Dermal papilla cells and melanocytes response to physiological oxygen levels depends on their interactions

Cell Proliferation. 2021;54:e13013.   | 1 of 11 https://doi.org/10.1111/cpr.13013 wileyonlinelibrary.com/journal/cpr 1 | INTRODUCTION Hair growth is mainly controlled by the dermal papilla (DP), the hair follicle (HF) inductive mesenchymal structure, whereas its pigmentation relies on the melanogenic activity of follicular melanocytes.1,2 These melanocytes represent the progeny of melanoblasts residing in the bulge, which proliferate and migrate to the hair bulb, surrounding the DP and starting to produce and transfer melanin to the keratinocytes of the growing shaft.37 Although the DP is considered the HF control centre, and its anatomical proximity with bulbar melanocytes implies a role also in hair pigmentation, little is known about the capacity of DP cells Received:16November2020 | Revised:15January2021 | Accepted:7February2021 DOI: 10.1111/cpr.13013 ORIGINAL ARTICLE Dermal papilla cells and melanocytes response to physiological oxygen levels depends on their interactions Carla M. Abreu1,2 | Rui L. Reis1,2 | Alexandra P. Marques1,2 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Cell ProliferationPublishedbyJohnWiley&SonsLtd. 13B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Guimarães, Portugal 2ICVS/3B’s–PTGovernmentAssociate Laboratory, Guimarães, Portugal Correspondence AlexandraP.Marques,3B’sResearch Group–Biomaterials,Biodegradablesand Biomimetics, University of Minho, Avepark 4805017 Barco, Guimarães, Portugal. Email: apmarque[email protected] Funding information Fundação para a Ciência e a Tecnologia, Grant/Award Number: PD/59/2013, PD/ BD/113800/2015 and IF/00945/2014; Consolidator Grant “ECM_INK”, Grant/ Award Number: ERC2016COG726061 Abstract Background: Human dermal papilla (DP) cells and melanocytes (hMel) are central players in hair growth and pigmentation, respectively. In hair follicles (HFs), oxygen (O2) levels average 5%, being coupled with the production of reactive oxygen species (ROS),necessarytopromotehairgrowth. Materials and Methods: DP cell and hMel proliferation and phenotype were studied under physiological (5%O2, physoxia) or atmospheric (21%O2, normoxia) oxygen levels. hMelDP cells interactions were studied in indirect coculture or by directly coculturing hMel with DP spheroids, to test whether their interaction affected the response to physoxia. Results: Physoxia decreased DP cell senescence and improved their secretome and phenotype, as well as hMel proliferation, migration, and tyrosinase activity. In indirectco-cultures,physoxiaaffectedDPcells’alkalinephosphatase(ALP)activitybut their signalling did not influence hMel proliferation or tyrosinase activity. Additionally, ROSproductionwashigherthaninmonoculturesbutadirectcorrelationbetween ROSgenerationandALPactivityinDPcellswasnotobserved.Inthe3Daggregates, where hMel are organized around the DP, both hMel tyrosinase and DP cells ALP activities,theirmainfunctionalindicators,plusROSproductionwerehigherinphysoxia than normoxia. Conclusions: Overall, we showed that the response to physoxia differs according to hMelDP cells interactions and that the microenvironment recreated when in direct contact favours their functions, which can be relevant for hair regeneration purposes. 2 of 11 | ABREU Et Al. to regulate melanocytes. Rodent studies indicate that DP cells can influence melanocytes proliferation/differentiation, migration and affect pigment formation and hair coat colour.8 In vitro studies confirmed a chemotactic effect of DP cellconditioned medium towards human melanocytes (hMel),5 further suggesting mediation of melanocytes location and migration in the HF by DP cells.Interestingly,DPcells’extracellularmatrix(ECM)wasalso suggested to stimulate tyrosinase activity,9,10 the ratelimiting step for melanin production.11 Oxygen (O2) is a basic component of the tissue's microenvironment, and their fluctuation can deeply affect cellular metabolism, signalling, proliferation, differentiation and reactive oxygen species(ROS)formation.12,13PhysiologicalROSplayaregulatoryrole in several cellular signalling events.1416 For example, melanogenesisitselfisaROSgeneratorcellularprocess,butmelanocyteshave mechanisms to cope with oxidative stress and avoid cellular damage. These include, among others, upregulation of the antioxidant response,17,18 and expression of the nuclear erythroid 2related factor (NRF2)19,20 or the Ataxia Telangiectasia Mutated (ATM) protein.21 In opposition, uncontrolled levels of ROS have been linked to the aetiopathogenesis of several conditions, including androgenetic alopecia and hair greying.22,23 It is well known that ROS accumulate at supraphysiological oxygen levels.24 Previous studies demonstrated that in cultures performed under 21% O2 (normoxia) both HF mesenchymal (DP and dermal sheath cells)25 and epithelial26 populations proliferate at lower rates than when respectively cultured at 6% O2 or 4% O2. Moreover, under hypoxic conditions (2% O2), DP cells viability, phenotype and inductivity are improved.23,27 Further, hMel proliferation and melanin production are also higher at 1%- 5% O2 than above normoxia.28 Overall, these studies indicate that low oxygen levels are beneficial for DP cells and melanocytes, which seem to agree with the oxygen tension measured in the human DP (4.0%- 5.2% O2)29 or skin (average 5.3% O2).30Nevertheless,theanagenhairbulbisaROS-enriched microenvironment,31inwhichROSdirectlyactivatesproliferation and differentiation programs, stimulating hair growth.32 Therefore, despite the involvement of oxygenassociated responses, potentially by the different cells implicated in hair growth is expected, it remains to be elucidated. Considering this, we investigated the response of hMel and DP cells to physiological oxygen levels (5% O2, physoxia) and if this response was influenced by their interaction, aiming at mirroring theirpotentialcommunicationwithintheHF.Wedemonstratethat under physiological culture conditions, together with an expected decreaseinROSlevels,bothDPcellsandhMelshowedincreased proliferative capacity and functionality. Interestingly, DP cells and hMel response to physoxia varied not only if these were cocultured, but also whether they were indirectly or directly interacting.WhenhMelandDPcellsweredirectlycontactingin3Dcell aggregates resembling their native organization, the microenvironment recreated under physoxia favoured their highly specialized functions. 2 | METHODS 2.1 | Cell culture DP cells were isolated33 from HF occipital scalp samples obtained from consenting patients who underwent hair transplantation procedures. DP cells were subcultured on bovine collagencoated (Sigma-Aldrich) surfaces in Dulbecco's modified eagle's medium (DMEM,Sigma-Aldrich)with10%foetalbovineserum(FBS)and1% antibioticantimycotic solution (Gibco). Neonatal foreskin hMel were purchased from Cell Applications (catalog no. 10405n) and cultured in the recommended HEM complete medium. Unless otherwise stated, the cell densities used to establish the monocultures were 2x104 cells/cm2 (DP cells and hMel). Physoxia cultures were established with 5% O2 in a hypoxic chamber (Coy O2 Control Glove Box; Coy Laboratory Products). Cells cultured under normoxia were used as controls. Cells were used up to passage 4 (hMel) or passage 7 (DP cells). ToassessthelinkbetweenROSandALP,DPcellswerecultured overnight and then treated with hydrogen peroxide (H2O2; 300 μΜ, PanReac AppliChem), with N-acetyl cysteine (NAC; 1 mM, Sigma- Aldrich) or with NAC for 2h before H2O2. 2.2 | Morphology and aggregation analysis After 3 days of culture, DP cells were fixed with 10%- formalin for 15 minutes at room temperature (RT) and their Factin filaments stainedwithPhalloidin-TRITC(0.1mg/mL,Sigma-Aldrich)for1hour at RT. Nuclei were counterstained with 4,6diamidino2phenylindole (DAPI) (1:1000, Biotium) for 15 minutes at RT. Images (six per triplicate) were acquired with an Axio Observer microscope (Zeiss) and used for the quantification of cell area, perimeter and major axis length, with the software module “MeasureObjectSizeShape” of CellProfilerTM 3.0.0.34 Cell aggregation was analysed after 7 days of culture, after labelling the cells' nuclei with DAPI. Images (ten per triplicate) were acquired (Axio Observer, Zeiss) and analysed with the CellProfiler 3.0.0TM module “RelabeledNuclei”. Cells were considered adjacent if the distance between their nuclei was below 8 pixels. Groups of 30 or more adjacent cells were counted as one aggregate. 2.3 | SenescenceassociatedβGalactosidase assay DP cells were seeded at a density of 1 x 104 cells/cm2 and cultured overnight. Next day, the cells medium was replaced by serumfree DMEM and DP cells were kept in culture for 5 days. The detection of senescence cells was performed using the Senescence βGalactosidase Staining Kit (Cell Signaling Technology) following manufacturer instructions. Images (12 per each triplicate) used to quantify the percentage of senescent cells were taken using an AxioVert.A1 microscope (Zeiss). | 3 of 11 ABREU Et Al. 2.4 | Quantification of collagenous and noncollagenous proteins The total amount of collagenous (COL) and noncollagenous (NCOL) proteinswerequantifiedusingtheSiriusRed/FastGreenStainingKit (Chondrex) according to the supplier instructions. DNA values were used to normalize data. 2.5 | Migration assay hMel (4 x 104 cells/cm2) were seeded in 8.0 µm pore size inserts (Corning) in nonsupplemented HEM medium, while complete medium was added to the bottom well. After 48 hours of culture under normoxia or physoxia, cells that migrated from the insert to the bottom well were fixed with 10% formalin (15 minutes, RT). Images were acquired with an AxioVert.A1 (Zeiss) for quantification (12 per triplicate) or with an Axio Observer (Zeiss) microscope after staining the cells' nuclei with DAPI. 2.6 | Tyrosinase activity quantification hMel were incubated for 5 minutes (ice) with 20 mmol/L Tris (hydroxyethyl) aminomethane (pH 7.5) containing 0.1% Triton X-100 and a protease inhibitor cocktail (1:100, Sigma-Aldrich). Cell lysates were then centrifuged at 14 500 rcf (10 minutes, 4°C) and 70 μL of the supernatant transferred into transparent 96well plates. As a substrate for tyrosinase, a 0.1% (wt/v) LDopa(Sigma-Aldrich)solutionwaspreparedinsodiumphosphate buffer (pH 6.8) and 140 μL were added to each well. Plates were incubated for 2 hours at 37°C, and the absorbance measured at 475nmusingamicroplatereader(SynergyHT,BioTek).Dataare presented as relative tyrosinase activity after normalization with DNA values. 2.7 | Cocultures DPcellsresuspendedinDMEMwith10%FBSwereseededata density of 2 x 104 cell/cm2 and cultured overnight before establishing the coculture with hMel, seeded at 2 x 104 cell/cm2 in HEM in 0.4 µm pore size inserts (Corning). Monocultures of each cell type were prepared as controls, either by culturing cells in their regular medium (DMEM or HEM), or in the medium used in to establish the coculture, DMEM with HEM at a 1:1 ratio (DMEM:HEM), to control possible medium effects. Direct cocultures were established by seeding 3 x 103 DP cells in round bottom ultralow attachment 96 wells (Corning) in 50 µL of DMEM with10%FBSfor24hoursbeforetheadditionof1.5x103 hMel resuspended in 25 µL of HEM medium. Both cocultures were performed for 3 days. 2.8 | DNA, active alkaline phosphatase and ROS quantification Cells were lysed in water with 0.01% sodium dodecyl sulphate. A 1hourincubationat37°Cwasfollowedbyfreezingat−80°C.DNA content was quantified using the QuantiT™ PicoGreen® dsDNA kit (ThermoFisherScientific),andROSlevelsweremeasuredusingthe OxiSelect™InvitroROS/RNSassaykit(CellBiolabsInc).Celllysates were also used to quantity active ALP levels in DP cells, using the Alkaline Phosphatase Detection Kit (Sigma-Aldrich). For DNA and ALP quantification in cell aggregates, a 5s sonication step (ice) was first performed to ensure the complete disintegration of the 3D aggregates prior quantification. All commercial kits were used according to the manufacturer instructions. DNA values were used to normalizeROSandALPresults. 2.9 | Alkaline phosphatase staining The detection of active ALP was performed by incubating formalinfixed DP cells (15 minutes, RT) for 20 minutes with a solution prepared with 5 μL of pnitroblue tetrazolium chloride (NBT) and 3.75 μL of 5bromo4chloro3indolyl phosphate (BCIP, Roche) in 1 mL of staining buffer [100 mmol/L NaCl, 100 mmol/L TrisHCl (pH 9.5) and 50 mmol/L MgCl2 in water]. Representative images were acquired with an AxioVert.A1 microscope (Zeiss). 2.10 | ELISA After coculture, inserts containing the hMel were removed and DP cells were cultured in serumfree DMEM for 24 hours. The supernatant of the cells was then collected, centrifuged (1000 g, 10 minutes) andsingle-usealiquotswerestoredat−80°C.Humanvascularendothelialgrowthfactor(VEGF)ELISADevelopmentKit(PeproTech) andhumanbonemorphogeneticprotein-2(BMP2)StandardELISA Development Kit (Petrotech) were then used following the manufacturer instructions to determine VEGF and BMP2 levels. DNA values were used to normalize data. 2.11 | Immunofluorescence staining DPhMel aggregates were fixed in 10%- formalin (overnight, 4°C), embedded in HistoGelTM (Thermo Scientific) and processed for paraffin inclusion. 4µm paraffinembedded sections were then dewaxed and heatmediated antigen retrieval was performed with sodiumcitratebuffer(pH6.0).Primaryantibodies(TableS1)were detected with Alexa Fluor®488/594 (1:500, Molecular Probes) secondary antibodies and nuclear counterstain was performed with DAPI. Images were acquired using an Axio Imager Z1m microscope (Zeiss). 4 of 11 | ABREU Et Al. Haematoxylinandeosin(H&E)stainingwasperformedaccording to standard protocols and representative images acquired with a DM750 microscope (Leica). Images were used to count cell nuclei and determine the DP cells/hMel ratio present within the cell aggregates for normalization of tyrosinase and ALP activity using the DNA amount of the corresponding cells. 2.12 | Statistical analysis Statisticalanalysisanddatavisualizationwereperformedusingthe GraphPad Prism 7.03. The D’Agostino & Pearson normality test was used to determine whether data followed a Gaussian distribution. Nonparametric data were analysed with a twotailed MannWhitneytest(twogroups,unpaired)orwithaFriedman(paired)or Kruskal-Wallistest(unpaired)whenmorethan2groupswerecompared.Parametricdatawereanalysedwithatwo-tailedStudent'st test (two groups, paired or unpaired). The comparison of more than two groups was performed with an ordinary (unpaired) or RM (paired) oneway ANOVA (one independent variable) or twoway ANOVA (two independent variables). Data are presented as mean ± standard errorofthemean(SEM).Fordatadisplayedasdotplots,blackdots represent data points and red bars indicate the mean. Differences with pvalues <.05 were considered significant. 3 | RESULTS 3.1 | Physoxia reduces the negative impact of in vitro culture conditions on DP cells DP cell cultures are typically established under normoxia, rapidly losing their native phenotype and intrinsic properties,35,36 including their key selfaggregation capacity.37,38 Moreover, they also have a short lifespan 39 in culture, which is accompanied by morphological changes such as shifting from a small polygonal morphology to a spindlelike shape,40,41 before acquiring an enlarged morphology.23 Therefore, we investigated whether those changes also occurred under physoxia to understand how the O2 level impacts DP cells’phenotypeinculture.DPcellsunderphysoxiadepictedapolygonal and less spindlelike shape and higher nucleitocytoplasm ratio, as demonstrated by the significant decrease in the cells’ area, perimeter and major axis length in comparison with normoxia (Figure 1AD). Physoxia also significantly decreased the percentage of senescent DP cells in culture (Figure 1E) and improved their aggregative capacity (Figure 1F). Moreover, it enhanced cell proliferation, albeit the DNA amount at day 3 was similar in normoxia and physoxia (Figure 1G). Interestingly, an opposite effect was observed regarding COL (Figure 1H) and NCOL (Figure 1I) protein secretion under physoxia, which was only beneficial after 3 days in culture. Altogether, these results suggest that physoxia promotes a healthier state in cultured DP cells, which featured characteristics typically associated with low passage cells. 3.2 | Physoxia enhances hMel migration, tyrosinase activity and proliferation within short culture times Although hMel are normally cultured under normoxia, there are indications that their proliferation and tyrosinase activity are favoured under lower oxygen tensions.28Wefoundthatphysoxiasignificantly increased both hMel migration (Figure 2A) and tyrosinase activity (Figure 2B), although this last effect was not sustained along with theculture.Similarly,significantlyhigherDNAlevelswereobserved for hMel cultured under physoxia at day 3 of culture (Figure 2C), suggesting an improved proliferative capacity. This effect was lost with the culture time, despite the high number of Ki67positive cells (Figure 2D). Physoxia did not seem to affect the morphology of hMel (Figure 2E). Collectively, these results indicate that physoxia supports hMel functional features better than normoxia but only for short culture periods. 3.3 | DP cell and hMel response to physoxia depends on their type of interaction Although residing in close vicinity in the hair bulb and having their functions coupled to anagen,42,43 little is known about how human DP cells and hMel interact and potentially affect each other's functionality. Knowing that physoxia individually improved hMel and DP functional features after 3 days in culture, we then explored its effect when these cells were indirectly cocultured (Figure 3A). The coculture with DP cells did not add to the increased hMel proliferation induced by physoxia (Figure 3B), in opposition to normoxia. Like for proliferation, coculture with DP cells under physoxia did not affect tyrosinase activity in hMel, contrarily to normoxia, which promoted a recovery from the negative effect of the coculture medium (Figure 3C). Regarding DP cells, they proliferated significantly more under physoxia than in normoxia but only when cultured in their conventional medium. Thus, the higher DNA amount detected in coculture might be due to the medium used. This is also sustained by the results similar to the control condition, both under physoxia and normoxia (Figure 3D). Active ALP levels in cocultured DP cells were not affected by physoxia but, as for proliferation, the medium used led to a significant increase of this inductive marker. In the presence of hMel,asignificantdecreaseofDPcells’activeALPwasobserved under physoxia, but not in normoxia (Figure 3E). In the HF, hMel and DP cells are separated only by a thin and permeable basal lamina.2 Therefore, we sought to investigate whether physoxia effects were different from those observed in the indirect cocultures, assuming a direct interaction between hMelandDPcells.WhenhMelwereculturedwithDPspheroids, they organized themselves around the spheroid, displaying a polarized position over onehalf of the DP spheroid, independently of the oxygen level (Figure 3F). Highly stable aggregates with similar DNA content were obtained (Figure 3G). Interestingly, the phenotype of both cell types was improved under physoxia, as | 5 of 11 ABREU Et Al. demonstrated by a significant increase of tyrosinase activity in hMel (Figure 3H) and by the higher amount of active ALP in DP cells (Figure 3I), their main functional markers, respectively. Moreover, the production of COL and NCOL proteins by aggregates cultured inphysoxiawassignificantlyhigherthaninnormoxia(Figure3J). Physoxia benefits hMel and DP cells functionality when both cell types are directly contacting. Interestingly, hMel response to physoxia does not seem to be indirectly affected by DP cells, whilehMelsignallingappearstohaveanimpactonDPcells’ALP activity. 3.4 | ROS generation due to hMel and DP cells interaction does not directly correlate with DP cells functionality Duringhairgrowthandpigmentation,thebulbisaROS-enriched environment31; therefore in addition to the functionality of hMel and DP cells, we addressed the involvement of ROS in their response. The production of ROS by hMel was significantly lower under physoxia, although this effect was significant only for the cocultures (Figure 4A). Moreover, hMel in coculture produced FIGURE 1 DP cells phenotype under physoxia. (A) Representative images of DP cells Factin cytoskeleton labelled with PhaloidinTRITC (left panel) and respective CellProfilerTM output (right panel) used to quantify morphological features such as (B) cell area, (C) perimeter and (D)majoraxislength.NucleiwerecounterstainedwithDAPI.SignificantdifferencesbetweenDPcellsculturedundernormoxia(21%O2) or physoxia (5% O2)wereanalysedusinganunpaired,two-tailedStudent'st test (n =3).Scalebar= 50 µm. (E) Representative images of the β-galactosidasestainingusedtoquantifythepercentageofsenescentDPcells.Significantdifferenceswereanalysedusingapaired,two- tailedStudent'st test (n =3).Scalebar= 50 µm. (F) Representative images of DAPIlabelled DP cells (left panel) and CellProfilerTM output of subsequent grouping of related nuclei (distance <8pixels)usedtocountthenumberofcellaggregates(≥30relatednuclei).Significant differenceswereanalysedusinganunpaired,two-tailedMann-Whitneytest(n=3).Scalebar= 200 µm. (G) DNA quantification used to evaluate cellular proliferation. Differences among oxygen levels at the same time point and differences for the same oxygen level along time wereanalysedusinganunpaired,two-tailedMann-Whitneytest(n= 3). Quantification of (h) COL and (i) NCOL proteins secretion. Results were analysed using an unpaired, twoway ANOVA. All data are presented as mean ±SEM,andstatisticaldifferencesareindicatedas *P <.05; **P <.01; ****P <.0001 6 of 11 | ABREU Et Al. significantlymoreROSthaninthecontrolconditions,regardless oftheoxygenlevel.PhysoxiaalsoledtoareductionofROSlevels in DP cells in comparison with normoxia, independent of the culture conditions (Figure 4B). The indirect coculture with hMel underphysoxiaalsoresultedinsignificantlyhigheramountsofROS thanincontrolconditions(Figure4B).Surprisingly,whencellswere directly cultured, ROS production in physoxia was significantly higher than in normoxia (Figure 4C), the opposite of what was observed in indirect cocultures. Considering that in indirect (Figures 3E and 4B) or direct (Figures 3I and 4C) cocultures the effect of physoxia over the amountofactiveALPandROSfollowedacommontrend,wethen investigated whether there was a correlation between these responses. For that, DP cells were treated with H2O2(exogenousROS) toincreasedoxidativestress,withtheROSinhibitorNACorboth. Treatment with H2O2 led to a significant decrease in the amount of DNA(Figure4D)butsurprisingly,itdidnotaffectROSintracellular levels in DP cells (Figure 4E) while a significant decrease of active ALP was observed (Figure 4F). Moreover, NAC pretreatment before H2O2additionfurtherenhancedROSproductionincomparisonwith H2O2 alone, but it also reduced the H2O2 effect on the amount of active ALP. Although it is not clear the mechanism by which H2O2 decreases ALP activity in DP cells, these results suggest that it is not directlycorrelatedwithanincreaseinROSlevels. 3.5 | Physoxia and 3D coculture supports DP cells and hMel phenotype To further explore both physoxia and hMel influence on DP cells hair regenerative potential, we looked at the production of a known promoter44,45 or inhibitor factor46 of hair induction, namely VEGF and BMP2, respectively. In physoxia, the amount of VEGF released by DP cells was significantly higher than in normoxia independently of the culture condition (Figure 5A). The coculture medium negatively impacted VEGF release by DP cells, which was not overcome with the coculture with hMel. In opposition, the amount of BMP2 in physoxia was significantly lower than in normoxia, for both cocultures and conventional DP culture medium (Figure 5B). As this effect was not seen in the control established with the coculture medium, it suggests that hMel presence was essential for the observed result. Considering that hMelDP cell aggregates better resemble the HF bulb than plain 2D cultures, and the observed advantages regarding their functionality under physoxia, we then investigated FIGURE 2 Characterization of hMel behaviour under physoxia. (A) Representative fluorescence microscopy images of DAPI labelled hMelthatmigratedover48handrespectivequantification.Scalebar= 100 µm. (B) Quantification of hMel tyrosinase activity (TYR). Relative values in comparison with cells cultured under 21% O2atday3ofculturearepresented.Significantdifferencesweredetermined by a paired, twoway ANOVA (n =3).(C)DNAquantificationusedtoassesshMelcellularproliferation.Significantdifferenceswere analysed using an unpaired, twoway ANOVA (n = 3). (D) Representative immunofluorescence images showing Ki67positive hMel. Nuclei werecounterstainedwithDAPI.Scalebar= 50 µm. (E) PMEL immunostaining showing hMel morphology. Nuclei were counterstained with DAPI.Scalebar= 50 µm.Significantdifferenceswereanalysedusingapaired,two-tailedStudent'st test (n = 3). All data are presented as mean ±SEM,andstatisticaldifferencesareindicatedas**P < .01; ****P < .0001 | 7 of 11 ABREU Et Al. the rescue of cell's nativelike phenotype under these conditions. The expression profile of different DP and hMel markers in the 3D aggregates (Figure 5C) showed that the cells display a phenotype similar to the native HF (Figure 5C, upper panel). The identification of specific markers of hMel— tyrosinase, PMEL, MelanA— and markers expressedalsoby DPcells—vimentinand S100—showedclear compartmentalization between the cell types. Moreover, Ki67 immunolabeling confirmed a low number of proliferative cells within both cellular compartments (Figure 5C). Noteworthy, tyrosinase expression in the cellular aggregates was higher than in conventionally culturedhMel (FigureS1). Moreover, theinvivo predominant V2isoform of versican,47 a DP inductive marker typically absent in2D-culturedcells(FigureS1)wasweaklyexpressedinthecellular aggregates, confirming what was previously described for DP spheroids.35 Overall, these results indicate that under physoxia DP cells’ inductive secretome is promoted which, in conjunction with 3Dculture conditions, allows an improved recovery of hMel and DP cell functional markers. 4 | DISCUSSION Besides ensuring overall cellular survival, oxygen levels are responsible for regulating a wide range of tissue functions, the reason why each organ, or even tissue, has its oxygenation status.12 In the HF and skin, oxygen ranges about 5% O2; however, the anagen hair bulb is a ROS-enriched microenvironment,31 which seems to indicate the involvement of multiple oxygenassociated responses, potentially by the different cells implicated in hair FIGURE 3 Physoxiaeffectsonco-culturedhMelandDPcells.(A)Schematicrepresentationoftheindirectco-culturesystemusedto study hMel and DP cells interactions, accompanied by images displaying the morphology of each cell type, respectively, immunolabelled withPMELandstainedwithPhalloidin-TRITC.NucleiwerecounterstainedwithDAPI.Scalebar= 50 µm. (B) DNA quantification used to assess hMel cell numbers in the coculture with DP cells, and in the respective homotypic controls— hMel cultured in standard conditions (HEM medium) or in the medium used for the coculture (DMEM:HEM). Data were analysed using an unpaired, oneway ANOVA (n = 3). (C) QuantificationofTYRinhMelinthedifferentcultureconditions.Statisticaldifferenceswerecalculatedusinganunpaired,Kruskal-Wallis test (n =3).Quantificationof(D)DNAand(E)activeALPofDPcellsco-culturedwithhMelandintherespectivecontrolmedia.Significant differences were analysed using a paired, Friedman test (n = 6 for DNA; n = 3 for active ALP). (F) Representative phase contrast (left panel) andH&E(rightpanel)imagesofthecellaggregatesformedafterdirectcultureofhMelwithDPspheroids.Scalebarsare100µm and 50 µm, respectively. (G) Quantification of DNA of the aggregates. Pvalueswerecalculatedusinganunpaired,two-tailedMann-Whitneytest (n =3).(H)QuantificationofTYRinhMelintheaggregates.Differenceswerecalculatedusingapaired,two-tailedStudent'st test (n = 3). (I) QuantificationofactiveALPintheDPspheroids.Apairedtwo-tailedStudent'st test was used to perform the statistical analysis (n =3).(J) QuantificationofCOLandNCOLproteinspresentintheaggregates.StatisticalanalysiswasperformedusingapairedWilcoxonsigned-rank test(COL)orapairedtwo-tailedStudent'st test (NCOL) (n = 4). Relative values are presented in comparison with the cells standard culture conditions at 21% O2 (C,D) or in comparison with aggregates formed at 21% O2 (H,I). All data are presented as mean ±SEMandstatistical differences are indicated as *P <.05; **P <.01; ***P <.001; ****P <.0001 8 of 11 | ABREU Et Al. growth. Therefore, we aimed to explore DP cells and hMel response to physiological oxygen levels, and if this response was influenced by either their indirect signalling or their direct contact, as it happens in the native tissue. Whenculturedatstandardatmosphericlevels,DPcellshavea characteristic short lifespan before entering growth arrest39 and gradually lose their native properties.1 This has been associated with premature senescence in vitro, triggered by excessive ROS production and oxidative stress.23WeshowthatDPcellscultured under physoxia featured an earlyculture morphology and phenotype, characterized by decreased senescence and increased proliferative and aggregative capacity, as previously observed for 2% O2 culture conditions.23Moreover,underphysoxiaROSlevels were lower, which is expected given the reduced oxygen availability for the mitochondrial respiratory chain,48themaintoxicROS producer.49WhenexposedtoexcessiveROS,thecapacityofDP cells to support hair growth is severely compromised and they lose their hair inductive ability.50 Interestingly, DP cells cultured under physoxia released higher amounts of VEGF and reduced levels of BMP2, which corresponds to the necessary trend observed during anagen induction and hair growth initiation,45,46,51,52 suggesting that physoxia stimulates DP cells inductive secretome. Further, also in agreement with what referred above to DP cells, hMel culturesunderphysoxiashowedlowerROSlevels.Thiswasalsoassociated with an increase of hMel migration, proliferative capacity and tyrosinase activity, confirming the results of a previous study showing higher hMel growth and pigmentation in cultures established under 1%- 5% O2.28 During the hair growth phase, hMel mature into tyrosinaseactive cells after migrating to the hair bulb and surrounding the DP, and indirect evidence suggests that DP may be involved in this process.11 In a previous study, DP cellconditioned medium was shown to enhance hMel tyrosinase activity under normoxic conditions.10 We show that hMel tyrosinase activity in the presence of DP cells and under physoxia was higher than in normoxia, yet similar to the homotypic controls, suggesting that the influence of DP cellproduced soluble factors on hMel do not surpass physoxia benefits. Interestingly, in our work hMel tyrosinase activity in the 3Daggregates cultured under physoxia also correlated with a higher ECM deposition, which is in agreement with a previous study10 that showed improved tyrosinase activity in both hMel directly cocultured with DP cells and with their ECM. These seem to support a link between hMel tyrosinase activity and DP cells ECM, in addition to their secretome. In the HF, DP cells are mitotically quiescent53,54 and the differentiated tyrosinaseactive hMel are less committed to cell division.6,55 In the indirect cocultures, hMel proliferated more in response to physoxia while DP cells remained unaffected, contrarily to the compromised proliferative capacity shown before for both DP cells and hMel cultured under normoxia.25,28Further,thecells’proliferative capacity in the 3D aggregates was lower than in the indirect cocultures that were established in 2D standard conditions, evidencing a mitotic profile that more closely emulates the native behaviour. FIGURE 4 ROSproductionbyco-culturedhMelandDPcellsandanalysisofitsassociationtoactiveALPinDPcells.ROSquantification in(A)hMeland(B)DPcellsco-culturedindirectlyandintherespectivecontrolconditions.Significantdifferenceswereanalysedusing an unpaired, oneway ANOVA (n =3).(C)AmountofROSintheDP-hMelcellaggregates.Statisticalanalysiswasperformedusinga paired,two-tailedStudent'st test (n = 3). (D) Quantification of the DNA amount in conventionally cultured DP cells (DMEM, 21% O2) in thepresenceoftheROS-scavengerNAC,H2O2 or the combination of both (NAC + H2O2). A paired Friedman test was used to identify significant differences (n =3).(E)QuantificationofROSproductionbyDPcellsafterNAC,H2O2orbothtreatments.Statisticalanalysiswas performed using a paired, oneway ANOVA (n = 3). (f) Representative light microscopy images of ALP staining in DP cells and respective quantificationofactiveALPlevels.Significantdifferenceswerecalculatedusingapaired,Friedmantest(n=3).Scalebar= 100 µm. Relative values are presented in comparison with the cells standard culture conditions at 21% O2 (A,B,E,F) or in comparison with aggregates formed at 21% O2 (C). All data is presented as mean ±SEMandstatisticaldifferencesareindicatedas*P < .05; **P < .01; ***P < .001; ****P < .0001 | 9 of 11 ABREU Et Al. Despite the generically accepted deleterious effects of supraphysiologicalROSaccumulation,thereisathinlineseparatingbeneficial and detrimental effects, which is associated to the tissues’ physiological ROS levels. In the hair bulb, a transient and physiological elevation of ROS31 is necessary to promote hair growth and differentiation programmes32 during anagen. In the indirect co-cultures,theamountofROSproducedwassignificantlyhigher than in the corresponding homotypic controls, independently of the oxygen level, which seems to suggest that cellular crosstalk in positivelyinfluencingROSformation.Intriguingly,ROSlevelsinthe indirect cocultures in physoxia were lower than in normoxia, but the opposite effect was observed in the direct cocultures (3D aggregates).WhilethelatterseemstocontradicttheexpectedlinkbetweentheavailableoxygenandROSformation,itisalsoknownthat spheroids represent a hypoxic environment.56,57 Previous work from Zheng and coworkers 27 showed that 2% O2improvedROSgeneration via nuclear NADPH oxidase 4, which was direct correlated with the increase of hair inductivity of human DP cells. Thus, the oxygen levels in the highly compact DPhMel aggregates are also likely to drop below 5% O2andleadtotheformationofROSbyan alternative mechanism, other than the one occurring in the indirect cocultures.24Moreover,ROSincreaseinthe3D-cellularaggregates may also be a consequence of the improved tyrosinase activity whichis,byitself,aROS-generatingoxidativestep.17,58 Moreover, albeit a direct correlation was not observed in flat cultures, in the 3D-cellaggregatesbothROSlevelsandALPactivitywerehigherin physoxia than in normoxia, demonstrating that in a more complex environment the interactions between hMel and DP cells can be better represented. 5 | CONCLUSIONS In summary, our results demonstrate that the recreation of the HF oxygenlevelsandtheassociateddecreaseofintracellularROSbenefit both hMel and DP cells, improving their proliferative capacity and functional features. Furthermore, we show that the type of interaction occurring between these cells also affects their response to physoxia and that, within the 3Daggregates, both hMel and DP celltypefunctionsandROSgenerationareincreased.Takentogether, our results demonstrate that hMelDP cells direct interaction under physiological oxygen levels has a superior capacity to recreate a microenvironment with features representative of the anagen bulb milieu, consequently enhancing their cellspecific functions. ACKNOWLEDGEMENTS WethankDrLucaGasperiniforhistechnicalassistanceintheimage analysis using the CellProfiler™ software. The authors also thank the financial support given by the European Research Council through the consolidator grant “ECM_INK” (ERC2016COG726061) and by FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the PD/59/2013, PD/ FIGURE 5 Phenotypicassessmentofco-culturedDPcellsandhMel.ELISAquantificationof(A)VEGFand(B)BMP2releasedbyDP cells indirectly cocultured with hMel and the respective control conditions. Results are shown as mean ±SEM.Significantdifferenceswere analysed using an unpaired, oneway ANOVA (n = 3) and are indicated as *P < .05; **P < .01; ***P < .001; ****P < .0001. (C) Representative immunofluorescence images showing the expression of different DP cells and hMel markers in the native hair follicle (HF) tissue and in the DP-HMelaggregatespreparedunderphysoxia.NucleiwerecounterstainedwithDAPI.WhitearrowheadsindicateKi67-positivecellsand dottedcirclesdelimitatetheDPspheroidarea.Scalebars= 50 μm