Astragaloside IV improves melanocyte differentiation from mouse bone marrow mesenchymal stem cells

Vitiligo results in an autoimmune disorder destructing skin pigment cells, melanocytes (Mcs). This study aimed to investigate whether Astragaloside IV (AIV) could efficiently induce differentiation of bone marrow mesenchymal stem cells (BMMSCs) into Mcs. BMMSCs were induced and differentiated into Mcs with 0.1, 0.2, and 0.4 mg/L AIV during 150-day. Morphologic changes of differentiated cells were observed. Levels of some melanocytic specific genes (TRP-1, TRP-2, MART-1, Mitf) were measured with quantitative polymerase chain reaction (qPCR) at 90, 120, and 150 days of induction. After 90-day induction, the differentiated cells with 0.4 mg/L AIV demonstrated the typical morphology of Mcs, positive 3,4 dihydroxyphenylalanine staining, and positive staining of TRP-1, TRP-2, MART-1, and Mitf. After 90and 120days’ induction with 0.4 mg/L AIV, TRP-1 expression was significantly elevated (p < 0.01), and TRP-2 expression was significantly increased in 0.4 mg/L AIV-treated group compared to negative control (p < 0.01), 0.1 mg/L (p < 0.01), and 0.2 mg/L (p < 0.01) AIV-treated groups. Moreover, MART-1 expression was significantly up-regulated in 0.4 mg/L AIV-treated group compared to negative control, but without difference compared to 0.1 mg/L (p > 0.05) and 0.2 mg/L (p > 0.05) AIV-treated groups. During 90 to 150day induction, there were no significant differences for Mitf levels between AIV-treated groups and negative control (p > 0.05). In conclusion, 90-day induction with 0.4 mg/L AIV up-regulated TRP-1, TRP-2, and MART-1 expression, indicating that AIV can efficiently induce Mcs differentiation from BMMSCs. These results provide experimental and theoretic evidence for AIV application in clinical vitiligo repigmentation treatment. Abbreviations Mc: melanocyte AIV: astragaloside IV BMMSC: bone marrow mesenchymal stem cell qPCR: quantitative polymerase chain reaction

90-day induction with 0.4 mg/L AIV up-regulated TRP-1, TRP-2, and MART-1 expression, indicating that AIV can efficiently induce Mcs differentiation from BMMSCs. These results provide experimental and theoretic evidence for AIV application in clinical vitiligo repigmentation treatment.

Introduction
Vitiligo, an acquired pigmentary disorder, is clinically characterized by skin macules with loss of pigmentation (Boniface et al., 2018). The affected skin macules turn white with sharp margins and usually locate in areas exposed to the sun. The etiology of vitiligo is still unclear; however, it is believed to be related to genetic susceptibility to environmental factors, resulting in an autoimmune disorder that destructs skin pigment cells, melanocytes (Mcs) (Ezzedine et al., 2015). The global incidence of vitiligo is around 1%, and some populations even reach the rates as high as 2-3% (Whitton et al., 2016;Osinubi et al., 2018). Currently, there is no available and effective treatment for treating vitiligo. The most common treatment is a steroid combining ultraviolet light or creAIV to promote repigmentation (Whitton et al., 2016;Passeron, 2017). De-pigmentation is only considered for patients who present with large areas of vitiligo clinically. Several surgical treatments are available that restore Mcs at the skin site (Razmi et al., 2019). Most surgical techniques utilize Mcs suspensions prepared from a skin biopsy, and these Mcs are then autologously transplanted (Donaparthi and Chopra, 2016). Bone marrow mesenchymal stem cells (BMMSCs) are members of the osteoblast lineage (Abdallah and Ali, 2020). BMMSCs have been demonstrated to be multi-potent and are capable of differentiating into various mesodermal and ectodermal lineages. This capability suggests that BMMSCs could be potentially used in therapeutic tissue repair procedures. In our earlier study (Mei et al., 2015), mouse BMMSCs were successfully differentiated into Mcs, of which the typical Mcs were verified not only by morphology but also by detection of Mcs specific cytoplasmic organelles, such as type III and IV melanosomes. This method could be applied to prepare an autologous Mcs suspension for transplantation in vitiligo treatment. However, the 180-day differentiation period may limit this application in the clinic.
Yi Qi Qu Bai (YQQB) granule was a traditional Chinese medicine made in our hospital, which has been used for vitiligo for many years and is primarily composed of Astragalusmembranaceus (Fisch.) Bunge (Fabaceae), Fructus Leonuri (Lamiaceae), Fructus Akebiae (Lardizabalaceae), and root of Caraganasinica (Bućhoz), Rehd. (Leguminosae). Astragalus membranaceus (AM) has been used to treat vitiligo patients in the progressive phase for nearly 30 years. In a prospective study (Zhang et al., 2017) of 233 segmental vitiligo patients, the effective rate and color-reverse rate of the patients who received combination therapy of YQQB granule and 308 nm-excimer laser were much higher than those who received only single 308 nm-excimer laser therapy.
Astragaloside IV (AIV) is characterized by a potential protective effect on the pathologies because of its wide range of biofunctional properties, including anti-inflammatory, antioxidant, anti-diabetic, anti-fibrotic, anti-viral, antiasthmatic functions. (Costa et al., 2019). In the current study, we investigated whether AIV, the main ingredients from AM, could more efficiently induce differentiation of BMMSCs into Mcs by optimizing the induction method, shortening differentiation times, and increasing the overall yield of desired differentiated cells. To improve the previous method of Mcs differentiation from BMMSCs, we incubated BMMSCs with AIV at varying concentrations (0.1-0.4 mg/L) in the differentiation medium trying to find out more efficient induction time. We also measured the levels of some melanocytic specific marker genes, including tyrosinase-related protein 1 (TRP-1), TRP-2, melanoma antigen recognized by T cells 1 (MART-1), and microphthalmia-associated transcription factor (Mitf), using quantitative real-time polymerase chain reaction (qPCR). The results of this study would provide evidence to support the application of AIV in Mc differentiation.

Differentiation of BMMSCs into Mcs
BMMSCs (1 × 10 6 /mL; passage 6) were seeded on 25 cm 2 cell culture plates (4 × 10 4 cells/cm 2 ) and cultured in Mcs differentiation medium containing Minimum Essential Medium Alpha Medium (MEM; Invitrogen, Carlsbad, CA, USA), 10% FBS and supplemented with 1.5 μg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL insulin (Sigma-Aldrich), 10 μg/mL transferrin (Invitrogen), 4 ng/mL basic fibroblast growth factor (FGF; Invitrogen) as a previous study described (Mei et al., 2015). The medium was changed every two days until the adherent cells reaching 90% confluent. The morphological characteristics of both primary and differentiated cells were observed under an inverted microscope (Leica, Wetzlar, Germany). In a preliminary experiment, we found that the induced BMMSCs demonstrated a lower survival rate in the culture media containing greater than 0.4 mg/L AIV (Suppl. Fig. S1). Thus, AIV was dissolved in Mcs differentiation medium at 0.4 mg/L.

Quantitative polymerase chain reaction (qPCR)
Total RNAs from B16 melanoma cells, induced cells, and BMMSCs (passage 6) were extracted with Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA (1 μg) was reversely transcribed into cDNA with M-MLV (Takara, Tokyo, Japan) and random primers in a final reaction volume of 20 μL. qPCR assay was performed using Sybergreen Master Mix (Takara). The primers for the qPCR are listed in Tab. 1. The thermal cycles of qPCR reaction were listed as followings: 95°C for 30 s, 45 cycles at 95°C for 5 s, 60°C for 30 s, 95°C for 1 min, and 79 cycles at 55-95°C for 4 s. The relative transcriptions of the targeting genes (TRP-1, TRP-2, MART-1, and Mitf) were quantified using the FTC3000HT Real-Time PCR System (Funglyn Biotech, Richmond Hill, Ontario, Canada). Each sample was analyzed at least for three repeats, and mouse ACTB (β-actin) was used as an internal reference gene for semiquantitative analysis.

Statistical analysis
Data were represented as mean ± standard deviation (SD) and analyzed using SPSS software (version: 20.0, IBM Corporation, Armonk, NY, USA). qPCR analysis was normalized to β-actin. Gene expression was analyzed by Student's t-test. Statistical significance was considered at P-values < 0.05 (two-sided).

Identification of BMMSCs with cellular immunostaining
The isolated BMMSCs were identified using the cellular immunostaining method (Fig. 1A). The results showed that the BMMSCs demonstrated CD44, CD90, CD105 positive staining and CD34, CD45 negative staining (Fig. 1B). Therefore, the isolated cells were characterized as BMMSCs.

Morphological changes during Mcs differentiation
During the initial induction period with 0.4 mg/L AIVcontaining differentiation buffer, primary BMMSCs were adherent and round in shape and were mixed with some other suspending cells ( Fig. 2A). The adherent cells presented as clonal expansion. At culture passage 3 (20-day induction), the cells were approximately 80-90% confluent and tended to gather in a monolayer on the bottom, which displayed a spindle-like shape with strong light refraction ( Fig. 2B). At 70-day induction, the cells still displayed fibroblast morphology with a spindle-like shape, large cell body, and abundant cell nuclei (Fig. 2C). In this phase, the cells proliferated rapidly, with a passage cycle of approximately 4-5 days. At 90-day induction, the proliferation of the differentiated cells gradually slowed, and the passage cycle extended to 7 days. The differentiated cells formed networks, which displayed a polygonal shape with distinct outlines and prolonged dendrites, typical morphology of Mcs (Fig. 2D).

DOPA positive stain in differentiated cells
The DOPA assay was used to identify active tyrosinase (TYR). TYR is a rate-limiting oxidase that controls melanin production in Mcs     in the nuclei, while the other markers were predominantly expressed in the cytoplasm (Fig. 4). These results further confirmed that cells undergoing 90-day induction with 0.4 mg/L AIV differentiated into Mcs. The Mcs differentiated in 0.1 mg/L and 0.2 mg/L AIV differentiation buffer for 90 days showed similar results (Suppl. Fig. S1C).

TRP-1 expression in differentiated Mcs supplemented with AIV
We used qPCR to measure the expression of the Mcs specific marker TRP-1 to determine the maturation state of the differentiated Mcs treated with different concentrations of AIV (Fig. 5A). At 150-day induction, TRP-1 expression was significantly increased in the 0.1, 0.2, and 0.4 mg/L AIVtreated cells compared to the control group (p < 0.01) and the differentiation medium group (p < 0.05), but still lower than that of the B16 positive control cells (p < 0.01) (Fig. 5A). The TRP-1 level in the 0.4 mg/L AIV group was higher compared to the 0.1 mg/L and 0.2 mg/L AIV groups (p < 0.05). TRP-1 expression levels between the 0.1 mg/L and 0.2 mg/L groups were not considerably different (p > 0.05). These data show that 150-day incubation with 0.1-0.4 mg/L AIV improves Mcs differentiation as evidenced by increased TRP-1 expression. At 90-and 120-day induction, TRP-1 expression was significantly elevated in the 0.4 mg/L AIV-treated cells, which was not only higher compared to the negative control group (p < 0.01) and the differentiation medium group (p < 0.01 in 90-day group and p < 0.05 in 120-day group), but also higher compared to the 0.1 mg/L AIV-treated (p < 0.01 in 90-day group and p < 0.05 in 120-day group) and 0.2 mg/L (p < 0.01 in 90-day group and p < 0.05 in 120-day group) AIV-treated groups, respectively (Fig. 5A). TRP-1 expression was still lower compared to the B16 positive control group (p < 0.01). TRP-1 expression in the 0.1 mg/L AIV and 0.2 mg/L AIV treated groups was higher compared to the non-induction (negative control) group (p < 0.01), however, they were not significantly different compared to the differentiation medium group (p > 0.05), and lower compared to B16 positive control group (p < 0.01; Fig. 5A).
In summary, these data suggest that from 90-to 120-day induction in differentiation medium supplemented with 0.4 mg/L AIV improves Mc differentiation, as evidenced by increased expression of TRP-1.

TRP-2 expression in differentiated Mcs supplemented with AIV
We analyzed expression of the Mcs markerTRP-2 in the differentiated Mcs treated with different concentrations of AIV using qPCR assay (Fig. 5B). At 120-to 150-day induction, TRP-2 expression was augmented in the 0.1, 0.2, and 0.4 mg/L AIV-treated groups compared to the control group (p < 0.01). In addition, the TRP-2 level in the 0.4 mg/L AIV-treated group was higher compared to the 0.1 mg/L and 0.2 mg/L AIV groups (p < 0.05 and p < 0.01, respectively) and the differentiation medium group (p < 0.01), but TRP-2 expression was still lower compared to the B16 positive control group (p < 0.01) (Fig. 5B). The TRP-2 levels in the 0.1 mg/L and 0.2 mg/L AIV groups were higher compared to the differentiation buffer group (p < 0.05 and p < 0.01, respectively). The levels between the 0.1 mg/L and 0.2 mg/L groups were not considerably different (p > 0.05). These data suggest that 120 to 150-day incubation with 0.2-0.4 mg/L AIV improves Mcs differentiation, as evidenced by increased TRP-2 expression.
At 90-day induction, TRP-2 expression was significantly increased in the 0.4 mg/L AIV-treated cells, which was higher compared to the negative control group (p < 0.01) and the differentiation medium group (p < 0.01), and also higher compared to the 0.1 mg/L (p < 0.01) and 0.2 mg/L (p < 0.01) AM-treated groups (Fig. 5B). However, TRP-2 expression was still lower compared to the B16 positive control group (p < 0.01). TRP-2 expression in the 0.2 mg/L AIV-treated group was higher compared to the negative control group (p < 0.01). There was no significant difference between the 0.1 mg/L and 0.2 mg/L groups (p > 0.05) (Fig. 5B).
In summary, these results indicate that 90-day induction with 0.2 and 0.4 mg/L AIV improves Mc differentiation, as evidenced by increased TRP-2 expression. The corresponding nuclei were stained with DAPI (middle panels). The corresponding merged images of antibody (Ab) and DAPI are presented in the right panels. As the images illustrated, Mitf was primarily detected in nuclei, while TRP1, TRP2, and MART-1 were predominantly expressed in the cytoplasm. (B) The statistical analysis for the Mcs biomarkers-stained cells.

MART-1 expression in differentiated Mcs supplemented with AIV
Gene expression of MART-1, another melanocytic marker protein, in the differentiated Mcs treated with different concentrations of AIV was measured using qPCR assay (Fig. 5C). At 120 to 150-day induction, MART-1 levels were amplified in the 0.1, 0.2 and 0.4 mg/L AIV-treated groups compared to the negative control group (p < 0.05, p < 0.01, and p < 0.01, respectively). Furthermore, MART-1 levels in the 0.4 mg/L AIV-treated group were elevated compared to the differentiation medium group (p < 0.05). However, there were no differences for MART-1 levels among the 0.1, 0.2 and 0.4 mg/L AIV-treated groups (p > 0.05). MART-1 levels in all AIV-treated groups were lower compared to the B16 positive control group (p < 0.01) (Fig. 5C). At 90-day induction, MART-1 expression was significantly elevated in the 0.4 mg/L AIV-treated cells, which was higher compared to the negative control group (p < 0.01) and the differentiation medium group (p < 0.05) but was not different compared to the 0.1 mg/L (p > 0.05) and 0.2 mg/L (p > 0.05) AIV-treated groups (Fig. 5C). However, MART-1 was lower compared to the B16 positive control group (p < 0.01). MART-1 expression in both the 0.1 mg/L and 0.2 mg/L AIV-treated groups was higher compared to the non-induction (negative control) group (P < 0.01) and differentiation medium group (p < 0.05). There was no significant difference between 0.1 mg/L and 0.2 mg/L groups (p > 0.05) (Fig. 5C).
In summary, these results indicate that 90-day induction with 0.4 mg/L AIV improves Mc differentiation, as evidenced by increased MART-1 expression.

Mitf expression in the differentiated Mcs supplemented with AIV
Finally, Mitf gene expression, which is limited in Mcs, osteoclasts, and mast cells (Hershey and Fisher, 2004), was measured in the differentiated Mcs treated with different concentrations of AIV using qPCR assay (Fig. 5D). At 90 to 150-day induction, Mitf levels were augmented in all AIVtreated groups compared to the negative control group (p < 0.01, except the 0.1 mg/L AIV-treated group for 90-day induction, where the p-value was less than 0.05). However, none of the AIV supplemental groups was significantly different compared to the differentiation medium group (p > 0.05) (Fig. 5D). These results indicate that Mitf expression is not increased by AIV supplementation.

Discussion
A surgical technique that involves autologous transplantation of Mcs has been established to treat patients with vitiligo (Donaparthi and Chopra, 2016). A crucial process in this technique is Mc differentiation. We have successfully differentiated Mcs from BMMSCs using hydrocortisone, insulin, transferrin, and FGF (Mei et al., 2015). However, the long induction time for differentiation (120-180 days) has limited the application of this technique. In this study, we applied a traditional Chinese medicine, AM, in combination with our previously described differentiation media to induce Mcs differentiation from BMMSCs. Morphological (Fig. 2), histochemical (DOPA) (Fig. 3), and immunofluorescence imaging (TRP-1, TRP-2, MART-1, and Mtif) (Fig. 4) analyses indicate that Mc differentiation can be optimized to a minimum of 90 days with 0.4 mg/L AIV. In addition, qPCR showed that expression levels of Mc markers, TRP-1, TRP-2, and MART-1, were significantly augmented (p < 0.01) compared to the negative control and the standard differentiation medium groups (p < 0.01). Another Mc marker, Mtif, was not affected by AIV-treatment and the standard differentiation medium group (p < 0.05) but was still higher compared to the negative (non-induction) controls (p < 0.05). These data suggest that treatment with 0.4 mg/L AIV for 90 days can increase TRP-1, TRP-2, and MART-1 expression. Collectively, morphology, immunocytochemistry, and immunofluorescence analyses suggest that 0.4 mg/L AIV decreases Mc differentiation time from 180 to 90 days when cells are cultured in the differentiation medium.
Mc proliferation and differentiation are primarily regulated by four different signaling transduction pathways including, diacylglycerol/protein kinase C (DAG/PKC), nitric oxide/cyclic GMP/protein kinase G (NO/cGMP/PKG), mitogen-activated protein kinase (MAPK), and cyclic AMP/protein kinase A (cAMP/PKA) (Halaban, 1994). Insulin, hydrocortisone, FGF, and transferrin, all of which were present in our differentiation medium, are non-carcinogenic factors that activate Mc differentiation via either the cAMP/PKA, MAPK, or NO/cGMP/PKG signaling pathways (Mei et al., 2015).
TYR is a rate-limiting enzyme that controls melanin production through hydroxylation of a monophenol and conversion of an o-diphenol to the corresponding o-quinone (Kumar et al., 2011). TRP-1 and TRP-2 are homologic enzymes of the TYR gene family, the function of which is to scavenge free radicals (Murisier and Beermann, 2006). In this study, 90-day culture in differentiation medium supplemented with 0.2-0.4 mg/L AIV increased TRP-1 and TRP-2 expression. Kawaguchi et al. (2012) found that DAG kinase (DGK) modulates TYR post-translation to regulate melanogenesis. This suggests that AIV may induce TYR activity in Mc differentiation through activation of the DAG/PKC pathway. It is widely speculated that AIV may be able to promote not only the expression of melanin biosynthesis-related proteins but also the differentiation of cells into intermediate melanoblasts. Future studies to investigate this differentiation process would be of particular interest.
MART-1 is a melanoma-associated antigen recognized by autologous cytotoxic T lymphocytes and is primarily presented on melanosomes and the endoplasmic reticulum, which plays an important role in the expression, stability, trafficking, and processing of the pre-melanosome protein as well as the formation of stage II melanosomes (Bandarchi et al., 2013). In this study, at 90-day induction of BMMSCs in differentiation medium supplemented with 0.4 mg/L AIV, MART-1 expression was significantly increased compared to the non-induced cells and the differentiation medium groups (Fig. 5C). This result suggests that AIV alone plays a primary role in regulating MART-1 expression. Indeed, Kono et al. (2006) found that inhibition of MAPK signaling with PD98059 or U0126 (MEK inhibitors) up-regulated MART-1 expression in melanoma cell lines, MU89 and MUX (a lower variant cell of MU89). These findings directly suggest that AIV may inactivate the MARK/MEK pathway to induce MART-1 expression during Mc differentiation.
Mitf highly expresses in malignant melanoma cells. Mitf regulates melanin production by binding to TYR, TRP-1, and TRP-2 promoters to trans-activating the expression of TYRrelated genes (Levy et al., 2006). Mitf expressed in the differentiated cells (either with or without AM) was relatively stable and significantly lower compared to the positive control cells (B16 cells) during the 150-day induction (Fig. 5D). These results indicate that AIV does not up-regulate Mitf expression. Low expression of Mitf is related to stem cell-like or invasive potential (Hartman and Czyz, 2015). In this sense, AIV may not be involved in arresting cell cycle but could play a role in regulating invasive cellular properties. In addition, lower Mitf expression may promote mild melanocyte differentiation, but not melanoma. Our data clearly showed that AIVinduced TRP-1, TRP-2, and MART-1 expression was significantly lower compared to expression in the B16 positive control cells. The differentiation conditions in this study are relatively mild and safe, and the significantly increased expression levels of TRP-1, TRP-2, MART-1, and Mitf were only found in the B16 melanoma cell line.
In the clinic of traditional Chinese medicine, AM (AIV) has been applied to control progressive vitiligo for many years, which could decrease VIDA scores without obvious adverse effects. Our data in this study provided experimental evidence that AIV has the potential to promote melanocyte differentiation from MSCs and melanin production. In some studies, it has been demonstrated that AIV can take effects in neural stem cell (NSCs) differentiation. Hu et al. (2016) investigated the effect of AIV on the proliferation and differentiation of the engrafted neural stem cells in Alzheimer's disease (AD) rat models. Their results indicated that AIV treatment resulted in improvements in learning and memory of AD models by promoting NSC proliferation and differentiation partly through the Notch signal pathway. These indicate that AIV plays a significant role in stem cell differentiation. Based on the results of our study, we speculate that AIV could improve Mc differentiation from mesenchymal stem cells in vitro as well.

Conclusions
We verified that supplementation of hydrocortisone, insulin, interferrin, and FGF-containing differentiation medium with 0.4 mg/L AIV improves Mcs differentiation by shortening the induction period from 180 to 90 days. Mcs differentiation was verified by morphology, immunocytochemistry, and immunofluorescence analyses, as well as by qPCR assay of melanocytic marker proteins (including TRP-1, TRP-2, and MART-1). Mitf expression was not induced by AIV, indicating that AIV is relatively safe. These results provide experimental and theoretic evidence for AIV application in clinical vitiligo repigmentation treatment.
Availability of Data and Materials: All data generated or analyzed during this study are included in this published article or are available from the corresponding author on reasonable request.