Promoting Immortalized Adipose-Derived Stem Cell Transdifferentiation and Proliferation into Neuronal-Like Cells through Consecutive 525 nm and 825 nm Photobiomodulation

Neuronal cells can be generated from adipose-derived stem cells (ADSCs) through biological or chemical inducers. Research has shown that this process may be optimized by the introduction of laser irradiation in the form of photobiomodulation (PBM) to cells. This in vitro study is aimed at generating neuronal-like cells with inducers, chemical or biological, and at furthermore treating these transdifferentiating cells with consecutive PBM of a 525 nm green (G) laser and 825 nm near-infrared (NIR) laser light with a fluence of 10 J/cm2. Cells were exposed to induction type 1 (IT1): 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM)+indomethacin (200 μM)+insulin (5 μg/ml) for 14 days, preinduced with β-mercaptoethanol (BME) (1 mM) for two days, and then incubated with IT2: β-hydroxyanisole (BHA) (100 μM)+retinoic acid (RA) (10-6 M)+epidermal growth factor (EGF) (10 ng/ml)+basic fibroblast growth factor (bFGF) (10 ng/ml) for 14 days and preinduced with β-mercaptoethanol (BME) (1 mM) for two days and then incubated with indomethacin (200 μM)+RA (1 μM)+forskolin (10 μM) for 14 days. The results were evaluated through morphological observations, viability, proliferation, and migration studies, 24 h, 48 h, and 7 days post-PBM. The protein detection of an early neuronal marker, neuron-specific enolase (NSE), and late, ciliary neurotrophic factor (CNTF), was determined with enzyme-linked immunosorbent assays (ELISAs). The genetic expression was also explored through real-time PCR. Results indicated differentiation in all experimental groups; however, cells that were preinduced showed higher proliferation and a higher differentiation rate than the group that was not preinduced. Within the preinduced groups, results indicated that cells treated with IT2 and consecutive PBM upregulated differentiation the most morphologically and physiologically.


Introduction
Within the field of regenerative medicine, stem cell (SC) technology has increasingly become a hot topic [1]. This is due to the self-renewal properties, expansion capabilities, and differentiation possibilities of SCs. Of these cells, adipose-derived stem cells (ADSCs) are becoming the cell type of choice due to their abundant availability and the ease at which they can be sourced through surgery that is not as invasive to donors as other cell types may require [2]. Furthermore, preliminary research performed in vivo and in vitro has shown that ADSCs can differentiate into multiple phenotypes, such as osteoblasts, or chondrocytes, depending on the inducers applied [3]. Specifically, ADSCs can transdifferentiate into neuron-like cells by applying a wide range of inducers that include forskolin, insulin, brain-derived growth factor (BDNF), or even retinoic acid (RA) [4]. ADSCs show potential for clinical use in the future to aid in the repair of mechanical brain injuries and neurodegenerative diseases [2,5,6]. This study used immortalized ADSCs (iADSCs) as previous studies have established iADSCs as an alternative model to isolated ADSCs while yielding similar results [7].
The metabolism of cells can be stimulated or inhibited by laser irradiation in the form of photobiomodulation (PBM) [8]. Whether the irradiation treatment is stimulatory or inhibitory is dependent on the laser parameters that are used. Wavelengths that range from visible to near-infrared (NIR) light (400-1100 nm) and the fluence (J/cm 2 ) are included in these parameters [2,[9][10][11][12]. It should be noted that 3-5 J/cm 2 is the most effective fluence for stimulating proliferation, whereas fluences higher than this can prove to be overstimulatory and fluences lower than this is ineffective [1,2]. Photobiomodulation has a stimulatory effect on mitochondrial chromophores, which in turn increases the activity of the electron transport chain and leads to an overall increase in adenosine triphosphate (ATP) production which upregulates the mitochondrial membrane potential (MMP) [13]. Various studies have shown that visible spectrum light, 450 nm-580 nm, stimulates an increase in differentiation from ADSCs into osteoblasts [1,14,15]. Furthermore, green laser irradiation has been shown to aid in the triggering of differentiation transcription factors [16]. While blue and green laser irradiation does not significantly stimulate proliferation, it has been shown in numerous studies that red and NIR laser light, such as 660 nm and 810 nm, upregulates proliferation significantly in ADSCs [8,9,17,18]. Near-infrared laser irradiation increases the homing abilities of ADSCs to injured sites by significantly increasing the migration of ADSCs [18,19]. The current study observed the effect on iADSC transdifferentiation into neuronal-like cells when a novel combination of consecutive laser light of 825 nm and 525 nm at 10 J/cm 2 was used. The effect of these irradiation parameters on morphology, migratory abilities, cellular viability, proliferation, and genetic expression was observed.

Photobiomodulation.
Once confluent, cultured iADSCs were seeded at a density of 1 × 10 5 onto 35 mm diameter culture dishes (Corning, 430165) in complete medium and maintained for 24 h to allow adhesion prior to irradiation. After this incubation period, cells were exposed to an 825 nm NIR Diode Laser (National Laser Centre of South Africa, SN 070900108) with a 1000 mA LaserSource (Arroyo Instruments, 4210) and subsequently to a 525 nm green (G) Diode Laser (National Laser Centre of South Africa, EN 60825-1 : 2007) with a 100-240 VAC, 47-63 Hz 5A LaserSource (OptoElectronics Tech.CO., LTD). The power of the laser output (mW) was measured using a FieldMate Laser Power Meter (Coherent, 1098297). Finally, a High-Sensitivity Thermopile Sensor PM3 (Coherent, 1098336) was used to determine the laser irradiation time based on fluency. The laser parameters can be seen in Table 1.
Two main study groups were used concerning PBM: (1) a control group that was not treated with laser light and (2) consecutive treatment with 825 nm and 525 nm (NIR-G) at 5 J/cm 2 each. The irradiation time of each laser wavelength was calculated with the following formula seen in the following equation:  3 Stem Cells International statistical analysis. The statistical difference between the standard and experimental groups was determined using a Student t-test. Within a specific period, the ANOVA repeated measure was used to determine statistical significance between the control and experimental groups, as well as the standard and all IT groups, and to compare the groups (controls with controls, etc.). Tables and graphs show statistical differences between the untreated controls and experimental groups as P0.05 ( * ), P0.01 ( * * ), and P0.001 ( * * * ), with dispersion bars representing standard error. Purple stars ( * ) indicated experimental samples compared to the control; black stars ( * ) indicated samples compared to the standard.

Characterization of Markers.
Flow cytometry was used to characterize the IT1, IT2, and IT3 NIR-G PBM-treated cells to establish whether iADSCs preserved or lost stemness towards neuronal-like cells and at what percentage this occurred. Three iADSC CD markers (CD44, CD90, and CD166) and two early (NeuN and NSE) and two late (MAP2 and Tau) neuronal markers were applied. The selected CD markers are widely recognized as markers for iADSC SC maintenance. In Figure 1, the standard group indicated high percentages of CD44, CD90, and CD166 at 65.3%, 74.6%, and 76.5%, respectively. A steady decline in SC expression was observed in the control groups. This decline was even more distinct in the experimental groups. Immortalized ADSCs treated with IT1 media and NIR-G PBM showed a significant (P < 0:05) decrease in CD44 (40.3%), CD90 (13.9%), and CD166 (13.5%) compared to the IT1 control which maintained CD44 at 40.3%, CD90 at 64.1%, and CD166 44.7%. Immortalized ADSCs treated with IT2 media and NIR-G PBM showed a significant (P < 0:05) decrease in CD44 (5.1%), CD90 (17.8%), and CD166 (16.3%) compared to the IT2 control group with CD44 (30.5%), CD90 (13%), and CD166 (5.4%), respectively. The IT3 NIR-G group also demonstrated a decrease in CD markers: CD44 (35.5%), CD90 (47.5%), and CD166 (40.5%) compared to its respective IT3 control group at CD44 (38.5%), CD90 (49.9%), and CD166 (38.7%). Cells treated with differentiation media and laser light indicated immunofluorescence for CD44, CD90, and CD166 SC markers ( Figure 2). The SC marker immunofluorescence results did not necessarily signify a lack of differentiation but could also have showed subpopulations of iADSCs. The expression of early and late neuronal markers was detected with flow cytometry to measure the efficacy of transdifferentiation through transdifferentiation media and PBM. In Figure 3, a small percentage of NeuN and NSE were detected in the standard sample (1.9% and 0.9%, respectively). The IT1 control group showed small increments of NeuN and NSE expression (4.1% and 0.9%). After IT1 NIR-G PBM treatment, large increases in NeuN and NSE were noted (8.3% and 12.0%, respectively). A small percentage of MAP2 and Tau were detected in the standard sample (1.1% and 0.4%, respectively). The IT1 control group showed a small upregulation in MAP2 and Tau expression (8.8% and 1.8%, respectively). After IT1 PBM treatment, large increases in MAP2 (17.1%) and Tau (17.1%) expression were observed compared to the standard and control   Tau   STANDARD  IT1 CONTROL  IT1 NIR-GREEN  IT2 NIR-GREEN  IT2 CONTROL  IT3 NIR-GREEN  IT3    iADSCs; untreated IT1 control cells, experimental IT1 iADSCs exposed to NIR-G PBM consecutively, untreated IT2 control cells, experimental IT2 iADSCs exposed to NIR-G PBM consecutively, untreated IT3 control cells, and experimental IT3 iADSCs exposed to NIR-G PBM consecutively. Scale bar: 100 μm. Morphological studies revealed that the standard group had the typical shape observed in healthy iADSCs with a smooth surface and spindle-like shape ( Figure 5). After 24 h, treated cells showed cytoplasmic extension. Following 48 h, the treated cells developed distinct changes in morphology; the formation of branches and refraction could be observed. After 7 days, cells transdifferentiated with NIR-G PBM showed morphology changes that were completely different to the standard untreated iADSCs.

Migration. Migration was measured with the "central
scratch method". The migration rate of each iADSC group was studied 0h, 24h, 48h, and 7 days post-PBM as the "wound" closed. Groups showed a linear growth pattern in homing progression. Cells treated with NIR-G PBM resulted in significant augmentation of cellular migration ( Figure 6). Statistical analysis of cell migration showed that the control and NIR-G PBM-treated iADSCs had a significant (P < 0:001) decrease in distance migrated post-PBM compared to the standard (Figure 7). Cell treated with IT1 NIR-G PBM showed the most significant (P < 0:001) decrease in scratch width of these groups. The results also indicated that neither transdifferentiation media nor PBM negatively impacted migration abilities in treated iADSCs.

Biochemical Analysis
3.3.1. Viability: MTT Assay. The MTT assay was used to determine the percentage cell viability after transdifferentiation and PBM. Results indicated no significant effects on the viability of the cells compared to the standard or control groups (Figure 8). High viability was maintained over time.  Shown here are the untreated standard iADSCs, untreated IT1 control cells, experimental IT1 iADSCs exposed to NIR-G PBM consecutively, untreated IT2 control cells, experimental IT2 iADSCs exposed to NIR-G PBM consecutively, untreated IT3 control cells, and experimental IT3 iADSCs exposed to NIR-G PBM consecutively. Scale bar: 500 μm.   Stem Cells International instead of reentering the cell cycle for proliferation at the G1 phase [21,22]. Immortalized ADSCs maintained high percentages (>98%) of DNA synthesis over time and irrespective of the treatment (Figure 9).

Protein Detection
3.4.1. ELISA. Immortalized ADSC protein detection of early, NSE, and late, CNTF, neuronal markers was determined by     Figure 10). Results indicated that IT1 NIR-G PBM-treated iADSCs had a significant increase in NSE (P < 0:001) and CNTF (P < 0:05). Induction type 2 PBM-treated cells had a significant increase in NSE (P < 0:01) and CNTF (P < 01) expression compared to the standard and control. Finally, iADSCs exposed to IT3 and consecutive PBM indicated a significant upregulation in NSE (P < 0:001) compared to the standard and a significant increase in CNTF compared to the standard (P < 0:001) and control (P < 0:01). Cells treated with IT2 and consecutive PBM showed the most NSE and CNTF expression increase compared to all the experimental groups; thus, this group was used for subsequent RT-PCR studies.

Genetic Expression
3.5.1. RT-PCR. This RT-PCR experiment was performed on IT2 NIR-G PBM-treated cells and subsequently compared to a control group treated with IT2 but not PBM. The RT-PCR array detected 84 genes associated with neurogenesis and transdifferentiation including the cell cycle, proliferation, migration, adhesive properties, and synapsis. Figure 11 is a heat map that indicates the RT-PCR results; the heat map related each gene to its degree of expression where blue showed low levels of gene expression and red showed high levels of gene expression. This RT-PCR array detected genes related to transdifferentiating cells which was indicative of successful transdifferentiation of IT2 NIR-G PBM of iADSCs into neuronal-like cells. Genes related to homing, including DCX and MDK, were also expressed, agreeing with the above migration study. A gene related to iADSC remodelling, BMP8B, for neuronal purposes was also expressed.

Discussion
Following analysis, results revealed that IT2 NIR-G (5.1%), IT1 NIR-G and IT2 control (13.9% and 13.0%, respectively), and IT2 NIR-G (5.4%) had the most significant (P < 0:05) decline in CD44, CD90, and CD166 expression compared to the standard and all experimental groups. At time of writing, no studies mention the effect of neuronal differentiation on CD [7,23,24]. Ashjian and coworkers, who formulated IT1, and Cardozo and coworkers characterized ADSCs prior to differentiation but did not perform CD marker characterization after differentiation [23,24]. A previous study using IT2 without PBM screened for CD90 prior to implementation of differentiation but did not comment on its detection following differentiation. Previous studies that also used RA, forskolin, and indomethacin for differentiation detected CD44, CD90, and CD166 prior to differentiation but were not tested for following induction [23,25,26]. Moraes and coworkers demonstrated the downregulation of CD44, CD90, and CD166 when mesenchymal SCs were differentiated into adipogenic and osteogenic lineages; this has not been performed in neuronal differentiation studies [27]. Ashjian and coworkers who formulated IT1 screened for NeuN, NSE, and MAP2 [23]. They detected an increase in NSE expression and observed that NeuN did not significantly increase compared to the control, and no late neuronal markers were detected. Cardozo and coworkers detected an upregulation in the expression of early neuronal markers TUB-III, GFAP, Nestin, and synaptophysin [24]. Cardozo and coworkers did not screen for NSE nor any late neuronal markers [24]. A study by Jang and coworkers transdifferentiated ASCs into neural-like cells through forskolin and observed an upregulation in NSE and MAP2 expression [26]. Immortalized ADSCs were transdifferentiated into neuronal-like cells with RA and resulted in NeuN and MAP2 expression [25]. Immunoreactivity with NeuN and NSE was significantly (P < 0:05) higher in iADSCs treated with IT2 NIR-G PBM (85.7% and 78.0%) compared to the other experimental groups. The detection of early neuronal markers is not unusual in neuronal transdifferentiation studies; however, the experimental groups showed an increase in early markers compared to each respective control group due to PBM stimulation [23,24]. Additionally, the late neuronal markers, MAP2 and Tau, were significantly (P < 0:05) expressed with IT2 NIR-G (79.0% and 84.0%). The expression of MAP2 and Tau as observed in this study differs from previous studies that induced transdifferentiation without PBM and did not detect any late neuronal markers [23,24]. This study demonstrated that PBM, combined with differentiation media, specifically, IT2 NIR-G-treated iADSCs, augmented neurogenesis and resulted in more mature neuronal-like cells compared to the standard and control groups. Flow cytometry confirmed that the IT2 NIR-G group expressed the highest percentage of early and late neuronal markers with the largest decrease in stemness.
It was observed that IT2 showed the largest change in morphology compared to the standard, IT1 and IT3. Furthermore, IT2 treated with consecutive NIR-G PBM showed the most distinct morphological changes. As described by Cardozo and coworkers, the shrinking towards the nucleus and extending branches was observed [28]. Additionally, the  Figure 10: Protein detection ELISA results of IT3 transdifferentiated iADSCs using NSE and CNTF 7 days following PBM. All treated iADSCs showed an increase in NSE and CNTF expression compared to the standard and control groups. 9 Stem Cells International bulbous formation of the cellular body with axon hillock similar formations as described by Singh and coworkers after transdifferentiating MSCs into neurons was also observed [29].
The homing ability of cells is a crucial element in transplanting transdifferentiated iADSCs to the site of injury [30]. The cell homing rate was measured with the "central scratch assay", and the distance that cells migrated to "heal" the artificial wound was determined. Overall, laser light stimulated the migration of the differentiated cells compared to the standard. When the different experimental groups were compared, cells treated with IT1 and NIR-G PBM indicated a significant (P < 0:001) decrease in wound closure compared to all other groups. Nöth and coworkers observed that cells migrated when exposed to IT1 [31]. Several studies have observed that the addition of RA, EGF, and/or bFGF to transdifferentiation media significantly upregulates cell migration [32][33][34][35]. Multiple studies have shown that cellular migration is augmented when applying PBM at 804 nm [36], 516 nm, and 635 nm [37]. It has been observed that SCs preconditioned with PBM displayed higher rates of migration once transplanted [38].
Transdifferentiated iADSC viability was determined with the MTT assay. Results indicated that cell viability was maintained irrespective of treatment; therefore, it was concluded that neither transdifferentiation nor PBM had detrimental effects on cell health. The outcome of IT1 differs to that of Ning and coworkers who observed apoptosis 24 h following IT [39]. However, cells exposed to transdifferentiation media containing EGF, bFGF, RA, or forskolin showed maintenance in cell viability [33,35,40,41]. It has been established that PBM does not have a significant stimulatory or inhibitory effect on iADSC viability [18,42]. Studies using visible laser light at 470 nm and 410 nm reported no detrimental effects on cellular viability [43,44]. Near-infrared laser light at 890 nm showed no effects on bone-marrow MSCs post-PBM [45]. Finally, iADSCs exposed to consecutive PBM at 825 nm and 525 nm have also showed no effects on cell viability [18].
EdU detection by flow cytometry was used to determine DNA synthesis in the S phase. As EdU (5-ethynyl-2 ′ -deoxyuridine) is analogous to thymidine and incorporates into DNA during active DNA replication, it can be used to detect the percentage of cells in the S phase [20,46], and it is also a more robust alternative to BrdU [46]. In this study, transdifferentiating cells were maintained in the S phase over time regardless of treatment; this is like the transdifferentiation EdU results of Chehrehasa and coworkers [47]. Previous studies have shown that cells exposed to transdifferentiation media containing indomethacin, insulin, and/or IBMX increased BrdU expression indicating DNA synthesis [48][49][50]. A decrease in the S phase was observed by Encinas and coworkers when SH-SY5Y neuroblastoma cells, treated with RA (10 μM) and BDNF (50 ng/ml), transdifferentiated into homogenous neuronal-like cells [51]. Additionally, the authors of a previous study transdifferentiated rat NPCs with (10 ng/ml) and EGF (10 ng/ml) and observed high expression of BrdU; this led to the conclusion that the NPCs were immature and in the S phase to synthesize DNA [52]. Furthermore, studies that transdifferentiated various cell types with bFGF, EGF, βME, RA, and/or BHA indicated a maintenance of high BrdU expression [51,53,54]. In this study,  10 Stem Cells International a decrease in S phase distribution was not observed; therefore, it was concluded that the iADSCs had not terminally transdifferentiated and were actively synthesizing DNA for proliferation.
Terminal transdifferentiation can be defined as the phase in the cell cycle in which proliferation stops for cellular specialization; thus, DNA synthesis decreases as cells commit towards terminal transdifferentiation [55]. Early, NSE, and late, CNTF, neuronal marker expression was detected by ELISA. The increase in NSE expression in IT1-, IT2-, and IT3-treated iADSCs was consistent with previ-ous studies [23][24][25][26]. A study by Ashjian and coworkers observed a significant increase in NSE after exposing isolated ADSCs to IT1 for 14 days without PBM treatment; however, they did not detect any late neuronal markers [23]. A study that incubated ADSCs for 14 days in IT2 media without PBM also observed NSE expression, but not CNTF [24]. Forskolin was used to transdifferentiate ASCs to neural-like cells and observed an upregulation in NSE, early, and MAP2, late neuronal markers [26]. Retinoic acid upregulated NeuN and MAP2 expression when ADSCs transdifferentiated into TNR Encourage neurite outgrowth and neural cell adhesion, proliferation and migration, axonal guidance, myelination, and synaptic plasticity 87.91 0.036105 [89] 11 Stem Cells International neuronal-like cells [25]. Multiple studies have observed an increase in late neuronal marker expression including MAP2, TUB-III, GFAP, or Nestin when ADSCs were transdifferentiated with RA, forskolin, or indomethacin [23,25,26]. Transdifferentiating studies performed without the use of PBM often showed little to no detection of late neuronal markers [7,23,24,56]. This study established the remarkable effects of laser light when applied for iADSC transdifferentiation purposes to neuronal-like cells. Table 2 is a summary of the functions of highly expressed genes with the corresponding P value and fold change compared to the control from the RT-PCR results. It was concluded that NIR-G PBM vastly increases the transdifferentiation capabilities of IT2-treated iADSCs.

Conclusion
Results from this study showed that PBM, specifically NIR-G PBM, increased the transdifferentiation abilities of iADSCs when combined with differentiation media. Morphology observations and characterization also showed the multipotent plasticity capabilities of iADSCs for neuronal-like transdifferentiation. Furthermore, immunofluorescent laser light augmented early and late neuronal marker expression while simultaneously decreasing the expression of SC markers. Biochemical assays showed that iADSCs maintained cell viability irrespective of treatment. Genetic expression of NSE and CNTF was detected through ELISA and showed augmentation in all experimental groups but was most augmented in the IT2 NIR-G PBM-treated group. The IT2 NIR-G group was overall the most augmented and following RT-PCR analysis showed an increase in genes pertaining to neurogenesis, transdifferentiation, and neuronal-like plasticity. In conclusion, optimization of the transdifferentiation process of iADSCs into neuronal-like cells was observed when consecutive 825 nm + 525 nm PBM was applied. Future directives to conclude on terminal neuronal differentiation and function should further be confirmed using electrophysiology and sodium ion channel protein expression. By optimizing the transdifferentiation process of ADMSCs, it can benefit future in vivo studies and clinical studies with regard to neurodegenerative disorders. Successful implementation of this protocol could potentially lead to the production of transplants to aid in the repair of mechanical brain injuries and neurodegenerative disorders. The use of consecutive PBM applied to transdifferentiation and maintenance iADSCs has great potential for the field of regenerative medicine.

Data Availability
The raw and analysed data used to support the findings of this study are available from the corresponding author upon request.