10.5826/dpc.1302a77
Introducion: Melanocyte progenitors are embryonically derived from the neural crest and subsequently get localized in hair follicles and epidermis to provide hair and skin pigmentation. These progenitor cells in hair follicles repeatedly proliferate and differentiate to maintain pigmentation. Vitiligo, a pigmentary disorder, is associated with loss of melanocytes. Repigmentation of vitiligo lesions mainly depends upon the proliferation, migration and differentiation of melanocyte stem cells (MelSCs) into functional melanocytes. Current study is designed to check the efficacy of lenalidomide, an imide drug in the differentiation of MelSCs into functional melanocytes.
Objectives: Aim of the study is to check the effect of lenalidomide in the proliferation, migration of cultured hair follicle derived melanocyte stem cells and their differentiation into functional melanocytes.
Methods: Primary culture of MelSCs was established from whisker hair of C57BL/6 mice. Proliferation and migration of cultured cells were done by MTT assay and boyden’s chamber migration assay, respectively. Effect of lenalidomide on the MelSCs differentiation was checked at gene level by qPCR &amp; protein expression was checked by immunocytochemistry.
Results: A significant increase in the migration of MelSCs in comparison to control was also observed. Lenalidomide treatment significantly increased the expression of melanocyte specific genes in cultured MelSCs as compared to control.
Conclusion: From the results we concluded that lenalidomide induce the proliferation and migration of MelSCs and accelerate the differentiation of MelSCs into functional melanocytes.
 
text/xml
2160-9381
2620
en
Ravinder Kumar
https://creativecommons.org/licenses/by-nc/4.0
Dermatology Practical & Conceptual
differentiation
Lenalidomide Augments Differentiation of Cultured Hair Follicle Derived Melanocyte Stem Cells Into Functional Melanocytes
Text.Serial.Journal
https://dpcj.org/index.php/dpc
Original Article
2023-04-29
2022-05-30
2023-04-29
2023-05-31
e2023077
Dermatol Pract Concept
Introducion: Melanocyte progenitors are embryonically derived from the neural crest and subsequently get localized in hair follicles and epidermis to provide hair and skin pigmentation. These progenitor cells in hair follicles repeatedly proliferate and differentiate to maintain pigmentation. Vitiligo, a pigmentary disorder, is associated with loss of melanocytes. Repigmentation of vitiligo lesions mainly depends upon the proliferation, migration and differentiation of melanocyte stem cells (MelSCs) into functional melanocytes. Current study is designed to check the efficacy of lenalidomide, an imide drug in the differentiation of MelSCs into functional melanocytes.
Objectives: Aim of the study is to check the effect of lenalidomide in the proliferation, migration of cultured hair follicle derived melanocyte stem cells and their differentiation into functional melanocytes.
Methods: Primary culture of MelSCs was established from whisker hair of C57BL/6 mice. Proliferation and migration of cultured cells were done by MTT assay and boyden’s chamber migration assay, respectively. Effect of lenalidomide on the MelSCs differentiation was checked at gene level by qPCR &amp; protein expression was checked by immunocytochemistry.
Results: A significant increase in the migration of MelSCs in comparison to control was also observed. Lenalidomide treatment significantly increased the expression of melanocyte specific genes in cultured MelSCs as compared to control.
Conclusion: From the results we concluded that lenalidomide induce the proliferation and migration of MelSCs and accelerate the differentiation of MelSCs into functional melanocytes.
 
Ravinder Kumar
2023/04/29
10.5826/dpc.1302a77
e2023077
Department of Zoology, Panjab University, Chandigarh, India.
2160-9381
differentiation
en
e2023077
Bertolotto C, Abbe P, Bille K, et al. Different cis-acting elements are involved in the regulation of TRP1 and TRP2promoter activities bycyclic AMP: pivotal role of M boxes (GTCATGTGCT)and of microphthalmia. Mol. Cell. Biol. 1998;18(2):694-702. doi: 10.1128/MCB.18.2.694.
Lenalidomide Augments Differentiation of Cultured Hair Follicle Derived Melanocyte Stem Cells Into Functional Melanocytes
https://dpcj.org/index.php/dpc/article/download/2620/1985
Lenalidomide Augments Differentiation of Cultured
 Hair Follicle Derived Melanocyte Stem Cells Into
               Functional Melanocytes
                     Uma Kumari1, Naveed Pervaiz1, Harjot Kaur1, Himani Sharma1,
                                Davinder Parsad2, Ravinder Kumar1


1 Department of Zoology, Panjab University, Chandigarh, India
2 Department of Dermatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India



Key words: lenalidomide, melanocyte stem cells, melanocyte, differentiation
Citation: Kumari U, Pervaiz N, Kaur H, Sharma H, Parsad D, Kumar R. Lenalidomide Augments Differentiation of Cultured Hair Follicle
Derived Melanocyte Stem Cells Into Functional Melanocytes. Dermatol Pract Concept. 2023;13(2):e2023077.
DOI: https://doi.org/10.5826/dpc.1302a77
Accepted: September 3, 2022; Published: April 2023
Copyright: ©2023 Kumari et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-
NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use,
distribution, and reproduction in any medium, provided the original authors and source are credited.
Funding: None.
Competing interests: None.
Authorship: All authors have contributed significantly to this publication.
Corresponding author: Ravinder Kumar, Department of Zoology, Panjab University, Chandigarh, India-160014.
E-mail: ravinder03@gmail.com/ravinder@pu.ac.in




                       Introduction: Melanocyte progenitors are embryonically derived from the neural crest and s­ ubsequently
  ABSTRACT             get localized in hair follicles and epidermis to provide hair and skin pigmentation. These progenitor
                       cells in hair follicles repeatedly proliferate and differentiate to maintain pigmentation. ­Vitiligo, a pig-
                       mentary disorder, is associated with loss of melanocytes. Repigmentation of vitiligo lesions mainly
                       depends upon the proliferation, migration and differentiation of melanocyte stem cells (MelSCs) into
                       functional melanocytes. The current study is designed to check the efficacy of l­enalidomide, an imide
                       drug in the differentiation of MelSCs into functional melanocytes.
                       Objectives:The aim of the study is to check the effect of lenalidomide in the proliferation, migration of
                       cultured hair follicle derived melanocyte stem cells and their differentiation into functional melanocytes.
                       Methods: Primary culture of MelSCs was established from whisker hair of C57BL/6 mice. Prolifera-
                       tion and migration of cultured cells were done by MTT assay and Boyden’s chamber migration assay,
                       respectively. Effect of lenalidomide on the MelSCs differentiation was checked at gene level by qPCR
                       and protein expression was checked by immunocytochemistry.
                       Results: A significant increase in the migration of MelSCs in comparison to control was also observed.
                       Lenalidomide treatment significantly increased the expression of melanocyte specific genes in cultured
                       MelSCs as compared to control.
                       Conclusions: From the results we concluded that lenalidomide induce the proliferation and migration
                       of MelSCs and accelerate the differentiation of MelSCs into functional melanocytes.



Original Article | Dermatol Pract Concept. 2023;13(2):e2023077                                                                        1
Introduction                                                      Methods
Hair follicle melanocyte stem cells (MelSCs) are present in       Ethical Statement
bulge or sub-bulge regions of hair and are important reser-       The study design was based on the guidelines set by the
voirs of melanocytes. They persist as self-renewing cells and     Institutional Animal Ethics committee, Panjab Univer-
play a very significant role in the pigmentation of hair and      sity, Chandigarh, India (Approval No: PU/45/99/CPCSEA/
skin [1]. They regenerate mature melanocytes for hair and         IAEC/2017/31). C57BL/6 mice were maintained as per the
skin in response to some kind of melanocyte loss under nor-       guiding principles of the Committee for the purpose of Con-
mal conditions and ultimately maintain the p
                                           ­ igmentation [2].     trol and Supervision of Experiments on Animals (CPCSEA).
The regeneration of functional melanocytes is influenced
by activation, proliferation and differentiation of these         Reagents
MelSCs [3]. Loss of functional melanocytes causes skin de-        The reagents used in the study include Dulbecco’s ­Modified
pigmentation and thus leads to depigmentory disorders like        Eagle Media (Cat. No. BE12-604F), Fetal Bovine Serum (Cat.
vitiligo [4,5]. Vitiligo is an autoimmune cutaneous disorder      No. 10270106), and Recombinant Human FGF-Basic pro-
that has grave psychosocial impact on the patient’s quality of    tein (Cat. No. PHG0264). The antibodies include ­Anti-mouse
life [6]. The mechanism of melanocyte disappearance in this       AF488-conjugated      CD34      (Cat.   No.     sc-7324AF488),
disease still remains unclear [7]. Several treatment modali-      PE-­
                                                                  ­  conjugated TRP2/ DCT (Cat.No. sc-74439PE), pri-
ties are available like the conventional medical, surgical and    mary anti-mouse tyrosinase (Cat. No. sc-20035) and ­nestin
physical means but none are completely satisfactory.              (Cat. N0 sc-23972) antibody and secondary antibody
    In the depigmented patches there is a loss of functional      anti-mouse IgG-FITC (sc-2010), purchased from Santa Cruz
melanocytes but hair follicle MelSCs are preserved. Therefore     Biotechnology.
this original source of regeneration in the hair follicle bulge
is being targeted to repopulates the vitiligo-depigmented         Isolation and Culture of MelSCs
epidermis with melanocytes [8]. Various studies have shown
                                                                  MelSCs culture was established from whisker hair cells of
that MelSCs in the outer root sheath played important part
                                                                  C57BL/6 mice at 4 weeks of age. Mice were euthanized
in the epidermal follicular repigmentation. However the
                                                                  and the upper lips containing vibrissae were dissected as
activation, proliferation, migration and differentiation of
                                                                  described by Gilanchi [12].It was rinsed with betadine and
MelSCs from the outer root sheath of the hair follicle require
                                                                  70% ethanol for 2 minutes and placed in phosphate buffer
more novel and effective strategies.
                                                                  saline (PBS). Hair follicles were separated from lip pad un-
    Here we aimed at investigating the potential therapeu-
                                                                  der sterile conditions and were incubated overnight at room
tic efficacy of lenalidomide as the possible repigmentation
                                                                  temperature in collagenase IV (1mg/ml). After incubation
strategy. Lenalidomide is an immunomodulatory drug that
                                                                  the digested hair follicles were pipetted thoroughly to ob-
has shown a clinical effect in several autoimmune and in-
                                                                  tain single cell suspension. Afterwards, the cell suspension
flammatory disorders. Recently, its effect was observed as
                                                                  was centrifuged at 1000 rpm for 5 minutes and the obtained
the hyperpigmentation of skin in black/African-American
                                                                  pellet was resuspended in a stem cell specific culture medium
patients suffering from multiple myeloma [9]. Interestingly,
                                                                  which contains 80% KnockOut™ DMEM/F12 medium,
in a case study it also led to progressive hair repigmentation
                                                                  20% KnockOut™ serum replacement, 200 mM L-glutamine,
in an old multiple myeloma patient [10]. Pervaiz et al [11]
                                                                  0.1 mM β-mercaptoethanol, 1% nonessential amino acids
also reported the inhibitory influence of this drug on the pro-
                                                                  and 4 ng/ml basic fibroblast growth factor (bFGF) [13].
gression of depigmented lesions in a vitiligo mouse model.
Therefore, the current study was designed to check the role
                                                                  Characterization of Cultured Spheres
of lenalidomide on the differentiation of murine hair folli-
                                                                  Cultured spheres were characterized for the presence of
cle derived melanocyte stem cells into pigment producing
                                                                  nestin which is a neural crest marker. Spheres were fixed in
melanocytes.
                                                                  chilled paraformaldehyde for 10 minutes and then washed
                                                                  in PBS. Blocking was performed overnight at 4o C with 3%

Objectives                                                        BSA in a humidified chamber. For nestin immunostaining,
                                                                  primary antibody (dilution 1:100) and secondary antibody
The aim of the study is to check the effect of lenalidomide in    (dilution 1:200) were used. After antibody incubation PBS
the proliferation, migration of cultured hair follicle derived    washing was done and spheres were then counterstained
melanocyte stem cells and their differentiation into func-        with DAPI. Spheres were washed again with PBS and exam-
tional melanocytes.                                               ined using confocal microscope.




2                                                                     Original Article | Dermatol Pract Concept. 2023;13(2):e2023077
Subculturing of Cells From Cultured Spheres                       treatment groups was added. Cells were kept at 37° C in
Subculturing was done for further propagation of cells. For       CO2 incubator for 18 hours. Afterwards, cells were fixed
that, spheres were easily detached by agitating the culture       with 4% formaldehyde and stained with 0.5% crystal violet.
plate. Spheres were then transferred to new culture wells,        The inside of each insert was swabbed using cotton swabs.
where they were cultured in media having DMEM, FBS and            Randomly selected fields were photographed, and the cells
recombinant human FGF-basic protein [14]. Next day cells          that had migrated were counted.
started coming out of the sphere and when the cells reached
to 80% confluency, they were further subcultured by tryp-         In Vitro Treatment of Melscs With Lenalidomide
sinization method using trypsin-EDTA.                             and Its Effect on Differentiation Into Functional
                                                                  Melanocytes
Characterization of Cells Obtained From Spheres                   Cultured MelSCs were treated with lenalidomide at con-
Cultured cells obtained from the spheres were characterized       centrations of 2.5 μM and 5 μM for 2 weeks. The media
by nestin, CD34, DCT and TYR immunostaining and DOPA              was changed in plate after every 48 hours. After 2 weeks
staining. For immunostaining, cells were fixed in chilled         of lenalidomide treatment, its effect on the differentiation
paraformaldehyde for 10 minutes and then washed in PBS.           of MelSCs into melanocytes was checked at both gene and
                                            o
Blocking was performed overnight at 4 C with 3% BSA in            protein level.
humidified chamber. Next day, the fixed cells were incubated
with fluorochrome conjugated antibodies CD34 and DCT              Quantitative Real-Time Polymerase Chain
for 3 hours at room temperature (dilution 1:150). For nestin      Reaction (qPCR)
and tyrosinase immunostaining, primary antibody (dilution         qPCR was performed to analyze the transcriptional gene ex-
1:100) and secondary antibody (dilution 1:200) were used          pression. RNA was isolated using Trizol reagent (Sigma) by
for 3 hours and 2 hours respectively. After PBS washing,          following manufacturer’s guidelines. Gel electrophoresis was
cells were then counterstained with DAPI. Cells were washed       performed after RNA isolation to assess its quality. Absor-
again with PBS and examined using confocal microscope.            bance at 260 and 280 nm measured on Nano-drop was used
For DOPA staining, washing of cultured cells was done with        to determine the concentration and purity of RNA. cDNA
1X PBS and then fixed with chilled paraformaldehyde for           was synthesized from the isolated RNA using iScriptTM
10 minutes. 10 mM L-DOPA was added to the cells and then          c-DNA synthesis kit (BIO-RAD). qPCR was performed us-
incubated at 37o C. Cells were examined hourly under the          ing SYBR(R) Green JumpStart (TM) TaqReadyMix (TM)
microscope for 4 hours.                                           master mix on a real time PCR system (Roche LC480) ac-
                                                                  cording to manufacturer’s guidelines. The forward and re-
Cell Proliferation Assay                                          verse sequences of primers are given in Table 1. The relative
Cell proliferation was checked by MTT assay by following          levels of mRNA expression were calculated by following the
the modified procedure of Mosmann [15].Cultured MelSCs            method of Livak and Schmittgen [16], normalized by β-actin.
      3
(5×10 ) were plated in the 96-well cell culture plate. Lenalid-
omide treatment was given to the cells at 2.5 μM and 5 μM         Immunostaining of Differentiated Melanocytes
concentrations, the untreated cells were considered as con-       After 14 days of differentiation assay MelSCs cells were
trol. After 24 hours of lenalidomide treatment, culture media     checked for melanocyte marker TYR and DCT by immu-
was removed from each well and 100μl of MTT (0.5 mg/ml)           nostaining (protocol discussed in characterization of cells
solution was added. The cells with MTT (0.5mg/ml) solution        obtained from spheres section). The fluorescence was quan-
                                    o
were incubated for 4 hours at 37 C till the formation of for-     tified using image-J software.
mazan crystals. After incubation 100 μl of DMSO was added
to each well and proper dissolution of formazan crystals was      Statistical Analysis
done. Optical density was evaluated using microplate spec-        Statistical analysis was performed by using GraphPad Prism
trophotometer (EPOCH) at 560 nm.                                  6 for windows version 6.01 (GraphPad Software, Inc.). Final
                                                                  results were shown as mean with standard deviation (mean
Boyden Chamber Migration Assay                                    ± SD). The data was examined for normality by the ­Shapiro–
Cultured MelSCs were starved for about six hours in serum         Wilk test. Two-tailed Student t test was done to calculate
                                                            4
free culture media. After the starvation period, 3 × 10 cells     the statistical significance between two groups, whereas
were plated in serum-free medium (no chemoattractant) in          One-way analysis of variance (one-way ANOVA) followed
the upper chamber. To the lower chamber, medium with              by post hoc Tukey test was used to calculate statistical sig-
serum for control group and serum plus lenalidomide for           nificance for more than two groups. P-values less than 0.05




Original Article | Dermatol Pract Concept. 2023;13(2):e2023077                                                               3
           Table 1. Primers used for qPCR, their Amplicon Length and Annealing Temperature.
                                                                                                  Amplicon         Annealing
    Gene                Forward primer                            Reverse primer                   length         temperature
 β-Actin   5’ GAATTGCTATGTGTCTGGGT 3’                5’ CATCTTCAAACCTCCATGATG 3’                     257bp             58oC
 TYR       5’ GGGCCCAAATTGTACAGAGA 3’                5’ ATGGGTGTTGACCCATTGTT 3’                      174bp             58oC
 TYRP1     5’ AAGTTCAATGGCCAGGTCAG 3’                5’ TCAGTGAGGAGAGGCTGGTT 3’                      157bp             58oC
 DCT       5’ AGCAGACGGAACACTGGACT 3’                5’ GCATCTGTGGAAGGGTTGTT 3’                      180bp             58oC
 MITF      5’ GGAACAGCAACGAGCTAAGG 3’ 5’ TGATGATCCGATTCACCAGA 3’                                     170bp             58oC
 SOX9      5’ CGACTACGCTGACCATCAGA 3’                5’ AGACTGGTTGTTCCCAGTGC 3’                      188bp             58oC
 SOX10     5’ AGCCCAGGTGAAGACAGAGA 3’ 5’ ATAGGGTCCTGAGGGCTGAT 3’                                     175bp             58oC



(*P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001)              Effect of Lenalidomide on the Differentiation
were considered as significant.                                   of MelSCs Into Functional Melanocytes
                                                                  The cultured cells derived from spheres were treated with
Results                                                           lenalidomide for 14 days to check its effect on the differenti-
                                                                  ation of MelSCs into functional melanocytes. After 14 days
Primary Culture and Characterization of MelSCs                    of treatment cell were checked for expression of melanocyte
MelSCs     were   cultured   from   whisker   hair   cells   of   specific genes. qPCR results showed that lenalidomide signifi-
C57BL/6mice. On the 5th day of cell plating, some of the          cantly enhanced the expression of melanocyte specific genes
cells started aggregating and forming spheres. A well-marked      (DCT, TYRP1, TYR, MITF, SOX10, SOX9) as compared
sphere took around 10 to 15 days in formation (Figure 1A).        to control. After lenalidomide treatment, the expression of
    Spheres were characterized for neural crest stem cells        TYR was found higher at 2.5 µM (1.902 ± 0.023 fold) and
marker nestin, and it was found that the cells of the spheres     5 µM concentrations (3.516 ± 0.269 fold) as compared to
showed positive staining for nestin (Figure 1D). For subcul-      control. Similarly, we observed a significant elevation in the
turing of MelSCs, spheres were taken out and plated in a          expression of TYRP1 (2.543 ± 0.08 fold) and (1.715 ± 0.104
new culture plate. The next day, the cells began to migrate       fold), DCT (1.786 ± 0.335 fold) and (3.516 ± 1.353 fold),
out of the spheres gradually (Figure 1B). These cells were        MITF (1.640 ± 0.080 fold) and (1.498 ± 0.188 fold), SOX9
than further characterized for specific stem cell marker          (2.738 ± 0.129 fold) and (1.166 ± 0.470 fold), and SOX10
nestin, CD34, and melanocyte stem cell marker, DCT and            (1.077 ± 0.408 fold) and (5.508 ± 2.203 fold) in 2.5 µM
melanocyte specific marker TYR. We observed that these            and 5 µM concentrations, respectively (Figure 3). Differen-
sphere-­derived cultured cells were nestin (Figure 1E) and        tiation of MelSCs into functional melanocyte was further
DCT (Figure 1G) positive and were negative for CD34               confirmed by protein expression of melanocyte specific genes
(­Figure 1F) and TYR (Figure 1H). Further, DOPA staining          TYR and DCT. It was found that lenalidomide treatment
was performed to check the presence of melanocyte contam-         significantly increased the protein expression of tyrosinase
ination in MelSCs culture. The results indicated that cells       in treated MelSCs. After 14 days of lenalidomide treatment,
were negative for DOPA, compared to melanocytes taken as          number of TYR positive cells were found significantly higher
positive control (Figure 1C).                                     at both 2.5 µM (14.33 times) and 5 µM concentration of
                                                                  lenalidomide (7.33 times) as compared to control. Similarly,
Effect of Lenalidomide on the Proliferation                       we observed a significant elevation in the expression of DCT
and Migration of MelSCs                                           (1.56 ± 0.1081 fold) and (1.33 ± 0.12 fold) in 2.5 µM and
Effect of lenalidomide was checked on the proliferation of        5 µM concentrations, respectively (Figure 4). Hence, the re-
MelSCs and we found that after 24 hours of treatment, it          sults from immunocytochemistry also showed that the pro-
increased the proliferation of MelSCs at both 2.5 µM and          tein expression of TYR and DCT was significantly higher in
5µM concentrations in comparison to untreated control, al-        lenalidomide treated cells in comparison to control (Figure 4).
though the increase was insignificant (Figure 2,A- and B).
Further it significantly enhanced the migration of MelSCs at
                                                                  Conclusions
both concentrations in comparison to control. The percent-
age of migrated cells increased by 88% and 95% in 2.5 µM          Vitiligo is an autoimmune disease characterized by cutane-
and 5 µM treated wells respectively in comparison to control      ous depigmentation caused by autoimmune-mediated dam-
(­Figure 2, C and D).                                             age of melanocytes. Despite continuous research is being



4                                                                     Original Article | Dermatol Pract Concept. 2023;13(2):e2023077
Figure 1. Representative images showing (A) primary culture of melanocyte stem cells at Day 0, Day 5, Day 10 and Day 15, (B) Subculture
of MelSCs at 3 hours and Day 1, (C) Dopa staining of melanocyte stem cells and melanocytes (taken as positive control) after 4 hours,
(D) ­Confocal imaging indicating nestin immunostaining for the characterization of MelSC spheres. Representative images from confocal
microscopy showing the characterization of MelSCs by immunostaining with (E) nestin, (F) CD34, (G) DCT, and (H) tyrosinase.


elucidated, a definitive cure still remains elusive. Therefore,      disorders and has shown therapeutic effects on various au-
many newer therapeutic options are being explored to im-             toimmune or inflammatory diseases. Dasanu et al [10] re-
prove the outcome; immunomodulators being the most re-               ported the first case of progressive hair repigmentation with
cent and exciting additions.                                         the use of this drug in an elderly patient of multiple myeloma.
    Lenalidomide is an FDA approved immunomodulatory                 Further they hypothesized that lenalidomide may be capable
imide drug which is currently being investigated in various          of stimulating migration and differentiation of melanocytes



Original Article | Dermatol Pract Concept. 2023;13(2):e2023077                                                                       5
     A                    PROLIFERATION OF MELANOCYTE STEM CELLS (24HR)                                                     B




                                                                                                                            Percentage Proliferation of MelSCs
                                                                                                                                                                 150
                        CONTROL                     LEN 2.5 µM                                     LEN 5 µM

                                                                                                                                                                 100



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     C               MIGRATION ASSAY OF MELANOCYTE STEM CELLS (18HR)                                                        D




                                                                                                                            Percentage migration of MelSCs
                                                                                                                                                                 250
                        CONTROL                     LEN 2.5 µM                                     LEN 5 µM
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Figure 2. (A) Representative pictures of cultured MelSCs. (B) Bar diagram showing the effect of lenalidomide (2.5 µM and 5 µM) on the
proliferation of MelSCs as compared to untreated control cells after 24 hours of treatment. (C) Light microscopic images depicting the mi-
gration of MelSCs in control, 2.5 µM and 5 µM lenalidomide treated cells at 18 hours. (D) Bar diagram displaying the effect of lenalidomide
(2.5 µM and 5 µM) on migration (at 18 hours) of MelSCs as compared to control. Data are presented as mean ± standard deviation; statistical
significance is shown by *P < 0.05, **P < 0.01.




  A                 3                                           B                 3                                                                                 C                6
                                                             (TYRP1 expression)




                                                                                                                                                                 (TYR expression)
 (DCT expression)




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(MITF expression)




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                                   LE




                                                                                                   LE




Figure 3. Bar diagram showing relative mRNA expression of (A) DCT (B) TYRP1 (C) TYR (D) MITF (E) SOX10 and (F) SOX9 in con-
trol, 2.5 µM lenalidomide and 5 µM lenalidomide treated MelSCs. The relative level of target gene expression was calculated by Livak
and Schmittgen method and normalized with β-actin. Data are presented as mean ± standard deviation; statistical significance is shown by
(*P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001).



6                                                                                                             Original Article | Dermatol Pract Concept. 2023;13(2):e2023077
                                                                            B

                                                                                               30




                                                                      2
                                                                         TYR + CELLS/MM
                                                                                               20



                                                                                               10



                                                                                                0
                                                                                                     CONTROL LEN 2.5 µM           LEN 5 µM

                                                                     D

                                                                                               2.5
                                                                   Relative intensity of DCT




                                                                                               2.0
                                                                         (fold change)




                                                                                               1.5

                                                                                               1.0

                                                                                               0.5

                                                                                               0.0
                                                                                                      CONTROL LEN 2.5 µM LEN 5 µM
Figure 4. Representative images from confocal microscopy showing the effect of lenalidomide at 2.5µM and 5µM concentrations on mela-
nocyte differentiation markers (A) TYR and (C) DCT in MelSCs after 14 days of treatment. (B) Bar diagrams depicting the TYR positive cells
after lenalidomide (2.5 µM and 5 µM) treatment in MelSCs. (D) Bar diagrams display the relative fold change of DCT in control and lenalido-
mide (2.5 µM and 5 µM) treated MelSCs after 14 days of their differentiation. Representative images of both proteins are from 3 independent
experiments that were quantified by using ImageJ software. Data are presented as mean ± standard deviation; statistical significance is shown
by *P < 0.05, ***P < 0.0001, ****P < 0.0001.



to promote repigmentation. Also, in a previous study from                         for neural crest cell marker, nestin. Nestin-positive neural
our laboratory we have shown that lenalidomide inhibited                          crest-derived stem cells have the ability to form neurospheres
the vitiligo progression in a vitiligo mouse model [11]. For                      [19,20]. The cultured spheres were later transferred to new
the repigmentation of vitiliginous skin, melanocyte reser-                        culture and cells started migrating out of these spheres and
voir in the hair follicles must be activated, differentiated                      were characterized for the various markers like nestin, CD34,
and migrated toward depigmented epidermis of vitiliginous                         tyrosinase, DCT and DOPA. The results showed that these
skin [17,18].                                                                     cells expressed nestin, a marker for neural progenitors that
    In this study effect of lenalidomide was directly checked                     is also expressed by stem cells of the hair follicle bulge [21].
on the cultured hair follicle derived melanocyte stem cells.                      Cultured cells were also positive for DCT, which is a marker
Cells from the murine whisker hair were isolated and cul-                         of MelSCs in hair matrix [22] but were negative for CD34.
tured. These cells formed spheres and were found positive                         CD34 negative neural crest derived melanocyte stem cells



Original Article | Dermatol Pract Concept. 2023;13(2):e2023077                                                                                  7
from hair follicle are primed for melanocyte differentiation         Kumar and Council of Scientific and Industrial Research,
[23]. As these sphere derived cells were negative for functional     New Delhi, India (09/135(0735)/2016-EMR-I) to grant fel-
melanocyte marker tyrosinase and DOPA, it confirmed that             lowship to Uma Kumari.
these cells were not functional melanocytes. Melanocytes are
also derived from the neural tube and neural-crest, and then
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Original Article | Dermatol Pract Concept. 2023;13(2):e2023077                                                                                    9
