiskon@upnm.edu.my Azizi MISKON, azizimiskon@upnm.edu.my azizimiskon@upnm.edu.my https://orcid.org/0000-0002-4267-4180 Bio-artificial Organ and Regenerative Medicine Unit, National Defense University of Malaysia, Kuala Lumpur57000, Malaysia Bio-artificial Organ and Regenerative Medicine Unit, National Defense University of Malaysia, Kuala Lumpur57000, Malaysia Cellular and Molecular Research Centre, Iran University of Medical Sciences, Tehran 1449614535, Iran Cellular and Molecular Research Centre, Iran University of Medical Sciences, Tehran 1449614535, Iran Institutes of Regenerative Medicine, Iran University of Medical Sciences, Tehran 1996714353, Iran Institutes of Regenerative Medicine, Iran University of Medical Sciences, Tehran 1996714353, Iran Stem Cell and Immunity Research Group, Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), Selangor43400, Malaysia Stem Cell and Immunity Research Group, Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), Selangor43400, Malaysia Mesenchymal stem/stromal cell (MSC)‍-based therapy has been regarded as one of the most revolutionary breakthroughs in the history of modern medicine owing to its myriad of immunoregulatory and regenerative properties. With the rapid progress in the fields of osteo- and musculoskeletal therapies, the demand for MSC-based treatment modalities is becoming increasingly prominent. In this endeavor, researchers around the world have devised new and innovative techniques to support the proliferation of MSCs while minimizing the loss of hallmark features of stem cells. One such example is electromagnetic field (EMF) exposure, which is an alternative approach with promising potential. In this review, we present a critical discourse on the efficiency, practicability, and limitations of some of the relevant methods, with insurmountable evidence backing the implementation of EMF as a feasible strategy for the clinically relevant expansion of MSCs. 在这篇综述中，我们对一些相关方法的效率、实用性和局限性进行了批判性讨论，并支持将电磁场（EMF）作为临床相关间充质干细胞（MSCs）扩展的可行策略。本文旨在寻找促进MSCs增殖和分化而没有细胞毒性和基因毒性作用的新疗法，可以为再生医学和组织工程领域带来新的思路。随着最近细胞培养方法的进步和专用生物反应器的出现，EMF疗法显然是进一步促进MSCs增殖和分化的一种有前途的方法。在过去，EMF疗法已被证实是一种成功用作治疗骨病的有效且非侵入性的方法。同时，各种研究结果证明了0.2–5.0 mT和15–75 Hz的EMF对不同治疗时间的MSCs增殖和分化的积极影响。尽管EMF暴露作为MSCs临床扩展的新策略已显示出巨大潜力，但有必要进一步检查EMF的核心特性，以确定在增强MSCs增殖的同时保持其分化潜能和干细胞的独特暴露范围。 Electromagnetic field Proliferation Mesenchymal stem cell Therapy 电磁场 增殖 间充质干细胞 治疗 1 Introduction 1 Discovered around 50 years ago in the bone marrow (BM), mesenchymal stem cells (MSCs) are a heterogeneous group of spindle-shaped cells with unique self-renewing and differentiation properties ( Friedenstein et al., 1966 Friedenstein et al., 1966 ). In many studies, MSCs have been observed to differentiate into distinct cells of the mesodermal lineage, including osteocytes, chondrocytes, and adipocytes ( Pittenger et al., 1999 Pittenger et al., 1999 ; Kolf et al., 2007 Kolf et al., 2007 ; Thibault et al., 2010 Thibault et al., 2010 ; Ang et al., 2014 Ang et al., 2014 ; Somoza et al., 2014 Somoza et al., 2014 ). Such propensity allows MSCs to be present almost ubiquitously at different sites within the human body, including but not limited to, adipose, umbilical cord, and amniotic fluid tissues ( el Omar et al., 2014 el Omar et al., 2014 ; de Francesco et al., 2015 de Francesco et al., 2015 ; Mushahary et al., 2018 Mushahary et al., 2018 ). In addition, MSCs have been associated with various other biologically significant qualities, such as exerting anti-tumorigenic effects ( Lu et al., 2008 Lu et al., 2008 ; Sarmadi et al., 2008, Sarmadi et al., 2008, 2020 2020 ; Bruno et al., 2013 Bruno et al., 2013 ), potent immunomodulatory activity ( Ramasamy et al., 2010 Ramasamy et al., 2010 ; Volarevic et al., 2017 Volarevic et al., 2017 ; Maqbool et al., 2020 Maqbool et al., 2020 ; Zhang et al., 2021 Zhang et al., 2021 ), and lower rates of immunogenicity ( Aggarwal and Pittenger, 2005 Aggarwal and Pittenger, 2005 ; Barry et al., 2005 Barry et al., 2005 ; Klyushnenkova et al., 2005 Klyushnenkova et al., 2005 ). These characteristics, coupled with fewer ethical cues ( Fung et al., 2017 Fung et al., 2017 ; Volarevic et al., 2018 Volarevic et al., 2018 ), have generated a widespread interest among scientists and researchers alike, thereby making MSCs one of the most clinically studied cells in the platform of experimental cell therapy. For instance, in musculoskeletal clinical trials, autologous and allogeneic transplantations of MSCs were found to yield better performance in terms of post-operative healing and functional outcomes ( Hashimoto et al., 2019 Hashimoto et al., 2019 ; Song et al., 2020 Song et al., 2020 ; Chung et al., 2021 Chung et al., 2021 ). Similar observations have been reported in several cases of psoriasis ( Lee et al., 2017 Lee et al., 2017 ; Chen MS et al., 2019 Chen MS et al., 2019 ; Wang et al., 2020 Wang et al., 2020 ), ischaemic and non-ischaemic cardiomyopathy ( Butler et al., 2017 Butler et al., 2017 ; Florea et al., 2017 Florea et al., 2017 ; Hare et al., 2017 Hare et al., 2017 ), and even in corona virus disease 2019 (COVID-19)‍-related pneumonia ( Leng et al., 2020 Leng et al., 2020 ; Liang et al., 2020 Liang et al., 2020 ; Shu et al., 2020 Shu et al., 2020 ; Tang et al., 2020 Tang et al., 2020 ). Further examples involve direct application of MSCs in the treatment and management of skin and sensory organ-related injuries ( Ude et al., 2018 Ude et al., 2018 ), post-kidney transplantation ( Erpicum et al., 2019 Erpicum et al., 2019 ), diabetes (types 1 and 2) ( Jiang et al., 2011 Jiang et al., 2011 ; Carlsson and Svahn, 2018 Carlsson and Svahn, 2018 ), graft-versus-host disease (GvHD) ( Kurtzberg et al., 2020 Kurtzberg et al., 2020 ), and multiple sclerosis ( Connick et al., 2012 Connick et al., 2012 ; Iacobaeus et al., 2019 Iacobaeus et al., 2019 ). Despite their numerous positive attributes, freshly isolated MSCs are rare and have high heterogeneity, and are in limited quantities because of factors such as age and gender ( Alt et al., 2012 Alt et al., 2012 ; Fossett and Khan, 2012 Fossett and Khan, 2012 ; Yang et al., 2018 Yang et al., 2018 ); therefore, an extensive expansion of the isolated cells is necessary to meet the growing demands of clinical experimental trials. However, extensive in vitro passages may undergo morphological, phenotypic, and genetic changes ( Yang et al., 2018 Yang et al., 2018 ). Considerable efforts have been made to enhance the proliferation of MSCs in vitro without modifying their capabilities of stemness and differentiation. These include providing additional supplements, mainly growth factors (ascorbic acid, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor with two subunits BB (PDGF-BB), and epidermal growth factor (EGF)) into their cultures ( Solchaga et al., 2005 Solchaga et al., 2005 ; Choi et al., 2008 Choi et al., 2008 ; Tamama et al., 2010 Tamama et al., 2010 ; Sun et al., 2013 Sun et al., 2013 ). Nevertheless, Gharibi and Hughes (2012) demonstrated that, even if given all of the supplementations, MSCs showed a marked decline in their differentiation ability and expression of stem cell genes after as few as 100 d of culture ( Gharibi and Hughes, 2012 Gharibi and Hughes, 2012 ). In this context, MSCs reaching senescence early would no longer be able to accommodate the need for translational medical therapies. This is because senescent cells, albeit in small numbers, could affect the functionalities of other organs and tissues through the release of secretomes containing proinflammatory molecules ( Childs et al., 2015 Childs et al., 2015 ). Thus, clinically practical and cost-effective protocols are imperative for the proper generation and maintenance of MSCs. 2 Potential of electromagnetic fields (EMFs) for MSC therapy 1 Over the last few years, EMF has garnered attention as one of the most promising tools in stem cell therapy ( Esposito et al., 2013 Esposito et al., 2013 ; Ongaro et al., 2014 Ongaro et al., 2014 ; Miskon et al., 2018 Miskon et al., 2018 ). EMF is a combination of electrical and magnetic fields, which can create non-ionized waves and non-thermal fields. Magnetic therapy encompasses a variety of approaches, which include EMF or pulsed EMF (PEMF). The latter is a type of electromagnetic therapy in which a small electrical current and low-frequency EMFs with specific waveforms and amplitudes, ranging between 6 and 500 Hz, are intermittently applied to the body ( Hu et al., 2020 Hu et al., 2020 ; Qiu et al., 2020 Qiu et al., 2020 ). The application of EMF on living cells was found to induce a variety of effects not only at the cellular level, but also at the genomic level. In one study, the overexpression of p27 (cyclin-dependent kinase inhibitor), due to extremely low-frequency (ELF)-EMF exposure, was found to lead to cell cycle arrest ( Geng et al., 2014 Geng et al., 2014 ), while in another study, PEMF caused significant changes in the expression of programmed cell-death-related genes during different stages of apoptosis and necrosis ( Kaszuba-Zwoinska et al., 2012 Kaszuba-Zwoinska et al., 2012 ). EMF exposure has also been linked to changes in DNA replication ( Cheng and Zou, 2006 Cheng and Zou, 2006 ) and cytokine expression ( Jasti et al., 2001 Jasti et al., 2001 ). As a form of therapy, EMF has been widely utilized in the treatment of bone fractures, as well as musculoskeletal and neuronal disorders, with high rates of success ( Shupak et al., 2003 Shupak et al., 2003 ). Interestingly, some findings suggested that, at predetermined time intervals and frequencies, EMF may also promote the proliferation of MSCs ( Sun et al., 2009 Sun et al., 2009 ; Fan et al., 2015 Fan et al., 2015 ; Miskon et al., 2018 Miskon et al., 2018 ) and could drive their cellular state of differentiation into chondrogenesis ( Parate et al., 2017 Parate et al., 2017 ), osteogenesis ( Kim et al., 2015 Kim et al., 2015 ), and even neurogenesis ( Seong et al., 2014 Seong et al., 2014 ). In light of these data, this paper aims to provide a concise review of current strategies employed for the culture and maintenance of MSCs in experimental laboratories. This includes a comprehensive analysis of factors and culture conditions related to long-term in vitro expansion of MSCs without compromising their "stemness." In addition, the underlying effects of EMF exposure on the proliferative, regenerative, and differentiation capacities of MSCs will be thoroughly discussed. Finally, we make brief evaluation of the deleterious impacts of EMF, if any, on cultures of MSCs. 2.1 Effects of EMFs on the proliferation of MSCs 2 In recent years, the effects of EMF exposure on the viability and proliferation of MSCs have been the topic of active research (Zhang et al., 2007, (Zhang et al., 2007, 2018 2018 ; Sun et al., 2009 Sun et al., 2009 ; Song et al., 2014a Song et al., 2014a ; Fan et al., 2015 Fan et al., 2015 ; Marędziak et al., 2017 Marędziak et al., 2017 ; Ehnert et al., 2018 Ehnert et al., 2018 ; Ferroni et al., 2018 Ferroni et al., 2018 ; Miskon et al., 2018 Miskon et al., 2018 ; Poh et al., 2018 Poh et al., 2018 ; Ross et al., 2018 Ross et al., 2018 ; Seo et al., 2018 Seo et al., 2018 ; Chen JY et al., 2019 Chen JY et al., 2019 ; Parate et al., 2020 Parate et al., 2020 ). In a study by Miskon et al. (2018) In a study by Miskon et al. (2018) , umbilical cord-derived MSCs were pulsed with EMF for 5 min, and the results indicated an increase in the numbers of monolayer and suspension MSCs by 1.39- and 2.41-fold, respectively, when compared with those of the control group after 5 d ( Miskon et al., 2018 Miskon et al., 2018 ). However, the underlying mechanism or EMF frequencies used in this study were not elucidated. In a different study, when BM-derived MSCs were exposed to 15 Hz of EMF for 8 h/d, Sun et al. (2009) Sun et al. (2009) reported a 59% and 40% increase in the viability of MSCs in low- (1000 cells/cm 2 ) and high-density (3000 cells/cm 2 ) seeded cultures, respectively. The reason for this could be due to the shortening of the lag phase, causing EMF to enhance the proliferation of MSCs during the exponential phase and leading the cells into the G2/M phase of the cell cycle. Nonetheless, enhanced cellular proliferation served as the outcome of increased DNA synthesis activity. This was made evident when exposing rat- and mouse-derived MSCs to 50 Hz of EMF for 4 h/d for three consecutive days. It was found that cell viability, DNA synthesis, and the proportion of MSCs in S phase in the EMF group were significantly elevated as compared with the SHAM group ( Fan et al., 2015 Fan et al., 2015 ). This finding was also in accordance with a recent study by Bloise et al. (2018) Bloise et al. (2018) who demonstrated increased DNA synthesis in PEMF-exposed BM-derived MSCs. The continuous expression of several cytokines is required for the process of MSC proliferation and differentiation, and the expression of these cytokines can be modulated by EMF. Some studies have shown that a shortened lag phase of the cell cycle induced by EMF may result in a higher cellular proliferation index, which could be mediated by the increased expression of cytokines ( Sun et al., 2009 Sun et al., 2009 ; Zhang et al., 2013 Zhang et al., 2013 ; Fan et al., 2015 Fan et al., 2015 ). MSCs exposed to EMF have exhibited the elevated messenger RNA (mRNA) expression of a group of hematopoietic cytokines, namely, macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), interleukin-7 (IL-7), and IL-11, while the mRNA expression of inflammatory cytokines, stromal cell-derived factor-1 (SDF-1), tumor necrosis factor-α (TNF-α), and IL-6 remained unchanged ( Fan et al., 2015 Fan et al., 2015 ). These inflammatory cytokines are a part of the soluble factors secreted upon the instigation of signaling pathways activated by BM cells ( Harmer et al., 2019 Harmer et al., 2019 ). This dynamic event might be due to the pattern and time points of EMF application; it was suggested that the elevation of the aforementioned cytokines might enhance the proliferation of MSCs ( Fan et al., 2015 Fan et al., 2015 ). The antigen expression profile of cytokines secreted by MSCs was further analyzed using an antibody fabricated array consisting of 120 antibodies reactive to 120 cytokines, where all MSCs were BM-derived ( Park et al., 2009 Park et al., 2009 ). The array was incubated with the secretion media of cells (from BM- and umbilical cord blood (UCB)‍-derived MSCs), followed by 1.5 h of incubation with biotin-conjugated antibodies. The hybridized antigen-antibody complexes were detected using streptavidin-conjugated peroxidase. A significant elevation of the signal was observed for six cytokines, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), vascular endothelial growth factor (VEGF), and osteoprotegerin (OPG), which was consistent across BM-derived MSC donors of all races, ages, and genders. This profile pattern was found to be similar to that of UCB-derived MSCs, with IL-6 exhibiting the strongest signal. Furthermore, IL-8 with known mitogenic and angiogenic potential showed the highest signal in UCB-derived MSCs. These observations further elucidate the molecular mechanism underlying MSCs ( Park et al., 2009 Park et al., 2009 ). Although the expression levels of M-CSF, SCF, thrombopoietin (TPO), LIF, IL-11, and IL-7 were observed to be significantly upregulated in EMF-exposed MSCs, the causative relationship leading towards MSC proliferation remains unclear. It is also interesting to note that the proliferative effects of EMF on MSCs extend even beyond days after initial exposure, which suggests that the triggered underlying mechanism of action is long-lasting ( Tu et al., 2018 Tu et al., 2018 ). The summary of recent studies on the proliferative effects of EMF and PEMF exposure on MSC culture is shown in Table 1 Table 1 and Fig. 1 Fig. 1 . EMF: electromagnetic field; PEMF: pulsed EMF; MSC: mesenchymal stem cell; BM: bone marrow; rBM-MSCs: rat BM-MSCs; hBM-MSCs: human BM-MSCs; hMSCs: human MSCs; hESCs: human epidermal stem cells; hUC-MSCs: human umbilical cord-derived MSCs; hAD-MSCs: human adipose-derived MSCs; LF: low-frequency; ELF: extremely LF; MF: magnetic field; SEMF: sinusoidal EMF. Study Biological model Type of exposure Exposure frequency (Hz) Exposure intensity (mT) Duration Outcome Jazayeri et al., 2017 rBM-MSCs LF-EMF 15 0.2 6 h/d for 5, 10, and 14 d Significantly increased MSC proliferation Sun et al., 2009 hBM-MSCs PEMF 15 8 h/d Expedited the proliferation of BM-MSCs Yan et al., 2010 hMSCs ELF-MF 50 20 Inhibited the growth of hMSCs Zhang et al., 2013 hESCs LF-EMF 1, 10, and 50 5 30 min/d for 3, 5, and 7 d Increased the proliferation hESCs Song et al., 2014b rBM-MSCs SEMF 15 1 1 h/d Increased MSC proliferation Song et al., 2014a BM-MSCs EMF 15 1 4 h/d Expedited the proliferation of BM-MSCs Fan et al, 2015 rBM-MSCs ELF-EMF 50 1 4 h/d Increased the proliferation of MSCs and upregulated haematopoietic cytokines Marędziaket al., 2017 hAD-MSCs Static MF 0.5 24 h/d for 7 d Improved the process of signaling associated with the proliferation of MSCs Miskon et al., 2018 hUC-MSCs EMF 300 s/d for 5 d Increased the proliferation of hUC-MSCs in suspension culture Ehnert et al., 2018 hAD-MSCs ELF-PEMF 16–24 6×10 -3 –282×10 -3 7 min/d, 5 times per week Significantly increased mitochondrial activity Ferroni et al., 2018 MSCs PEMF 1, 3, 15, and 30 d Significantly increased MSC proliferation Seo et al., 2018 BM-MSCs PEMF 50 1 1 h/d for 5, 7, and 10 d Slight changes though not significant Bloise et al., 2018 hBM-MSCs PEMF 75 2 10 min/d for1–15 d Significantly increased MSC proliferation Poh et al., 2018 hAD-MSCs ELF-PEMF 26 3 d No significant changes Ross et al., 2018 hMSCs ELF-EMF 5 0.4 20 min/d, 3 times per weekfor 2 weeks Slight changes though not significant Tu et al., 2018 rBM-MSCs SEMF 15 1 4 h/d Significantly increased MSC proliferation Zhang et al., 2018 BM-MSCs EMF 7.5, 15, 30, 50, and 75 1 24 h Increased MSC migration but no proliferative changes Chen et al., 2019a rBM-MSCs SEMF 15 1 4 h/d for 7 d Significantly increased MSC proliferation Parate et al., 2020 MSCs PEMF 15 2 30 min No significant changes It was proposed that PEMF exposure may have triggered the release of free ions such as potassium ion (K + ) and calcium ion (Ca 2+ ) from the smooth endoplasmic reticulum (ER), which can subsequently affect the activity of activated K + channels, leading to enhanced cellular progression from G1 to S phases ( Jazayeri et al., 2017 Jazayeri et al., 2017 ). For instance, Ca 2+ has been regarded as an important second messenger in a given cell, as it regulates a plethora of signal transduction in critical processes, such as adenosine triphosphate (ATP) synthesis, apoptosis, cellular motility, gene expression, and proliferation. In a study it was demonstrated that intracellular Ca 2+ signaling mediates insulin-like growth factor-1 (IGF-1)-induced proliferation in MSCs ( Wu et al., 2020 Wu et al., 2020 ). As non-excitable cells, Ca 2+ oscillations in MSCs are typically initiated by the upregulation of inositol-1,‍4,‍5-triphosphate receptor 2 (IP 3 R2) and sarco/endoplasmic reticulum Ca 2+ -ATPase 3 (SERCA3), resulting in Ca 2+ release from the ER. It was outlined that Ca 2+ oscillations may increase the levels of cyclins A and E that regulate cell cycle progression and proliferation ( Hanna et al., 2017 Hanna et al., 2017 ). Moreover, it was reported that elevated levels of extracellular Ca 2+ may also directly promote MSC proliferation through the induced expression of FGF-2 and transforming growth factor-‍β1 (TGF-‍β1), as well as cell cycle regulator c-Jun ( Lee et al., 2018 Lee et al., 2018 ). Given that cellular exposure to EMF has been shown to dramatically increase Ca 2+ influx though enhanced presynaptic calcium channel expression, the proposed notion that the increased proliferation of EMF-exposed MSCs is mediated by the alterations of its Ca 2+ current is well supported ( Sun et al., 2016 Sun et al., 2016 ). Some investigators also proposed the function of signal transduction pathway as responsible for the proliferative effects of EMF exposure on MSC cultures. In their study, Fan et al. (2015) In their study, Fan et al. (2015) proposed that the induced FGF-2 may inhibit cellular senescence and promote cellular proliferation via the phosphoinositide 3-kinase (PI3K)/serine-threonine kinase (AKT)-mouse double minute 2 (MDM2) signaling pathway. This notion is in agreement with recent studies that proposed the involvement of PI3K/AKT signaling as one of key players in the maintenance of self-renewal and stemness of MSCs ( Fong et al., 2007 Fong et al., 2007 ; Schaefer et al., 2020 Schaefer et al., 2020 ). The in-depth explanation regarding EMF interaction with biological cells may open a new perspective, in particular in the application of EMF to MSCs, as depicted in Fig. 2 Fig. 2 . Though multiple studies have provided evidence on the proliferative effects of EMF on MSC cultures, some papers presented contradicting findings ( Yan et al., 2010 Yan et al., 2010 ). These discrepancies are not immediately clear, though some scholars described that the effects of EMF depend on the cell type, cell state, frequency, amplitude, and exposure time ( Ivancsits et al., 2005 Ivancsits et al., 2005 ; Zhang et al., 2007 Zhang et al., 2007 ; Song et al., 2014b Song et al., 2014b ). Prolonged EMF exposure, for instance, could negatively impact the viability and proliferation of MSCs. This has been made evident by Marmotti et al. (2018) who investigated the optimal exposure duration of EMF onto MSC culture per day and the longest time period of exposure it could withstand before exhibiting a decreasing trend in cell survival. Additionally, this study reaffirms the findings of earlier works that had also provided evidence supporting that the observed proliferative effects of MSCs post-EMF exposure are greatly time duration-dependent ( Song et al., 2014b Song et al., 2014b ). 2.2 Effects of EMF on the differentiation of MSCs 2 Another important goal of the EMF exposure strategy is to positively reinforce the differentiative capability of MSCs. As mentioned previously, EMF therapy has been used to treat a broad range of diseases, such as non-union bone fractures, osteoporosis, and pseudarthrosis, as well as for cartilage and tendon repair ( Shupak et al., 2003 Shupak et al., 2003 ; Assiotis et al., 2012 Assiotis et al., 2012 ; Zhou et al., 2012 Zhou et al., 2012 ). Although the exact mechanism by which EMF stimulation induces lineage commitment within MSCs remains inconclusive, it has been well established that Runt-related transcription factor 2 ( Runx2 )/core-binding factor α1 ( Cbfa1 ) and osterix ( Sp7 ) serve as predominant transcription factors that drive MSC osteogenic differentiation, while SRY-box transcription 9 ( Sox9 ) and the modulation of the Wnt/β‍-catenin signaling pathway control chondrogenesis in cultures ( Ross et al., 2015 Ross et al., 2015 ). Interestingly, the expression levels of osteoblastic marker genes Runx2 and osteocalcin ( Ocn ) were significantly elevated when the MSCs were subjected to daily PEMF exposure when compared with unexposed cultures prior to transitioning to the mineralization phase between Day 10 and Day 14 ( Jazayeri et al., 2017 Jazayeri et al., 2017 ). Other studies have also highlighted the phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase A (PKA) signaling pathways in osteogenesis induction by EMF as a result of elevated cyclic adenosine monophosphate (cAMP) levels. Yong et al. (2016) Yong et al. (2016) have shown that pretreatment of MSCs with ERK and PKA inhibitors prior to PEMF exposure resulted in the significant inhibition of the induction of osteogenic markers Runx2 , bone morphogenetic protein 2 ( BMP2 ), and Ocn . In a different study, increased osteogenic differentiation through PEMF exposure was accompanied by the strong expression of osteogenic markers including osteonectin, osteopontin, collagen I, and collagen III. It is noteworthy that alkaline phosphatase (ALP) activity was observed to be upregulated followed by the downregulation of MSC markers cluster of differentiation 90 (CD90), CD73, and CD105 in cells ( Kim et al., 2015 Kim et al., 2015 ). In other examples, EMF was employed for cartilage and tendon repair via the chondrogenic and tenogenic differentiation potentials of MSCs, respectively, with varying degrees of success ( Haddad et al., 2007 Haddad et al., 2007 ; de Girolamo et al., 2013 de Girolamo et al., 2013 ). Although several studies observed little to no effect of EMF stimulation during chondrification (Mayer-Wagner et al., 2011; Esposito et al., 2013 Esposito et al., 2013 ; Ongaro et al., 2015 Ongaro et al., 2015 ), Parate et al. (2017) Parate et al. (2017) showed that brief single exposure of low-amplitude EMF worked best at inducing the chondrogenesis of MSCs. Obvious differences in the stimulation protocols or techniques used by different research groups may explain these inconsistent and conflicting results. Past studies had longer exposure time (hours per day for days or weeks), whereas Parate et al. (2017) applied EMF only once for 10 min for the chondrogenic stimulation of BM-derived MSCs. The major players implicated in the chondrogenic developmental process are Ca 2+ influx and the mechanosensitive transient receptor potential (TRP) channels TRPC1 and TRPV4 ( Parate et al., 2017 Parate et al., 2017 ). With respect to the in vitro tendon commitment of umbilical cord-derived MSCs, exposure to PEMF for different periods leads to the increased syntheses of scleraxis and collagen type I. Moreover, an increase in the amounts of IL-10 and VEGF throughout the culture suggested the possibility of an indirect mechanism of tenogenic activation initiated by EMF for the healing and repair of tendons (Marmotti et al., 2018). EMF exposure may also influence the differentiation of MSCs into non-mesodermal lineage cells, such as neurons and astrocyte cells ( Jeong et al., 2017 Jeong et al., 2017 ; Asadian et al., 2021 Asadian et al., 2021 ). At an intensity of 400 µT, BM-MSC can reach the highest degree of neuronal differentiation for EMF groups with sinusoidal frequencies of 50 and 75 Hz, while astrocytes are frequency-dependent, and 75 Hz square and 75 Hz EMF produce the most significant effect on the differentiation of BM-MSC to astrocytes ( Asadian et al., 2021 Asadian et al., 2021 ). In another study of astrocytes, ELF-EMF at 50 Hz upregulated sirtuin1 ( SIRT1 ), promoting the astrocytic differentiation of human BM-MSCs (hBM-MSCs) through the regulation of downstream molecules of SIRT1 (HES1 and MASH1) ( Jeong et al., 2017 Jeong et al., 2017 ). In addition to the use of PEMF, in bone tissue engineering applications, the incorporation of PEMF into titanium dioxide (TiO 2 ) surfaces cultured with hBM-MSCs in osteogenic medium also showed a good combination. This fusion is evidenced by the increased expression of early- and end-stage osteogenic genes and proteins (such as ALP, COL-I , OCN , and OPN ) in PEMF-stimulated hBM-MSCs. Interestingly, the fact that osteogenesis significantly increased was further supported by the findings that cells treated with PEMF/TiO 2 produced secretions into media containing higher amounts of BMP2, decorin (DCN), and collagen type I, compared with untreated controls ( Bloise et al., 2018 Bloise et al., 2018 ). Table 2 Table 2 shows a collection of studies on the effects of EMFs on the differentiation capacity of MSCs, and Fig. 3 Fig. 3 depicts the effects of EMF on the differentiation of MSCs. EMF: electromagnetic field; PEMF: pulsed EMF; MSC: mesenchymal stem cell; hMSCs: human MSCs; WJ-MSCs: Wharton's Jelly-derived MSCs; AD-MSCs: adipose-derived MSCs; BM: bone marrow; hBM-MSCs: human BM-MSCs; hSSCs/BMSCs: human skeletal stem cells/bone marrow stem cells; LF: low-frequency; ELF: extremely LF; SELF-MFs: sinusoidal ELF magnetic fields; SEMF: sinusoidal EMF. Study Biological model Type of exposure Exposure frequency (Hz) Exposure intensity (mT) Duration Outcome Mayer-Wagner et al., 2011 hMSCs Homogeneous SELF-MFs 5 3 weeks EMF might be a way to stimulate and maintain the chondrogenesis of hMSCs Esposito et al., 2013 WJ-MSCs PEMF 75 (±5%) 1.8–3.0 8 h/d for 21 d Significant early differentiation of WJ-MSCs into cartilaginous tissue Ongaro et al., 2015 BM-MSC and AD-MSCs PEMF 75 1500 PEMF stimulates the osteogenic differentiation of both BM-MSCs and AD-MSCs Kim et al., 2015 hBM-MSCs EMF 45 1 2× every 8 h/d for 7 d Significantly increased osteogenic differentiation Ross et al., 2015 hSSCs/BMSCs LF-EMF Significantly enhanced osteogenic and chondrogenic differentiation of hSSCs/BMSCs Yong et al., 2016 Rat MSCs SEMF 15 1 8 h/d for 6 d EMF promoted the osteogenic differentiation of sinus MSC Parate et al., 2017 MSCs PEMF 15 2 10 min/d Significantly increased chondrogenic differentiation Jazayeri et al., 2017 MSCs PEMF 15 0.2 6 h/d for 10 d Significantly increased osteogenic differentiation Jeong et al., 2017 hBM-MSCs ELF-EMF 50 1 12 d Significantly promoted astrocytic differentiation Bloise et al., 2018 hBM-MSCs PEMF 75 2 10 min/dfor 7 and 28 d Significantly increased osteogenic differentiation Asadian et al., 2021 BM-MSCs SEMF 50 and 75 0.4 1 h/d for 7 d Significantly increased differentiation to neurons and astrocyte cells 2.3 Effects of EMF on the immunoregulatory and regenerative activity of MSCs 2 MSCs exhibit potent immunomodulatory activity on almost all kinds of immune cells. This activity allows MSCs to act as an attractive tool for ameliorating autoimmune diseases ( de Bari, 2015 de Bari, 2015 ). For instance, the intravenous injection of MSCs derived from umbilical cord blood could constitute a therapeutic option for patients with rheumatoid arthritis (RA) through the reduction of pro-inflammatory cytokine levels, such as those of IL-‍1β, IL-6, IL-8, and TNF-‍α ( Park et al., 2018 Park et al., 2018 ). MSCs activated by lipopolysaccharide (LPS) and exposed to 5.1 Hz EMF for 5 min showed the declined production of pro-inflammatory molecules (IL-1b, IL-6, and IL-17A) and enhanced secretions of anti-inflammatory cytokines (IL-3, IL-4, and IL-10) ( Ross et al., 2019 Ross et al., 2019 ). It was recently shown that EMF affects tissue regeneration and wound healing processes ( Pesce et al., 2013 Pesce et al., 2013 ; Geng et al., 2014 Geng et al., 2014 ). This includes reductions in total healing time, recurrence rate, wound size, as well as enhancement of the re-epithelialization process ( Ieran et al., 1990 Ieran et al., 1990 ; Ross and Harrison, 2013 Ross and Harrison, 2013 ; Cheing et al., 2014 Cheing et al., 2014 ). Zhang et al. (2018) Zhang et al. (2018) employed different EMF frequencies (range of 7.5–75 Hz) to evaluate the migration activity of MSCs. They reported that EMF promoted MSC migration by accumulating intracellular Ca 2+‍ . The activation of focal adhesion kinase (FAK) leads to the formation of focal contacts and Rho GTPase, which mediates the organization of the cytoskeleton via the FAK/Rho GTPase signaling pathways. Moreover, Wang et al. (2014) Moreover, Wang et al. (2014) investigated the effects of EMF on rat osteoblast adhesion on implant surfaces (titanium), and found that EMF stimulation significantly increased the initial osteoblast adhesion to these surfaces. Furthermore, EMF promotes directional migration and adhesion for a variety of cells involved in regenerative medicine, tissue engineering, and wound healing ( Ross, 2017 Ross, 2017 ). 3 Cytotoxic and genotoxic effects of EMF on MSCs 1 Safety and efficacy are two of the major facets of clinical-based trials and continue to serve as the obligatory criteria for any form of therapy intended for mass consumption ( Bernardo and Fibbe, 2012 Bernardo and Fibbe, 2012 ; Wang et al., 2013 Wang et al., 2013 ; Freitag et al., 2016 Freitag et al., 2016 ). EMF and its impacts on MSCs are a good example of emerging solutions for many ailments. Because of the negative connotations revolving around EMF and the effects of radiation, concerns have been raised regarding its usage at the therapeutic level ( D'Angelo et al., 2015 D'Angelo et al., 2015 ). To determine whether EMF is either dangerous or harmless, certain parameters such as amplitude, frequency, and exposure time are needed to be considered ( Carpenter and Ayrapetyan, 1994 Carpenter and Ayrapetyan, 1994 ; Miskon and Uslama, 2011 Miskon and Uslama, 2011 ). Two studies have shown that EMF exposure within a specified range does not cause any cytotoxic effects in MSCs. In the first, Kim et al. (2015) In the first, Kim et al. (2015) reported no morphological changes or necrosis to human BM-derived MSCs during osteogenesis upon exposure to 45 Hz of EMF (16 h/d with intervals) for a duration of 7 d. Furthermore, the lactate dehydrogenase (LDH) activity of MSCs treated with EMF did not increase, suggesting no damage to the cell membrane due to radio sensitization. Another study conducted by Ross et al. (2018) tested the cyto/genotoxic activity of 5 Hz, 0.4 mT ELF-EMF on human MSCs for 20 min/d three times a week for two weeks. Their experiment involved cell viability and proliferation assays along with karyotype analysis, and the results showed no evidence of cytotoxicity or genotoxic chromosomal breaks in the nucleus of MSCs after EMF exposure. Nevertheless, the abovementioned findings are limited to EMF exposure within a small range of frequency, and the influence of other contributing variables, namely amplitude and length of exposure, remains to be investigated. Thus, the critical evaluation and optimization of the said parameters are vital prior to introducing EMF in the clinical therapy of MSCs. 4 Current strategies to trigger the proliferation of MSCs 1 During the clinical administration of MSCs, certain features such as suitable dose, proper routes of delivery, and correct functionalities of cells within the system are essential to ensure the most effective form of therapy ( Kurtz, 2008 Kurtz, 2008 ; Golpanian et al., 2016 Golpanian et al., 2016 ; Florea et al., 2017 Florea et al., 2017 ). Nonetheless, primary human MSCs do not grow indefinitely in culture; hence an adequate repertoire of MSCs is necessary prior to conducting clinical-based transplantations. To achieve this aim, previous studies have performed the in-depth assessment and standardization of many aspects of MSC culture, including the type of culture media used, cell-plating density, addition of growth factors, and selection of appropriate culture flasks ( Sotiropoulou et al., 2006 Sotiropoulou et al., 2006 ; Bernardo et al., 2007 Bernardo et al., 2007 ; Ikebe and Suzuki, 2014 Ikebe and Suzuki, 2014 ). Regardless, it is worth mentioning that supplementation techniques and culture conditions are two of the most extensively studied areas on MSCs. The term culture conditions/environments here refer strictly to the physicochemical components of cell culture (temperature, pH, oxygen concentration, etc.). Thus, from this point onwards, focus will be given primarily to studies on these two parameters. The proliferation and survival of MSCs depend heavily on the type and amount of nutrients contained in the culture media, which has been widely investigated in a number of recent studies ( Solchaga et al., 2005 Solchaga et al., 2005 ; Choi et al., 2008 Choi et al., 2008 ; Tamama et al., 2010 Tamama et al., 2010 ; Sun et al., 2013 Sun et al., 2013 ). The basic FGF (b-FGF) is one of the key ingredients for MSC culture due to its varied effector actions including the enhancement of proliferation and immunosuppressive activity of MSCs ( Bianchi et al., 2003 Bianchi et al., 2003 ; Sotiropoulou et al., 2006 Sotiropoulou et al., 2006 ; Ikebe and Suzuki, 2014 Ikebe and Suzuki, 2014 ). Besides b-FGF, other factors that may further ameliorate the expansion of MSCs are IGF ( Doucet et al., 2005 Doucet et al., 2005 ), EGF ( Tamama et al., 2010 Tamama et al., 2010 ), PDGF ( Kumar et al., 2010 Kumar et al., 2010 ), IL-6 ( Pricola et al., 2009 Pricola et al., 2009 ), and TGF-‍β ( Doucet et al., 2005 Doucet et al., 2005 ; Ng et al., 2008 Ng et al., 2008 ). However, whether MSCs could maintain their stem cell phenotype and differentiation potential following long-term culture with these supplements remains to be conclusive, since many of these studies were carried out within a short period of time. Some factors promoting cell division may elicit unwanted bystander impacts that could act on certain signaling pathways. The mitogen PDGF, for instance, could indirectly prevent the differentiation of MSCs (Pytlík et al., 2011). Furthermore, it has been shown that the addition of b-FGF could trigger a dose-dependent upregulation of histocompatibility markers, especially that of class I molecules ( Sotiropoulou et al., 2006 Sotiropoulou et al., 2006 ). This will inevitably render the cultured MSCs more prone to rejection during clinical transfer as a result of immune incompatibility. When it comes to the viability and multipotency of stem cells, local niche conditions are deemed highly influential. Likewise, MSCs are sensitive to various types of physicochemical and mechanical changes in their microenvironment ( Winer et al., 2009 Winer et al., 2009 ; Kelly and Jacobs, 2010 Kelly and Jacobs, 2010 ). With regard to this matter, several studies have highlighted oxygen concentration as a major determinant of stem cell proliferation, as well as the maintenance of pluripotency ( Ezashi et al., 2005 Ezashi et al., 2005 ; Ma et al., 2009 Ma et al., 2009 ; Widowati et al., 2014 Widowati et al., 2014 ). In the case of MSCs, it was found that the low-oxygen (the volume fraction of O 2 is 2.5%) condition significantly increased the cell proliferation kinetics with reduced population doubling time ( Widowati et al., 2014 Widowati et al., 2014 ). This is in accordance with the observation made by Haque et al. (2013), which concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and the expression of chemokine receptors during in vitro expansion, and eventually increase efficiency of MSC-based regenerative therapies. Hence, by manipulating the oxygenic settings of cells, the quantity and quality of MSCs may be improved for clinical use. One drawback to this approach is that the role of oxygen tension during MSC differentiation is largely influenced by the applied cell isolation technique, the presence of growth factors, and other physiologic requirements. Moreover, substantial discrepancies exist in the differentiation capacities of hypoxic MSCs derived from in vitro and in vivo experimentations ( Ma et al., 2009 Ma et al., 2009 ). Although seemingly promising, further laboratory validations are vital for a complete understanding of the effects of oxygen content on driving the underlying molecular mechanism of cell specialization. Apart from oxygen concentration, mechanical strain and vibrations have also been associated with the modulation of MSCs, especially during cellular proliferation and differentiation. The response of MSCs to physical stimulation is influenced by the amount of frequency, acceleration, and duration used ( McClarren and Olabisi, 2018 McClarren and Olabisi, 2018 ). Low-intensity vibration (LIV) is usually applied to cells within the range of 10 and 800 Hz, which enhances their structure, leading to increased cellular proliferation and differentiation ( Touchstone et al., 2019 Touchstone et al., 2019 ). When compared with the control group, the application of LIV on MSCs (about twice daily for 20 min at 90 Hz) increased the cell doubling rate by 28%, reduced the activity of β‍-galactosidase by about 39% in (related to senescence), and improved the rates of osteogenesis and adipogenesis at earlier passages ( Bas et al., 2020 Bas et al., 2020 ). The application of LIV may also be beneficial for restoring the diminished proliferation of MSCs ( Touchstone et al., 2019 Touchstone et al., 2019 ). Notwithstanding these findings, the reactivity of MSCs to mechanical loading appears to lack consistency and gave varying results in different culture environments, either in tissue culture plastics or two/three-dimensional scaffolds ( McClarren and Olabisi, 2018 McClarren and Olabisi, 2018 ). 5 Conclusions 1 Finding novel treatments to promote MSC proliferation and differentiation without cytotoxic and genotoxic effects can bring new ideas to the field of regenerative medicine and tissue engineering. With the recent advancement of cell culture methods and the emergence of specialized bioreactors, EMF therapy is clearly a promising method to further promote the proliferation and differentiation of MSCs. In the past, EMF therapy was successfully used as an effective, non-invasive approach for the treatment of bone diseases. 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Arch Med Res , 43 ( 4 ): 274 - 282 . https://doi.org/10.1016/j.arcmed.2012.06.002 https://doi.org/10.1016/j.arcmed.2012.06.002 Effects of pulsed electromagnetic fields on bone mass and Wnt/β‍-catenin signaling pathway in ovariectomized rats Haslinda Abdul HAMID drafted and wrote the manuscript with contribution from Azizi MISKON, Rajesh RAMASAMY, Vahid Hosseinpour SARMADI, and Vivek PRASAD. Azizi MISKON conceived the original idea. All authors have read and approved the final manuscript, and therefore, take responsibility for the integrity and security of the data. This research was supported by the Malaysian Fundamental Research Grant Scheme (No. FRGS/2/2013/SKK01/UPNM/02/1). The authors fully acknowledge the Ministry of Higher Education (MOHE) Malaysia for the funding approval and allowing this important research to come to fruition. The authors would like to thank the staff and students from the Stem Cell Research Laboratory, Genetics and Regenerative Medicine Research Centre, and Immunology Unit, Department of Pathology in Universiti Putra Malaysia for their knowledge and expertise. We also wish to acknowledge the librarian from the National Defence University of Malaysia for assistance in the provision of literary materials. 10.1631/jzus.B2100443 1673-1581（2022）01-0042-16 This research was supported by the Malaysian Fundamental Research Grant Scheme (No. FRGS/2/2013/SKK01/UPNM/02/1). 2021-05-21 2021-08-22 2022-01-15 2022-04-19 2021-12-17 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2021 2022 浙江大学出版社 Haslinda Abdul HAMID, Vahid Hosseinpour SARMADI, Vivek PRASAD, Rajesh RAMASAMY, and Azizi MISKON declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 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