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. Author manuscript; available in PMC: 2023 Dec 20.
Published in final edited form as: Pigment Cell Melanoma Res. 2023 Jan 3;36(2):232–245. doi: 10.1111/pcmr.13077

Induced pluripotent stem cells reprogramming overcomes technical limitations for highly pigmented adult melanocyte amplification and integration in 3D skin model

Catherine Cohen 1, Virginie Flouret 1, Meenhard Herlyn 2, Mizuho Fukunaga-Kalabis 2, Ling Li 2, Françoise Bernerd 1
PMCID: PMC10731472  NIHMSID: NIHMS1951956  PMID: 36478412

Abstract

Understanding pigmentation regulations taking into account the original skin color type is important to address pigmentary disorders. Biological models including adult melanocytes from different phenotypes allow to perform fine-tuned explorative studies and support discovery of treatments adapted to populations’ skin color. However, technical challenges arise when trying to not only isolate but also amplify melanocytes from highly pigmented adult skin. To bypass the initial isolation and growth of cutaneous melanocytes, we harvested and expanded fibroblasts from light and dark skin donors and reprogrammed them into iPSC, which were then differentiated into melanocytes. The resulting melanocyte populations displayed high purity, genomic stability, and strong proliferative capacity, the latter being a critical parameter for dark skin cells. The iPSC-derived melanocyte strains expressed lineage-specific markers and could be successfully integrated into reconstructed skin equivalent models, revealing pigmentation status according to the native phenotype. In both monolayer cultures and 3D skin models, the induced melanocytes demonstrated responsiveness to promelanogenic stimuli. The data demonstrate that the iPSC-derived melanocytes with high proliferative capacity maintain their pigmentation genotype and phenotypic properties up to a proper integration into 3D skin equivalents, even for highly pigmented cells.

Keywords: induced pluripotent stem cells, lineage specification, melanocyte pigmentation, reconstructed skin model, skin color

1 |. INTRODUCTION

The major phenotypical trait observable in humans arises from the diversity of skin color which is ranging from lightly to heavily pigmented. A growing attention has to be given to differentially pigmented phenotypes in order to improve knowledge on skin pigmentary disorders and discover treatments more suited to various populations (Halder & Nootheti, 2003).

The skin pigmentation process is regulated by complex mechanisms (Serre et al., 2018) but mostly, relies on biological activity of the melanocyte, the cell type responsible for melanin synthesis. Skin color phenotype depends on quality and quantity of melanin pigment polymers from two different groups, the dark eumelanins and the reddish pheomelanins (Del Bino et al., 2015) and also depends on melanosome size and organization (Hurbain et al., 2018). While constitutive skin pigmentation and sun-induced melanogenesis are under the control of intrinsic melanocyte regulation, they are also influenced by extracellular signals coming from surrounding cells such as keratinocytes and fibroblasts, leading to a global regulatory microenvironment (Duval et al., 2014; Wang et al., 2017).

To conduct experimental studies on pigmentation according to skin phenotype, a biological system having the potential to reproduce skin color is essential. Three-dimensional (3D) pigmented skin models allow to rebuild the epidermal melanin unit over a fibroblast-populated dermal compartment, thus the three main cutaneous cell types, keratinocytes, melanocytes, and fibroblasts, can interact within a homeostatic tissue organization (Duval et al., 2014; Supp et al., 2020). Organotypic models are more physiological than classical monolayered cell cultures and able to display the cutaneous pigmentation depending on the skin type of the melanocyte donor.

Primary adult human melanocytes cell banking raises several technical issues. Often, studies addressing melanogenesis are conducted with mouse melanocyte cell lines like Melan-A, murine melanoma cells such as the B16 strain or human cells from neonatal foreskin, because they are easy to handle and proliferate rapidly in culture. However, when using cells from neonatal foreskin the donor’s skin color phenotype is difficult to determine in contrast to those from adult skin samples. Therefore, the use of adult melanocytes is better suitable for investigation into pigmentation processes operating in mature skins from light to dark.

Melanocytes from adult skin biopsies can be isolated and amplified with success in about 10% of cases (Hsu & Herlyn, 1996) and the few harvested melanocytes need to be passaged many times to create a working cell bank. However, adult melanocytes undergo a drastic reduction of their proliferative rate after 7–10 passages compared to neonatal cell source (Abdel-Malek et al., 1994). In addition, the phenotype of origin is also impacting because it has been shown that cultured melanocytes from dark origin reach replicative senescence earlier than those from light skin (Hsu & Herlyn, 1996). Many studies on media composition have been performed to enhance melanocyte proliferation using components impacting tyrosinase activity and melanin content (i.e. PTTU, tyrosinases inhibitors, phorbol esters) (Boyce et al., 2017; Kim et al., 2016; Swope et al., 1995). However, this raises the question of persisting impacts after with-drawing these molecules. Hence, getting large amount of adult dark melanocytes without impairing their constitutive pigmentation remains a major challenge.

To overcome these drawbacks, another method of melanocyte production and expansion was explored in order to obtain melanocytes from dark skin individuals.

The approach was to generate human induced pluripotent stem cells (iPSC) and differentiate them into melanocytes. Using the four ‘Yamanaka’ transcription factors OSKM (Oct4, Sox2, Klf4, c-Myc) adult differentiated cells can be reprogrammed into pluripotent stem state, a cellular status very similar to the one of embryonic stem cells (ESC) possessing an unlimited growth potential and broad differentiation potential (Takahashi & Yamanaka, 2006). Differentiation protocols (Yamane et al., 1999) recapitulating the specification from human embryonic stem cells (hESC) to neural crest cells and to epidermal pigmented melanocytes were refined in 2006 when the benefit of adding Wnt3a was demonstrated (Fang et al., 2006). Then, the protocol originally developed for hESC was shown to be effective for human iPSC (Ohta et al., 2011). This alternative overcomes melanocyte isolation and amplification constraints by producing cells with higher proliferation rates due to telomere elongation that takes place during iPSC reprogramming (Plikus et al., 2017). The attempts to integrate iPSC-derived melanocytes into reconstructed models were not conclusive and did not specifically address cells from dark skin donors (Gledhill et al., 2015; Jones et al., 2013; Mica et al., 2013).

The objective of the present work was to evaluate the success rate of this technique when applied to several cell donors and to also validate it for cells from very dark skin in quest of a proliferative rate compatible with cell banking. The second step was to verify whether the generated melanocytes had kept the pigmented phenotype of origin throughout the processes of iPSC generation and iPSC derivation into pigmented melanocytes. To this end, experimentations were conducted in parallel with adult cells from light origin (standing for controls). Adult dermal fibroblasts were isolated from different skin color phenotypes (three light and three dark individuals) as this is a cell type that can be easily obtained. Original skin phenotypes were qualified by spectrocolorimetric measurements and melanin content of the skin biopsies (Del Bino & Bernerd, 2013). Fibroblast strains were reprogrammed into iPSC using Sendaï virus vectors and committed into the melanocytic lineage according to the literature (Fang et al., 2006). The derived melanocytes strains were qualified using specific markers and were tested for proliferative rate and purity of cell population. Finally, induced melanocyte strains were assessed for correct integration into the pigmented reconstructed skin model and ability to respond to known propigmenting stimuli.

2 |. MATERIALS AND METHODS

2.1 |. Fibroblasts isolation and culture

Normal human skin biopsies were obtained from mammary surgical residues of six adult females between 18 and 37 years old, after written informed consent from the donors according to the principles expressed in the Declaration of Helsinki and in article L.1243–4 of the French Public Health Code. Given their special nature, surgical residues are subject to specific legislation included in the French Code of Public Health (anonymity, gratuity, sanitary/safety rules…). This legislation does not require prior authorization by an ethics committee for sampling or use of surgical waste. Pigmentation level of skin samples was determined through calculation of the Individual Typology Angle (ITA°) based on colorimetric parameters L* and b* using Check III spectrocolorimeter (Datacolor, Montreuil, France) and/or quantification of melanin content from Fontana Masson stained skin sections as described (Del Bino & Bernerd, 2013). Normal human fibroblasts (NHF) were then isolated using the skin explant method, and amplified in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) + 10% fetal calf serum (FCS). The strains were amplified until passage 5 before iPSC reprogramming.

2.2 |. Induced pluripotent stem cells generation and characterization

Induced pluripotent stem cells (iPSC) were reprogrammed from fibroblasts at passage P5 following the CytoTune-iPS 2.0 Sendai Reprogramming Kit recommendations (Thermo Fisher Scientific) using transcription factor constructs for hKlf4, hOct3/4, hSox2 and hc-Myc (KOSM). Reprogrammed fibroblasts (100–200,000 cells) were harvested at day 7 and transferred onto MEF feeder in DMEM/15% FBS medium. Media was switched on day 8 to a mixture of 50% of DMEM/15% FBS and 50% hESC media composed of DMEM F12 medium (Thermo Fisher Scientific)/20% Knockout Serum Replacement (KOSR), 200 mM L-glutamine, 1X MEM-Non Essential Amino Acids and 10 ng/ml bFGF, then changed on day 9 to 100% hESC media, and refreshed every day. Each colony was picked and transferred to another MEF layer for expansion into individual iPSC lines. Serial cultivation of iPSC lines was performed for 4 to 5 months before cell freezing. This expansion period was achieved to negatively select the mutated cell (Hussein et al., 2011) and avoid potential genetic instability. After over 15 passages, iPSC cells were subjected to initial characterization. Pluripotency markers of iPSC were characterized by FACS analysis for TRA-1–60 and SSEA4, and RT-qPCR analysis for hNANOG, hREX1, hDNMT3B, endogenous hOCT4 and endogenous hSOX2. Pluripotency and trilineage differentiation potential of iPSC were assessed by the formation of embryoid bodies (EB) and their gene profile analysis with Taqman® hPSC Scorecard™ assay (Thermo Fisher Scientific). Additional information is provided in Supplemental Materials and Methods (Appendix S1).

2.3 |. Induced pluripotent stem cells derivation into induced melanocyte

Melanocytes were differentiated from iPSC after generation of embryoid bodies (EBs) as previously described (Fang et al., 2006). EBs were collected and plated on Geltrex® (Thermo Fisher Scientific) coated flasks in melanocyte differentiation medium named Mel-1, see detailed composition in supplemental Materials and Methods. After a culture time varying for each cell line from 4 to 6 weeks in Mel-1 medium, the first derived melanocytes (iMc P0) appeared as EBs outgrowth. Then, all iMc strains were serially passaged for 4 weeks for amplification and then Mel-1 medium was replaced by M254 culture medium with HMGS-2 supplement (both from Cascade Biologics, Thermo Fisher Scientific) supplemented with 10 ng/ml TPA (Sigma-Aldich).

2.4 |. Melanocytes cell culture

Cell amplification of iMc strains and normal human melanocyte (NHM) strains i.e. NHM-D2 (from donor D2) and NHM-D3 (from donor D3) was achieved in M254 medium supplemented with HMGS-2 supplement and 10 ng/ml 12-O-tetradecanoyl-phorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBU) (Sigma-Aldrich). Cells were cryopreserved after harvesting, centrifugation, resuspension into 92% vol/vol FBS and 8% vol/vol dimethylsulfoxide in cryovials cooled at −1°C/min. Neonatal NHM strains, used as primary melanocyte controls, were isolated from foreskin and cultivated in a defined melanocyte growth medium M2 (Promocell) until passage 4 for cell amplification. The conditions of skin sample collection were similar to those for fibroblasts obtention.

2.5 |. Pigmented reconstructed skin model (PRS)

The PRS model is a pigmented skin reconstructed in vitro on a living dermal equivalent and performed as previously described (Duval et al., 2014) deprived of Cholera Toxin. For details of methodology see Supplemental Materials and Methods Appendix (S1). Briefly, normal human keratinocytes (NHK) and melanocytes (NHM or iMc) were co-seeded on the top of a dermal equivalent consisting of normal human fibroblasts (NHF) embedded in a collagen I contracted lattice. After a 7 days’ immersion phase, the 3D system was raised at the air–liquid interface for 11 days (emersion phase), allowing for epidermal stratification and differentiation. For melanogenic stimulation, the model was treated during all the emersion phase by adding 50 nM α-melanocyte-stimulating hormone (αMSH; Sigma) to culture medium. For dermal equivalent without fibroblasts, dermal reconstructs were subjected to osmotic shock in water. For conducting comparative studies only the melanocyte component varied while strains of NHK and NHF remained constant. Isolation and amplification of these two cell strains were performed as described in Duval et al. (2012).

2.6 |. Immunohistochemical labeling and FACS analysis on iMc strains

For immunofluorescence (IF) characterization, iMc were cultivated on Millicell chamber (Merck-Millipore, Merck KGaA), and fixed with methanol. Cell suspensions for cytometric analysis were fixed using Intracellular Fixation & Permeabilization Buffer Set (Thermofisher). The following primary antibodies: P-MEL17 clone NKI-beteb, P-MEL17 clone HMB45 and TRP1 clone TA-99 followed by the specific secondary antibodies: Alexa 488 anti-Mouse IgG2b, IgG1 or IgG2a respectively. Details are provided in Supplemental Materials and Methods (Appendix S1).

2.7 |. Analysis of melanin quantity and quality in iMc strains

iMc strains (at P15) were sub-cultured to obtain around 2 million cells. Two primary neonatal melanocyte strains (foreskin) were performed in parallel as reference for each color group. For each strain, the quantifications of total amount of melanin were carried out using the Soluene solubilization technique and the melanin types were assessed using the HPLC dosage of products of degradation from eumelanin and phaeomelanin (Del Bino et al., 2015). Details are provided in Supplemental Materials and Methods (Appendix S1).

2.8 |. Characterization of PRS

To compare the level of pigmentation between PRS samples, the Luminance parameter (L*) was measured by a Check III spectrocolorimeter (Datacolor; CIELAB 1976 system, for D65 lighting. 10°observer). It quantifies the level of gray, ranging from deep black (value 0) to white (value 100). For each PRS sample, four consecutive L* measurements covering the whole sample surface were averaged. In each experiment, one result was the mean L* value ± standard deviation (SD) of three PRS samples. Each sample of PRS was then cut into four quarters. One quarter was fixed with 4% formaldehyde (Carlo Erba) and embedded in paraffin for quality control of skin reconstruction by HES (Hematoxylin Eosin Saffron) staining, and for histological detection of melanin (dark and brown spots) by Fontana Masson (FM) staining combined with fast red counterstaining. Images of sections were captured with a Nanozoomer laser scanner microscope (Hamamatsu Photonics). An in house image analysis software was used to evaluate the melanin content (Xiao et al., 2019), which corresponds to a melanin density assessed by the ratio of epidermal melanin area to the whole viable epidermal area. A second quarter was separated with forceps to obtain the epidermal sheet. Melanocytes were stained using the primary antibody TRP1 and Alexa488 anti-Mouse IgG2a. Epidermal sheets were captured with the Nanozoomer scanner, then melanocyte counting per mm2 was performed using Histolab software (Microvision Instruments). The third quarter was used to perform DOPA reaction on separated epidermal sheet to reveal the melanogenic potential of cells. The fourth quarter was embedded in Tissue Tek and frozen in liquid nitrogen. Then, 5 μm cryo-sections were stained using primary antibody against Tyrosinase clone T311 dilution 1/50 (Leica Biosystem, Cat #NCL-L-TYROS) followed by anti-Mouse-HRP (kit Impress-HRP MP-7500, Vector Laboratories) for immunohistochemistry. Images were acquired using microscope at 20-fold magnification. Details are provided in Supplemental Materials and Methods (Appendix S1).

2.9 |. Statistics

Statistical analysis was performed on the mean ± standard deviation (SD) from replicate sample determinations using the Wilcoxon-Mann–Whitney test with a p value <.05 considered as significant.

3 |. RESULTS

3.1 |. Amplification limitation for highly pigmented primary melanocytes

Expanding adult melanocytes from dark adult skin is a difficult task as these cells highly loaded with melanin pigments poorly proliferate. For example, melanocytes amplification from D1 donor failed. The attempts of amplification of primary melanocytes from the two other dark donors (D2, D3) in classical culture conditions (without tyrosinase inhibitors) are exemplified in Figure 1. Proliferation kinetics recorded from passage 1 to 5 evidenced very low amplification yields for the two strains and a cumulated population doubling below 2 after 3 weeks and four passages. Moreover, the step of freezing/thawing was very impactive on proliferation rates (dotted lines in Figure 1). After defrosting, cells showed a marked slowdown in proliferation compared to cells continuously sub-cultured. The very limited dark-skinned melanocyte numbers obtained after biopsy isolations did not allow to repeat the growth evaluation experiment. This raises the point that melanocyte cell banking from dark-skinned adults is severely constrained.

FIGURE 1.

FIGURE 1

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Cultivation of adult normal melanocytes from highly pigmented biopsies. Representative proliferation curves for primary normal human melanocyte (NHM) of dark origin in M254 Cascade medium (see section 2 Materials & Methods). Cells obtained from donor D2 (red line) and D3 (blue line) were grown from passage 1 to 5. Dotted lines represent cell growth after a freezing/thawing step (tagged with *).

3.2 |. Successful generation of iPSC from light and dark-skinned donors

To overcome this obstacle and obtain access to growing adult melanocytes, we initiated the generation of melanocytes derived from iPSC obtained from dark-skinned donors. To allow accurate comparisons and robustness between different melanogenic status, the process was also conducted in parallel with light-skinned donor cells. Hence, two groups of skin samples were specified for light (L1, L2, L3) and dark (D1, D2, D3) skins. For each skin biopsy, constitutive pigmentation status was monitored by the ITA° value of Individual Typology Angle (Chardon et al., 1991) and/or the melanin content value (Figure 2). Fontana Masson staining illustrates obvious differences in melanin content. Then, a fibroblast strain was established from each skin biopsy (see Materials & Methods) and amplified until passage 5 for iPSC reprogramming.

FIGURE 2.

FIGURE 2

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Characterization of original skin biopsies. Donor age, ITA° (individual typology angle) value (when available), melanin quantification on Fontana Masson-stained sections are indicated for each donor. Two groups of skin color typologies light and dark were determined according to the above parameters.

Fibroblast reprogramming was achieved using replication incompetent Sendai virus vector for OSKM transcription factors, leading to iPSC lines as described in Materials and Methods and Supplemental Materials and Methods. The first iPSC colonies emerged around day 12, but a period of 3–4 weeks was requested for each colony to reach a sufficient size. After this phase, and for each fibroblast strain, four iPSC clones were chosen for amplification by colony picking and sub-cultured using classical iPSC procedures. The 24 strains were cultured over 5 months’ period for expansion and genomic stabilization as recommended (Hussein et al., 2011; Yoshihara et al., 2017). When pluripotency markers were characterized, cell lines derived from the same donor showed very similar results (data not shown) leading to the selection of one line per donor for derivation into melanocyte lineage. FACS analyses of pluripotency markers TRA-1–60 and SSEA4 (Figure S1) determined that the purity of iPSC strains was excellent, between 93.9% and 96.8% for six selected iPSC cell lines at P10. Gene expression analyses for hNanog, hRex1, hDNMT3B, endogenous hOct4, and endogenous hSOX2 validated a high expression level for these pluripotency genes in each iPSC cell line. They were comparable to gene expression levels found in the H9 embryonic stem cell line used as a positive control and were undetectable in the corresponding original fibroblasts. Figure S2 displays results for six selected iPSC lines. Embryoid Bodies were generated from 24 iPSC cell lines and their pluripotency was confirmed through the RT-PCR data obtained using the TaqMan® hPSC Scorecard™ assay. This assay revealed for each iPSC line the potential of differentiation toward the three germ layers and the downregulation of self-renewal genes achieved at the EB stage (Figure S3). Hence, the reprogramming process was successful for generating iPSC lines in a reproducible manner.

3.3 |. Melanocytes induced from iPSC

3.3.1 |. Validation of the melanocytic identity of induced melanocytes

The steps from iPSC cell lines to EB generation and melanocyte derivation using specific media are detailed in Materials and Methods and in supplemental Materials and Methods. The melanocytes coming out of EBs were picked up (Passage 0) and after short sub-culture (Passage 6), the strains of iMc demonstrated the typical dendritic morphology of melanocytes (Figure 3a). The six strains were then confirmed to be positively labeled for the markers P-MEL17 (HMB45 and NKI-beteb) or TRP1 showing their melanocytic nature (Figure 3b). Hence the procedure allowed to obtain the melanocytic cell type in adequation with the typical cell morphology and the biomarkers of melanin synthesis. Interestingly, the iMc pellets obtained after centrifugation revealed the differences between the strain groups, iMc-D pellets being darker than iMc-L pellets, in line with the original donor skin color type (Figure 3a). The melanocytic identity of the six iMc strains was further confirmed by assessing the expression of eight characteristic genes by qRT-PCR (TYR, TYRP1, MITF, PMEL, MLANA, EDNRB, SOX10, PAX3; Figure S4A). In all iMc strains these genes were expressed, as can be seen in a primary normal human melanocyte strain (NHM) of intermediate pigmentation, thus supporting the melanocytic identity of the iMc strains. The data also highlighted that the TYR gene coding for tyrosinase, the enzyme initiating the melanin production process, was found expressed at a higher level (strong effect size) in the dark strains than in the light (Figure S4B).

FIGURE 3.

FIGURE 3

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Induced melanocytes from iPSC. (a) Pictures of the six iMc strains cultured in monolayers. Amplification until P6 has been run in parallel. All iMc display a dendritic morphology characteristic of melanocyte cell type, scale bar =100 μm. Respective cell pellets showing melanin content with darker pellets for iMc from darker skins (iMc-D1, iMc-D2, iMc-D3). (b) Characterization of iMc by immunolabeling using antibodies directed against P-Mel17 (HMB45, or NKI-beteb) and TRP1. All iMc are positive for the melanocytic markers, scale bar = 50 μm

3.3.2 |. Proliferative capacities and purity of iMc

All iMc strains exhibited strong proliferative properties (Figure 4a). During sub-culture from P6 to P12, they demonstrated similar kinetics except for iMc-D2 which displayed a slight delay in population doubling time. Noticeably, the level of pigmentation of cell pellets was conserved at P12. After cell amplification it was mandatory to verify the quality of cell populations. Conducting FACS analyses for melanogenic markers on each strain at passage 13 (Figure 4b) revealed a high level of purity for each melanocyte population, between 96.85% and 100%.

FIGURE 4.

FIGURE 4

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Proliferative activity, cell purity and melanins analysis of iMc strains. (a) Growth curves of the iMc strains. Each strain was sub-cultivated for six passages (P6 to P12). Population doubling (PD) was calculated as follows: LOG10 (harvested cells number/seeded cells number) / LOG10(2). PD was obtained for each passage and cumulated PD was plotted along time for each iMc strain. Pictures of cell pellets obtained at passage 12 showing darker pellets for iMc from dark skin donors. (b) Cytometric analysis of melanocytic proteins P-Mel17 (HMB45 and NKI-beteb) and TRP1. Notice the high purity level of all iMc strains. Illustration of P-Mel17 (NKI-beteb) FACS plots is shown for iMc-L3 and iMc-D3. (c) Melanins analysis of iMc strains in parallel with two NHM neonatal reference strains showed results for Total melanin (TM expressed in μg/million cells) by spectrophotometry or HPLC and the linear correlation between them. The strains were submitted to HPLC dosages (expressed in ng/million cells) for PTCA and PDCA (reflecting DHICA and DHI moieties) from eumelanin and TTCA (reflecting benzothiazole moiety) from phaeomelanin. The dosages of these markers were coherent with the phenotypical groups.

3.3.3 |. Melanin analysis of iMc strains

The quantity and nature of melanins were assessed for iMc strains in parallel with two primary neonatal melanocyte strains (NHM) standing as references for phenotypical groups L and D (Figure 4C). The two different techniques for Total Melanin (TM) assessment, spectrophotometry and HPLC showed an excellent correlation that strengthens the results. TM values for strains of light origin were in a lower order of magnitude than the dark ones. In line, HPLC dosages of PTCA and PDCA (respectively, the DHICA and DHI moieties in eumelanin) and of TTCA (benzothiazole moiety in phaeomelanin) showed the same trend. Indeed, data obtained for iMc strains were in line with the corresponding reference NHM of skin color group.

3.3.4 |. Genomic stability of iMc

The ultimate verification was to ensure the cell genomic stability throughout the entire process. Molecular karyotyping was carried out by comparative genomic hybridization (CGH) between each iMc strain at passage 13 and the corresponding fibroblasts obtained from female donors (Figure S5). Results showed negligible gene copy number variations (CNV) except that three iMc strains (L2, L3, D3) had lost one X chromosome.

3.4 |. Integration of iMc into 3D pigmented reconstructed skin (PRS) model

The best way to reveal the potential of iMc strains in vitro was to integrate them into reconstructed skin. For robustness the six iMc strains integration into PRS were tested in parallel in the same set of experiments. Figure 5 demonstrates successful incorporation into the skin reconstructs regardless of the iMc strain used. The sections depict correct epidermal organization and terminal differentiation up to a Stratum Corneum layer. HES-stained cross sections validated the quality of each model reconstruct. Fontana Masson staining revealed in each PRS the presence of melanin-producing cells displaying elongated dendrites positioned at the basal layer and the presence of melanin pigment located within epidermal keratinocytes. Melanin quantification revealed the level of pigment in PRS and interestingly indicated higher values for PRS reconstructed with iMc of dark skin origin compared to that of light origin. TRP1 green fluorescent staining of epidermal sheet enabled to visualize melanocytes within the entire basal layer and allowed to measure their density. This parameter was similar in all strains except iMc-D2, being integrated at lower density. DOPA reaction demonstrated the constitutive melanogenic enzymatic activity and more active melanocytes were revealed as darker, such as the three iMc-D strains. Tyrosinase labeling confirmed the integration of six functionally pigmenting cell strains at the basal layer of the epidermis. Lastly, all iMc strains exhibited normal physiologic melanocyte behavior once integrated in organotypic constructs. To exemplify the advantage of using iMc versus primary adult dark-skinned melanocytes, we compared PRS reconstructed with iMc-D3 and NHM-D3 (adult primary melanocytes from the same donor). Figure 6 illustrates that iMc-D3 allowed to get a similar level of melanocyte integration as a neonatal NHM of dark skin origin, whereas NHM-D3 integrated much lower. Cell counting from TRP1 labeling confirmed the important integration limitations when using primary adult NHM of dark origin compared to the corresponding iMc strain.

FIGURE 5.

FIGURE 5

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Characterization of PRS model after iMc integration. Representative results from one experiment where all iMc strains were performed in parallel. Results were replicated within a total of three independent experiments. Histology quality control after HES staining: All samples displayed a correct 3D organization with fully differentiated epidermis and stratum corneum (pink), scale bar = 50 μm. Fontana Masson staining of skin sections reveals melanin as black dots quantified by the ratio of the surface covered by melanin over the epidermal surface and expressed by mean ± SD of the triplicate samples. Higher magnification shows melanocytes with dendrites and melanin pigment within epidermal keratinocytes, scale bar = 50 μm. TRP1 immunostaining on epidermal sheets highlights melanocytes (green cells) homogeneously distributed at the basal layer, scale bar = 100 μm. Pictures indicate the iMc counting values as mean ± SD cell/mm2 calculated from each triplicate sample. DOPA reaction reveals more intense DOPA activity for cells of dark origin, scale bar = 50 μm. Immunostaining of tyrosinase on skin sections outlines in red the iMc localized at the basal layer of the epidermis, scale bar = 50 μm. Results were confirmed in other experiments (n = 3 for each strain).

FIGURE 6.

FIGURE 6

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Integration of iMc, neonatal primary melanocyte and adult primary melanocyte from dark origin in 3D skin model. Illustration of comparative integration of iMc-D3 (induced melanocyte from D3 adult biopsy), of neonatal NHM (a primary melanocyte from dark origin) and NHM-D3 (adult primary melanocyte from D3 biopsy). HES staining displays the correct skin reconstruction. Fontana Masson staining labels melanin production. Measurement of melanin density was calculated by ratio of the surface covered by melanin over the epidermal surface. DOPA reaction illustrates the strong DOPA activity of melanocytes. TRP1 labeling at the dermal epidermal junction of PRS enables melanocyte (green) counts per mm2. Notice the very low amount of NHM-D3 integrated in PRS. All conditions were performed in triplicates and numbers indicate means and standard deviations.

3.5 |. iMc responsiveness to melanogenic stimuli

iMc strains were tested for their ability to respond to Forskolin, a melanogenic stimulator that increases cAMP level (Duval et al., 2012; Newton et al., 2007). Melanin concentration was assessed after 48 h’ incubation with 10 μM Forskolin (Figure 7a). At baseline, the mean melanin concentration of the dark strains (iMc-D) was significantly higher than that from the light strains (iMc-L; Figure 7b). Forskolin stimulation induced significant increase in melanin content for both iMc-L and iMc-D.

FIGURE 7.

FIGURE 7

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Functionality of iMc strains under melanogenic stimulation. Representative results from one experiment where all iMc strains were performed in parallel. Results were replicated in two independent experiments. (a) iMc strains were incubated for 3 days in PDBU supplemented culture medium before stimulation with 10 μM Forskolin for 48 h. After cell lysis, centrifugation, melanin pellet solubilization in NaOH 1 N, samples were deposited in a microplate for optical density (OD) reading at 405 nm. (b) Graph shows resulting melanin concentration means ± SD for iMc-L and iMc-D groups stimulated by Forskolin versus control (each group includes 3 donors × 2 replicates). Significant differences are indicated between conditions (*p < .05). (c) Macroscopic view of triplicate PRS samples under basal (ctrl) or stimulated conditions by αMSH 50 nM or fibroblast deletion (w/o fib), scale bar = 15 mm. (d) Graph of corresponding spectrocolorimetric luminance values expressed as mean value ± SD from each donor’s PRS triplicates. (e) Graph of luminance values expressed as mean values ± SD of samples from light (iMc-L) or dark (iMc-D) origin (each group includes 3 donors × 3 PRS replicates). (f) Graph of melanin density quantified on PRS sections stained by Fontana Masson, expressed as mean ± SD for each iMc strain condition. (g) Graph of melanin density expressed as mean value ± SD of samples from light (iMc-L) or dark (iMc-D) origin (each group includes 3 donors × 3 PRS replicates). (h) Pictures from representative Fontana Masson staining of PRS samples from light (L3) or dark (D3) origin. Melanin contents (black dots) are increased after melanogenesis stimulation, scale bar = 50 μm.

A validation assessment of the melanogenic function was performed in the 3D skin model to ensure that the iMc strains adequately respond to various stimuli in a more physiological environment. To activate the MC1R pathway, the PRS model was stimulated by 50 nM αMSH (Herraiz et al., 2017). In a second condition, PRS samples were cultivated in the absence of fibroblasts in the dermal compartment (referred as “w/o Fib”, see Supplemental Materials and Methods). This condition helped to reveal the pigmenting potential of iMc because fibroblasts are known to down-regulate melanin synthesis through the release of soluble factors (Duval et al., 2014; Wang et al., 2017). Pigmentation levels can be observed in Figure 7c with macroscopic views of triplicate PRS samples, along with the respective Luminance (lightness) values of control versus stimulated conditions (Figure 7d). These data illustrated some variability among the six strains. Nevertheless, a significant difference between the two skin color sources was revealed in Figure 7e when regrouping Luminance means by iMc origin (L and D). The “w/o Fib” condition induced a decrease in Luminance in both light and dark origin PRS; αMSH condition induced this effect only in the dark skin color group. Melanin content measurements were recorded in all samples (Figure 7f) and means of melanin content by iMc from L or D origin are shown in Figure 7g. PRS-D had a melanin content significantly higher than PRS-L. Similarly, to Luminance, αMSH induced measurable melanin increase only in the PRS-D group. The “w/o Fib” condition induced the highest increase in melanin content whatever the skin color type. However, in the PRS-D group an important SD can be observed due to the low iMc-D2 melanin content value. FM staining of PRS integrating iMc-L3 and iMc-D3 (Figure 7h) were representative of the results. More intensely stained melanocytes and melanin pigment can be observed in the conditions featuring iMc of dark origin compared with the light ones. In agreement with melanin content the highest staining appeared in the “w/o Fib” culture condition.

4 |. DISCUSSION

Building cell stocks of adult primary melanocytes especially from dark skin donors is a difficult challenge. In regular culture conditions, these cell populations proliferate very slowly and as illustrated in the present study, it is still an issue to restart their growth after thawing. To overcome this challenge, the feasibility of iPSC reprogramming and derivation was tested to generate adult melanocyte cell banks. A feasibility study has been developed with multiple donors to gather more robust results. Each skin color group (light or dark) was originated from three different individuals to consider interpersonal variability. Accurate selection of lightly or darkly pigmented skin biopsies was essential, each biopsy being selected by colorimetric assessment of Individual Typology Angle (ITA°) as well as melanin content. The dark skin biopsies displayed higher melanin content than the light ones. Six fibroblast strains were isolated from the six skin samples and subjected to iPSC reprogramming using viral vectors. To encompass the possible cell line variability arising from the iPSC reprogramming process, four isogenic iPSC lines were isolated from each fibroblastic cell strain. For somatic cell reprogramming a very wide range of technologies are available (Rony et al., 2015) i.e. viral or non-viral delivery of reprogramming factors OSKM, direct transfection of the factors as well as their mRNAs, some specific miRNAs and more recently some small chemical compounds acting on epigenetic or signaling pathways. Our approach is based on non-integrating mRNA Sendai virus vectors, which are widely used because they disappear from transfected cells after few passages without genomic footprint. Reprogrammed cell clones were obtained from all six fibroblast strains and gave rise to 24 pure populations of iPSC displaying key pluripotency markers. As iPSC can be kept frozen, they allow a sustainable cell sourcing for each donor.

The pluripotent state provides the cells an opportunity to differentiate into any cell type of the organism. iPSC generate differentiated cells with the same genetic background (Jang et al., 2011) so that for pathological cells, it is possible to create specific genetic disease in vitro models of considerable interest for pharmacological targets discovery and therapeutic compounds screening (Yahata et al., 2011).

Due to animal welfare concerns, the pluripotent state of iPSC was validated avoiding the teratoma formation test in mice, using instead the Scorecard™ assay based on 94 genes qPCR microarray analysis (Fergus et al., 2016). Each iPSC line displayed the potential to differentiate into the three germ layers, this specification occurred during embryoid body (EB) formation. For cells derived from iPSC, the disappearance of the pluripotency potential has to be demonstrated for safety profiles (Attwood & Edel, 2019), which is mandatory when they are intended for cell therapy. The TaqMan® hPSC Scorecard™ assay validated that cells of EBs derived from the 24 iPSC strains had lost the expression of pluripotency genes, such as hDNMT3B, NANOG, POU5F1 and SOX2. The test confirmed that after EBs formation, cells are not pluripotent stem cells.

The melanocyte derivation protocol starting from EBs is based on previous work (Fang et al., 2006; Ohta et al., 2011; Yamane et al., 1999). The step of EBs generation and selection before differentiation was instrumental to prevent contamination by residual iPSC, a very serious concern in cell therapy (Suman et al., 2019). The generation of six melanocyte strains was confirmed by specific melanogenic protein labeling (TRP1, pMEL17) and expression of key genes by RT-qPCR assessment (TRP1, TYR, MITF, PMEL, MLANA, EDNRB, SOX10 and PAX3). Cytometric analyses demonstrated that the protocol provides pure iMc populations, measured at 97% purity and higher. The derivation procedure used appears to be far more effective than the one employed by Nissan’s team (Nissan et al., 2011) when iMc were obtained unintentionally during the process of keratinocyte derivation from human iPSC.

To generate cell banks, each iMc strain was subcultured for cell expansion. After 14 population doublings, the strains continued to proliferate actively with proliferation curves very close together except for iMc-D2 which displays a slightly lower proliferation. The proliferative rate is a direct consequence of the derivation from iPSC, which are rapidly dividing cells that have elongated telomeres and self-renewing potential (Liu et al., 2019). Although it has been shown that a high melanin content in melanocyte accelerate senescence by terminal differentiation and proliferative arrest (Bandyopadhyay & Medrano, 2000), light or dark origin of the cell donor, closely related with melanin content, did not seem to affect the proliferative curves of the iMc strains. Hence, it could be hypothesized that the reprogramming process provides derived cells with some capacity to compensate for ultimate differentiation.

Melanin analysis, in terms of quantity and quality (eumelanin and phaeomelanin) was performed to study iMc strains in parallel to the neonatal reference NHM of skin color type. All the data revealed that strains of light origin had lower amount of melanins compared to the dark iMc strains, being very consistent with the skin color group of origin (Del Bino et al., 2015). This demonstrated that iMc strains are competent to synthesize melanin polymers in quantities and qualities similar to NHM.

The iPSC literature suggests that genomic instability can arise in iPSC clones (Yoshihara et al., 2017). For this reason, a long-term expansion (4 to 5 months) was performed for each iPSC line and molecular karyotyping (CGH array) of iMc in comparison to their respective original fibroblast was performed. It demonstrated negligible genomic modifications, except for X-chromosome monosomy that appeared in three strains. It has been previously described (Guttenbach et al., 1995; Kim et al., 2014) that hESC lines derived from females have frequently lost part of or the entire X chromosome, arising from long-term cultivation. Because one X chromosome is normally silenced in adult female cells, that loss may not affect cell behavior.

After addressing the identity and the quality of iMc, we verified their correct incorporation into a reconstructed skin model. Integrated into the course of a newly built epidermis, iMc appeared correctly settled at the dermal epidermal junction allowing proper interactions with the neighboring keratinocytes and fibroblasts. They were shown to be DOPA reactive, positive for tyrosinase and TRP1, producing melanosomes labeled for P-Mel17 and decorated with melanin (Fontana Masson staining) similarly to that of normal primary melanocytes. Attempts were made in the past to introduce iPSC-derived melanocytes into human skin models resulting in very poorly organized epidermal reconstruction and only rare integrations of induced melanocytes (Gledhill et al., 2015; Jones et al., 2013; Mica et al., 2013). No previous experiment succeeded in properly integrating highly melanogenic iPSC-derived melanocytes.

From macroscopic micrographs, skin model samples appeared consistent with the original skin color phenotype for five strains, while a low pigmentation for iMc-D2 strain appeared inconsistent. During PRS preparation a step of melanocyte proliferation takes place in the immersion phase. As iMc-D2 demonstrated a lower proliferative rate, it led to a reduced melanocyte integration in PRS responsible for this lower basal pigmentation. However, DOPA reaction and Forskolin stimulation revealed that iMc-D2 has a strong melanogenic potential comparable to iMc-D1 and iMc-D3, consistent with its original phenotype.

To assess biologic responsiveness of the iMc strains, monolayer cultures were submitted to the pro-pigmenting agent Forskolin. They all responded by increasing their melanin content, still more prominently for the dark iMc strains. To reveal melanogenic potential in a more physiological context, PRS were stimulated by αMSH triggering MC1R signaling pathway or by using PRS deleted of the fibroblastic population (Duval et al., 2014). It appeared that melanocytes of light skin origin in 3D skin reconstructs did not respond to αMSH as reported earlier (Hedley et al., 2002) while melanocytes from dark origin triggered melanin production as previously shown (Duval et al., 2012). In the fibroblast lacking condition, melanin content was increased for both skin color types, showing intense pigmentation for dark origin iMc. Experimental results confirmed that all iMc strains were functional to activate melanogenesis.

In summary, the iPSC technology allowed overcoming difficulties to obtain and amplify adult heavily pigmented melanocytes. The methodology appeared very effective and highly reproducible for the six iMc strains production independently of their skin color phenotype and resulted in cell banking of pure melanocyte populations. After successful integration into reconstructed skin, cell strains were shown to be competent to elicit a melanogenic response to various stimuli, in line with donors’ phenotype. By using the operated process, it becomes possible to address pigmentation regulation mechanisms or explore in vitro specific therapeutic approaches more appropriate to pigmentation groups.

Supplementary Material

Suppl Figures
Suppl Materials and Methods

Significance.

Accessing cell populations of adult darkly pigmented melanocytes is a difficult task. To bypass this obstacle, iPSC reprogramming technology was used together with melanocyte derivation protocols to produce induced melanocytes. The differentiated cells were pure populations of actively proliferating melanocytes, they were correctly integrated in 3D skin models and produced pigmentation gradients in keeping with the donor color phenotype. The results offer new opportunities for research using highly pigmented melanocytes, for cell therapy dedicated to skin pigmentation restoration and for pigmentary disorders.

ACKNOWLEDGMENTS

This research was funded by L’Oréal Research and Innovation. CC, VF and FB are full time employees of L’Oréal Research and Innovation. MH had received honoraria for consultancy from L’Oréal Research and Innovation until 2016. Studies were supported in part by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation to M.H. The authors would like to warmly thank Professor Shosuke Ito from Fujita Health University School of Health Sciences (Aichi, Japan) for his expertise and for chemical analysis of melanins. The authors wish to acknowledge Thérèse Baldeweck for the conception and development of the melanin image analysis software. They are also grateful to Claire Marionnet, Emilie Warrick, Sandra del Bino, Juliette Sok, Christelle Golebiewski, and Diane-Lore Vieu for kind advice and help.

Funding information

L’Oréal Research and Innovation

Footnotes

SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

REFERENCES

  1. Abdel-Malek ZA, Swope VB, Nordlund JJ, & Medrano EE (1994). Proliferation and propagation of human melanocytes in vitro are affected by donor age and anatomical site. Pigment Cell Research, 7(2), 116–122. 10.1111/j.1600-0749.1994.tb00035.x [DOI] [PubMed] [Google Scholar]
  2. Attwood SW, & Edel MJ (2019). iPS-cell technology and the problem of genetic instability-can it ever Be safe for clinical use? Journal of Clinical Medicine, 8(3), 288. 10.3390/jcm8030288 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bandyopadhyay D, & Medrano EE (2000). Melanin accumulation accelerates melanocyte senescence by a mechanism involving p16INK4a/CDK4/pRB and E2F1. Annals of the New York Academy of Sciences, 908, 71–84. 10.1111/j.1749-6632.2000.tb06637.x [DOI] [PubMed] [Google Scholar]
  4. Boyce ST, Lloyd CM, Kleiner MC, Swope VB, Abdel-Malek Z, & Supp DM (2017). Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal-epidermal engineered skin substitutes. Pigment Cell & Melanoma Research, 30(6), 531–540. 10.1111/pcmr.12609 [DOI] [PubMed] [Google Scholar]
  5. Chardon A, Cretois I, & Hourseau C (1991). Skin colour typology and suntanning pathways. International Journal of Cosmetic Science, 13(4), 191–208. 10.1111/j.1467-2494.1991.tb00561.x [DOI] [PubMed] [Google Scholar]
  6. Del Bino S, & Bernerd F (2013). Variations in skin colour and the biological consequences of ultraviolet radiation exposure. The British Journal of Dermatology, 169(Suppl 3), 33–40. 10.1111/bjd.12529 [DOI] [PubMed] [Google Scholar]
  7. Del Bino S, Ito S, Sok J, Nakanishi Y, Bastien P, Wakamatsu K, & Bernerd F (2015). Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio. Pigment Cell & Melanoma Research, 28(6), 707–717. 10.1111/pcmr.12410 [DOI] [PubMed] [Google Scholar]
  8. Duval C, Chagnoleau C, Pouradier F, Sextius P, Condom E, & Bernerd F (2012). Human skin model containing melanocytes: Essential role of keratinocyte growth factor for constitutive pigmentation-functional response to alpha-melanocyte stimulating hormone and forskolin. Tissue Engineering. Part C, Methods, 18(12), 947–957. 10.1089/ten.TEC.2011.0676 [DOI] [PubMed] [Google Scholar]
  9. Duval C, Cohen C, Chagnoleau C, Flouret V, Bourreau E, & Bernerd F (2014). Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: Impact of photo-aging. PLoS One, 9(12), e114182. 10.1371/journal.pone.0114182 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fang D, Leishear K, Nguyen TK, Finko R, Cai K, Fukunaga M, Li L, Brafford PA, Kulp AN, Xu X, Smalley KS, & Herlyn M (2006). Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells, 24(7), 1668–1677. 10.1634/stemcells.2005-0414 [DOI] [PubMed] [Google Scholar]
  11. Fergus J, Quintanilla R, & Lakshmipathy U (2016). Characterizing pluripotent stem cells using the TaqMan® hPSC scorecard(TM) panel. Methods in Molecular Biology, 1307, 25–37. 10.1007/7651_2014_109 [DOI] [PubMed] [Google Scholar]
  12. Gledhill K, Guo Z, Umegaki-Arao N, Higgins CA, Itoh M, & Christiano AM (2015). Melanin transfer in human 3D skin equivalents generated exclusively from induced pluripotent stem cells. PLoS One, 10(8), e0136713. 10.1371/journal.pone.0136713 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guttenbach M, Koschorz B, Bernthaler U, Grimm T, & Schmid M (1995). Sex chromosome loss and aging: in situ hybridization studies on human interphase nuclei. American Journal of Human Genetics, 57(5), 1143–1150. [PMC free article] [PubMed] [Google Scholar]
  14. Halder RM, & Nootheti PK (2003). Ethnic skin disorders overview. Journal of the American Academy of Dermatology, 48(6 Suppl), S143–S148. 10.1067/mjd.2003.274 [DOI] [PubMed] [Google Scholar]
  15. Hedley SJ, Layton C, Heaton M, Chakrabarty KH, Dawson RA, Gawkrodger DJ, & MacNeil S (2002). Fibroblasts play a regulatory role in the control of pigmentation in reconstructed human skin from skin types I and II. Pigment Cell Research, 15(1), 49–56. 10.1034/j.1600-0749.2002.00067.x [DOI] [PubMed] [Google Scholar]
  16. Herraiz C, Garcia-Borron JC, Jiménez-Cervantes C, & Olivares C (2017). MC1R signaling. Intracellular partners and pathophysio-logical implications. Biochimica et Biophysica Acta -Molecular Basis of Disease, 1863(10 Pt A), 2448–2461. 10.1016/j.bbadis.2017.02.027 [DOI] [PubMed] [Google Scholar]
  17. Hsu MY, & Herlyn M (1996). Cultivation of normal human epidermal melanocytes. Methods in Molecular Medicine, 2, 9–20. 10.1385/0-89603-335-x:9 [DOI] [PubMed] [Google Scholar]
  18. Hurbain I, Romao M, Sextius P, Bourreau E, Marchal C, Bernerd F, Duval C, & Raposo G (2018). Melanosome distribution in keratinocytes in different skin types: Melanosome clusters are not degradative organelles. The Journal of Investigative Dermatology, 138(3), 647–656. 10.1016/j.jid.2017.09.039 [DOI] [PubMed] [Google Scholar]
  19. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Närvä E, Ng S, Sourour M, Hämäläinen R, Olsson C, Lundin K, Mikkola M, Trokovic R, Peitz M, Brüstle O, Bazett-Jones DP, Alitalo K, Lahesmaa R, Nagy A, & Otonkoski T (2011). Copy number variation and selection during reprogramming to pluripotency. Nature, 471(7336), 58–62. 10.1038/nature09871 [DOI] [PubMed] [Google Scholar]
  20. Jang J, Kang HC, Kim HS, Kim JY, Huh YJ, Kim DS, Yoo JE, Lee JA, Lim B, Lee J, Yoon TM, Park IH, Hwang DY, Daley GQ, & Kim DW (2011). Induced pluripotent stem cell models from X-linked adrenoleukodystrophy patients. Annals of Neurology, 70(3), 402–409. 10.1002/ana.22486 [DOI] [PubMed] [Google Scholar]
  21. Jones JC, Sabatini K, Liao X, Tran HT, Lynch CL, Morey RE, Glenn-Pratola V, Boscolo FS, Yang Q, Parast MM, Liu Y, Peterson SE, Laurent LC, Loring JF, & Wang YC (2013). Melanocytes derived from transgene-free human induced pluripotent stem cells. The Journal of Investigative Dermatology, 133(8), 2104–2108. 10.1038/jid.2013.139 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim KY, Hysolli E, Tanaka Y, Wang B, Jung YW, Pan X, Weissman SM, & Park IH (2014). X chromosome of female cells shows dynamic changes in status during human somatic cell reprogramming. Stem Cell Reports, 2(6), 896–909. 10.1016/j.stemcr.2014.04.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim YH, Park JI, Myung CH, Lee JE, Bang S, Chang SE, & Hwang JS (2016). 1-Phenyl-3-(2-thiazolyl)-2-thiourea inhibits melanogenesis via a dual-action mechanism. Archives of Dermatological Research, 308(7), 473–479. 10.1007/s00403-016-1659-2 [DOI] [PubMed] [Google Scholar]
  24. Liu L, Michowski W, Kolodziejczyk A, & Sicinski P (2019). The cell cycle in stem cell proliferation, pluripotency and differentiation. Nature Cell Biology, 21(9), 1060–1067. 10.1038/s41556-019-0384-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mica Y, Lee G, Chambers SM, Tomishima MJ, & Studer L (2013). Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell Reports, 3(4), 1140–1152. 10.1016/j.celrep.2013.03.025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Newton RA, Cook AL, Roberts DW, Leonard JH, & Sturm RA (2007). Post-transcriptional regulation of melanin biosynthetic enzymes by cAMP and resveratrol in human melanocytes. The Journal of Investigative Dermatology, 127(9), 2216–2227. 10.1038/sj.jid.5700840 [DOI] [PubMed] [Google Scholar]
  27. Nissan X, Larribere L, Saidani M, Hurbain I, Delevoye C, Feteira J, Lemaitre G, Peschanski M, & Baldeschi C (2011). Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis. Proceedings of the National Academy of Sciences of the United States of America, 108(36), 14861–14866. 10.1073/pnas.1019070108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ohta S, Imaizumi Y, Okada Y, Akamatsu W, Kuwahara R, Ohyama M, Amagai M, Matsuzaki Y, Yamanaka S, Okano H, & Kawakami Y (2011). Generation of human melanocytes from induced pluripotent stem cells. PLoS One, 6(1), e16182. 10.1371/journal.pone.0016182 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Plikus MV, Guerrero-Juarez CF, Ito M, Li YR, Dedhia PH, Zheng Y, Shao M, Gay DL, Ramos R, Hsi TC, Oh JW, Wang X, Ramirez A, Konopelski SE, Elzein A, Wang A, Supapannachart RJ, Lee HL, Lim CH, … Cotsarelis G (2017). Regeneration of fat cells from myofibroblasts during wound healing. Science, 355(6326), 748–752. 10.1126/science.aai8792 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rony IK, Baten A, Bloomfield JA, Islam ME, Billah MM, & Islam KD (2015). Inducing pluripotency in vitro: Recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming. Cell Proliferation, 48(2), 140–156. 10.1111/cpr.12162 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Serre C, Busuttil V, & Botto JM (2018). Intrinsic and extrinsic regulation of human skin melanogenesis and pigmentation. International Journal of Cosmetic Science, 40(4), 328–347. 10.1111/ics.12466 [DOI] [PubMed] [Google Scholar]
  32. Suman S, Domingues A, Ratajczak J, & Ratajczak MZ (2019). Potential clinical applications of stem cells in regenerative medicine. Advances in Experimental Medicine and Biology, 1201, 1–22. 10.1007/978-3-030-31206-0_1 [DOI] [PubMed] [Google Scholar]
  33. Supp DM, Hahn JM, Lloyd CM, Combs KA, Swope VB, Abdel-Malek Z, & Boyce ST (2020). Light or dark pigmentation of engineered skin substitutes containing melanocytes protects against ultraviolet light-induced DNA damage In vivo. Journal of Burn Care & Research, 41(4), 751–760. 10.1093/jbcr/iraa029 [DOI] [PubMed] [Google Scholar]
  34. Swope VB, Medrano EE, Smalara D, & Abdel-Malek ZA (1995). Long-term proliferation of human melanocytes is supported by the physiologic mitogens alpha-melanotropin, endothelin-1, and basic fibroblast growth factor. Experimental Cell Research, 217(2), 453–459. 10.1006/excr.1995.1109 [DOI] [PubMed] [Google Scholar]
  35. Takahashi K, & Yamanaka S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663–676. 10.1016/j.cell.2006.07.024 [DOI] [PubMed] [Google Scholar]
  36. Wang Y, Viennet C, Robin S, Berthon JY, He L, & Humbert P (2017). Precise role of dermal fibroblasts on melanocyte pigmentation. Journal of Dermatological Science, 88(2), 159–166. 10.1016/j.jdermsci.2017.06.018 [DOI] [PubMed] [Google Scholar]
  37. Xiao Y, Decencière E, Velasco-Forero S, Burdin H, Bornschlogl T, Bernerd F, Warrick E, Baldeweck T (2019). A new color augmentation method for deep learning segmentation of histological images. IEEE 16th International Symposium on Biomedical Imaging (ISBI 2019) (pp. 886–890). 10.1109/ISBI.2019.8759591 [DOI] [Google Scholar]
  38. Yahata N, Asai M, Kitaoka S, Takahashi K, Asaka I, Hioki H, Warrick E, & Inoue H (2011). Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer’s disease. PLoS One, 6(9), e25788. 10.1371/journal.pone.0025788 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yamane T, Hayashi S, Mizoguchi M, Yamazaki H, & Kunisada T (1999). Derivation of melanocytes from embryonic stem cells in culture. Developmental Dynamics, 216(4–5), 450–458. [DOI] [PubMed] [Google Scholar]
  40. Yoshihara M, Hayashizaki Y, & Murakawa Y (2017). Genomic instability of iPSCs: Challenges towards their clinical applications. Stem Cell Reviews and Reports, 13(1), 7–16. 10.1007/s12015-016-9680-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Suppl Figures
NIHMS1951956-supplement-Suppl_Figures.pdf (4.1MB, pdf)
Suppl Materials and Methods
NIHMS1951956-supplement-Suppl_Materials_and_Methods.docx (648.7KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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