Mobilizing Transit-Amplifying Cell-Derived Ectopic ...Mobilizing Transit-Amplifying Cell-Derived Ectopic Progenitors Prevents Hair Loss from Chemotherapy or Radiation Therapy Wen-Yen

Molecular and Cellular Pathobiology

Mobilizing Transit-Amplifying Cell-DerivedEctopic Progenitors Prevents Hair Loss fromChemotherapy or Radiation TherapyWen-Yen Huang1, Shih-Fan Lai1,2, Hsien-Yi Chiu1,3,4, Michael Chang5,Maksim V. Plikus6, Chih-Chieh Chan4, You-Tzung Chen7, Po-Nien Tsao8,9,Tsung-Lin Yang9,10,11, Hsuan-Shu Lee12,13, Peter Chi14,15, and Sung-Jan Lin1,4,9,11

Abstract

Genotoxicity-induced hair loss from chemotherapy andradiotherapy is often encountered in cancer treatment, andthere is a lack of effective treatment. In growing hair follicles(HF), quiescent stem cells (SC) are maintained in the bulgeregion, and hair bulbs at the base contain rapidly dividing, yetgenotoxicity-sensitive transit-amplifying cells (TAC) that main-tain hair growth. How genotoxicity-induced HF injury isrepaired remains unclear. We report here that HFs mobilizeectopic progenitors from distinct TAC compartments for regen-eration in adaptation to the severity of dystrophy induced byionizing radiation (IR). Specifically, after low-dose IR, keratin5þ basal hair bulb progenitors, rather than bulge SCs, werequickly activated to replenish matrix cells and regenerated allconcentric layers of HFs, demonstrating their plasticity. After

high-dose IR, when both matrix and hair bulb cells weredepleted, the surviving outer root sheath cells rapidly acquiredan SC-like state and fueled HF regeneration. Their progeny thenhomed back to SC niche and supported new cycles of HFgrowth. We also revealed that IR induced HF dystrophy andhair loss and suppressed WNT signaling in a p53- and dose-dependent manner. Augmenting WNT signaling attenuated thesuppressive effect of p53 and enhanced ectopic progenitorproliferation after genotoxic injury, thereby preventing bothIR- and cyclophosphamide-induced alopecia. Hence, targetedactivation of TAC-derived progenitor cells, rather than quies-cent bulge SCs, for anagen HF repair can be a potentialapproach to prevent hair loss from chemotherapy and radio-therapy. Cancer Res; 77(22); 6083–96. �2017 AACR.

IntroductionChemotherapy and radiotherapy are widely employed in the

treatment of various cancers (1, 2). Both ionizing radiation (IR)and a substantial proportion of chemotherapeutic agents exerttheir treatment effects against cancer cells by inducing DNAdamage (1–3). Despite extensive efforts made to minimize off-target injuries, damage tonormal tissues is still inevitable (2, 4–6).Hair loss is a common side effect (7–9). It brings psychosocialstress, compromises patients' sense of personal identity, and evenjeopardizes the willingness for treatment (8, 10). Prevention ofsuch hair loss is still an unmet clinical need (7, 8).

Hair follicles (HF) are a dynamic organ that undergo lifelonggrowth cycles, consisting of anagen (active growth), catagen(regression), and telogen (relative rest) phases (Fig. 1A; ref. 9).Both telogen and anagenHFs share an upper permanent segment,spanning from the follicular infundibulum to the bulge (Fig. 1A;refs. 9, 11–13). The structures below the bulge are not permanent(Fig. 1A; refs. 9, 11–13). In telogen, the lower segment shrinks to aminimum structure of secondary hair germ (SHG; refs. 9, 11, 12).In anagen, the lower segment expands dramatically into a longcylinder where distinct populations of transit-amplifying cells(TAC) reside. Among them, outer root sheath (ORS) cells, locatedimmediately below the bulge, are connectedwith an enlarged hairbulb where hair matrix germinative cells surrounding the dermalpapilla (DP) actively multiply to generate concentric cellularlayers of distinct differentiations to support hair elongation(9, 13–15). As at any given time, the majority of human scalp

1Institute of Biomedical Engineering, College of Medicine and College of Engi-neering, National Taiwan University, Taipei, Taiwan. 2Division of RadiationOncology, Department of Oncology, National Taiwan University Hospital andCollege of Medicine, Taipei, Taiwan. 3Department of Dermatology, Hsin-ChuBranch, National Taiwan University Hospital, Hsin-Chu City, Taiwan. 4Depart-ment of Dermatology, National Taiwan University Hospital and College ofMedicine, Taipei, Taiwan. 5Sophie Davis School of Biomedical Education, CityUniversity of New York, New York, New York. 6Department of Developmentaland Cell Biology, Sue and Bill Gross Stem Cell Research Center and Center forComplex Biological Systems, University of California, Irvine, Irvine, California.7Institute of Medical Genomics and Proteomics, National Taiwan UniversityCollege of Medicine, Taipei, Taiwan. 8Department of Pediatrics, National TaiwanUniversity Hospital and College of Medicine, Taipei, Taiwan. 9Research Centerfor Developmental Biology and Regenerative Medicine, National Taiwan Uni-versity, Taipei, Taiwan. 10Department of Otolaryngology, National Taiwan Uni-versity Hospital and College of Medicine, Taipei, Taiwan. 11Graduate Institute ofClinical Medicine, National Taiwan University College of Medicine, Taipei, Tai-wan. 12Department of Internal Medicine, National Taiwan University Hospital andCollege of Medicine, Taipei, Taiwan. 13Institute of Biotechnology, NationalTaiwan University, Taipei, Taiwan. 14Institute of Biochemical Sciences, Collegeof Life Science, National Taiwan University, Taipei, Taiwan. 15Institute of Bio-logical Chemistry, Academia Sinica, Taipei, Taiwan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Sung-Jan Lin, National Taiwan University, 1, Section 1,Jen-Ai Road, Taipei 100, Taiwan. Phone: 8862-2356-2141; Fax: 8862-2393-4177;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0667

�2017 American Association for Cancer Research.

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Figure 1.

Dystrophic changes and regenerative activities inHFs after IR exposure.A,Mouse hair cycle and introduction of IR injury. Bg, bulge;Mx,matrix; PD, postnatal day; PW,postnatal week; SG, sebaceous gland. B, IR-induced hair loss (n ¼ 10 in each dose). C–E, Histology and quantification of HF lengths and matrix cell numbers.F and G, Apoptosis detected by TUNEL staining and quantification of apoptotic matrix cells. H and I, Cell proliferation mapped by BrdUrd and quantificationof BrdUrdþ matrix cells. J, Apoptosis detected by cleaved caspase-3. Statistical significance was determined by one-way ANOVA, followed by Bonferronimultiple comparison test. Blue � , P < 0.05, 2 Gy versus 0 Gy; green � , P < 0.05, 5.5 Gy versus 0 Gy; #, P < 0.05, 2 Gy versus 5.5 Gy. Error bars, mean� SEM. Dashedline, DP. Scale bar, 75 mm in C; 25 mm in F, H, and J.

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HFs are in anagen (9), this highly proliferative nature makesanagen HFs one of the most sensitive organs to genotoxic injury(7, 16, 17).

The slow-cycling long-lived HF stem cells (HFSC) are constant-ly preserved in the bulge around hair cycles (11, 12, 18). Inaddition to these quiescent bulge SCs (BgSC), telogen HFs houseanother population of more active SCs in SHG [SHG stem cells(ShgSC); refs, 11, 12, 19, 20]. During transition from telogen toanagen, ShgSCs proliferate first to support the formation of theinitial hair bulbs (11, 12, 19). This is followed by the activation ofquiescent BgSCs a few days later, which contribute to the upperORS (11, 12, 19). In contrast, the highly expanded lower segmentof anagen HFs lacks long-lived SCs (9, 18).

With the presence of TACs only in the lower segment, how areanagen HFs repaired to resume the ongoing anagen hair growthfollowing genotoxic injuries? As quiescent BgSCs are relativelyresistant to genotoxic insults (21), such repair might be drivenby BgSCs. However, it would require complete HF involutionand lengthy resetting of the hair cycle. This possibility is con-trasted by the fact that DNA-damaged anagen HFs often bypasstelogen involution and resume hair production without cycleresetting (8, 17). Such observations suggest that, in contrast to thetelogen-to-anagen regeneration that relies on the activation ofBgSCs and ShgSCs, anagen HFs can mobilize other progenitorcells for repair.

In this work, we attempt to explore the mechanisms and mapthe progenitor sources underlying the regenerative responses ofanagen HF repair following ionizing radiation (IR) injury. Weprovide evidence that HFs are able to employ ectopic progenitorcells from distinct TAC compartments for repair in adaptation tothe severity of genotoxic damage. We also demonstrate thatefficient mobilization of TAC-derived ectopic progenitor cells foranagen HF repair can be a strategy to prevent hair loss fromchemotherapy and radiotherapy.

Materials and MethodsMice

All animal experiments were approved by the InstitutionalAnimal Care and Use Committee of National Taiwan University.K5CreERmice were provided by C.M. Chen (22), Lgr5EGFP-Ires-CreERT2

micewere fromH.Clevers (23), andK19CreERmicewere fromG.Gu(24). p53-null mice, Ctnnb1flox/flox mice (25) and R26LSLtdTomato

mice were from The Jackson Laboratory. C57BL/6 mice were fromTaiwanNational LaboratoryAnimalCenter (Taipei, Taiwan). For IRand invasive experiments, animalswere anesthetizedby tiletamine-zolazepam (Telazol).

Radiation exposureThe dorsal hair of femalemice at postnatal day 30was carefully

shaved by an electric shaver. Around 2 days later when dorsal HFswere in early full anagen (�postnatal day 32), single doses (2 or5.5 Gy) of g irradiation were given from the dorsal side by a 137Cssource (dose rate 3.37 Gy/minute, g irradiator IBL 637 from CISBio International). For comparison, littermate control with thesame genetic background was used. Mice were consistently irra-diated in the afternoon.

Lineage-tracing experimentTo label basal cells and BgSCs, K5CreER/þ; R26LSLtdTomato/þ and

K19CreER/þ; R26LSLtdTomato/þmice received a single intraperitoneal

injection of tamoxifen (Sigma; 0.1mg/g of bodyweight) 24 hoursprior to irradiation. To label cells in the lower segment of epithe-lial strand at 5.5 Gy of IR, Lgr5EGFP-Ires-CreERT2/þ; R26LSLtdTomato/þ

mice received a single dose of tamoxifen (0.05 mg/g of bodyweight) at 48 hours after radiation.

Inhibition of Wnt/b-catenin signalingInhibition of Wnt/b-catenin signaling in the epithelium was

achieved by the conditional deletion of Ctnnb1 in K5CreER/CreER;Ctnnb1flox/flox mice by tamoxifen (0.1 mg/g body weight) at 6and 12 hours after irradiation or by using specific inhibitorsIWR1and IWP2 (Sigma). Both IWR1and IWP2were reconstitutedin DMSO, and DMSO was used as a control. Mice were subcu-taneously injected with IWR1 and IWP2 at 48, 72, and 96 hourspostirradiation, totaling 12.5 mg/g body weight for each dose.Skin was harvested at day 5 for examination.

Histology, immunostaining, and TUNEL stainingSkin specimens were fixed at 4�C overnight either in formalin–

acetic–alcohol solution for paraffin embedment or 4% parafor-maldehyde for OCT (Sakura Finetek) embedment. Specimenswere sectioned and stained with hematoxylin and eosin (H&E).Apoptotic cells were detected by DeadEnd Fluorometric TUNELSystem (Promega). Cryosections were used to visualize tdTomatofluorescence of lineage tracing. IHC and immunofluorescencestaining were performed with routine antigen retrieval as sug-gested by the antibodymanufacturers. Super Sensitive IHCDetec-tion Systems (BioGenex) were used for the detection of horse-radish peroxidase activity. The antibodies used were described inSupplementary Table S1.

Image acquisition and quantificationAll fluorescent images were acquired on the confocal micro-

scope (SP5, Leica). To quantify bromodeoxyuridine-positive(BrdUrdþ) and TUNELþ cells, we acquired 1,024 � 1,024 pixelssequential scanswitha63�oil immersionobjective lens (1.4NA).Hair matrix was counted as epithelial cells below the top of DP.Germinative cells are epithelial cells adjacent toDP, andbasal hairbulb cells are basal cells located on the outer surface of the hairbulb abutting dermal sheaths.

Quantification of g-H2AX fociDNA double-strand breaks were determined by g-H2AX foci as

described previously (26). Briefly, the 3-dimensional fluorescentimages were reconstituted by approximately 10 to 15 images ofserial two-dimensional Z-stacks of confocal images. Data wereanalyzed using the softwareMetaMorph 7.7. Foci withinHoechst-stained nucleus were scored by the value of pixels after the focusthreshold was set manually. Five pixels were considered as astandard for single DNA double-strand breaks based on the fewdiscrete g-H2AX foci generated in the nucleus of unirradiatedcontrol specimen. Because extensive g-H2AX foci appeared afterirradiation, individual foci could not be distinguished accurately.We compared the total value of positive pixels within the nucleusin selected cells with the standard 5 pixels/focus to estimate thenumber of foci in an entire nuclear region.

RNA sequencing analysisKeratinocytes of irradiated skin were collected at different time

points after irradiation. Total RNA from keratinocytes wasextracted using TRI reagent solution (Thermo Fisher Scientific)

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for the following RNA-sequencing (RNA-seq) library preparationand sequencing. All procedures were carried out according to theprotocol from Illumina. The libraries were sequenced on theIllumina NextSeq 500 platform using 75 single-end base pairstrategy, and 10 million reads per sample were generated. GeneOntology (GO) andKEGGanalyseswere performedusingDAVID(https://david.ncifcrf.gov/; ref. 27).

Quantitative RT-PCRTotal RNA from keratinocytes of irradiated skin collected at

different time points was extracted using TRI reagent solution(Thermo Fisher Scientific). The RNA was reverse transcribedinto cDNA using RevertAid H Minus First Strand cDNA Syn-thesis Kit (Thermo Fisher Scientific). In bead implantationexperiment, HF epithelial cells were isolated from the HFssurrounding Wnt3a and bovine serum albumin (BSA)-soakedbeads, and the RNA was extracted and cDNA was amplified byusing REPLI-g WTA Single Cell Kit (Qiagen). Quantitative PCRanalysis was performed using SYBR Green qPCR Master Mix(Thermo Fisher Scientific) on an ABI 7900HT Real-Time PCRSystem (Applied Biosystems). Sequences of gene-specific pri-mers used were described in Supplementary Table S2.

FACSThe dorsal skin of 7-week-old female mice were used for the

sorting of inactive ShgSCs. For the sorting of activated ShgSCs,dorsal hairs of 7-week-old female mice were plucked to activateShgSCs 2 days before skin specimen collection (28). The dorsalskin of 32-day-oldmice was irradiated with 5.5 Gy of IR, and skinspecimenwas collected at 72hours. Cell preparation for FACSwasperformed as described previously (29). The following antibodieswere used: CD34-FITC (eBioscience, 11-0341, 1:50), Sca1-PE-Cy7(eBioscience, 25-5981, 1:50) and P-cadherin-PE (R&D Systems,FAB761P, 1:100). FACSAria III sorter equipped with Diva soft-ware (BDBiosciences)wasused for sorting. Total RNA fromsortedcells was extracted usingMessageBOOSTER cDNA Synthesis fromCell Lysates Kit (Epicentre).

Protein administration experimentIntracutaneous implantation of protein-coated beads was per-

formed as described previously (30).HumanWnt3a recombinantprotein (R&D Systems) was resuspended in 1 mg/mL BSA solu-tion at 1 mg/mL. Affinity affi-gel blue gel beads (Bio-Rad) werethen suspended in 5 mL protein solution, either control (1mg/mLBSA) or experimental (1 mg/mL Wnt3a), at 4�C for 2 hours.Approximately 100 beads were implanted into skin immediatelyafter irradiation. Mice were sacrificed at different time pointspostirradiation. To visualizeHFs, skin specimenswere dehydratedin ethanol with graded concentrations and then immersed inxylene until it became transparent. The skin specimens were thenfurther processed for histologic examination and immunofluo-rescence staining.

Statistical analysisStatistical comparison was performed using the software

Prism (GraphPad). An unpaired Student t test was used tocompare datasets with two groups. To compare three or moregroups, we performed one-way ANOVA followed by Bonfer-roni multiple comparison. Data were presented as mean � SE.P values were considered statistically significant when lessthan 0.05.

ResultsDose-dependent HF dystrophy after IR and two tempospatiallydistinct regenerative attempts

To characterize the effect of IR, mice were irradiated with 2 and5.5 Gy on postnatal day 32 when dorsal HFs were in early fullanagen (Fig. 1A). In 5 to 10 days, there was a dose-dependent hairloss andHFdystrophy (Fig. 1B andC; Supplementary Fig. S1A). At2 Gy, the initial reduction of matrix cells and HF shortening wererecoveredbydays 3 and4 (Fig. 1C–E). At 5.5Gy,HFsprogressivelyshrank to slender epithelial strands by day 3, followed by resto-ration of anagen morphology and length between days 5 and 7(Fig. 1C andD). Aroundday10,HFsof theunirradiated, 2Gy, and5.5 Gy groups entered catagen, indicated by decreased cell pro-liferation and increased apoptosis (Fig. 1C and F–I). Afterwards,HFs could resume a new anagen in 2 and 5.5 Gy groups (Sup-plementary Fig. S1A), indicating the hair loss was transient.

We then analyzed cell death and proliferation. TUNEL stainingand cleaved caspase-3 staining were performed to detect apopto-sis, and both assays exhibited a similar trend (Fig. 1F, G and J).Apoptosis was more extensive and persistent after 5.5 than 2 Gy(Fig. 1F, G and J). After 2 Gy exposure, cell proliferation almostentirely halted after 6 hours and then increased at 12 and 48 hours(Fig. 1H and I). This early proliferative response successfullyrestored hair bulb structures and HF length between 72 and 96hours (Fig. 1C and D). After 5.5 Gy exposure, a similar regener-ative response, albeit with lower proliferation, was observedbetween 12 and 48 hours (Fig. 1H and I). However, proliferationalmost entirely ceased again at 72 hours. ProgressiveHF shrinkagebetween 0 and 72 hours indicated failure of the early regenerativeattempt to compensate for themore severe cell death (Fig. 1C, F, Gand J). Importantly, a second late proliferative attempt wasobserved in the lower tip of HFs at 96 hours (Fig. 1H and I) andled to HF elongation and restoration of hair bulbs (Fig. 1C–E).

Next, we examined whether these regenerative attempts led toproduction of mature hair shafts. At 2 Gy, differentiation towardinner root sheath and hair cortex was only transiently disruptedbetween 36 and 48 hours (Supplementary Fig. S1B). At 5.5 Gy, alengthier and more severe disruption was induced, yet differen-tiation was eventually restored (Supplementary Fig. S1C).

These results show that, depending on the severity of IRdamage, HFs activate two distinct regenerative responses: earlyregenerative attempt between 12 and 48 hours and late regener-ative attempt after 72 hours.

K5þ basal hair bulb keratinocytes preferentially proliferateduring the early regenerative attempt

Next, we tried to identify cells contributing to early regenerativeattempts. In normal hair bulbs, keratin 5 (K5) is exclusivelyexpressed in basal cells (Fig. 2A, 0 hour). However, 12 to 36hours after 2 Gy exposure, K5þ cells extended into the suprabasalpositions (Fig. 2A and B). TUNEL showed that, after 2 Gy,apoptosis was more prominent in suprabasal K5� matrix andgerminative cells thanK5þ cells (Fig. 2A, C, andD; SupplementaryFig. S2A) and that apoptosis of K5þ cells only slightly increased(Fig. 2C). BrdUrd pulse labeling showed these K5þ cells wereproliferative (Fig. 2E, yellow arrowheads, and F). During the sameperiod, proliferation of K5� suprabasal matrix cells nearly ceasedat 6hours andwas progressively restored only by 48hours (Fig. 2Eand G). Analysis of double-stranded DNA breaks by g-H2AXexpression also showed faster repair of DNA damage in K5þ basal

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hair bulb cells (Supplementary Fig. S2B and S2B0). Together, basalK5þ cells are more resistant to IR than suprabasal K5�matrix andgerminative cells and become proliferative to support early regen-erative attempts.

A total of 5.5 Gy exposure induced a similar increase insuprabasal K5þ cells (Fig. 2A and B), yet their proliferation wasdelayed compared with the 2 Gy group (Fig. 2E and F). Notably,higher apoptosis in both K5þ and K5� cells was detected after

5.5 Gy during the early regenerative attempt (Fig. 2A, whitearrowheads, C and D). K5þ cells also showed more persistentexpression of DNA damage markers (Fig. 2H; Supplementary Fig.S2C and S2C0). These data suggest that K5þ cells and their progenysustain catastrophic IR injury. Without sufficient resupply fromthe K5þ compartment, K5� matrix cells, which also proliferatepoorly (Fig. 2E and G), undergo eventual collapse, leading tofailure of the early regenerative attempt.

Figure 2.

Apoptosis, proliferation, and DNA damage of hair bulb cells during the early regenerative attempt. Mice were pulse labeled with BrdUrd 1 hour before sampling.A, Double staining with K5 and TUNEL. B–D, Quantification of K5 and TUNEL expression in A. B, Proportion of K5þ cells in the matrix. C, Proportion of K5þ

TUNELþmatrix cells. More severe apoptosis of K5þ cells was observed in 5.5 Gy than 2 Gy. D, Proportion of K5� TUNELþmatrix cells. E, Double staining with K5 andBrdUrd. F and G, Quantification of K5 and BrdUrd expression in E. F, Proportion of K5þ BrdUrdþ matrix cells. G, Proportion of K5� BrdUrdþ matrix cells.Compared with 2 Gy, 5.5 Gy of IR induced more prolonged reduction of proliferation in K5� matrix cells. H, Quantification of g-H2AX foci in basal hair bulb cells.Statistical significance was determined by one-way ANOVA, followed by Bonferroni multiple comparison test. Blue � , P < 0.05, 2 Gy versus 0 Gy; green� , P < 0.05, 5.5 Gy versus 0 Gy; #, P < 0.05, 2 Gy versus 5.5 Gy. Error bars, mean � SEM. Dashed line, DP. Scale bar, 25 mm.






K5þ basal hair bulb cells replenish germinative population andcontribute to all layers of recovered HFs

To further confirm the cellular source for early regenerativeattempts (Fig. 3A), we lineage traced K5þ cells in K5CreER;R26LSLtdTomato mice (Fig. 3B). Injection with tamoxifen 24hours prior to IR allowed successful and exclusive labeling ofbasal, but not germinative cells in the hair bulb (Fig. 3B, 0hour). After 2 Gy exposure, labeled cells expanded into supra-basal positions at 12 hours (Fig. 3B, yellow arrowhead),replenished the germinative compartment (Fig. 3B, whitearrowheads) and contributed to all layers of repaired hairbulbs, including hair shafts (Fig. 3B, white arrow), between24 and 36 hours. In control HFs, most of the labeled cellsremained in the basal position even after 36 hours (Fig. 3B).Quantitatively, progeny of K5þ lineage progressively increased,accounting for about 60% of all matrix cells at 36 hours (Fig.3C). The data underscore that, following IR, K5þ basal hairbulb cells display high lineage plasticity.

Next, we determined the contribution of BgSCs. Neither apo-ptosis (Fig. 3D) nor proliferation (Fig. 3E) was detected in thebulge. Lineage tracing in K19CreER; R26LSLtdTomato mice did notshow BgSC expansion (Fig. 3F). The results indicate that BgSCssurvive 2 Gy IR injury but do not actively contribute to earlyregenerative attempts.

K5þ ORS cells are remodeled into the epithelial strand duringthe late regenerative attempt

The results above show that the remaining short epithelialstrand at 72 hours serves as a platform for the late regenerativeattempt. To determine the origin of the epithelial strand, weperformed lineage tracing in K5CreER; R26LSLtdTomato mice(Fig. 4A) following tamoxifen administration 24 hours prior toIR. This protocol labeled basal cells in theORS in addition to basalcells of the bulb (Fig. 4A). As HFs regressed between 24 and 72hours, a larger portion of the epithelial strand became composedof the K5þ progeny (Fig. 4A). As K5þ basal hair bulb cells fail tosurvive 5.5 Gy injury, we conclude that the epithelial strandoriginates from the K5þ ORS cells that are more radioresistantand that basal ORS progeny fuel successful HF regenerationduring late regenerative attempts (Fig. 4A).

Lower tip cells acquire a stem cell–like property and undergostepwise activation with BgSCs for the late regenerative attempt

After 5.5 Gy, cell proliferation largely halted at 72 hours(Figs. 1H and I and 4B). By 84 hours, proliferation was firstresumed in P-cadherinþ lower tip cells (Fig. 4B, white arrow-heads). At 96 hours, more lower tip cells proliferated as theycontinued to regenerate new hair bulbs (Fig. 4B). We did notdetect apoptosis of BgSCs (Fig. 4C). Continuous BrdUrd labeling

Figure 3.

Cell origin for the early regenerative attempt after 2 Gy of IR. A, Possible cell origin. B and C, Lineage tracing in K5CreER; R26LSLtdTomato mice and quantification oflabeled cells in the hair matrix. B, After 2 Gy, labeled cells expanded suprabasally (yellow arrowhead), replenished germinative cells (white arrowheads),and formed hew hair shafts (white arrow). Tam, tamoxifen. C, Proportion of tdTomatoþ matrix cells. Statistical significance was determined by Student t test.� , P < 0.05. Error bars, mean � SEM. D, Analysis of BgSC apoptosis by TUNEL. E, Continuous BrdUrd labeling for analysis of BgSC proliferation. F, Lineagetracing in K19CreER; R26LSLtdTomato mice. Dashed line, DP; Bg, bulge. Scale bar, 25 mm.

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Figure 4.

Remodeling of ORS cells for the late regenerative attempt after 5.5 Gy of IR.A, Lineage tracing in K5CreER; R26LSLtdTomatomice. B, Cell proliferationmapped by pulseBrdUrd labeling. The P-cadherinþ lower tip cells started to proliferate at 84 hours (white arrowheads) and regenerated new hair bulbs at 96 hours. C,Analysis of BgSC apoptosis by TUNEL. D, Continuous BrdUrd labeling showed that BgSCs did not proliferate until day 5 (white arrowheads). E, Lineage tracing ofBgSCs in K19CreER; R26LSLtdTomato mice. Labeled BgSC progeny did not move out of the bulge until day 5. F, Immunofluorescence of P-cadherin and Sox9.Similar to SHG (yellow arrowheads), P-cadherinþ lower tip cells were also positive for Sox9 (white arrowheads). G, Lgr5 promoter-driven EGFP expression inLgr5EGFP-Ires-CreERT2 mice. The lower tip cells were positive for EGFP. H, Expression of ShgSC-specific genes. Similar to activated ShgSCs, several specificgenes were upregulated in lower tip cells at 72 hours. I, Possible cell origin for late regenerative attempts. J, Lineage tracing in Lgr5EGFP-Ires-CreERT2; R26LSLtdTomato

mice. Error bars, mean � SEM. Scale bar, 25 mm.






showed that BgSCs did not start proliferating until day 5 post-IR(Fig. 4D, white arrowheads), when the new hair bulb alreadyregenerated (Fig. 1C). Lineage tracing of BgSCs in K19CreER;R26LSLtdTomato mice showed that BgSCs contributed to the supra-bulbar ORS cells right below the bulge, but not to the regeneratedhair bulbs (Fig. 4E). These results indicate that the lower tip cellsfuel the initial late regenerative attempt. This parallels the stepwiseactivation of epithelial progenitor populations during physiolog-ic telogen-to-anagen transition, when ShgSCs, rather than quies-cent BgSCs, fuel early HF growth (12, 19).

We considered that cells in the lower segment of the epithelialstrand acquire SC-like characteristics. We compared the epithelialstrand structure of 5.5 Gy–irradiated anagen HFs with normaltelogen HFs. Typical BgSCmarkers, CD34 and K15 (31, 32), werestill maintained in the bulge (Supplementary Fig. S3A). However,cells at the lower end of the epithelial strand were CD34/K15double-negative, yet positive for P-cadherin, Sox9, and Lgr5promoter activity, markers of the normal telogen ShgSCs (Fig.4F and G; Supplementary Fig. S3A; refs. 12, 20, 33). Lack of AE13,AE15, and K75 keratin expression shows that lower epithelialstrand cells do not prematurely differentiate toward inner anagenHF structures (Supplementary Fig. S3A). We then FACS-isolatedthese lower tip cells using a Sca1low CD34low P-cadherinhigh

marker profile (Supplementary Fig. S3B; ref. 12) and examinedthe expression of known ShgSC genes (12). We found Ccnb1,Clca1, Sox4, and Sox6 were also upregulated in the sorted lowertip cells at 72 hours (Fig. 4H). Taken together, these results showthat following 5.5Gy IR injury, ORS cells were remodeled into theepithelial strand whose lower tip cells acquired a ShgSC-likeprogenitor property.

To strengthen the evidence for the cellular dynamics notedabove (Fig. 4I), we performed additional lineage tracing usingLgr5EGFP-Ires-CreERT2 mice, which allowed for specific tracing of thelower tip cells (Fig. 4J; Supplementary Fig. S4). Consistent withthe prior report (34), in unirradiated Lgr5EGFP-Ires-CreERT2 mice,ORS cells and suprabasal hair bulb cells were labeled aftertamoxifen induction (Fig. 4J, left). In the 5.5 Gy group, althoughthe epithelial strand was negative for Lgr5 by immunostaining(Supplementary Fig. S3A), its lower portionwasdistinctly positivefor the Lgr5 promoter activity (Fig. 4G). Twenty-four hours aftertamoxifen injection to Lgr5EGFP-Ires-CreERT2; R26LSLtdTomato mice at48 hours after 5.5 Gy, labeled cells were exclusively found in thelower portion of the epithelial strands of all HFs examined (n ¼50; Fig. 4J, right), with higher incidence in the lower tip. Theselabeled Lgr5 progeny contributed to theORS, the entire hair bulb,and hair shafts of the repaired HFs (Fig. 4J, right; SupplementaryFig. S4).

Further tracing showed that the progeny of labeled Lgr5 cellshomed back to the SHG and bulge when repaired HFs eventuallytransitioned to telogen (d17, Fig. 4J, right), and that in the nextcycle, they formed the new lower segment of the anagen HFs(d35, Fig. 4J, right). These results reveal expanded plasticity ofORS cells following IR and underscore IR-induced acquisition ofSC properties.

Genotoxic injury disrupts WNT signaling that is required forlate regenerative attempt

Because HF regeneration from ORS-derived progenitors repre-sents a novel repair mechanism, we aimed to clarify its molecularbasis. First, we compared epithelial cell transcriptomes at differenttime points before and after 5.5 Gy of IR by RNA-seq. GO

enrichment analysis revealed several biological processes alteredfollowing IR (Fig. 5A). We screened for signaling pathways down-regulated between 0 and 24 hours and upregulated between 24and 72hours.We found thatWNT and hedgehog pathwaysfit thistrend (Supplementary Table S3). Hedgehog signaling has beenshown to be downregulated by cyclophosphamide in growingHFs (35).

WNT signaling is activated in ShgSCs at the onset of physiologicanagen and is crucial for normal hair cycle progression (36–38).The transcript and protein levels of Lef1, a downstream mediatorof WNT signaling in HFs, were prominently suppressed after IRand restored only by 96 hours post-IR (Fig. 5B and C). This wasaccompanied by an increase in nuclear b-catenin in the lower tipcells 72 to 96 hours post-IR (Fig. 5D).WNT ligands are secreted byHF epithelium and help to maintain proper epithelial–mesen-chymal interaction during anagen (36, 39–41). We found thatWnt3a levels were diminished between 24 and 48 hours post-IR,but later rebounded from 72 hours onward at the base of theepithelial strand (Fig. 5E and F). These results show that 5.5 Gydisrupts WNT signaling and that its reactivation correlates withlate regenerative attempts. In comparison, after 2 Gy exposure,levels of Wnt3a and Lef1 were suppressed only transiently andpartially (Supplementary Fig. S5A–S5D). Therefore, higher IRdoses induce more severe suppression of WNT signaling.

To determine whether WNT signaling reactivation is requiredfor late regenerative attempts, we pharmacologically disruptedWNT ligand secretion with IWR1 and IWP2 inhibitors and foundthat the late regenerative attempt was attenuated (Fig. 5G). Wealso disrupted b-catenin inK5CreER; Ctnnb1flox/floxmice after 5.5 Gyof IR and found that the late regenerative attempt was abolished(Fig. 5H). Therefore, reactivation ofWNT signaling is essential forthe late regenerative attempt. This parallels the requirement forWNT signaling during normal HFSC activation upon telogen-to-anagen transition (36, 38).

p53 is required for IR-inducedHFdystrophy and suppression ofWNT signaling

p53 has been shown to mediate pathologic changes of che-motherapy-induced hair loss and IR-induced injury to otherorgans (42, 43). Its role in IR-induced HF dystrophy remainsunclear. We found that p53 was induced by IR in hair bulbs, butnot DP, and that p53 expression was more extensive and persis-tent at 5.5 Gy than 2 Gy (Fig. 6A). Furthermore, matrix apoptosis,suppression of proliferation, HF shrinkage, and hair loss were notinduced in p53-null mice by IR (Fig. 6B–E; Supplementary Fig.S6A–S6C). In wild-type mice, IR increased expression of down-stream target genes of p53, Noxa, Bax, and p21, whereas Pumaexpression was slightly decreased from 6 to 48 hours and tran-siently elevated at 72 hours (Fig. 6F). In p53-null mice, thebaseline expression of Bax and p21 was higher than wild-typemice, while Noxa and Puma were lower than wild-type mice (Fig.6F). After IR, comparedwithwild-typemice, decreased expressionof Bax, Noxa, and Puma was observed in p53-null mice, and p21was only slightly increased at 24 hours. Thismight help to explainthe decreased apoptosis and unsuppressed cell proliferation afterIR in p53-null mice.

Comparison of 2- and 5.5 Gy–induced injuries revealed acorrelation between the duration of p53 activation and moresevere and persistent WNT signaling suppression, suggestingthat p53 might be involved in mediating IR-induced WNTsuppression. We found that the basal mRNA expression of

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Wnt3a and Lef1 at 0 hour was significantly higher in p53-nullmice than in p53 wild-type mice (Fig. 6G). The higher WNTsignaling activity in p53-null mice is consistent with the priorreport showing the inhibition of WNT signaling by p53 (44). Inp53 wild-type mice, both Wnt3a and Lef1 expressions wereprominently suppressed by 5.5 Gy of IR and were restoredagain at 72 and 96 hours, respectively. In p53-null mice,although the Wnt3a expression was decreased after IR, it wasstill higher than that in p53 wild-type mice at 6, 24, and 96hours. Lef1 expression was not downregulated until 24 hours,but its levels in p53-null mice were much higher than that ofp53 wild-type at all time points. Consistent with this, we foundthat protein expression of Wnt3a and Lef1 was not suppressedby IR in p53-null mice (Fig. 6H and I). Judging from Lef1expression, WNT signaling was maintained at a higher level inp53-null mice, and this might contribute in part to the atten-uated HF dystrophy after IR.

Local delivery of WNT ligands prevents IR andcyclophosphamide-induced alopecia by enhancing ectopicprogenitor cell proliferation

AsWNT ligands produced by anagenHF epithelium are knownto maintain anagen progression (36, 38, 39) and as WNT signal-ing undergoes early suppression after IR, we posited that main-tainingWNT signalingmight promoteHF regeneration, in part bybypassing the suppressive effect of p53 on cell proliferation. Totest this, we delivered Wnt3a-soaked beads into the 5.5 Gyirradiated skin (Fig. 7A).We found that hairs continued to emergein the Wnt3a-treated skin (Fig. 7A; Supplementary Fig. S6D) withreduced HF dystrophy and faster restoration of anagen structures(Fig. 7B; Supplementary Fig. S6E and S6F). Wnt3a treatmentpreserved Lef1 expression (Supplementary Fig. S7A), but did notprevent p53 activation and apoptosis (Fig. 7C and D; Supple-mentary Fig. S7B). The latter was only slightly decreased at 24hours post-IR (Fig. 7C and D). Analysis of gene expression of HF

Figure 5.

WNT signaling is suppressed by IR, and its reactivation is required for late regenerative attempts. A, Cellular processes affected by 5.5 Gy based on the comparativeGO-enrichment analysis of the transcriptomes of the epithelial cells. B and C, Quantitative RT-PCR and immunofluorescence for Lef1 expression after5.5 Gy of IR. D, Immunofluorescence of b-catenin. Nuclear localization of b-catenin was detected in the lower tips between 72 and 96 hours. E and F, QuantitativeRT-PCR and immunofluorescence for Wnt3a after 5.5 Gy of IR. G, Inhibitor for WNT secretion and WNT signaling by IWP2 and IWR1, respectively. H,Inhibition of epithelial WNT/b-catenin signaling by ablating Ctnnb1 in keratinocytes of K5CreER; Ctnnb1f/f mice. � , P < 0.05, compared with 0 hour, by Studentt test. Error bars, mean � SEM. Scale bar, 25 mm in C, D, and F; 75 mm in G and H.






epithelial cells fromWnt3a-treated and control (BSA)-treated skinshowed that Wnt3a treatment significantly inhibited the upregu-lation of Bax, Noxa, and p21, but increased the expression of Puma

after IR (Fig. 7E). The upregulation of proapoptotic Puma withdownregulation of proapoptotic Bax and Noxa might help toexplain that Wnt3a did not largely suppress IR-induced apoptosis

Figure 6.

WNT ligand production and WNT signaling are not inhibited in p53-null mice after 5.5 Gy of IR. A, IHC for p53. B, Apoptosis detected by TUNEL staining.C, Cell proliferation mapped by BrdUrd pulse labeling. D, IR-induced hair loss was not observed in p53-null mice 5 days after IR. E, Histology showed no dystrophicchanges in HFs of p53-null mice after IR. F, Quantitative RT-PCR for Bax, Noxa, Puma, and p21 expression in epithelial cells of p53 WT and p53-null mice after5.5 Gy of IR, respectively. G, Quantitative RT-PCR for Wnt3a and Lef1 expression in epithelial cells of p53 WT and p53-null mice after 5.5 Gy of IR. H,Immunofluorescence for Wnt3a. I, Immunofluorescence for Lef1. Dashed line, DP. Scale bar, 75 mm. Blue � , P < 0.05, compared with 0 hour of p53 WT; green� , P < 0.05, compared with 0 hour of p53 null; #, P < 0.05, p53 WT versus p53 null. Error bars, mean � SEM. WT, wild type.

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Figure 7.

Enhancement of WNT signaling prevents IR and cyclophosphamide-induced alopecia by boosting the early regenerative attempt. A, Subcutaneous injection ofWnt3a-soaked beads (blue beads) maintained hair growth after 5.5 Gy of IR. Dashed circles, bead-implanted area. B, Histology of Wnt3a and BSA-treatedHFs. C and D, Apoptosis detected by TUNEL and quantification of TUNELþ matrix cells. � , P < 0.05. E, Quantitative RT-PCR for p53 transcriptional targets in HFepithelial cells in Wnt3a-treated skin. Blue � , P < 0.05, compared with 0 hour of BSA treatment; green �, P < 0.05, compared with 0 hour of Wnt3atreatment. F and G, Pulse BrdUrd labeling and quantification of K5þ BrdUrdþ matrix cells. Wnt3a increased K5þ cell proliferation (white arrows). � , P < 0.05. H,Cyclophosphamide (CYP) treatment. Hair loss was observed 5 days after treatment. I, Wnt3a-soaked beads (blue beads) maintained hair growth in theirvicinity after cyclophosphamide treatment. J, Anagen HF repair. Depiction of cell origin and cell dynamics for early and late regenerative attempts for anagen HFrepair following IR injury. Dashed lines in C and F, DP. Scale bar, 75 mm in B; 25 mm in C and F.






(Fig. 7D). The suppression of p21 expression, an important p53target that induces cell-cycle arrest, by Wnt3a treatment (Fig. 7E)might promote regenerative proliferation. Indeed, comparedwithcontrol, an early increase of basal K5þ cell proliferation wasobserved at 24 hours, and cell proliferation within the hair matrixwas maintained at a higher level for at least 72 hours (Fig. 7F,white arrows, and G; Supplementary Fig. S7C). Importantly, thenumber of matrix cells was increased in the Wnt3a-treated HF(Supplementary Fig. S7D).

We also tested whether this approach can prevent chemother-apy-induced alopecia. Cyclophosphamide administered on post-natal day 32 also induced extensive hair loss on day 5 (Fig. 7H).Similarly, local delivery of Wnt3a-soaked beads reduced hair lossafter cyclophosphamide treatment (Fig. 7I). These results dem-onstrate that maintaining WNT signaling can prevent hair lossfrom genotoxic injury by enhancing the regenerative cell prolif-eration of K5þ basal hair bulb progenitors.

DiscussionOur results show that, rather than deploying long-lived bulge

SCs, IR-damaged anagen HFs mobilize extra-bulge TAC-derivedprogenitor cells for repair without having to reset their hair cycle(Fig. 7J). This ability of HFs to mobilize distinct extra-bulgeprogenitors for anagen HF repair also suggests a new therapeuticstrategy for hair loss that relies on TACs.

In hair bulbs, germinative and basal bulb cells are thought torepresent two distinct lineages within the TAC population (14).Proliferative germinative cells fuel hair shaft elongation(14, 45), while basal hair bulb cells (also referred to as lowerproximal cup cells) are speculated to maintain the shape of hairbulbs and not to contribute to the inner HF structures (14). Weshow that K5þ basal cells serve as SCs for rapid repair of the HFbulb and that their concealed plasticity is quickly unveiledupon injury. Previously, colony-forming epithelial SCs wereshown to be present in the hair bulbs (46). It is likely that K5þ

basal hair bulb cells can be a source of colony-forming cells.The employment of local multipotent progenitors within theTACs shortens the time needed for regeneration, and thisstrategy is advantageous over regeneration via BgSCs. Theemployment of BgSCs would require intricate signaling relaysand create a significant time delay during their downwardmigration toward injured hair bulbs.

We also revealed a novel role for ORS cells of the TAC pool forregeneration. HFs suffering from amore dystrophic change regen-erate from the surviving ORS cells. Although our lineage studiesare not able to directly differentiate between K5þ basal bulb cellsandK5þ basalORS cells, the two cell populations are known to bedistinct in origin and function during anagen (14). K5þ hair bulbcells broadly apoptose after 5.5 Gy, indicating that radioresistantK5þ ORS cells are the most likely origin for the epithelial strand.Although targeted depletion of SCs can allow differentiated cellsto gain SC-like properties uponmigration into the physiologic SCniche (47, 48), it remains unknown whether acquisition of SC-like properties can occur ectopically outside the physiologic SCniche. Here, we showed that ORS cells can directly acquire a SC-like state ectopically after IR. As DP cells are able to convertinterfollicular keratinocytes into follicular cells for HF neogenesisupon close contact (49), the close proximity of lower tip cells toDP suggested that DP cells might play an important role here byeither directly reprogramming ORS cells into a SC-like state or

providing signals for the dedifferentiation of ORS cells throughshort-range interaction.

The lower tip cells in the epithelial strand not only expressmarkers characteristic of ShgSCs, but also behave like them.Similar to the telogen-to-anagen transition (12), the late regen-erative attempt displays the step-wise activation of progenitorcells: lower tip cells are activated first to regenerate hair bulbs,followed by the activation of BgSCs to resupply upper ORS. Theircontribution to the entire hair bulb and all bulb-derived differ-entiated structures confirms their multipotency. Furthermore,their progeny home back to the SC niche at the end of the repairedhair cycle and contribute to the new hair bulb andORS during thefollowing physiologic hair growth cycle.

The activation of p53, a central mediator of pathologic changesfollowing IR injuries in other organs (43, 50), is required forchemotherapy-induced HF dystrophy (42). We revealed its keyrole in the response ofHFs to IR and also uncovered a complicatedcross-talk between IR, p53, and WNT signaling. We found that IRinduces p53 expression in the hair bulb in a dose-dependentmanner. In p53-null mice, neither apoptosis nor dystrophy wasinduced, indicating the essential role of p53 in IR-induced hairloss. AsWNT signaling is not inhibited by IR in p53-null mice, ourresults suggest that p53 may have dual roles in both inducingapoptosis and suppressing regeneration-promoting WNT signal-ing following IR injury. We demonstrated that augmenting WNTsignaling could attenuate the suppressive effect of p53 on cellproliferation, likely through inhibition of p53-responsive upre-gulation of p21, to enhance the regenerative program of ectopicprogenitors from TACs following the injury of IR and cyclophos-phamide. Because the effect of applied Wnt3a protein is transientand localized, it potentially represents a new, low-risk clinicalstrategy to reduce hair loss after genotoxic injury.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design:W.-Y. Huang, S.-F. Lai, H.-Y. Chiu, M. Chang, S.-J. LinDevelopment of methodology: W.-Y. Huang, S.-J. LinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W.-Y. Huang, C.-C. Chan, T.-L. Yang, S.-J. LinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W.-Y. Huang, H.-Y. Chiu, P.-N. Tsao, T.-L. Yang,S.-J. LinWriting, review, and/or revision of the manuscript:W.-Y. Huang, H.-Y. Chiu,M.V. Plikus, P.-N. Tsao, P. Chi, S.-J. LinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.-F. Lai, H.-Y. Chiu, M. Chang, C.-C. Chan,Y.-T. Chen, S.-J. LinStudy supervision: H.-Y. Chiu, S.-J. LinOther (supporting specific transgenic mice): H.-S. Lee

AcknowledgmentsWe thank the staff of the imaging core and the Flow Cytometric Analyzing

and Sorting Core at the First Core Lab, National Taiwan University College ofMedicine, and the staff of the 8th Core Lab, Department of Medical Research,National Taiwan University Hospital for technical support. The authors alsothank the members of the S.J. Lin laboratory for their discussion andDrs. Hironobu Fujiwara, George Cotsarelis, and Ralf Paus for their discussion.

Grant SupportThis work was supported by Taiwan Bio-Development Foundation (TBF; to

S.J. Lin), a TaiwanNational Health Research Institutes grant (EX104-10410EI toS.J. Lin), TaiwanMinistryofScienceandTechnologygrants (105-2627-M-002-010

Huang et al.





to S.J. Lin; 105-2314-B-002-073-MY4 to P. Chi; and 106-2314-B-002-133-MY3to S.F. Lai), National Taiwan University Hospital grants (105-S3010, 104-P04 toS.J. Lin), NIH NIAMS grants (R01-AR067273 and R01-AR069653 toM.V. Plikus)and Pew Charitable Trust (to M.V. Plikus).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 5, 2017; revised August 2, 2017; accepted September 14,2017; published OnlineFirst September 22, 2017.

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2017;77:6083-6096. Published OnlineFirst September 22, 2017.Cancer Res Wen-Yen Huang, Shih-Fan Lai, Hsien-Yi Chiu, et al. Prevents Hair Loss from Chemotherapy or Radiation TherapyMobilizing Transit-Amplifying Cell-Derived Ectopic Progenitors

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