Introduction

Stem cells (SCs) are touted as a unique cell source to alleviate various diseases. These cells are in null phenotype and have the potential to proliferate, preserve self-renewal activity, and differentiate into different multiple cell phenotypes. It seems that numerous stimuli and key signals could hamper/harness these bioactivities [1, 2]. Along with these descriptions, SC therapy paves a way for a promising treatment approach in the cases that conventional modalities are ineffective [3, 4]. Based on the previous data, SCs are classified into embryonic stem cells (ESCs), adult stem cells (ASCs), and fetal stem cells (FSCs). It has been demonstrated that SCs encompass heterogeneous populations not only given to their origins but also in the case of trans-differentiation capacity [5, 6].

ESCs are defined as primordial cells to be used in modern biology and human medicine [7]. ESCs are originated from the inner cell mass of human blastocysts during embryogenesis. In the term of differentiation, ESCs display dramatic potential of self-renewal and differentiation with potency to generate all cell types in vivo [8]. Despite a high potential differentiation capacity, the application of ESCs is limited according to immune cells response [9].

In contrast to ESCs, ASCs are present in various tissues especially adult bone marrow, etc. Compared to the ESCs, these cells possess only limited differentiation capacity with an orientation to specific cell lineages. Two main types of bone marrow-derived SCs include hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) which have been used extensively by authorities in the field of tissue engineering and SC biology [10, 11]. In most of these experiments, the underlying mechanisms participating in cell orientation promote differentiation by genetic elements and protein effectors.

Effect of methylation/demethylation in SC differentiation

The gradual conversion of a single fertilized egg into the formation of a complete organism is an intricate process procedure. In addition to the production of numerous cell number, it requires the acquisition of functional activity and morphologies in a well-designed 3D niche [12]. Cell differentiation is defined as a crucial process in which distinct cell phenotype acquires specific function and profound alterations in the gene profile [13] (Table 1). In this regard, trans-differentiation of SCs is the most common changes seen inside the body [23]. Numerous experiments showed that differentiation of embryonal cells happens in cells at the G1 phase. In this phase, cells are sensitive to external signals and the accumulation of distinct transcription factors that promote cell differentiation [24, 25]. In addition to numerous de novo molecular adaptations, chromatin modification involved a pattern termed bivalent domains. The region Lys27 methylation related to pluripotency consisted of smaller regions of Lys4 methylation participate in chromatin modification. Therefore, methylation of these regions is commonly seen during differentiation of ESCs [26]. During ESC differentiation, most of the bivalent regions become monovalent and devoid of activating and inhibiting factors [26, 27]. The change of transcriptional and methylation pattern has a fundamental role in cell conversion to the specific cell type [28]. In addition to the complete gene suppression and/or expression during SC differentiation toward various lineages, some reversible modification could affect gene activity. Of these changes, epigenetic programming commonly happens inside the SCs committed to specific lineage [13].

Table 1 Effect of laser on cell differentiation
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The epigenetic modification includes changes in gene bioactivity without any alterations on structure and sequence. These modifications are viewed as the reversible, heritable events and mostly comprise of methylation, acetylation, and histone modification [29] (Fig. 1). DNA methylation is conceived as the common investigated epigenetically event with a highly significant role in controlling gene activity during SC differentiation [30, 31]. This phenomenon has critical effects on the stemness maintenance and the preservation of lineage orientation toward various cell types, and in some circumstances, it is described as an epigenetic memory of SCs [23].

Fig. 1
figure 1

The direct and indirect effects of low-level laser irradiation on target DNA and genome pool. A prolonged and or high irradiation doses could contribute to DNA damage and irreversible outcomes

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To methylate gene, the methyl group is added by DNA methyltransferases (DNMTs) on carbon 5 of the cytosine into CpG sequences during SC development and differentiation [32]. There are two types of DNMTs. Dnmt1 is a basic enzyme and targets nucleotide cytosines at hemimethylated CpG sites [33]. This enzyme plays a key role in SC differentiation and self-renewal pre and postnatal periods [34]. In addition to type 1 Dnmt, Dnmt3a and 3b also take part in the methylation of DNA by the converting unmethylated sites into methylate form [35]. Of note, epigenetic procedures can regulate gene expressions which accompanied with modification of chromatin structure in specific phases of SCs entered to development and differentiation by two pathways that include the prohibition binding specific transcription factors and collaboration of proteins containing methyl-CpG-binding domain (MBD) that eventually suppress the target genes [32, 36]. Experiments show that DNA methylation could be also termed epigenetic programming because of developmental genes silencing and activation of tissue-specific genes. In contrast, the epigenetic pattern can be modulated in phenomena called reprogramming and anaplastic changes. Under these conditions, epigenetic factors become silent, contributing to the acquisition of developmental potential [13]. Therefore, one could hypothesize that there is a reverse relationship between methylation and multipotentiality in SCs. Commensurate with this claim, various experiments have verified the inherent role of epigenetic modifications on SC differentiation by using chromatin-modifying drugs in order to investigate the differentiation capacity of SCs to several lineages [29]. Demethylation of the promoter is accompanied by activation of specific genes in various stage of the development process. It should mention that while differentiating, promoters become more methylated [34]. Epigenetic modifications on gene promoters may lead to dual influences, having said that it facilitates (or avoid) the recruitment of additional chromatin modifying enzymes or transcriptional regulators that would drive SC differentiation [29]. In addition to modulation of epigenetic indices in distinct cell types, experiments show that remarkable quantity of epigenetic information inside somatic cells is transferred to daughter cells. In ESCs, the balance between hypo/hypermethylation of genetic pool could pre-determine cell fate and differentiation into specific lineages. The decrease of methylation rate, for example in ESCs, could trigger cell orientation to specific lineage and expression of differentiation-associated markers [37]. SC renewal and differentiation requires selective transcriptome profile and is promoted by an inevitable dialog between transcription factors and epigenetic modulators, changing the structure of chromatin and packaged eukaryotic genome. Epigenetic changes cause to the regulation of the memory of active and silent gene states and determine SC fates [38]. Therefore, the impact of epigenetic modifications in the maintenance/loss of the multipotentiality and differentiation process is an area of intense care in SC biology [35]. In addition to adult SCs, DNA methylation is commonly seen in the embryonic stage. The blastocyst stage is characterized by a high rate of epigenetic activities such as DNA methylation, inactivation of X chromosomes, and remodeling of chromatin.

Conducted researches related to in vitro fertilization evaluation have surprisingly indicated the existence of multiple epigenetic aberrations in human embryos at the early-stage development, inspiring the fact that expanded ESCs differ in the light of epigenetic status; thereby, these unique characteristics contribute to diverse differentiation capacity. However, a vivid consensus can be inspired regarding distinct epigenetic features in various hESC cell lines. Of note, the conditions which these cells are expanded possibly alter epigenetic status. Despite these comments, consolidate data exist neither about the stability of ESCs epigenetic profile after prolonged in vitro culture nor how it may change as the cells differentiate along different developmental pathways [39].

Natural effects of photobiomodulation

Low-level laser therapy (LLLT) or photobiomodulation (PBM) is defined as a photochemical process relies on production of low intensity of light by lasers [40]. By using LLLT, low power irradiation is used on the target biological sites without heat generation, so it is also touted as “cold laser” therapy in the field of cancer and regeneration [41]. Regarding different side effects of conventional treatments, scientists and researchers try to find novel treatment modalities in order to limit unwanted consequences in candidate patients. In this regard, LLLT is one of the promising procedures which accompanied with trivial side effects on body surface such as cutaneous irritation, itching, and redness [42]. In contrast to other modalities, the advantages of LLLT hugely outweigh the existing negligible drawbacks and disadvantages [43]. To evaluate the efficacy of LLLT, various parameters related to laser type and target tissue consistency or cell response should be noted. As a matter of fact, the wavelength of the laser, power density, exposure time, and energy density is critical [44]. The suitable wavelengths for LLLT are restricted to specific ranges at red and near infrared at the wavelength of 600–1070 nm. It should be mentioned that shorter wavelengths (< 600 nm) are efficiently absorbed by tissue chromophores. Wavelengths in the range of 600–700 nm are prominently effective for the treatment of target sites located near to cutaneous tissue while wavelengths between 780 and 950 nm have the potential to reach beneath tissues. Of note, the ranges between 700 and 770 nm contain less biochemical activity. In wavelengths more than 1200 nm, water molecules have significant absorption. Therefore, roughly 810 nm is conceived as the optimum wavelength in this treatment technique [41, 45].

After the absorbance of LLLT by chromophores, it stimulates the movement of ground state electrons to higher energy orbits. In this case, stimulated electron is transported through biological transmembrane carriers such as cytochrome C oxidase and delivered to the ultimate electron acceptors. This process creates a proton gradient which assists in ATP production. Furthermore, LLLT contributes to an enhanced reactive oxygen species (ROS) production and the activation of multiple transcription factors [46] (Fig. 1). LLLT endeavors to preserve SCs cellular DNA from unwanted modifications included chromosomal deletion or translocation. It exerts splendid impacts on SCs functions and bioactivity such as survival rate, migration, and adhesion. In line with these comments, LLLT could direct SCs to distinct activity and phenotype. In the next step, we aim to debate more about the potency of LLLT on SC biology, trans-differentiation into specific lineages, or preserving stemness feature by the modulation methylation rate.

One of the well-accepted and substantial mechanisms of LLLT is a photochemical process in mitochondria following receiving of photons which cause to increase the activity of cytochrome C oxidase, nitric oxide (NO) release and production of ATP, and changes in the intracellular content of signaling molecules such as calcium ions and ROS [47]. ROS are classified as Janus face mediators that are useful in low contents while detrimental effects are evident at high concentrations and these effects depend on the feature and protocol LLLT. In a better word, low levels of ROS are generated via the normal metabolism rate in mitochondria. When the basal mitochondrial membrane potential is changed, the cellular content of ROS can be modulated.

In cells under normal condition, the direct action of photons by Cox promotes changes in mitochondrial membrane potential and ROS accumulation. After a decrease of mitochondrial membrane potential due to current oxidative stress, excitotoxicity, and suppression of electron transport, light captivation increases mitochondrial membrane potential toward normal levels while ROS generation is halted [48]. Of note, ROS levels have a pivotal impact on regulating cellular bioactivities such as self-renewal capacity, cellular division, and differentiation. For example, at trivial levels, ROS perform as secondary messengers along with various signaling pathways regulate proliferation, survival, and differentiation of SCs [49]. Furthermore, proper levels of ROS within SCs assist to keep their potency. In contrast, the uncontrolled levels of ROS could hamper cellular hemostasis and thereby inhibit physiological biochemical reactions correlated with stemness and normal bioactivities [50].

Laser for cell differentiation

SC trans-differentiation into different cell types is requisite to reconstitute the injured tissues [17]. LLLT can modulate SC bioactivity by the increase of migration, cellular division, and survival rate, as well as promotion of proteins related to differentiation [51]. Therefore, SC differentiation attitude can be affected by specific stimulators such as LLLT [52]. Through recent decades, different investigations have been published related to LLLT efficacy in induction of differentiation. Despite a few numbers of researches on cell differentiation induced by laser irradiation, positive effects of LLLT on differentiation have approximately been illustrated. In some cases, there are no evident differences in differentiation properties pre- and post-irradiation [53].

In an experiment done by Kim and co-workers, they did not find a noticeable difference between differentiation rate of laser-treated adipose-derived mesenchymal stem cells by He–Ne cold laser (632.8 nm) and non-irradiated cells [14]. It is proposed that differentiation of MSCs is based on light source quality and feature, laser intensity, and light exposure treatment protocol. Based on the released data, the minimum exposure time point was near to 4 weeks [18]. In one study, MSCs were seeded on lyophilized collagen sponge Gingistat and subjected to 1064-nm irradiation provided by Nd:YAG laser and, 14 days after irradiation, differentiation was dramatically [15].

LLLT has potential to trans-differentiate into various cell lineages. LLLT (He–Ne laser functions as an osteogenesis-inducing factor with a continuous mode (at the wavelength of 632.8 nm) and evaluated previously on osteoblast differentiation of MSCs. In irradiated MSCs, alkaline phosphatase (ALP) activity, as an osteoblast differentiation indicator, was promoted in the early stages following laser treatment. The profound decrease in ALP activity was seen in the later stages, suggesting cellular maturation and bone formation [16]. In addition to cell differentiation, the proliferation of human osteoblast cells has positively affected by LLLT (He–Ne laser, at 632.8 nm) [22]. The application of growth factors is the main method to induce cell differentiation into various lineages. Growth factors (GF) are polypeptides which play an important role in differentiation. Low-intensity laser irradiation and GF can lead to differentiation of ADSCs into smooth muscle cells [17].

Although there is a positive relation in the majority of studies, it is believed that light source and laser irradiation does not eventuate to significant differentiation in MSCs. So auxiliary agents such as non-coherent red LED of BMSC specimens with osteogenic medium improved differentiation rate and GF [17, 53].

Human bone marrow–derived MSCs (BMSCs) irradiated by 810 nm gallium aluminum arsenide (GaAlA) laser at energy densities of 3 or 6 J/cm2 acquired neuron-like phenotype at days 1, 3, and 5. The use of GaAlAs at energy densities of 2 or 4 J/cm2 promoted osteoblast-like phenotype. In fact, low-level laser irradiation (LLLI) acts in a dose-dependent manner to promote BMSC-to-osteoblasts differentiation. In support of this claim, ALP activity was significantly promoted in the 4 J/cm2 treated LLLI compared to the 2 J/cm2 counterpart [18]. It also described that LLLI provided by indium–gallium–arsenate–phosphate (InGaAsP) diode laser at 635 nm and 5 J/cm2 could dictate myogenic differentiation of BMSCs [19]. These data confirm the potential role of LLLI energy density on phenotype acquisition and trans-differentiation quality. In addition to energy density, the entity and wavelength of LLLI could direct cell polarization toward various lineages. In an empirical study, the impact of four multiple wavelengths (420, 540, 660, and 810 nm) at an energy density of 3 J/cm2 was examined on human ASCs. LLLI at the wavelength of prompt osteoblast differentiation compared to 660 and 810 nm. Biochemical analyses confirmed an increase in intracellular calcium level at 420 and 540 nm [20]. Regarding the wavelength value in inducing cell differentiation, it is better to mention that near infrared light enhances cytochrome C oxidase activity while abrogates the bioactivity of nitric oxide synthetase. The activation of cytochrome C oxidase, in turn, increases mitochondrial membrane integrity and ATP production. Concomitant with these changes, the profound shift in metabolic profile from glycolytic to oxidative status is induced by light exposure, conceiving as an important factor in osteogenic differentiation [20]. LLLI not only promotes BMSC differentiation toward osteoblast-like phenotype but also decreases the expression of adipocyte-related markers in BMSCs. Molecular analysis revealed the critical role of β-catenin in cells exposed to the LLLI compared to the non-treated control. LLLI has the ability to increase the levels of β-catenin which further promotes Wnt signaling pathway by engaging APN receptors located inside the nucleus. Totally, LLLI increases the β-catenin transport to the nucleus. It seems that the activation of β-catenin/Wnt axis induces the osteogenic capacity of BMSCs [21].

Effect of LLLI on SC differentiation by modulating methylation

Similar to numerous modulators, it is proved that the epigenetic mechanisms are apparently regulated via external clues such as laser irradiation. During LLLT, cellular mechanisms including DNA synthesis and gene expression, cell proliferation, and differentiation are prominently changed. Therefore, the critical role of epigenetic mechanisms in the molecular responses through LLLT is supported [54]. In addition to numerous LLLI effects, some adverse outcomes were also evident. The ionizing radiation of the body was found to result in the induction of acute myeloid leukemia (r-AML); meanwhile, LLLI could also promote DNA damage in each cell inside the body [55,56,57]. Therefore, one could hypothesize that both somatic and progenitor cells are efficiently affected by LLLI. The potency of laser irradiation was extensively investigated on the global DNA methylation and the expression of Dnmt1 and Dnmt3a genes in a rat model of skin wound healing. Laser-emitted diodes were used in animals at a wavelength of 604 nm in two different doses 0.8 and 1.6 J/cm2. Total DNA methylation reduction was evident in the experimental condition, confirming DNA methylation during the healing process. Therefore, epigenetic mechanisms participate in the regeneration of epithelial tissues. In support of these changes, the expression of Dnmt3a was induced at a density of 1.6 J/cm2 compared to the control [54]. De Farias and colleagues explored the impact of photobiomodulation (InGaAlP laser, 660 nm, 4 J/cm2, 4 s) on the site of histone 3 acetylation (acH3) and NF-κB expression on ulcer healing in the oral cavity. In situ irradiation not only modulated the levels of acH3 and NF-κB but also increased migration and differentiation of keratinocytes [58] (Fig. 2).

Fig. 2
figure 2

The potency of low-level laser irradiation on the epigenetic pattern of target genes to change acetylation and methylation. These changes participate in the differentiation of stem cells toward specific lineages

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Conclusion

Manipulation of DNA methylation by several modalities such as LLLT is considered to yield various therapeutic outcomes. In light of accessibility, ability to adapt to different therapeutic conditions and potential therapeutic properties according to wavelength, energy intensity, and LLLT could be applied for the modulation of various pathologies and injuries. Given the small number of studies related to the impact of LLLT on SC methylation, further investigations are needed to address precisely the restorative effects. For further insurance, it can be fruitful to examine the expression of differentiation-related genes and methylation rate in the development of SCs.