WO2022212790A1 - Methods of modulating hair growth - Google Patents

Methods of modulating hair growth Download PDF

Info

Publication number
WO2022212790A1
WO2022212790A1 PCT/US2022/022956 US2022022956W WO2022212790A1 WO 2022212790 A1 WO2022212790 A1 WO 2022212790A1 US 2022022956 W US2022022956 W US 2022022956W WO 2022212790 A1 WO2022212790 A1 WO 2022212790A1
Authority
WO
WIPO (PCT)
Prior art keywords
mice
agent
hair
gas6
adx
Prior art date
Application number
PCT/US2022/022956
Other languages
French (fr)
Inventor
Ya-Chieh HSU
Sekyu CHOI
Original Assignee
President And Fellows Of Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Publication of WO2022212790A1 publication Critical patent/WO2022212790A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Stem cells are regulated by intrinsic regulators as well as by extrinsic signals from the niche 1 4 .
  • systemic factors regulate stem cell behaviors to couple tissue regeneration with diverse bodily changes remains poorly understood.
  • Hair follicle stem cell HFSCs are located at the bulge and at the hair germ and are quiescent except during early anagen, when they proliferate transiently to initiate tissue regeneration 6,7 .
  • Chronic stress which triggers an increase in corticosterone secretion from the adrenal glands, has been anecdotally associated with hair loss in humans 8 .
  • mice showed that the topical application of betamethasone — a steroid that inhibits glucocorticoid receptor (GR), the receptor for corticosterone — inhibits entry of the hair follicle into anagen 9 , and overexpression of GR at the embryonic stage leads to underdeveloped hair follicles 10 .
  • adrenalectomy which removes the source of corticosterone — accelerates hair growth in rats, rabbits and minks 11 13 .
  • Some aspects of the disclosure are related to methods of modulating hair growth or methods of modulating hair follicle stem cell (HFSC) activation in an individual in need thereof.
  • the methods comprise promoting or increasing hair growth.
  • the methods comprise inhibiting or decreasing hair growth.
  • Some aspects of the disclosure are directed to methods of promoting or increasing hair growth in an individual in need thereof comprising administering to the individual an effective amount of an agent that increases Gas6 expression.
  • the agent increases the Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway.
  • TAM interaction or pathway is an AXL interaction or pathway, a Tyro3 interaction or pathway, and/or a Mertk interaction or pathway.
  • the agent suppresses BMP signaling, e.g., the agent is noggin.
  • the agent increases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl .
  • the agent is administered using an AAV vector, e.g., AAV8.
  • the agent is administered through intradermal injection.
  • hair growth is increased by at least 10%, 15%, 20%, or 25% relative to a suitable control.
  • Some aspects of the disclosure are directed to methods of decreasing hair growth in an individual in need thereof comprising administering to the individual an effective amount of an agent that decreases or inhibits Gas6 expression.
  • the agent decreases the Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway.
  • TAM interaction or pathway is an AXL interaction or pathway, a Tyro3 interaction or pathway, and/or a Mertk interaction or pathway.
  • the agent comprises an AXL inhibitor, e.g., the agent is R428.
  • the agent activates BMP signaling.
  • the agent decreases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl.
  • the agent is administered using an AAV vector (e.g., AAV8).
  • the agent is administered through intradermal injection.
  • hair growth is decreased by at least 10%, 15%,
  • RNAi RNA interference
  • FIGS. 1A-1F demonstrate that removal of adrenal glands activates HFSCs.
  • FIG. 1A shows sham and ADX mice were shaved and monitored for hair coat recovery. Quantifications represent the percentage of back skin that is covered by regrown hairs.
  • FIG. IB shows 5-Ethynyl-2'-deoxyuridine (EdU) and P-cadherin (PC AD) staining in hair follicles from sham and ADX mice (P49-P58) with quantifications. Yellow dashed lines, bulge; white dashed lines, hair germ or hair bulb (HB); solid lines, DP.
  • FIG. 1A shows sham and ADX mice were shaved and monitored for hair coat recovery. Quantifications represent the percentage of back skin that is covered by regrown hairs.
  • FIG. IB shows 5-Ethynyl-2'-deoxyuridine (EdU) and P-cadherin (PC AD) staining in hair follicles
  • FIG. 1C show EdU and CD34 staining in late anagen (AnaV) (sham, PI 10; ADX, P62) and mid catagen (CatV) (sham, P122; ADX, P74). Yellow dashed lines indicate the bulge (Bu), white dashed lines indicate the rest of the hair follicle, ORSup indicates the upper outer root sheath.
  • FIG. ID shows hair cycle length in sham and ADX mice.
  • FIG. IE provides the number of hair cycles of sham and ADX mice from P60 to P513 (see also FIG. 5).
  • FIG. IF provides representative hair regrowth status of 22-month-old sham and ADX mice. Telo, telogen; Ana, anagen; Cat, catagen.
  • FIGS. 2A-2D demonstrate corticosterone derived from the adrenal gland regulates HFSC quiescence.
  • FIG. 2A provides plasma corticosterone levels in different mice 2 weeks after feeding corticosterone (CORT) or vehicle (Veh).
  • FIG. 2B shows hair cycle progression (left) and haematoxylin and eosin (H&E) staining (right) of skin from sham+veh, ADX+veh and ADX+CORT mice. CORT, corticosterone. Scale bar, 50 pm.
  • FIG. 2C shows hair cycle progression of C57BL/6 mice fed with vehicle or corticosterone.
  • FIG. 2D shows hair cycle progression in C57BL/6 mice subjected to chronic unpredictable stress and in non-stressed control mice.
  • FIGS. 3A-3G demonstrate corticosterone acts on the DP to regulate HFSC quiescence.
  • FIG. 3A shows Pdgfra-CreER depletes GR efficiently in the DP and in dermal fibroblasts (DF).
  • FIG. 3B shows hair cycle progression of control and Pdgfra- CreER;GR fl/fl mice.
  • FIG. 3C provides immunohistochemical analyses (YFP and PCAD) of skin from Sox2-CreER;R26-lsl-YFP mice, showing the presence of YFP in the DP of guard hair follicles but not zigzag hair follicles.
  • FIG. 3A shows Pdgfra-CreER depletes GR efficiently in the DP and in dermal fibroblasts (DF).
  • FIG. 3B shows hair cycle progression of control and Pdgfra- CreER;GR fl/fl mice.
  • FIG. 3C provides immunohistochemical analyses (YFP and
  • FIG. 3D provides H&E staining of skin from control and Sox2-CreER;GR fl/fl mice.
  • the arrowhead indicates an anagen guard hair follicle surrounded by telogen hair follicles.
  • FIG. 3E provides surface view (side (left) and top (right)) showing accelerated anagen only in the guard hairs of the Sox2-CreER;GR fl/fl mice (shaved at P45; imaged at P67).
  • FIG 3F shows RNA-seq workflow.
  • FIG. 3G provides Gene Ontology enrichment analysis of 121 shared differentially expressed genes in HFSCs comparing sham vs. ADX, ADX vs. ADX+CORT, and control vs.
  • FIGS. 4A-4E demonstrate that Gas6 overexpression counteracts the inhibitory effect of corticosterone.
  • FIG. 4A provides Gas6 expression visualized by in situ hybridization in early telogen of sham, ADX, and ADX+CORT mice, and negative control expression in sham mice. Quantification shows Gas6 signals in the DP. Bold dashed lines, bulge; thin dashed lines, hair germ; solid lines, DP.
  • FIG. 4B shows cultured HFSCs in the presence or absence of GAS6.
  • FIG. 4C shows intradermal injection of AAV-CAG-Gas6, but not AAV-CAG-GFP, induces anagen in injection sites. Dashed circles indicate AAV injection areas.
  • FIG. 4A provides Gas6 expression visualized by in situ hybridization in early telogen of sham, ADX, and ADX+CORT mice, and negative control expression in sham mice. Quantification shows Gas6 signals in the DP. Bold dashed lines, bulge;
  • FIGS. 4D provides AAV-mediated expression of GFP or Gas6 in mice subjected to chronic unpredictable stress or corticosterone feeding.
  • FIGS. 4E provides a model summarizing the main findings of this study. When corticosterone levels decrease, an increase in Gas6 expression promotes HFSC activation and anagen entry. Conversely, when corticosterone levels are increased, Gas6 expression is inhibited and HFSCs stay in prolonged quiescence. Scale bar (FIGS. 4A, 4C), 50 pm. Data are mean + s.e.m. *P ⁇ 0.05, ****P ⁇ 0.0001. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
  • FIGS. 5A-5L demonstrate hair cycle progression in ADX mice over time.
  • FIG. 5A shows hair cycle with immunohistochemical analyses (PCAD) in sham and ADX mice.
  • FIG. 5B shows hair cycle progression in sham male and ADX male mice.
  • FIG. 5C provides a schematic depicting HFSCs in anagen and telogen. The upper ORS of anagen hair follicles contributes to the new bulge and hair germ (HG) of the following telogen hair follicles. See refs. 14 15 for details.
  • FIG. 5D provides the ORS length in the zigzag hairs of sham (PI 13) and ADX (P65) mice during late anagen. The brackets indicate the ORS length below the bulge.
  • FIG. 5A shows hair cycle with immunohistochemical analyses (PCAD) in sham and ADX mice.
  • FIG. 5B shows hair cycle progression in sham male and ADX male mice.
  • FIG. 5C provides a schematic
  • FIG. 5E provides the hair shaft length of each hair subtype in sham and ADX mice after anagen.
  • FIG. 5F shows H&E staining at P65 of skin from sham and ADX mice.
  • FIG. 5G shows immunohistochemical analyses (Sox9 and CD34) in telogen (telo), late anagen (AnaV), and mid catagen (CatV) hair follicles. Yellow dashed lines, bulge; white dashed lines, HG (telo), hair follicle (AnaV, CatV); solid line, DP.
  • telogen telo
  • AnaV late anagen
  • CatV mid catagen
  • FIG. 51 Left shows H&E staining in the late anagen skin of sham and ADX mice with quantification of the epidermal thickness (E).
  • FIG. 5J shows representative hair regrowth status of sham and ADX mice from P60 to P549.
  • FIG. 5K shows duration of telogen in sham and ADX mice.
  • FIG. 5L provides H&E staining of skin from young sham, aged sham, and aged ADX mice with quantification of the number of hair follicles per mm. Yellow dashed lines, bulge; white dashed lines, HG, solid lines, DP. Telo, telogen; Ana, anagen; Cat, catagen. Scale bars, 50 pm (FIGS. 5A, 5D, 5F-5I, 5L), 1 mm (FIG. 5E). Data are mean ⁇ s.e.m. *P ⁇ 0.05, ****P ⁇ 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
  • FIGS. 6A-6M demonstrate corticosterone restores normal hair cycle progression in ADX mice.
  • FIG. 6A provides H&E staining of the skin of 21 -month- old sham and ADX mice.
  • FIG. 6B shows morphology of each hair subtype from the skin of 18-month-old sham and ADX mice.
  • FIG. 6C Left provides immunohistochemical analyses (CD34 and PCAD) of telogen hair follicles in the skin of sham and ADX mice at 22 months old, showing normal hair follicle morphology and comparable stem cell numbers.
  • FIG. 6C Middle provides quantification of the number of bulge and hair germ cells per HF.
  • FIG. 6C Right shows the percentage of HFSCs in epithelial fraction by FACS.
  • FIG. 6D shows hormones from the adrenal gland and plasma levels of corticosterone in P45 sham and ADX mice.
  • FIG. 6E shows plasma levels of noradrenaline and adrenaline measured by LC-MS/MS at P45 (10 days after surgery) in sham and ADX mice.
  • FIG. 6F Left provides an experimental design to test if supplying corticosterone rescues ADX phenotypes.
  • FIG. 6F Right shows hair cycle progression of sham mice fed with vehicle (sham+veh) or ADX mice fed with corticosterone (ADX+CORT).
  • FIG. 6G provides plasma corticosterone levels at P62 in C57BL/6 mice after a week’s feeding with vehicle or corticosterone.
  • FIG. 6H Top provides experimental design for 3 days of corticosterone feeding.
  • FIG. 6H Bottom shows the percentage of hair regrowth of the back skin at P38.
  • FIG. 61 shows hair cycle progression of C57BL/6 male mice fed with vehicle or corticosterone. Corticosterone feeding prolonged telogen as long as corticosterone was provided to the mice (both male and female).
  • FIG. 6J shows body weight of C57BL/6 mice fed with vehicle or corticosterone from P83 to PI 18.
  • FIG. 6K Left provides H&E staining in the skin of vehicle and corticosterone-fed mice.
  • FIG. 6K, Middle and right provide quantification of the thickness of dermis (middle) and dermal adipose layer (right).
  • FIG. 6L provides immunohistochemical analysis (active caspase 3 (aCAS3) and PCAD) in vehicle- and corticosterone-fed mice. Dashed lines, epidermis and hair follicles.
  • FIG. 6M Left provides experimental design to test the effect of corticosterone withdrawal.
  • FIG. 6M Right shows hair cycle progression of C57BL/6 mice after completion of 3 weeks of vehicle or corticosterone feeding. Scale bars (a, b, c, k, 1), 50 pm. Data are mean ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
  • FIGS. 7A-7F demonstrate removal of the adrenal glands in stressed or aged mice leads to hair follicle regeneration.
  • FIG. 7A provides plasma corticosterone levels at P62 in non-stressed control and stressed mice.
  • FIGS. 7B-7C show H&E staining (FIG. 7B) and immunohistochemical analyses (active caspase3 (aCAS3) and PCAD) (FIG. 7C) in control and stressed mice. Dashed lines, epidermis and hair follicles.
  • FIG. 7D shows stressed sham (sham+stress) and stressed ADX (ADX+stress) mice were monitored for hair coat recovery. Quantification shows the percentage of back skin that is covered by newly regenerated hairs.
  • FIG. 7E provides plasma levels of corticosterone in young mice (P46, P77, and P98) and aged mice (P427 and P581).
  • FIG. 7F shows sham and ADX operations were performed on aged mice (P521). The mice were shaved and monitored for hair coat recovery from P521 to P574. Scale bars (FIGS. 7B-7C), 50 mih. Data are mean ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ 0.01,
  • FIGS. 8A-8H demonstrate GR depletion in different cell types in the skin.
  • FIG. 8A Top shows K15-CrePGR depletes GR efficiently in HFSCs.
  • FIG. 8A Bottom shows immunohistochemical analysis (GR and CD 140a) of telogen hair follicle in the skin of control and K 15-CrcPGR;GR ll/fl mice.
  • FIG. 8B shows hair cycle progression of control and K15-CrePGR;GR fl/fl mice.
  • FIG. 8C shows immunohistochemical analyses (GR and PCAD) of telogen hair follicles in the skin of control and Pdgfra-CreER;GR fl/fl mice, showing that Pdgfra-CreER depletes GR efficiently in the dermal fibroblasts and DP.
  • FIG. 8A Top shows K15-CrePGR depletes GR efficiently in HFSCs.
  • FIG. 8A Bottom shows immunohistochemical analysis (GR and CD 140a) of telogen hair follicle in the skin of control and K
  • FIG. 8D shows immunocolocalization (EdU and CD34) in control and Pdgfra-CrcER;GR ll/fl hair follicles after tamoxifen administration.
  • EdU incorporation reveals premature HFSC activation in the hair follicles of Pdgfra-CreER;GR fl/fl mice.
  • FIG. 8E provides a comparison of EdU localization in bulge and upper ORS in late anagen (AnaV) of control (PI 24) and Pdgfra-CreER;GR fl/fl (P73) mice.
  • FIG. 8F shows representative hair regeneration status of control and Pdgfra-CreER;GR fl/fl mice from P73 to P205, with quantification of the number of hair cycles.
  • FIG. 8G shows immunocolocalization (EdU and CD34) in infundibulum (IF), junctional zone (JZ), sebaceous gland (SG), mid ORS (ORSmid), lower ORS (ORSlow), and matrix (Mx) of late anagen (AnaVI) hair follicles in control and Pdgfra-CrcER;GR ll/fl mice during late anagen with quantifications.
  • FIG. 8G shows immunocolocalization (EdU and CD34) in infundibulum (IF), junctional zone (JZ), sebaceous gland (SG), mid ORS (ORSmid), lower ORS (ORSlow), and matrix (Mx) of late anagen (AnaVI) hair follicles in control and Pdgfra-CrcER
  • FIG. 8H Top shows H&E staining in the late anagen skin of control and Pdgfra-CreERiGR ⁇ mice, with quantification of the thickness of epidermis (E).
  • FIG. 8H Bottom shows immunocolocalization (EdU and DAPI) in interfollicular epidermis (IFE) and dermis in control and Pdgfra-CreER;GR fl/fl mice. Scale bars (FIGS. 8A, 8C-8E, 8G-8H), 50 pm. Yellow dashed lines, bulge (FIG. 8A, 8C-8E); white dashed lines, hair germ (FIG. 8A, 8C, 8D), the rest of hair follicles (FIG.
  • FIGS. 9A-9G demonstrate corticosterone acts on the DP.
  • FIG. 9 A provides RT-qPCR of GR from DP and DF.
  • FIG. 9B provides immunohistochemical analyses (YFP and DAPI) of anagen skin from Sox2-CreER;R26-lsl-YFP mice. Left, the arrowhead indicates an anagen guard hair follicle with YFP+ DP cells. Right, quantification of the percentage of YFP + and YFP- DP in Sox2-CreER;R26-lsl-YFP. Only guard hair follicles have YFP + DP.
  • FIG. 9 A provides RT-qPCR of GR from DP and DF.
  • FIG. 9B provides immunohistochemical analyses (YFP and DAPI) of anagen skin from Sox2-CreER;R26-lsl-YFP mice. Left, the arrowhead indicates an anagen guard hair follicle with YFP+ DP
  • FIG. 9C provides immunohistochemical analyses (GR and DAPI) of skin from control and Sox2-CreER;GR fl/fl mice. Dashed lines, epidermis and hair follicles; solid line, DP. The arrowhead indicates the DP of Sox2-CreER;GR fl/fl guard hairs.
  • FIG. 9D shows representative hair regeneration status of control and Sox2-CreER;GR fl/fl mice from P45 to P160. Quantification shows the number of hair cycles for guard hairs and other hairs in control and Sox2- CreER;GR fl/fl mice.
  • FIG. 9E shows comparison of the hair bulb diameter in late anagen (AnaV) in the skin of control (P120) and Sox2-CreER;GR fl/fl (P67) mice. Yellow lines indicate the hair bulb diameter.
  • the arrowhead denotes minor hyper thickening of the Sox2-CreER;GR fl/fl hair follicle around the ORS, probably because the dermis has not expanded to accommodate the extra proliferation from HFSCs.
  • FIG. 9F shows immunocolocalization of phosphohistone H3 (pHH3) and CD34 in bulge and upper ORS, middle ORS (ORSmid), lower ORS (ORSlow) and matrix (Mx) of late anagen (AnaV) guard hair follicles in control and Sox2-CreER;GR fl/fl mice.
  • the white arrowhead denotes a thickened region in a Sox2-CreER;GR fl/fl hair follicle, probably due to excessive proliferation from HFSCs. Yellow dashed lines, bulge; white dashed lines, the rest of the hair follicle.
  • FIG. 9G shows hair shaft length of guard hairs in control and Sox2-CreER;GR fl/fl mice after anagen. Scale bars, 50 pm (FIGS. 9B, 9C, 9E, 9F), 1 mm (FIGS. 9D, 9G). Data are mean ⁇ s.e.m.
  • FIGS. 10A-10F demonstrate differential gene expression in HFSCs of control, ADX and dermal GR-knockout mice.
  • FIG. 10A shows sample clustering based on Pearson’s correlation of transcriptomes in HFSCs from sham, ADX, and ADX+CORT as well as control and Pdgfra-CreER;GR fl/fl mice.
  • FIG. 10B provides principal component analysis (PCA) comparing the transcriptome of HFSCs from sham, ADX, ADX+CORT, control and Pdgfra-CreERiGR ⁇ mice.
  • PCA principal component analysis
  • IOC provides heat map of log2 fold change of gene expression of 121 common genes among ADX (versus sham), Pdgfra-CreER;GR fl/fl (versus control), and ADX+CORT (versus ADX). Cell-cycle-related genes are noted in orange.
  • FIG. 10D Left provides a heat map of log2 fold change of gene expression of transcription factors (Foxcl, Lhx2, Foxpl, Nfatcl), key signalling factors (Fgfl8), or downstream readout of key signalling factors (Idl for BMP pathway, Axin2 for WNT pathway, Glil for SHH pathway) known to regulate HFSC quiescence.
  • FIG. 10D Left provides a heat map of log2 fold change of gene expression of transcription factors (Foxcl, Lhx2, Foxpl, Nfatcl), key signalling factors (Fgfl8), or downstream readout of key signalling factors (Idl for BMP pathway, Axin2 for WNT pathway, Glil for
  • FIGS. 10E-10F provide RT-qPCR of genes related to cell-cycle machineries and cytokinesis from telogen HFSCs of sham and ADX mice (FIG. 10E) and control and Pdgfra-CreER;GR fl/fl mice (FIG. 10F).
  • Data are mean ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Source Data For statistics, sample sizes and numbers of replications, see Methods.
  • FIGS. 11A-11J demonstrate the expression of cell-cycle -related genes in HFSCs.
  • FIGS. 1 lA-1 IB show RT-qPCR of genes related to cell-cycle machineries and cytokinesis using telogen HFSCs from vehicle and corticosterone-fed mice (FIG. 11A) and control and stressed mice (FIG. 11B).
  • FIGS. 11C-11D show RT-qPCR of genes related to cell-cycle machineries and cytokinesis in telogen epidermis from sham and ADX mice (FIG. 11C) and vehicle and corticosterone-fed mice (FIG. 11D).
  • FIG. 1 lA-1 IB show RT-qPCR of genes related to cell-cycle machineries and cytokinesis using telogen HFSCs from vehicle and corticosterone-fed mice (FIG. 11A) and control and stressed mice (FIG. 11B).
  • FIGS. 11C-11D show RT-qPCR of genes related
  • FIGS. 11E-11E show experimental workflow of the differentially expressed genes (DEGs, > 1.5-fold, Padj ⁇ 0.05) from DP cells of sham and ADX mice, as well as control and Pdgfra-CreERjGR ⁇ mice.
  • FIGS. 11F-11G show immunohistochemical analysis (PCAD) of skin samples from sham and ADX (FIG. 1 IF) or control and Pdgfra- CreER;GR fl/fl (FIG. 11G) mice used in RNA-seq experiments to validate hair cycle (all telogen). Dashed lines, epidermis and hair follicles.
  • FIG. 11H Top shows FACS strategies for isolating DP cells for RNA-seq 24,44 .
  • FIG. 11H Bottom shows the expression levels of cell-type-specific signature genes (DP, fibroblasts, HFSCs and mast cells) in FACS-purified DP cells.
  • TPM transcripts per million.
  • FIG. 1 II shows sample clustering based on Pearson’s correlation of transcriptomes in DP of sham and ADX mice (left), as well as control and Pdgfra-CreER;GR fl/fl mice (right).
  • FIG. 11 J provides heat maps of the differentially expressed genes (DEGs, > 1.5-fold,
  • Padj ⁇ 0.05 from FACS-purified DP cells of sham and ADX mice (left) or control and Pdgfra-CreERjGR ⁇ mice (right). Scale bars (FIGS. 11F-11G), 50 pm. Data are mean ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
  • FIGS. 12A-12H demonstrate transcriptome analysis and secretome analysis identified GAS6 as a secreted factor suppressed by systemic corticosterone in the DP.
  • FIG. 12A shows secretome analysis identifying common secreted factors from DEGs (> 1.5-fold, Padj ⁇ 0.05) in ADX and Pdgfra-CreER;GR fl/fl DP cells identified by RNA- seq.
  • FIGS. 12B-12C show expression levels of shared differentially expressed secreted factors as TPM, in the DP cells of ADX (FIG. 12B) and Pdgfra-CrcER;GR ll/fl (FIG. 12C) mice.
  • FIG. 12A shows secretome analysis identifying common secreted factors from DEGs (> 1.5-fold, Padj ⁇ 0.05) in ADX and Pdgfra-CreER;GR fl/fl DP cells identified by RNA- seq.
  • FIGS. 12B-12C show
  • FIG. 12D Top shows negative control and Gas6 mRNA expression by in situ hybridization in late anagen (AnaV) and mid catagen (CatV) skin of sham and ADX mice.
  • FIG. 12D Bottom shows quantification of Gas6 mRNA in the DP. Dashed lines, hair follicle; solid lines, DP.
  • FIG. 12E provides a representative image of negative control and Axl mRNA expression by in situ hybridization in telogen skin. Yellow dashed lines: bulge; white dashed lines: hair germ (top). RT-qPCR of Axl, Tgfbrl, Bmprla, Nfatcl and PPIB from HFSCs and epidermal stem cells (EpSCs) of control mice (P83) (bottom).
  • FIG. 12F provides representative images of negative control and Axl mRNA expression by in situ hybridization in late anagen skin.
  • FIG. 12G shows the expression levels (as TPM) of genes encoding TAM receptors (Tyro3, Axl and Mertk) in HFSCs.
  • FIG. 12H Feft provides a schematic of the GAS6-AXF receptor tyrosine kinase pathway.
  • R428 is a selective inhibitor of AXF tyrosine kinase activity 43 .
  • FIG. 12H Right shows colony-formation assays of cultured HFSCs in R428 or GAS6 with R428 with quantifications. Scale bars (d-f), 50 pm. Data are mean ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
  • FIGS. 13 A- 13 J demonstrate analyses of skin changes upon Gas6 overexpression or treatment with an AXF inhibitor.
  • FIG. 13A shows immunohistochemical analysis (GFP and PCAD) of PBS -injected second telogen skin and AAV-GFP-injected second telogen skin. Dashed lines, epidermis and hair follicles; solid lines: DP.
  • FIG. 13B provides RT-qPCR of Gas6 from dermal fibroblasts of PBS -injected second telogen skin (control) and AAV-CAG-Gas6- injected second telogen skin.
  • FIG. 13C shows precocious HFSC activation in mice injected with AAV-CAG-Gas6 shown by EdU incorporation.
  • FIG. 13D shows comparison of EdU and CD34 localization in bulge and upper outer root sheath (ORS) in late anagen (AnaV) (control, D50 after injection; GAS6, D17 after injection).
  • FIG 13E provides H&E staining of late anagen (AnaVI) skin (control, D53 after injection; GAS6, D20 after injection). Quantification of the ORS length in the zigzag hairs of control and AAV-CAG-Gas66-injected mice during late anagen. Brackets indicate the ORS length below the bulge.
  • FIG. 13F shows immunocolocalization (EdU and CD34) in infundibulum (IF), junctional zone (JZ), sebaceous gland (SG), mid ORS (ORSmid), lower ORS (ORSlow) and matrix (Mx) of late anagen (AnaVI) hair follicles in control and AAV-CAG-Gas6-injected mice with quantifications. Dashed lines outline hair follicles.
  • FIG. 13G, Top shows H&E staining in the late anagen skin of control and AAV-CAG-Gas6-injected mice with quantification of the thickness of epidermis (E).
  • FIG. 13G Bottom shows immunocolocalization (EdU and DAPI) in interfollicular epidermis (IFE) and dermis in control and AAV-CAG-Gas6-injected mice with quantifications.
  • FIG. 13H provides RT-qPCR of genes related to HFSC proliferation in HFSCs of second telogen skin.
  • FIG. 131 shows hair cycle progression of sham and ADX mice treated with ethanol topically, or ADX mice treated with R428 in ethanol.
  • FIG. 13 J shows RT-qPCR of genes related to cell-cycle machineries and cytokinesis from HFSCs of sham and ADX mice treated with ethanol topically, or ADX mice treated for 7 days with R428 (in ethanol) topically.
  • FIGS. 14A-14G demonstrate interactions between systemic corticosterone and local BMP signalling.
  • FIG. 14A shows in situ hybridization of negative control and Gas6 mRNA expression in different conditions, including P49 (early telogen) of sham mice, P80 (late telogen) of sham mice, P49 of ADX mice, P49 of in sham mice injected with AAV-CAG-noggin (noggin in sham), and P49 in ADX mice after injection of AAV-CAG-noggin (noggin in ADX). Quantifications show in situ hybridization signal intensities in the DP. The model shows how changes in corticosterone and BMP signalling influence Gas6 levels in the DP.
  • FIG. 14A shows in situ hybridization of negative control and Gas6 mRNA expression in different conditions, including P49 (early telogen) of sham mice, P80 (late telogen) of sham mice, P49 of ADX mice, P49 of in
  • FIG. 14B provides RT-qPCR of Gas6 from the DP of P49 (early telogen) and P80 (late telogen) of sham mice, and P49 of ADX mice, noggin in sham mice, and noggin in ADX mice.
  • FIG. 14C shows hair cycle progression in sham, ADX, noggin in sham, and noggin in ADX mice with quantifications. Dashed circles indicate AAV-CAG-noggin injection areas.
  • FIG. 14D shows control and Pdgfra-CreERiGR ⁇ mice were subjected to chronic unpredictable stress from P55. Quantification shows the percentage of hair regrowth at P107.
  • FIG. 14C shows hair cycle progression in sham, ADX, noggin in sham, and noggin in ADX mice with quantifications. Dashed circles indicate AAV-CAG-noggin injection areas.
  • FIG. 14D shows control and Pdgfra-CreERiGR ⁇ mice were subject
  • FIG. 14E provides RT-qPCR of Gas6 from DP cells of control (P83) and stressed (P83) mice (left), and vehicle (P83) and corticosterone-fed (P83) mice (right).
  • FIG. 14F shows in situ hybridization of Gas6 in vehicle (P83, late telogen) and corticosterone-fed (P83) mice, with quantification of in situ signals in DP.
  • FIG. 14G provides a model of corticosterone regulation of telogen length. In normal conditions, corticosterone levels remain constant, but BMP levels naturally decrease as telogen progresses, until a point is reached at which Gas6 levels are sufficiently increased so as to drive HFSCs out of quiescence.
  • This dynamic can be altered by changing either the corticosterone level or the BMP level. If corticosterone levels decrease, the sum of inhibitory cues on Gas6 falls below a critical threshold sooner in telogen, leading to increased Gas6 levels and precocious anagen entry. In the case of stress, age, or corticosterone feeding, increased corticosterone levels reduce Gas6 levels to below the critical threshold, leading to an extended telogen. Dashed lines, hair follicles; solid lines, DP. Scale bars (a, f), 50 pm. Data are mean ⁇ s.e.m. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
  • TAM Gas6-Tyro3/Axl/Mertk
  • aspects of the disclosure are related to methods of modulating hair growth and HFSC activation in an individual in need thereof. This includes administering an agent that modulates a Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway (e.g., turns the pathway on or off).
  • TAM Gas6-Tyro3/Axl/Mertk
  • the TAM interaction or pathway is an AXL, a Tyro3, or a Mertk interaction or pathway.
  • modulate is used consistently with its use in the art, i.e., meaning to cause or facilitate a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of a process, pathway, or phenomenon.
  • a “modulator” is an agent that causes or facilitates a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.
  • agent means any compound or substance such as, but not limited to, a small molecule, nucleic acid (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, polypeptide, peptide, drug, ion, etc.
  • nucleic acid e.g., RNAi agents, antisense oligonucleotide, aptamers
  • lipids e.g., RNAi agents, antisense oligonucleotide, aptamers
  • polysaccharides e.g., polypeptide, peptide, drug, ion, etc.
  • an “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-pro teinaceous entities.
  • an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc.
  • agents are small molecules having a chemical moiety.
  • chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof.
  • agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • agents may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the agent.
  • An agent may be at least partly purified.
  • an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments.
  • an agent may be provided as a salt, ester, hydrate, or solvate.
  • an agent is cell-permeable, e.g., within the range of typical agents that are taken up by cells and act intracellularly, e.g., within mammalian cells, to produce a biological effect.
  • Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z- isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)- isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated.
  • Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable.
  • “decrease”, “reduced”, “reduction”, “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
  • “reduced”, “reduction”, “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • the terms “increased”, “increase”, “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase”, “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • statically significant refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker.
  • 2SD two standard deviation
  • concentration of the marker refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
  • the agent modulates Gas6 activity or expression. In some embodiments, the agent modulates the interaction of Gas6 with theTyro3/Axl/Mertk (TAM) pathway. In some embodiments, the agent modulates the interaction of Gas6 with AXL. In some embodiments, the agent modulates the interaction of Gas6 with Tyro3. In some embodiments, the agent modulates the interaction of Gas6 with Mertk. In some embodiments, the agent (e.g., a chemical agent) modulates the Gas6-AXL pathway.
  • TAM Tyro3/Axl/Mertk
  • the agent increases Gas6 activity or expression. In some embodiments, the agent increases Gas6 activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, e.g., relative to a suitable control. In some aspects, the agent increases Gas6 activity by at least 5%. In some aspects, the agent increases Gas6 activity by at least 10%. In some aspects, the agent increases Gas6 activity by at least 15%. In some aspects, the agent increases Gas6 activity by at least 20%. In certain aspects, the agent increases Gas6 activity by at least 25%.
  • the agent decreases Gas6 activity or expression. In some embodiments, the agent decreases Gas6 activity by at least 5%, 10%, 15%,
  • the agent decreases Gas6 activity by at least 5%. In some aspects, the agent decreases Gas6 activity by at least 10%. In some aspects, the agent decreases Gas6 activity by at least 15%. In some aspects, the agent decreases Gas6 activity by at least 20%. In certain aspects, the agent decreases Gas6 activity by at least 25%.
  • the agent increases hair growth or HFSC activation. In certain aspects, the agent increases hair growth. In certain aspects, the agent increases HFSC activation. In some embodiments, the agent increases hair growth or HFSC activation relative to a suitable control. In some embodiments, the agent increases hair growth or HFSC activation by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%. In some aspects, the agent increases hair growth or HFSC activation by at least 5%. In some aspects, the agent increases hair growth or HFSC activation by at least 10%. In some aspects, the agent increases hair growth or HFSC activation by at least 15%. In some aspects, the agent increases hair growth or HFSC activation by at least 20%. In certain aspects, the agent increases hair growth by at least 25%. In certain embodiments, the agent increases HFSC activation by at least 25%.
  • the agent decreases hair growth or HFSC activation. In certain aspects, the agent decreases hair growth. In certain aspects, the agent decreases HFSC activation. In some embodiments, the agent decreases hair growth or HFSC activation relative to a suitable control. In some embodiments, the agent decreases hair growth or HFSC activation by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%. In some aspects, the agent decreases hair growth or HFSC activation by at least 5%. In some aspects, the agent decreases hair growth or HFSC activation by at least 10%. In some aspects, the agent decreases hair growth or HFSC activation by at least 15%. In some aspects, the agent decreases hair growth or HFSC activation by at least 20%. In certain aspects, the agent decreases hair growth by at least 25%. In certain embodiments, the agent decreases HFSC activation by at least 25%.
  • the agent modulates AXL activity or expression. In some embodiments, the agent increases AXL activity or expression. In some embodiments, the agent increases Gas6 activity or expression. In some embodiments, the agent inhibits bone morphogenetic protein (BMP) signaling.
  • BMP signaling inhibitors include noggin, chordin, gremlin, follistatin, sclerostin, and chordin. In one embodiment, a BMP signaling inhibitor comprises noggin.
  • the agent increases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl . In some embodiments, the agent increases expression of one or more genes selected from the group consisting of Ccnbl , Rad51, and Cdkl .
  • the agent decreases AXL activity or expression.
  • AXL inhibitors include R428 or BGB324, crizotinib, bosutinib, cabozantinib, sunitinib, foretinib, merestinib, and glesatinib.
  • an AXL inhibitor comprises R428.
  • the agent inhibits or decreases Gas6 activity or expression.
  • the agent decreases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl.
  • the agent decreases expression of one or more genes selected from the group consisting of Ccnbl, Rad51, and Cdkl.
  • the agent is administered using a delivery system.
  • An acceptable delivery system may be a delivery system known to those of skill in the art.
  • the delivery system is a viral vector delivery system.
  • the viral vector delivery system comprises adenoviruses, adeno- associated viruses, retroviruses (e.g., lentiviruses), vaccinia virus and other poxviruses, herpesviruses (e.g., herpes simplex vims), or others.
  • the delivery system comprises an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • an AAV has an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Anc80, or PHP.eB. Any AAV serotype, or modified AAV serotype, may be used as appropriate and is not limited. In certain embodiments, the AAV is AAV8.
  • a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap.
  • a packaging cell line that stably supplies rep and cap is transfected (transiently or stably) with a construct encoding the transgene flanked by ITRs.
  • AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus.
  • helper vims i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase
  • helper functions i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase
  • helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level.
  • the transgene flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors.
  • Some embodiments of the present invention relate to methods of treatment or prevention for a disease or condition, such as a skin or hair condition, disease, or disorder, by the delivery of a pharmaceutical composition comprising an effective amount of the agents described herein.
  • An effective amount of the pharmaceutical composition is an amount sufficient to prevent, slow, inhibit, or ameliorate a disease or disorder in a subject to whom the composition is administered.
  • the methods stimulate or promote hair growth or HFSC activation. In some embodiments, hair growth or HFSC activation is stimulated or promoted by increasing Gas6 expression or activation. In some embodiments, hair growth or HFSC activation is stimulated or promoted by decreasing or inhibiting BMP signaling. In some embodiments, the methods comprise administering an agent that increases Gas6 expression or activation. In some embodiments, the methods comprise administering an agent that inhibits or suppresses BMP signaling (e.g., noggin). In some embodiments, the methods comprise administering an AXL activator.
  • the methods decrease or inhibit hair growth or HFSC activation. In some embodiments, hair growth or HFSC activation is decreased or inhibited by decreasing Gas6 expression. In some embodiments, the methods comprise administering an agent that decreases Gas6 expression or activation. In some embodiments, the methods comprise administering an AXL inhibitor (e.g., R428). In some embodiments, the methods comprise administering an activator of BMP signaling.
  • the method increases hair growth or HFSC activation under stress conditions.
  • the stress condition is characterized by elevated corticosterone or hair loss such as a hair loss condition.
  • the hair loss condition is telogen effluvium.
  • telogen effluvium refers to a form of temporary hair loss that may occur after stress, a shock, or a traumatic event and occurs on the top of the scalp.
  • a delivery system comprising an agent is administered to a subject.
  • the terms “subject” and “individual” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and/or to whom treatment, including prophylactic treatment, with the cells as described herein, is provided.
  • the term subject refers to that specific animal.
  • subject also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish.
  • the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like.
  • a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like.
  • fur growth may be modulated (i.e., increased or decreased) by modulating the Gas6/AXL pathway.
  • the terms “treat,” “treatment” or “treating”, in reference to a subject includes amelioration, cure, and/or maintenance of a cure (i.e., the prevention or delay of relapse and/or reducing the likelihood of recurrence) of a disorder.
  • Treatment after a disorder has started aims to reduce, ameliorate or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, to slow the rate of progression, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse).
  • Treating encompasses administration of an agent that may not have an effect on the disorder by itself but increases the efficacy of a second agent administered to the subject.
  • a suitable dose and therapeutic regimen may vary depending upon the specific agent used, the mode of delivery of the compound, and whether it is used alone or in combination.
  • agents disclosed herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
  • routes including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
  • Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracranial, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
  • the agent is administered through intradermal injection.
  • compositions and agents are formulated into solutions, suspensions, lotions, sprays, shampoos, hair conditions, serums, patches, wipes, gels, hydrogels, powders, patches, impregnated pads, emulsions, vesicular dispersions, sprays, aerosols, foams, ointments, tinctures, salves, gels, cleansing soaps, cleansing cakes, or creams as generally known in the art.
  • the formulation can be, e.g., in a multi-use or single-use applicator.
  • Topical administration can include the application of the pharmaceutical or cosmetic compositions to the scalp and/or hair.
  • an “effective amount” or “effective dose” of an agent refers to the amount sufficient to achieve a desired biological and/or pharmacological effect, e.g., when delivered to a cell or organism according to a selected administration form, route, and/or schedule.
  • the absolute amount of a particular agent or composition that is effective may vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc.
  • an “effective amount” may be contacted with cells or administered in a single dose, or through use of multiple doses, in various embodiments.
  • a biological effect may be, e.g., reducing expression or activity of one or more gene products, reducing activity of a metabolic pathway or reaction, or reducing cell proliferation or survival of cells.
  • a single additional agent or multiple additional agents or treatment modalities may be co-administered (at the same or differing time points and/or via the same or differing routes of administration and/or on the same or a differing dosing schedule).
  • the dosage, administration schedule and method of administering the agent are not limited.
  • a reduced dose may be used when two or more agents are administered in combination either concomitantly or sequentially.
  • a maximum tolerated dose may be used, that is, the highest safe and tolerable dose according to sound medical judgment.
  • compositions comprise one or more agents or compositions that have therapeutic utility, and a pharmaceutically acceptable carrier, e.g., a carrier that facilitates delivery of agents or compositions.
  • a pharmaceutically acceptable carrier e.g., a carrier that facilitates delivery of agents or compositions.
  • Agents and pharmaceutical compositions disclosed herein may be administered by any suitable means such as topically, orally, intranasally, intradermally, subcutaneously, intramuscularly, intravenously, intra-arterially, parenterally, intraperitoneally, intrathecally, intratracheally, ocularly, sublingually, vaginally, rectally, dermally, or as an aerosol.
  • the pharmaceutical compositions typically comprise a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid vehicles, fillers, diluents, or encapsulating substances which are suitable for administration to a human or non-human animal.
  • a pharmaceutically-acceptable carrier is a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • compatible means that the components of the pharmaceutical compositions are capable of being comingled with an agent, and with each other, in a manner such that there is no interaction which would substantially reduce the pharmaceutical efficacy of the pharmaceutical composition under ordinary use situations.
  • Pharmaceutically-acceptable carriers should be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the human or non-human animal being treated.
  • the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
  • the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum.
  • Numerical values include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”.
  • Approximately or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value).
  • HFSC hair follicle stem cell
  • corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth.
  • Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term.
  • both adrenal glands were surgically removed from C57BL/6 mice on post-natal day (P) 35, a time before mice enter an extended second telogen (FIG. 5A).
  • P post-natal day
  • the second telogen began at around P43-P45 and lasted on average 58 + 2 days (mean + s.e.m.).
  • ADX mice both male and female — had a significantly shorter telogen (around 11 days), with precocious activation starting from the hair germ (FIGS. 1A-1B, FIGS. 5A-5B).
  • HFSCs proliferate only in early anagen and quickly resume quiescence by mid-anagen 7 .
  • HFSCs located in the bulge and in the upper outer root sheath (ORS) 14,15 remained proliferative in late anagen (FIG. 1C, FIG. 5C).
  • ORS outer root sheath
  • hair follicles in the ADX mice were longer than those at the same stage of anagen in sham control mice (FIG. 5D).
  • the hair shaft in ADX mice also becomes longer, probably due to the enhanced HFSC proliferation, ADX mice did not display gross abnormalities in their skin and showed similar expression of HFSC markers to control mice (FIGS. 5E-5I).
  • telogen phases became progressively longer (second telogen, 58 + 2 days; third telogen, 75 + 2 days; fourth telogen, 95 + 4 days), and entry into anagen became sporadic and asynchronized (FIGS. 1D-1E, FIGS. 5J- 5K).
  • telogen phases became progressively longer (second telogen, 58 + 2 days; third telogen, 75 + 2 days; fourth telogen, 95 + 4 days), and entry into anagen became sporadic and asynchronized (FIGS. 1D-1E, FIGS. 5J- 5K).
  • hair follicles in ADX mice stayed in telogen for only about 2 weeks, and they repeatedly entered anagen in a synchronized fashion (FIG. 5J).
  • ADX mice Over an approximately 16-month period after adrenalectomy, ADX mice went through around 10 synchronized hair cycles in their back skin, whereas the hair follicles of sham mice entered anagen asynchronously and rarely, and only about 3 hair cycles were completed within 16 months (FIG. IE). Telogen became around 30 days long in 17- 18-month-old ADX mice, which is still significantly shorter than the length of the second telogen in young sham mice (which begins at around 1.5 months old), and is in sharp contrast to that of old sham mice at the same age 16,17 (FIG. 5K). At 18-22 months of age, ADX mice had a hair coat density similar to that of young mice, and showed robust anagen entry (FIG. IF, FIGS.
  • corticosterone The adrenal gland produces several hormones, including corticosterone, adrenaline (also known as epinephrine), noradrenaline (also known as norepinephrine) and aldosterone 18 (FIG. 6D).
  • the levels of each hormone were quantified and it was found that corticosterone showed the most substantial decrease — becoming barely detectable — in ADX mice compared with control mice (FIGS. 6D-6E).
  • Supplementation with corticosterone effectively suppressed the aberrant activation of HFSCs in ADX mice (FIGS. 2A-2B, FIG. 6F).
  • corticosterone secreted from the adrenal glands acts as a key systemic regulator to suppress anagen entry under normal physiological conditions. Increased corticosterone levels inhibit anagen
  • corticosterone levels were given supplementary corticosterone in their drinking water 19 , leading to enhanced levels of circulating corticosterone (FIG. 2C, FIG. 6G).
  • Transient increases in corticosterone levels had a minimal effect on the hair cycle (FIG. 6H), but long-term supplementation prolonged the telogen phase (FIG. 2C, FIG. 61).
  • Corticosterone-fed mice did not display obvious changes in weight, skin thickness or apoptosis (FIGS. 6J-6L). When corticosterone was removed, mice were able to enter anagen (FIG. 6M), which suggests that the effect of corticosterone on HFSCs is reversible.
  • Corticosterone acts on dermal papillae
  • Dermal fibroblasts are a heterogenous population that surrounds the HFSCs. Some fibroblast subpopulations, including the dermal papillae (DP) and adipocyte precursor cells, can regulate HFSC activity 22,23 .
  • DP dermal papillae
  • adipocyte precursor cells can regulate HFSC activity 22,23 .
  • GR was depleted using Pdgfra-CreER, a driver expressed in the majority of fibroblast populations including DP and adipocyte precursor cells 24 (FIG. 3A, FIG. 8C).
  • Pdgfra- crcER;GR ll/fl mice displayed precocious anagen entry, and their bulge and upper ORS continued to proliferate in late anagen (FIG. 3B, FIGS. 8D-8E).
  • Pdgfra-crcER;GR n/l1 mice like ADX mice — also entered anagen repeatedly, with short telogen phases in between (FIGS. 8F-8H).
  • mice displayed precocious anagen entry and entered more rounds of the hair cycle only in guard hair follicles, not in other hair follicles in which Sox2- CreER was not active (FIGS. 3D-3E, FIG. 9D).
  • Late anagen (Anagen V-Anagen IV) Sox2-CreER;GR fl/fl guard hair follicles were also more proliferative in their bulge and upper ORS regions and grew longer hair shafts (FIGS. 9E-9G). Together, these results indicate that DP has a key role in mediating the effect of corticosterone on prolonging telogen.
  • RNA sequencing was performed on HFSCs purified by fluorescence-activated cell sorting (FACS) from sham, ADX, ADX + corticosterone, Pdgfra-CreER control and Pdgfra-CreER;GR fl/fl mice (all in telogen).
  • FACS fluorescence-activated cell sorting
  • RNA-seq showed that genes encoding cell-cycle and cell-division machineries are upregulated in the HFSCs of ADX mice despite their being phenotypically quiescent, which suggests that the HFSCs are primed for activation. Moreover, these cell-cycle-related genes were downregulated to levels similar to those in sham controls when the ADX mice were fed with corticosterone, indicating that these molecular changes are downstream of corticosterone (FIG. IOC).
  • RNA-seq data further supported the idea that dermal fibroblasts relay the effect of corticosterone to HFSCs (FIG. IOC).
  • Gas6 is upregulated in the DP upon adrenalectomy
  • RNA-seq was conducted using FACS-enriched DP cells from sham and ADX mice.
  • RNA-seq was also conducted of DP from control and Pdgfra-CreER;GR fl/fl mice (FIG. 11E).
  • FIGS. 11F-11H To avoid identifying changes that simply track with the different stages of the hair cycle, all samples were taken at telogen (FIGS. 11F-11H). From these, differentially expressed genes were identified (1.5-fold change, P adj ⁇ 0.05) in DP upon adrenalectomy or dermal GR depletion (FIGS. 11I-11J).
  • Gas6 encodes a gamma-carboxyglutamic acid- containing secreted protein and predominantly binds to AXL, a member of the TYR03, AXL and MERTK (TAM) family of receptor tyrosine kinases 31 . It was confirmed by in situ hybridization that Gas6 is upregulated in the DP of ADX mice, and returns to baseline levels when ADX mice are fed with corticosterone (FIG. 4A). In late anagen, Gas6 is downregulated in the DP of control mice, but its expression levels remain high in the DP of ADX mice. In catagen, Gas6 is down-regulated both in control and in ADX mice (FIG. 12D).
  • HFSCs at the bulge and at the hair germ express Axl — the gene encoding the GAS6 receptor — whereas the matrix and epidermis have significantly lower levels of Axl (FIGS. 12E-12F). Of all three TAM receptors, Axl is most highly expressed in HFSCs (FIG. 12G).
  • GAS6 relays the effect of corticosterone
  • HFSCs were purified by FACS, plated in culture, and recombinant GAS6 was added to the media. HFSCs formed more colonies in the presence of GAS6 (FIG. 4B). Moreover, blocking AXL activity with the AXL- specific inhibitor R428 inhibited the ability of GAS 6 to promote HFSC proliferation in vitro (FIG. 12H).
  • CAG-GFP and CAG-Gas6 were generated and packaged into adeno-associated viruses (AAVs) and these AAVs were injected into the skin (FIG. 4C, FIG. 13A).
  • RT-qPCR confirmed that Gas6 transcripts were upregulated in the dermal fibroblasts in the AAV-injected regions (FIG. 13B).
  • AAV- CAG-Gas6 injection promoted precocious anagen entry at the site of injection (FIG. 4C).
  • HFSCs continued to proliferate at late anagen when Gas6 was overexpressed, but cells in no other compartment did (FIGS. 13C-13G).
  • the seven shared core genes that were upregulated in ADX mice also became upregulated in HFSCs when Gas6 was overexpressed (FIG. 13H).
  • Cdkl, Ccnbl and Rad51 have been reported as downstream targets of the GAS6-AXL pathway 32-34 , which supports the idea that GAS6 relays the effects of corticosterone to HFSCs.
  • Gas6 expression in the DP is low in early (refractory) telogen and is upregulated in late (competent) telogen (FIG. 14A), in agreement with previously reported DP microarray data 6 .
  • corticosterone levels are relatively constant at different telogen stages (FIG. 7E)
  • additional upstream signals such as bone morphogenetic protein (BMP) signalling — might also regulate Gas6 levels.
  • BMPs are secreted from fibroblasts and dermal adipocytes to promote HFSC quiescence, and are present at high levels in early telogen but low levels in late telogen 4,6 — opposite to the trend that was observed for Gas6.
  • Stem cells integrate both local and systemic inputs to couple tissue regeneration with the overall physiological state of the animal (Supplementary Discussion).
  • a mechanism was identified by which a systemic factor regulates a stem cell population by inhibiting a niche factor.
  • Previous findings have suggested several mechanisms by which acute stress affects the biology of melanocyte stem cells or hair follicles 35-37 .
  • the results now suggest that chronic stress delays anagen entry through a distinct mechanism.
  • the findings not only reveal important regulators of HFSC quiescence and activation at both local and systemic levels, but also identify the cellular and molecular mechanisms by which chronic stress influences the hair cycle (FIG. 14G).
  • tissue-regeneration capacity of HFSCs remains robust even after significantly increased rounds of anagen entry throughout life. It might therefore be possible to exploit the ability of HFSCs to promote hair-follicle regeneration by modulating the corticosterone-GAS6 axis.
  • mice were randomly assigned to control or experimental groups whenever possible, except in experiments that required specific genotypes, for which littermate controls were used.
  • LC-MS/MS liquid chromatography coupled to tandem mass spectrometry
  • RNA-seq library preparation and sequencing experimenters were blinded to experimental conditions. Blinding was not possible in mouse studies when specific genotypes or surgical models had to be identified according to experimental designs.
  • mice C57BL/6J, GRTM 8 , K15-CrePGR 39 , Pdgfra-CreER 40 , Sox2-CreER 41 and R26- lsl-YFP 42 mice were obtained from The Jackson Laboratory. Given the inherent differences in the timing of the hair cycle in wild-type male and female mice — and that male mice start to fight after P70, which confounds hair cycle analyses — data was presented from female mice for most experiments. However, consistent trends were observed in both males and females. For ADX and corticosterone-fed mice, results from both male and female mice were included, and the data are presented separately (female data: FIGS. 1A, 2C; male data: FIG. 5B, FIG. 61).
  • mice For GR-knockout experiments, sex-matched littermates without Cre or without GR M were used as controls. All the control mice received the same dose of tamoxifen (in the case of Pdgfra-CreER and Sox2-CreER) or RU486 (in the case of K15-CrePGR) as the experimental mice. The mice were maintained in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were approved by the Institutional Animal Care and Use Committee at Harvard University or the Icahn School of Medicine at Mount Sinai.
  • mice were housed in individually ventilated cages at a maximum density of five mice per cage with Nestlet bedding and a red hut for enrichment and kept on a 12 h-12 h light-dark cycle. Room temperature was maintained at 22 °C ⁇ 1 °C with 30-70% humidity. Mice were fed ad libitum with rodent diet (Prolab IsoPro RMH 30005P75) and water. None of the mice were involved in any previous procedures before the study.
  • C57BL/6J mice were anaesthetized and small incisions were made on the back skin directly above each adrenal gland. Both adrenal glands were removed with a pair of curved forceps. Sham mice underwent the same procedures as the ADX mice, except their adrenal glands were not removed. Because adrenal glands also secrete aldosterone to regulate salt balance, drinking water was supplemented with 1% w/v saline solution for both ADX and sham mice.
  • RU486 (TCI America, M1732; 4%) in ethanol was used to induce K15- CrePGR. RU486 was applied topically once per day for 10 days to K15- CrcPGR;GR ll/fl mice and sex-matched, littermate controls.
  • Tamoxifen (Millipore Sigma, T5648) was dissolved in corn oil to a final concentration of 20 mg ml -1 and was used to induce CreER recombinase. Tamoxifen was injected into Pdgfra- CreER;GR fl/fl , Sox2-CreER;GR fl/fl , and sex-matched, littermate controls intraperitoneally once per day for 4-6 days.
  • R428 43 (APExBIO, A8329; 2 mM in ethanol) was applied to ADX mice topically once a day.
  • EdU Liprobe Corporation, 10540; 25 mg kg -1
  • AAVs were produced as described previously 1,44 and injected directly into the dermis through intradermal injections.
  • Two-month-old C57BL/6J mice were injected with AAV-CAG-GFP, AAV-CAG-Gas6, or AAV- CAG-noggin (5 x 10 10 genome copy numbers per mouse).
  • Hair cycle progression was documented by standardized photographs at the start of each experiment and weekly thereafter. Anagen was determined by darkening of the skin followed by hair growth as previously described 5,45 . The back skin of mice was shaved with an electric clipper to reveal skin colour changes and hair coat recovery. Once the hair coat recovery reached about 90% of the back skin, the mice were shaved again to monitor the entry into next anagen. To assess the length of each hair cycle phase (telogen, anagen, catagen) of sham and ADX mice (P43-P140), skin colour changes were documented every 2-3 days. The length of each hair cycle was quantified as described previously 4 and was confirmed by histological section analysis.
  • mice were shaved before ADX surgery or tamoxifen treatment to trim off the original hair coat. The mice were then followed through a complete anagen cycle and hair shafts (actual hairs) were plucked in the following telogen phase after anagen was completed. Individual hair shafts were sorted according to hair type (based on their unique banding patterns), and the shaft lengths were measured and analysed under a Keyence BX-700 microscope with 4x, 20x or 40x objectives. Chronic corticosterone feeding
  • Corticosterone 35 mg ml -1 (Millipore Sigma, C2505) was dissolved in 0.45% hydroxypropyl-P-cyclodextrin and added to the drinking water during the entire corticosterone feeding period 19 . Corticosterone water was changed every 3 days to prevent degradation. Control mice received vehicle water (0.45% hydroxypropyl-b- cyclodextrin).
  • Chronic unpredictable stress was adapted from protocols described previously 20,21 .
  • C57BL/6 mice, sham, ADX, Pdgfra-CrcER;GR ll/fl mice, and their littermate controls were exposed to diverse stressors for 9 weeks. Two of the following stressors were applied each day in a randomized fashion: cage tilt, isolation, crowding, damp bedding, rapid light-dark changes, restraining, empty cage, and three cage changes.
  • Blood corticosterone levels were measured by ELISA (ARBOR assays, K014- Hl) according to the manufacturer’s instructions. Serum was collected by the tail clip bleeding method into heparinized tubes (Microvette CB 300 LH (16.443.100) or Microvette 300 LH (20.1309.100), Sarstedt) between 10:00 and 12:00.
  • Blood adrenaline and noradrenaline were measured by LC-MS/MS.
  • a stable- isotope-labelled internal standard (d 6 -adrenaline, Cambridge Isotope Laboratories, E- 077) was used for absolute quantification.
  • the standards for the HPLC system were prepared using a catecholamine mixture (adrenaline and noradrenaline) (Millipore Sigma, C-109). All samples were analysed on an Agilent 6460 Triple Quadrupole with an Agilent 1290 Infinity LC system.
  • the slides were then blocked in blocking buffer (5% donkey serum, 1% bovine serum albumin, 2% cold-water fish gelatin in 0.3% Triton X-100 in PBS) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C, and incubated with secondary antibodies for 2-4 h at room temperature.
  • blocking buffer 5% donkey serum, 1% bovine serum albumin, 2% cold-water fish gelatin in 0.3% Triton X-100 in PBS
  • CD34 eBioscience, 14-0341-82, 1:100
  • CD140a R&D Systems, AF1062, 1:100
  • P-Cadherin R&D Systems, AF761, 1:400
  • GFP Abeam, ab290, 1:5000
  • cleaved caspase-3 Cell Signaling Technology, 9661S, 1:300
  • Sox9 Millipore Sigma, AB5535, 1:500
  • phosphorylated histone H3 Cell Signaling Technology, 3377S, 1:500
  • glucocorticoid receptor Cell Signaling Technology, 3660S, 1:100
  • DAPI was used as a counterstain for the nucleus.
  • Cell proliferation assays were performed using a Click-It EdU Proliferation kit (Thermo Fisher Scientific, C10337) according to the manufacturer’s instructions. Haematoxylin and eosin (H&E) staining was performed according to standard protocols.
  • RNAscope 2.5 HD detection kit (Red) (322360, Advanced Cell Diagnostics) with Gas6 probe (450941, Advanced Cell Diagnostics), Axl probe (450931, Advanced Cell Diagnostics), or negative control probe (bacterial gene DapB, 310043, Advanced Cell Diagnostics) according to the manufacturer’s protocol. Mean pixel intensities were measured using ImageJ (v.l.52h).
  • Dermal cells were isolated as described 6,24,44,46 ’ 47 .
  • Mouse dorsal skin was dissected and treated with collagenase in Hank’s Balanced Salt Solution for 20-30 min at 37 °C on an orbital shaker.
  • the dermal fraction was collected by scraping followed by centrifugation at 300g for 10 min.
  • Dermal single-cell suspensions were obtained after 0.25% trypsin treatment for 10-20 min at 37 °C followed by filtering and centrifugation. Samples were stained for 30 min on ice.
  • CD140a-biotin eBioscience, 13-1401-82, 1:250
  • CD45-eFluor450 eBioscience, 48-0451-82; 1:250
  • CD31-PE-Cy7 eBioscience, 25-0311-81, 1:200
  • Sca-l-PerCP-Cy5.5 eBioscience, 45-5981-82, 1:1000
  • CD24-FITC eBioscience, 11-0242-82; 1:250
  • Streptavidin-APC eBioscience, 17-4317-82, 1:500.
  • DAPI was used to exclude dead cells.
  • DP cells were enriched as
  • the FACS strategy was further validated by the enrichment of DP signature genes 46 48 (FIG. 11H).
  • mice dorsal skin was dissected and the fat layer was removed using a surgical scalpel.
  • the skin was incubated in trypsin-EDTA at 37 °C for 35-45 min on an orbital shaker.
  • a single-cell suspension was obtained by scraping the epidermal side and filtering. Cells were centrifuged for 8 min at 350g at 4 °C, resuspended in 5% fetal bovine serum, and stained for 30-40 min.
  • CD49f integrated protein alpha 6
  • CD34-eFluor660 eBioscience, 50-0341-82, 1:100
  • Sca-l-PerCP-Cy5.5 eBioscience, 45-5981-82, 1:1,000
  • CD45-eFluor450 eBioscience, 48-0451-82, 1:250
  • the HFSCs were isolated as CD45 _ integrin alpha 6 + CD34 + Sca-l _ cells
  • the epidermal stem cells were isolated as CD45 _ integrin alpha 6 + CD34 _ Sca-l + cells, as described previouslyl,17.
  • FACS plots see Supplementary Figure 1. The data were analysed with FACSDiva (BD Biosciences, v.8.0.2) and FlowJo (FlowJo LLC, v.10.0.7).
  • RNA sequencing and computational analysis were performed using the Superscript IV VILO Master Mix with ezDNase Enzyme (Thermo Fisher Scientific, 11766050). Quantitative PCR was performed using power SYBR Green dye (Thermo Fisher Scientific, 4368706) on a QuantStudio 6 Flex Real-Time PCR system. Ct values were normalized to an internal control (b-actin).
  • RNA sequencing libraries were prepared using 1 ng of total RNA as input.
  • a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, 634888) was used for cDNA synthesis, with a 10-cycle PCR enrichment.
  • Sequencing libraries were made using Illumina’s Nextera XT Library Prep kit (Illumina, FC-131-1024). Single read sequencing reads for DP samples were obtained using the Illumina NextSeq 500 platform, and aligned to the mouse reference genome (mmlO) using Salmon (v.1.55) 49 . Paired-end sequencing reads for HFSC samples were trimmed with Trim Galore!
  • Transcripts per kilobase million were used to determine the expression levels of the AXL receptor shown in FIG. 12G.
  • Signal peptide prediction and secretome analysis were performed on differentially expressed genes using the Phobius web-accessible tool 55 (phobius.sbc.su.se/) and the secretome knowledge base available at the MetazSecKB web-accessible tool 56 .
  • FACS -purified HFSCs were plated on mitomycin C (P212121, M920 Re treated J2 fibroblast feeders at a density of 10,000 cells per well in 12-well plates in E media supplemented with 15% (v/v) serum and 0.3 mM calcium (calcium chloride; Millipore Sigma, C3881) as described in previous studies 57 .
  • E medium was supplemented with recombinant mouse GAS6 protein (R&D systems, 8310-GS) at 500 ng ml -1 .
  • R428-treatment experiments E medium was supplemented with R428 (APExBIO, A8329) at 1 mM. Cells were fixed and stained with 1% (w/v) Rhodamine B (Millipore Sigma, R6626). Colony diameter was measured from scanned images of plates using ImageJ (v.l.52h). Imaging and image analysis
  • Images were obtained with a Zeiss LSM 880 confocal microscope with a 20x air objective or 40x oil-based objective (Carl Zeiss) or a Keyence BX-700 epifluorescence microscope with 4x, 20x or 40x objective (Keyence). Images are presented as maximum intensity projection images or a single Z stack. Images were further processed and assembled into panels using Adobe Photoshop (v.21.2.4) and Adobe Illustrator (v.24.3).
  • FIG. 1A, n 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. IB, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 1C, n 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. ID, n 5 mice per condition. The experiments were performed twice with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. IB, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results.
  • FIG. 1C, n 30 hair follicles from 5 mice per
  • FIG. 2A, n 3 mice per condition. The experiments were performed three times with similar results.
  • One-way ANOVA with Tukey’ s two-sided multiple comparisons test.
  • FIG. 2B, Left, n 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 2C, n 5 mice per condition.
  • FIG. 3A, n 3 biologically independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIG. 3B, n 6 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 3C The experiments were performed three times with similar results.
  • FIG. 3D The experiments were performed three times with similar results.
  • FIG. 3E, n 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 3G, n 2 biological replicates from each condition. The experiments were performed once. One-sided Fisher’s exact test with FDR correction.
  • FIG. 4A, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. One-way ANOVA with Tukey’s two- sided multiple comparisons test.
  • FIG. 4B, n 3 biologically independent samples per condition. The experiments were performed three times with similar results. Two- sided unpaired t-test.
  • FIG. 4C, Left, n 5 mice per condition. The experiments were performed three times with similar results. Right, the experiments were performed three times with similar results.
  • FIG. 4D, n 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 5 A The experiments were performed three times with similar results.
  • FIG. 5B, n 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 5D, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t- test.
  • FIG. 5F The experiments were performed three times with similar results.
  • FIG. 5G The experiments were performed three times with similar results.
  • FIG. 5H, n 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 51, n 30 skin regions from 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 5K, n 5 mice per condition.
  • FIG. 5L, n 40 skin regions from 4 mice per condition. The experiments were performed twice with similar results.
  • FIG. 6A The experiments were performed three times with similar results.
  • FIG. 6D, n 3 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
  • FIG. 6E, n 3 mice per condition.
  • FIG. 8B, n 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 8C The experiments were performed three times with similar results.
  • FIG. 8D, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 8E, n 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 8F Two-way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 8G, n 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results.
  • FIG. 8H, n 30 skin regions from 5 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
  • FIG. 9A, n 2 biological independent samples per condition. The experiments were performed twice with similar results.
  • FIG. 9B, n 118, 139, 149 hair follicles per mouse from 3 mice. The experiments were performed three times with similar results.
  • FIG. 9D, n 3 mice per condition. The experiments were performed twice with similar results.
  • FIG. 9E, n 20 guard hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
  • FIG. 9A, n 2 biological independent samples per condition. The experiments were performed twice with similar results.
  • FIG. 9B, n 118, 139, 149 hair follicles per mouse from 3 mice. The experiments were performed three times with similar results.
  • FIGS. 10A-10D, n 2 biological independent samples for each condition. The experiments were performed once.
  • FIG. 10E, n 4 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIG. 10F, n 3 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIGS. 1 lA-1 IB, n 3 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIGS. 1 lC-1 ID, n 4 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIGS. 1 lF-11G The experiments were performed three times with similar results.
  • FIG. 11H, n 3 biological independent samples. The experiments were performed once.
  • FIGS lll- ll J, n 2 biological independent samples for each condition. The experiments were performed once.
  • FIG. 12A, n 2 biological independent samples for each condition. The experiments were performed once.
  • FIGS. 12B-12C, n 2 bio-logical independent samples for each condition. The experiments were performed once. Two-sided Wald test with multiple testing adjustments using the Benjamini-Hochberg method in DESeq2.
  • FIG. 12D, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 12E, Top, the experiments were performed three times with similar results. Bottom, n 4 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIG. 12F The experiments were performed three times with similar results.
  • FIG. 12G, n 2 biological independent samples per condition. The experiments were performed once.
  • FIG. 12H, n 3 biological independent samples per condition. The experiments were performed three times with similar results.
  • FIG. 13A The experiments were performed four times with similar results.
  • FIG. 13B, n 2 biologically independent samples per condition. The experiments were performed twice with similar results.
  • FIG. 13C, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two- way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 13D, n 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons.
  • FIG. 13E, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t- test.
  • FIG. 13B, n 2 biologically independent samples per condition. The experiments were performed twice with similar results.
  • FIG. 13C, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two- way ANOVA with Bonferroni’s two-sided
  • FIG. 14A, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. One-way ANOVA with Tukey’s two- sided multiple comparisons.
  • FIG. 14B, n 3 biologically independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIG. 14C, n 5 per condition. The experiments were performed three times with similar results. Two-way ANOVA repeated measures with Bonferroni’s two-sided multiple comparisons.
  • FIG. 14D, n 5 mice per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
  • FIG. 14F, n 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
  • E. FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential. Proc. Natl Acad. Sci. USA 113, E1506-E1515 (2016).
  • Greco, V. et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4, 155-169 (2009).

Abstract

Disclosed herein are methods for modulating hair growth and hair follicle stem cell activation in an individual in need thereof. In some embodiments, the methods increase hair growth. In other embodiments, the methods decrease hair growth.

Description

METHODS OF MODULATING HAIR GROWTH
RELATED APPLICATIONS
This application is related to and claims the benefit of U.S. Provisional Application No. 63/169,042, filed March 31, 2021. The entire teachings of the applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Stem cells are regulated by intrinsic regulators as well as by extrinsic signals from the niche1 4. However, how systemic factors regulate stem cell behaviors to couple tissue regeneration with diverse bodily changes remains poorly understood.
The hair follicle cycles between rest (telogen) and growth (anagen) phases5. Hair follicle stem cell HFSCs are located at the bulge and at the hair germ and are quiescent except during early anagen, when they proliferate transiently to initiate tissue regeneration6,7. Chronic stress, which triggers an increase in corticosterone secretion from the adrenal glands, has been anecdotally associated with hair loss in humans8. Early work in mice showed that the topical application of betamethasone — a steroid that inhibits glucocorticoid receptor (GR), the receptor for corticosterone — inhibits entry of the hair follicle into anagen9, and overexpression of GR at the embryonic stage leads to underdeveloped hair follicles10. By contrast, adrenalectomy — which removes the source of corticosterone — accelerates hair growth in rats, rabbits and minks11 13. Despite these observations, the cellular and molecular mechanisms by which corticosterone regulates tissue regeneration remain largely unexplored. SUMMARY OF THE INVENTION
Some aspects of the disclosure are related to methods of modulating hair growth or methods of modulating hair follicle stem cell (HFSC) activation in an individual in need thereof. In some embodiments, the methods comprise promoting or increasing hair growth. In some embodiments, the methods comprise inhibiting or decreasing hair growth.
Some aspects of the disclosure are directed to methods of promoting or increasing hair growth in an individual in need thereof comprising administering to the individual an effective amount of an agent that increases Gas6 expression.
In some embodiments, the agent increases the Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway. In some embodiments, the TAM interaction or pathway is an AXL interaction or pathway, a Tyro3 interaction or pathway, and/or a Mertk interaction or pathway. In some embodiments, the agent suppresses BMP signaling, e.g., the agent is noggin. In some embodiments, the agent increases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl .
In some embodiments, the agent is administered using an AAV vector, e.g., AAV8. In some embodiments, the agent is administered through intradermal injection. In some embodiments, hair growth is increased by at least 10%, 15%, 20%, or 25% relative to a suitable control.
Some aspects of the disclosure are directed to methods of decreasing hair growth in an individual in need thereof comprising administering to the individual an effective amount of an agent that decreases or inhibits Gas6 expression.
In some embodiments, the agent decreases the Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway. In some embodiments, the TAM interaction or pathway is an AXL interaction or pathway, a Tyro3 interaction or pathway, and/or a Mertk interaction or pathway. In some embodiments, the agent comprises an AXL inhibitor, e.g., the agent is R428. In some embodiments, the agent activates BMP signaling. In some embodiments, the agent decreases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl.
In some embodiments, the agent is administered using an AAV vector (e.g., AAV8). In some embodiments, the agent is administered through intradermal injection. In some embodiments, hair growth is decreased by at least 10%, 15%,
20%, or 25% relative to a suitable control. The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et ah, (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R.I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V.A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1, 2010, ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein. The above discussed features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention in conjunction with the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1F demonstrate that removal of adrenal glands activates HFSCs. FIG. 1A shows sham and ADX mice were shaved and monitored for hair coat recovery. Quantifications represent the percentage of back skin that is covered by regrown hairs. FIG. IB shows 5-Ethynyl-2'-deoxyuridine (EdU) and P-cadherin (PC AD) staining in hair follicles from sham and ADX mice (P49-P58) with quantifications. Yellow dashed lines, bulge; white dashed lines, hair germ or hair bulb (HB); solid lines, DP. FIG. 1C show EdU and CD34 staining in late anagen (AnaV) (sham, PI 10; ADX, P62) and mid catagen (CatV) (sham, P122; ADX, P74). Yellow dashed lines indicate the bulge (Bu), white dashed lines indicate the rest of the hair follicle, ORSup indicates the upper outer root sheath. FIG. ID shows hair cycle length in sham and ADX mice. FIG. IE provides the number of hair cycles of sham and ADX mice from P60 to P513 (see also FIG. 5). FIG. IF provides representative hair regrowth status of 22-month-old sham and ADX mice. Telo, telogen; Ana, anagen; Cat, catagen. Scale bars (FIGS. 1B-1C), 50 pm. Data are mean ± s.e.m. **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 2A-2D demonstrate corticosterone derived from the adrenal gland regulates HFSC quiescence. FIG. 2A provides plasma corticosterone levels in different mice 2 weeks after feeding corticosterone (CORT) or vehicle (Veh). FIG. 2B shows hair cycle progression (left) and haematoxylin and eosin (H&E) staining (right) of skin from sham+veh, ADX+veh and ADX+CORT mice. CORT, corticosterone. Scale bar, 50 pm. FIG. 2C shows hair cycle progression of C57BL/6 mice fed with vehicle or corticosterone. FIG. 2D shows hair cycle progression in C57BL/6 mice subjected to chronic unpredictable stress and in non-stressed control mice. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 3A-3G demonstrate corticosterone acts on the DP to regulate HFSC quiescence. FIG. 3A shows Pdgfra-CreER depletes GR efficiently in the DP and in dermal fibroblasts (DF). FIG. 3B shows hair cycle progression of control and Pdgfra- CreER;GRfl/fl mice. FIG. 3C provides immunohistochemical analyses (YFP and PCAD) of skin from Sox2-CreER;R26-lsl-YFP mice, showing the presence of YFP in the DP of guard hair follicles but not zigzag hair follicles. FIG. 3D provides H&E staining of skin from control and Sox2-CreER;GRfl/fl mice. The arrowhead indicates an anagen guard hair follicle surrounded by telogen hair follicles. FIG. 3E provides surface view (side (left) and top (right)) showing accelerated anagen only in the guard hairs of the Sox2-CreER;GRfl/fl mice (shaved at P45; imaged at P67). FIG 3F shows RNA-seq workflow. FIG. 3G provides Gene Ontology enrichment analysis of 121 shared differentially expressed genes in HFSCs comparing sham vs. ADX, ADX vs. ADX+CORT, and control vs. Pdgfra-CreER;GRfl/fl (see also FIG. 10). Scale bars, 50 pm (FIGS. 3C-3D), 1 mm (FIG. 3E). Data are mean ± s.e.m. **P < 0.01,
****P < 0.0001. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 4A-4E demonstrate that Gas6 overexpression counteracts the inhibitory effect of corticosterone. FIG. 4A provides Gas6 expression visualized by in situ hybridization in early telogen of sham, ADX, and ADX+CORT mice, and negative control expression in sham mice. Quantification shows Gas6 signals in the DP. Bold dashed lines, bulge; thin dashed lines, hair germ; solid lines, DP. FIG. 4B shows cultured HFSCs in the presence or absence of GAS6. FIG. 4C shows intradermal injection of AAV-CAG-Gas6, but not AAV-CAG-GFP, induces anagen in injection sites. Dashed circles indicate AAV injection areas. FIG. 4D provides AAV-mediated expression of GFP or Gas6 in mice subjected to chronic unpredictable stress or corticosterone feeding. FIGS. 4E provides a model summarizing the main findings of this study. When corticosterone levels decrease, an increase in Gas6 expression promotes HFSC activation and anagen entry. Conversely, when corticosterone levels are increased, Gas6 expression is inhibited and HFSCs stay in prolonged quiescence. Scale bar (FIGS. 4A, 4C), 50 pm. Data are mean + s.e.m. *P < 0.05, ****P < 0.0001. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 5A-5L demonstrate hair cycle progression in ADX mice over time. FIG. 5A shows hair cycle with immunohistochemical analyses (PCAD) in sham and ADX mice. FIG. 5B shows hair cycle progression in sham male and ADX male mice. FIG. 5C provides a schematic depicting HFSCs in anagen and telogen. The upper ORS of anagen hair follicles contributes to the new bulge and hair germ (HG) of the following telogen hair follicles. See refs.14 15 for details. FIG. 5D provides the ORS length in the zigzag hairs of sham (PI 13) and ADX (P65) mice during late anagen. The brackets indicate the ORS length below the bulge. FIG. 5E provides the hair shaft length of each hair subtype in sham and ADX mice after anagen. FIG. 5F shows H&E staining at P65 of skin from sham and ADX mice. FIG. 5G shows immunohistochemical analyses (Sox9 and CD34) in telogen (telo), late anagen (AnaV), and mid catagen (CatV) hair follicles. Yellow dashed lines, bulge; white dashed lines, HG (telo), hair follicle (AnaV, CatV); solid line, DP. FIG. 5H shows immunocolocalization (EdU and CD34) in infundibulum (IF), junctional zone (JZ), sebaceous gland (SG), mid ORS (ORSmid), lower ORS (ORSlow) and matrix (Mx) of late anagen (AnaVI) hair follicles. The dashed lines outline the hair follicle. FIG. 51, Left shows H&E staining in the late anagen skin of sham and ADX mice with quantification of the epidermal thickness (E). FIG. 51, Right shows immunocolocalization (EdU and DAPI) in interfollicular epidermis (IFE) and dermis. Dashed lines indicate the boundary between the epidermis and the dermis. FIG. 5J shows representative hair regrowth status of sham and ADX mice from P60 to P549. FIG. 5K shows duration of telogen in sham and ADX mice. FIG. 5L provides H&E staining of skin from young sham, aged sham, and aged ADX mice with quantification of the number of hair follicles per mm. Yellow dashed lines, bulge; white dashed lines, HG, solid lines, DP. Telo, telogen; Ana, anagen; Cat, catagen. Scale bars, 50 pm (FIGS. 5A, 5D, 5F-5I, 5L), 1 mm (FIG. 5E). Data are mean ± s.e.m. *P < 0.05, ****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 6A-6M demonstrate corticosterone restores normal hair cycle progression in ADX mice. FIG. 6A provides H&E staining of the skin of 21 -month- old sham and ADX mice. FIG. 6B shows morphology of each hair subtype from the skin of 18-month-old sham and ADX mice. FIG. 6C, Left provides immunohistochemical analyses (CD34 and PCAD) of telogen hair follicles in the skin of sham and ADX mice at 22 months old, showing normal hair follicle morphology and comparable stem cell numbers. FIG. 6C, Middle provides quantification of the number of bulge and hair germ cells per HF. FIG. 6C, Right shows the percentage of HFSCs in epithelial fraction by FACS. FIG. 6D shows hormones from the adrenal gland and plasma levels of corticosterone in P45 sham and ADX mice. FIG. 6E shows plasma levels of noradrenaline and adrenaline measured by LC-MS/MS at P45 (10 days after surgery) in sham and ADX mice. FIG. 6F, Left provides an experimental design to test if supplying corticosterone rescues ADX phenotypes. FIG. 6F, Right shows hair cycle progression of sham mice fed with vehicle (sham+veh) or ADX mice fed with corticosterone (ADX+CORT). FIG. 6G provides plasma corticosterone levels at P62 in C57BL/6 mice after a week’s feeding with vehicle or corticosterone. FIG. 6H, Top provides experimental design for 3 days of corticosterone feeding. FIG. 6H, Bottom shows the percentage of hair regrowth of the back skin at P38. FIG. 61 shows hair cycle progression of C57BL/6 male mice fed with vehicle or corticosterone. Corticosterone feeding prolonged telogen as long as corticosterone was provided to the mice (both male and female). FIG. 6J shows body weight of C57BL/6 mice fed with vehicle or corticosterone from P83 to PI 18. FIG. 6K, Left provides H&E staining in the skin of vehicle and corticosterone-fed mice. FIG. 6K, Middle and right provide quantification of the thickness of dermis (middle) and dermal adipose layer (right). D, dermis; A, adipose layer. FIG. 6L provides immunohistochemical analysis (active caspase 3 (aCAS3) and PCAD) in vehicle- and corticosterone-fed mice. Dashed lines, epidermis and hair follicles. FIG. 6M, Left provides experimental design to test the effect of corticosterone withdrawal. FIG. 6M, Right shows hair cycle progression of C57BL/6 mice after completion of 3 weeks of vehicle or corticosterone feeding. Scale bars (a, b, c, k, 1), 50 pm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 7A-7F demonstrate removal of the adrenal glands in stressed or aged mice leads to hair follicle regeneration. FIG. 7A provides plasma corticosterone levels at P62 in non-stressed control and stressed mice. FIGS. 7B-7C show H&E staining (FIG. 7B) and immunohistochemical analyses (active caspase3 (aCAS3) and PCAD) (FIG. 7C) in control and stressed mice. Dashed lines, epidermis and hair follicles.
FIG. 7D shows stressed sham (sham+stress) and stressed ADX (ADX+stress) mice were monitored for hair coat recovery. Quantification shows the percentage of back skin that is covered by newly regenerated hairs. FIG. 7E provides plasma levels of corticosterone in young mice (P46, P77, and P98) and aged mice (P427 and P581). FIG. 7F shows sham and ADX operations were performed on aged mice (P521). The mice were shaved and monitored for hair coat recovery from P521 to P574. Scale bars (FIGS. 7B-7C), 50 mih. Data are mean ± s.e.m. *P < 0.05, **P < 0.01,
****P < 0.0001. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 8A-8H demonstrate GR depletion in different cell types in the skin.
FIG. 8A, Top shows K15-CrePGR depletes GR efficiently in HFSCs. FIG. 8A, Bottom shows immunohistochemical analysis (GR and CD 140a) of telogen hair follicle in the skin of control and K 15-CrcPGR;GRll/fl mice. FIG. 8B shows hair cycle progression of control and K15-CrePGR;GRfl/fl mice. FIG. 8C shows immunohistochemical analyses (GR and PCAD) of telogen hair follicles in the skin of control and Pdgfra-CreER;GRfl/fl mice, showing that Pdgfra-CreER depletes GR efficiently in the dermal fibroblasts and DP. FIG. 8D shows immunocolocalization (EdU and CD34) in control and Pdgfra-CrcER;GRll/fl hair follicles after tamoxifen administration. EdU incorporation reveals premature HFSC activation in the hair follicles of Pdgfra-CreER;GRfl/fl mice. FIG. 8E provides a comparison of EdU localization in bulge and upper ORS in late anagen (AnaV) of control (PI 24) and Pdgfra-CreER;GRfl/fl(P73) mice. FIG. 8F shows representative hair regeneration status of control and Pdgfra-CreER;GRfl/fl mice from P73 to P205, with quantification of the number of hair cycles. FIG. 8G shows immunocolocalization (EdU and CD34) in infundibulum (IF), junctional zone (JZ), sebaceous gland (SG), mid ORS (ORSmid), lower ORS (ORSlow), and matrix (Mx) of late anagen (AnaVI) hair follicles in control and Pdgfra-CrcER;GRll/fl mice during late anagen with quantifications. FIG. 8H, Top shows H&E staining in the late anagen skin of control and Pdgfra-CreERiGR^ mice, with quantification of the thickness of epidermis (E). FIG. 8H, Bottom shows immunocolocalization (EdU and DAPI) in interfollicular epidermis (IFE) and dermis in control and Pdgfra-CreER;GRfl/fl mice. Scale bars (FIGS. 8A, 8C-8E, 8G-8H), 50 pm. Yellow dashed lines, bulge (FIG. 8A, 8C-8E); white dashed lines, hair germ (FIG. 8A, 8C, 8D), the rest of hair follicles (FIG. 8E, 8G), or the boundary between the epidermis and the dermis (FIG. 8H); solid white line, DP (FIGS. 8A, 8C, 8D). Data are mean ± s.e.m. **P < 0.01, ***P < 0.001,
****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 9A-9G demonstrate corticosterone acts on the DP. FIG. 9 A provides RT-qPCR of GR from DP and DF. FIG. 9B provides immunohistochemical analyses (YFP and DAPI) of anagen skin from Sox2-CreER;R26-lsl-YFP mice. Left, the arrowhead indicates an anagen guard hair follicle with YFP+ DP cells. Right, quantification of the percentage of YFP+ and YFP- DP in Sox2-CreER;R26-lsl-YFP. Only guard hair follicles have YFP+ DP. FIG. 9C provides immunohistochemical analyses (GR and DAPI) of skin from control and Sox2-CreER;GRfl/fl mice. Dashed lines, epidermis and hair follicles; solid line, DP. The arrowhead indicates the DP of Sox2-CreER;GRfl/fl guard hairs. FIG. 9D shows representative hair regeneration status of control and Sox2-CreER;GR fl/flmice from P45 to P160. Quantification shows the number of hair cycles for guard hairs and other hairs in control and Sox2- CreER;GRfl/fl mice. FIG. 9E shows comparison of the hair bulb diameter in late anagen (AnaV) in the skin of control (P120) and Sox2-CreER;GRfl/fl (P67) mice. Yellow lines indicate the hair bulb diameter. The arrowhead denotes minor hyper thickening of the Sox2-CreER;GRfl/flhair follicle around the ORS, probably because the dermis has not expanded to accommodate the extra proliferation from HFSCs. FIG. 9F shows immunocolocalization of phosphohistone H3 (pHH3) and CD34 in bulge and upper ORS, middle ORS (ORSmid), lower ORS (ORSlow) and matrix (Mx) of late anagen (AnaV) guard hair follicles in control and Sox2-CreER;GRfl/fl mice. The white arrowhead denotes a thickened region in a Sox2-CreER;GRfl/flhair follicle, probably due to excessive proliferation from HFSCs. Yellow dashed lines, bulge; white dashed lines, the rest of the hair follicle. FIG. 9G shows hair shaft length of guard hairs in control and Sox2-CreER;GRfl/fl mice after anagen. Scale bars, 50 pm (FIGS. 9B, 9C, 9E, 9F), 1 mm (FIGS. 9D, 9G). Data are mean ± s.e.m.
****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 10A-10F demonstrate differential gene expression in HFSCs of control, ADX and dermal GR-knockout mice. FIG. 10A shows sample clustering based on Pearson’s correlation of transcriptomes in HFSCs from sham, ADX, and ADX+CORT as well as control and Pdgfra-CreER;GRfl/fl mice. FIG. 10B provides principal component analysis (PCA) comparing the transcriptome of HFSCs from sham, ADX, ADX+CORT, control and Pdgfra-CreERiGR^ mice. FIG. IOC provides heat map of log2 fold change of gene expression of 121 common genes among ADX (versus sham), Pdgfra-CreER;GRfl/fl (versus control), and ADX+CORT (versus ADX). Cell-cycle-related genes are noted in orange. FIG. 10D, Left provides a heat map of log2 fold change of gene expression of transcription factors (Foxcl, Lhx2, Foxpl, Nfatcl), key signalling factors (Fgfl8), or downstream readout of key signalling factors (Idl for BMP pathway, Axin2 for WNT pathway, Glil for SHH pathway) known to regulate HFSC quiescence. FIG. 10D, Right provides a heat map of log2 fold change of gene expression of 7 core genes related to cell-cycle machineries and cytokinesis. FIGS. 10E-10F provide RT-qPCR of genes related to cell-cycle machineries and cytokinesis from telogen HFSCs of sham and ADX mice (FIG. 10E) and control and Pdgfra-CreER;GRfl/fl mice (FIG. 10F). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 11A-11J demonstrate the expression of cell-cycle -related genes in HFSCs. FIGS. 1 lA-1 IB show RT-qPCR of genes related to cell-cycle machineries and cytokinesis using telogen HFSCs from vehicle and corticosterone-fed mice (FIG. 11A) and control and stressed mice (FIG. 11B). FIGS. 11C-11D show RT-qPCR of genes related to cell-cycle machineries and cytokinesis in telogen epidermis from sham and ADX mice (FIG. 11C) and vehicle and corticosterone-fed mice (FIG. 11D). FIG. 11E shows experimental workflow of the differentially expressed genes (DEGs, > 1.5-fold, Padj < 0.05) from DP cells of sham and ADX mice, as well as control and Pdgfra-CreERjGR^ mice. FIGS. 11F-11G show immunohistochemical analysis (PCAD) of skin samples from sham and ADX (FIG. 1 IF) or control and Pdgfra- CreER;GRfl/fl (FIG. 11G) mice used in RNA-seq experiments to validate hair cycle (all telogen). Dashed lines, epidermis and hair follicles. FIG. 11H, Top shows FACS strategies for isolating DP cells for RNA-seq24,44. FIG. 11H, Bottom shows the expression levels of cell-type- specific signature genes (DP, fibroblasts, HFSCs and mast cells) in FACS-purified DP cells. TPM, transcripts per million. FIG. 1 II shows sample clustering based on Pearson’s correlation of transcriptomes in DP of sham and ADX mice (left), as well as control and Pdgfra-CreER;GRfl/fl mice (right). FIG. 11 J provides heat maps of the differentially expressed genes (DEGs, > 1.5-fold,
Padj < 0.05) from FACS-purified DP cells of sham and ADX mice (left) or control and Pdgfra-CreERjGR^ mice (right). Scale bars (FIGS. 11F-11G), 50 pm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 12A-12H demonstrate transcriptome analysis and secretome analysis identified GAS6 as a secreted factor suppressed by systemic corticosterone in the DP. FIG. 12A shows secretome analysis identifying common secreted factors from DEGs (> 1.5-fold, Padj < 0.05) in ADX and Pdgfra-CreER;GRfl/fl DP cells identified by RNA- seq. FIGS. 12B-12C show expression levels of shared differentially expressed secreted factors as TPM, in the DP cells of ADX (FIG. 12B) and Pdgfra-CrcER;GRll/fl (FIG. 12C) mice. FIG. 12D, Top shows negative control and Gas6 mRNA expression by in situ hybridization in late anagen (AnaV) and mid catagen (CatV) skin of sham and ADX mice. FIG. 12D, Bottom shows quantification of Gas6 mRNA in the DP. Dashed lines, hair follicle; solid lines, DP. FIG. 12E provides a representative image of negative control and Axl mRNA expression by in situ hybridization in telogen skin. Yellow dashed lines: bulge; white dashed lines: hair germ (top). RT-qPCR of Axl, Tgfbrl, Bmprla, Nfatcl and PPIB from HFSCs and epidermal stem cells (EpSCs) of control mice (P83) (bottom). FIG. 12F provides representative images of negative control and Axl mRNA expression by in situ hybridization in late anagen skin.
Yellow dashed lines: bulge; white dashed lines: epidermis and hair follicles. FIG. 12G shows the expression levels (as TPM) of genes encoding TAM receptors (Tyro3, Axl and Mertk) in HFSCs. FIG. 12H, Feft provides a schematic of the GAS6-AXF receptor tyrosine kinase pathway. R428 is a selective inhibitor of AXF tyrosine kinase activity43. FIG. 12H, Right shows colony-formation assays of cultured HFSCs in R428 or GAS6 with R428 with quantifications. Scale bars (d-f), 50 pm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 13 A- 13 J demonstrate analyses of skin changes upon Gas6 overexpression or treatment with an AXF inhibitor. FIG. 13A shows immunohistochemical analysis (GFP and PCAD) of PBS -injected second telogen skin and AAV-GFP-injected second telogen skin. Dashed lines, epidermis and hair follicles; solid lines: DP. FIG. 13B provides RT-qPCR of Gas6 from dermal fibroblasts of PBS -injected second telogen skin (control) and AAV-CAG-Gas6- injected second telogen skin. FIG. 13C shows precocious HFSC activation in mice injected with AAV-CAG-Gas6 shown by EdU incorporation. Immunocolocalization (EdU and CD34) in control and AAV-CAG-Gas6-injected skin after AAV injection (D3 to D9). FIG. 13D shows comparison of EdU and CD34 localization in bulge and upper outer root sheath (ORS) in late anagen (AnaV) (control, D50 after injection; GAS6, D17 after injection). FIG 13E provides H&E staining of late anagen (AnaVI) skin (control, D53 after injection; GAS6, D20 after injection). Quantification of the ORS length in the zigzag hairs of control and AAV-CAG-Gas66-injected mice during late anagen. Brackets indicate the ORS length below the bulge. FIG. 13F shows immunocolocalization (EdU and CD34) in infundibulum (IF), junctional zone (JZ), sebaceous gland (SG), mid ORS (ORSmid), lower ORS (ORSlow) and matrix (Mx) of late anagen (AnaVI) hair follicles in control and AAV-CAG-Gas6-injected mice with quantifications. Dashed lines outline hair follicles. FIG. 13G, Top shows H&E staining in the late anagen skin of control and AAV-CAG-Gas6-injected mice with quantification of the thickness of epidermis (E). FIG. 13G, Bottom shows immunocolocalization (EdU and DAPI) in interfollicular epidermis (IFE) and dermis in control and AAV-CAG-Gas6-injected mice with quantifications. FIG. 13H provides RT-qPCR of genes related to HFSC proliferation in HFSCs of second telogen skin. FIG. 131 shows hair cycle progression of sham and ADX mice treated with ethanol topically, or ADX mice treated with R428 in ethanol. FIG. 13 J shows RT-qPCR of genes related to cell-cycle machineries and cytokinesis from HFSCs of sham and ADX mice treated with ethanol topically, or ADX mice treated for 7 days with R428 (in ethanol) topically. Yellow dashed lines, bulge (FIGS. 13C-13D); white dashed lines, hair germ (FIG. 13C), the rest of the hair follicle (FIGS. 13D, 13F), or the boundary of epidermis and dermis (FIG. 13G); solid white line, DP (FIG. 13C). EtOH, ethanol. Scale bars (FIGS. 13A, 13C-13G), 50 pm. Data are mean ± s.e.m.
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
FIGS. 14A-14G demonstrate interactions between systemic corticosterone and local BMP signalling. FIG. 14A shows in situ hybridization of negative control and Gas6 mRNA expression in different conditions, including P49 (early telogen) of sham mice, P80 (late telogen) of sham mice, P49 of ADX mice, P49 of in sham mice injected with AAV-CAG-noggin (noggin in sham), and P49 in ADX mice after injection of AAV-CAG-noggin (noggin in ADX). Quantifications show in situ hybridization signal intensities in the DP. The model shows how changes in corticosterone and BMP signalling influence Gas6 levels in the DP. FIG. 14B provides RT-qPCR of Gas6 from the DP of P49 (early telogen) and P80 (late telogen) of sham mice, and P49 of ADX mice, noggin in sham mice, and noggin in ADX mice. FIG. 14C shows hair cycle progression in sham, ADX, noggin in sham, and noggin in ADX mice with quantifications. Dashed circles indicate AAV-CAG-noggin injection areas. FIG. 14D shows control and Pdgfra-CreERiGR^ mice were subjected to chronic unpredictable stress from P55. Quantification shows the percentage of hair regrowth at P107. FIG. 14E provides RT-qPCR of Gas6 from DP cells of control (P83) and stressed (P83) mice (left), and vehicle (P83) and corticosterone-fed (P83) mice (right). FIG. 14F shows in situ hybridization of Gas6 in vehicle (P83, late telogen) and corticosterone-fed (P83) mice, with quantification of in situ signals in DP. FIG. 14G provides a model of corticosterone regulation of telogen length. In normal conditions, corticosterone levels remain constant, but BMP levels naturally decrease as telogen progresses, until a point is reached at which Gas6 levels are sufficiently increased so as to drive HFSCs out of quiescence. This dynamic can be altered by changing either the corticosterone level or the BMP level. If corticosterone levels decrease, the sum of inhibitory cues on Gas6 falls below a critical threshold sooner in telogen, leading to increased Gas6 levels and precocious anagen entry. In the case of stress, age, or corticosterone feeding, increased corticosterone levels reduce Gas6 levels to below the critical threshold, leading to an extended telogen. Dashed lines, hair follicles; solid lines, DP. Scale bars (a, f), 50 pm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. For exact P values, see Source Data. For statistics, sample sizes and numbers of replications, see Methods.
DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein are methods for modulating hair growth and increasing hair follicle stem cell activation in an individual in need thereof. This includes administering an agent that modulates a Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway.
Aspects of the disclosure are related to methods of modulating hair growth and HFSC activation in an individual in need thereof. This includes administering an agent that modulates a Gas6-Tyro3/Axl/Mertk (TAM) interaction or pathway (e.g., turns the pathway on or off). In some embodiments, the TAM interaction or pathway is an AXL, a Tyro3, or a Mertk interaction or pathway.
The term “modulate” is used consistently with its use in the art, i.e., meaning to cause or facilitate a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of a process, pathway, or phenomenon. A “modulator” is an agent that causes or facilitates a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.
The term “agent” as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-pro teinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In certain embodiments, agents are small molecules having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds. In general, agents may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the agent. An agent may be at least partly purified. In some embodiments an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments an agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments an agent is cell-permeable, e.g., within the range of typical agents that are taken up by cells and act intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z- isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)- isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable.
The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction”, “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
The terms “increased”, “increase”, “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase”, “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
In some embodiments, the agent modulates Gas6 activity or expression. In some embodiments, the agent modulates the interaction of Gas6 with theTyro3/Axl/Mertk (TAM) pathway. In some embodiments, the agent modulates the interaction of Gas6 with AXL. In some embodiments, the agent modulates the interaction of Gas6 with Tyro3. In some embodiments, the agent modulates the interaction of Gas6 with Mertk. In some embodiments, the agent (e.g., a chemical agent) modulates the Gas6-AXL pathway.
In some embodiments, the agent increases Gas6 activity or expression. In some embodiments, the agent increases Gas6 activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, e.g., relative to a suitable control. In some aspects, the agent increases Gas6 activity by at least 5%. In some aspects, the agent increases Gas6 activity by at least 10%. In some aspects, the agent increases Gas6 activity by at least 15%. In some aspects, the agent increases Gas6 activity by at least 20%. In certain aspects, the agent increases Gas6 activity by at least 25%.
In some embodiments, the agent decreases Gas6 activity or expression. In some embodiments, the agent decreases Gas6 activity by at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95%, e.g., relative to a suitable control. In some aspects, the agent decreases Gas6 activity by at least 5%. In some aspects, the agent decreases Gas6 activity by at least 10%. In some aspects, the agent decreases Gas6 activity by at least 15%. In some aspects, the agent decreases Gas6 activity by at least 20%. In certain aspects, the agent decreases Gas6 activity by at least 25%.
In some embodiments, the agent increases hair growth or HFSC activation. In certain aspects, the agent increases hair growth. In certain aspects, the agent increases HFSC activation. In some embodiments, the agent increases hair growth or HFSC activation relative to a suitable control. In some embodiments, the agent increases hair growth or HFSC activation by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%. In some aspects, the agent increases hair growth or HFSC activation by at least 5%. In some aspects, the agent increases hair growth or HFSC activation by at least 10%. In some aspects, the agent increases hair growth or HFSC activation by at least 15%. In some aspects, the agent increases hair growth or HFSC activation by at least 20%. In certain aspects, the agent increases hair growth by at least 25%. In certain embodiments, the agent increases HFSC activation by at least 25%.
In some embodiments, the agent decreases hair growth or HFSC activation. In certain aspects, the agent decreases hair growth. In certain aspects, the agent decreases HFSC activation. In some embodiments, the agent decreases hair growth or HFSC activation relative to a suitable control. In some embodiments, the agent decreases hair growth or HFSC activation by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%. In some aspects, the agent decreases hair growth or HFSC activation by at least 5%. In some aspects, the agent decreases hair growth or HFSC activation by at least 10%. In some aspects, the agent decreases hair growth or HFSC activation by at least 15%. In some aspects, the agent decreases hair growth or HFSC activation by at least 20%. In certain aspects, the agent decreases hair growth by at least 25%. In certain embodiments, the agent decreases HFSC activation by at least 25%.
In some embodiments, the agent modulates AXL activity or expression. In some embodiments, the agent increases AXL activity or expression. In some embodiments, the agent increases Gas6 activity or expression. In some embodiments, the agent inhibits bone morphogenetic protein (BMP) signaling. Non-limiting examples of BMP signaling inhibitors include noggin, chordin, gremlin, follistatin, sclerostin, and chordin. In one embodiment, a BMP signaling inhibitor comprises noggin. In some embodiments, the agent increases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl . In some embodiments, the agent increases expression of one or more genes selected from the group consisting of Ccnbl , Rad51, and Cdkl .
In some embodiments, the agent decreases AXL activity or expression. Non limiting examples of AXL inhibitors include R428 or BGB324, crizotinib, bosutinib, cabozantinib, sunitinib, foretinib, merestinib, and glesatinib. In one embodiment, an AXL inhibitor comprises R428. In some embodiments, the agent inhibits or decreases Gas6 activity or expression. In some embodiments, increases BMP signaling. In some embodiments, the agent decreases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl.
In some embodiments, the agent decreases expression of one or more genes selected from the group consisting of Ccnbl, Rad51, and Cdkl.
In some embodiments, the agent is administered using a delivery system. An acceptable delivery system may be a delivery system known to those of skill in the art. In some embodiments, the delivery system is a viral vector delivery system. In some aspects, the viral vector delivery system comprises adenoviruses, adeno- associated viruses, retroviruses (e.g., lentiviruses), vaccinia virus and other poxviruses, herpesviruses (e.g., herpes simplex vims), or others. In one embodiment, the delivery system comprises an adeno-associated virus (AAV). In some embodiments, an AAV has an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Anc80, or PHP.eB. Any AAV serotype, or modified AAV serotype, may be used as appropriate and is not limited. In certain embodiments, the AAV is AAV8.
Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, e.g., US Patent 7790449; US Patent 7282199; WO 2003/042397; WO 2005/033321, WO 2006/110689; and US 7588772 B2, the contents of which are incorporated herein by reference in their entirety. In one system, a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transfected (transiently or stably) with a construct encoding the transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus. More recently, systems have been developed that do not require infection with helper vims to recover the AAV - the required helper functions (i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system. In these newer systems, the helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level. In yet another system, the transgene flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al,
2009, "Adenovirus- adeno-associated virus hybrid for large-scale recombinant adeno- associated virus production," Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.
Some embodiments of the present invention relate to methods of treatment or prevention for a disease or condition, such as a skin or hair condition, disease, or disorder, by the delivery of a pharmaceutical composition comprising an effective amount of the agents described herein. An effective amount of the pharmaceutical composition is an amount sufficient to prevent, slow, inhibit, or ameliorate a disease or disorder in a subject to whom the composition is administered.
In some embodiments, the methods stimulate or promote hair growth or HFSC activation. In some embodiments, hair growth or HFSC activation is stimulated or promoted by increasing Gas6 expression or activation. In some embodiments, hair growth or HFSC activation is stimulated or promoted by decreasing or inhibiting BMP signaling. In some embodiments, the methods comprise administering an agent that increases Gas6 expression or activation. In some embodiments, the methods comprise administering an agent that inhibits or suppresses BMP signaling (e.g., noggin). In some embodiments, the methods comprise administering an AXL activator.
In some embodiments, the methods decrease or inhibit hair growth or HFSC activation. In some embodiments, hair growth or HFSC activation is decreased or inhibited by decreasing Gas6 expression. In some embodiments, the methods comprise administering an agent that decreases Gas6 expression or activation. In some embodiments, the methods comprise administering an AXL inhibitor (e.g., R428). In some embodiments, the methods comprise administering an activator of BMP signaling.
In some embodiments, the method increases hair growth or HFSC activation under stress conditions. In some embodiments, the stress condition is characterized by elevated corticosterone or hair loss such as a hair loss condition. In some embodiments, the hair loss condition is telogen effluvium. As used herein, “telogen effluvium” refers to a form of temporary hair loss that may occur after stress, a shock, or a traumatic event and occurs on the top of the scalp.
In some embodiments, a delivery system comprising an agent is administered to a subject. As used herein, the terms “subject” and “individual” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and/or to whom treatment, including prophylactic treatment, with the cells as described herein, is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human subject, the term subject refers to that specific animal. The “non-human animals” and “non-human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like. In some embodiments, where the subject is an animal (e.g., a sheep, mink, rabbit, etc.) fur growth may be modulated (i.e., increased or decreased) by modulating the Gas6/AXL pathway.
As used herein, the terms “treat,” “treatment” or “treating”, in reference to a subject, includes amelioration, cure, and/or maintenance of a cure (i.e., the prevention or delay of relapse and/or reducing the likelihood of recurrence) of a disorder. Treatment after a disorder has started aims to reduce, ameliorate or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, to slow the rate of progression, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse). Treating encompasses administration of an agent that may not have an effect on the disorder by itself but increases the efficacy of a second agent administered to the subject. A suitable dose and therapeutic regimen may vary depending upon the specific agent used, the mode of delivery of the compound, and whether it is used alone or in combination.
The agents disclosed herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracranial, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In certain embodiments, the agent is administered through intradermal injection.
For topical application, the compositions and agents are formulated into solutions, suspensions, lotions, sprays, shampoos, hair conditions, serums, patches, wipes, gels, hydrogels, powders, patches, impregnated pads, emulsions, vesicular dispersions, sprays, aerosols, foams, ointments, tinctures, salves, gels, cleansing soaps, cleansing cakes, or creams as generally known in the art. The formulation can be, e.g., in a multi-use or single-use applicator. Topical administration can include the application of the pharmaceutical or cosmetic compositions to the scalp and/or hair.
An “effective amount” or “effective dose” of an agent (or composition containing such agent) refers to the amount sufficient to achieve a desired biological and/or pharmacological effect, e.g., when delivered to a cell or organism according to a selected administration form, route, and/or schedule. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular agent or composition that is effective may vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. Those of ordinary skill in the art will further understand that an “effective amount” may be contacted with cells or administered in a single dose, or through use of multiple doses, in various embodiments. A biological effect may be, e.g., reducing expression or activity of one or more gene products, reducing activity of a metabolic pathway or reaction, or reducing cell proliferation or survival of cells.
A single additional agent or multiple additional agents or treatment modalities may be co-administered (at the same or differing time points and/or via the same or differing routes of administration and/or on the same or a differing dosing schedule).
The dosage, administration schedule and method of administering the agent are not limited. In certain embodiments a reduced dose may be used when two or more agents are administered in combination either concomitantly or sequentially.
The absolute amount will depend upon a variety of factors including other treatment(s), the number of doses and the individual patient parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, a maximum tolerated dose may be used, that is, the highest safe and tolerable dose according to sound medical judgment.
As used herein, pharmaceutical compositions comprise one or more agents or compositions that have therapeutic utility, and a pharmaceutically acceptable carrier, e.g., a carrier that facilitates delivery of agents or compositions. Agents and pharmaceutical compositions disclosed herein may be administered by any suitable means such as topically, orally, intranasally, intradermally, subcutaneously, intramuscularly, intravenously, intra-arterially, parenterally, intraperitoneally, intrathecally, intratracheally, ocularly, sublingually, vaginally, rectally, dermally, or as an aerosol.
In addition to the active agent(s), the pharmaceutical compositions typically comprise a pharmaceutically-acceptable carrier. The term “pharmaceutically- acceptable carrier”, as used herein, means one or more compatible solid or liquid vehicles, fillers, diluents, or encapsulating substances which are suitable for administration to a human or non-human animal. In preferred embodiments, a pharmaceutically-acceptable carrier is a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “compatible”, as used herein, means that the components of the pharmaceutical compositions are capable of being comingled with an agent, and with each other, in a manner such that there is no interaction which would substantially reduce the pharmaceutical efficacy of the pharmaceutical composition under ordinary use situations. Pharmaceutically-acceptable carriers should be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the human or non-human animal being treated.
Figure imgf000024_0001
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.
Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”.
“Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value).
It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated”.
Examples
Chronic, sustained exposure to stressors can profoundly affect tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here it is reported that the stress hormone corticosterone — which is derived from the adrenal gland and is the rodent equivalent of cortisol in humans — regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. In the absence of systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, increased levels of corticosterone prolong HFSC quiescence and maintain hair follicles in an extended resting phase. Mechanistically, corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term.
Results
Adrenalectomy activates HFSCs
To determine how hormones from the adrenal glands affect hair cycle, both adrenal glands were surgically removed from C57BL/6 mice on post-natal day (P) 35, a time before mice enter an extended second telogen (FIG. 5A). In sham-operated (sham) wild-type mice, the second telogen began at around P43-P45 and lasted on average 58 + 2 days (mean + s.e.m.). By contrast, adrenalectomized (ADX) mice — both male and female — had a significantly shorter telogen (around 11 days), with precocious activation starting from the hair germ (FIGS. 1A-1B, FIGS. 5A-5B). Normally, HFSCs proliferate only in early anagen and quickly resume quiescence by mid-anagen7. In ADX mice, however, HFSCs located in the bulge and in the upper outer root sheath (ORS)14,15 remained proliferative in late anagen (FIG. 1C, FIG. 5C). By late anagen, hair follicles in the ADX mice were longer than those at the same stage of anagen in sham control mice (FIG. 5D). Although the hair shaft in ADX mice also becomes longer, probably due to the enhanced HFSC proliferation, ADX mice did not display gross abnormalities in their skin and showed similar expression of HFSC markers to control mice (FIGS. 5E-5I). For both sham and ADX mice, HFSCs in the bulge and in the upper ORS resumed quiescence in the catagen phase, and the durations of the anagen and catagen phases were similar in the two groups of mice (FIGS. 1C- ID). These data suggest that HFSCs in ADX mice spend less time in quiescence. ADX mice enter anagen continuously
Monitoring the hair cycle of sham and ADX mice for an extended period of time revealed that, in sham mice, the telogen phases became progressively longer (second telogen, 58 + 2 days; third telogen, 75 + 2 days; fourth telogen, 95 + 4 days), and entry into anagen became sporadic and asynchronized (FIGS. 1D-1E, FIGS. 5J- 5K). By contrast, hair follicles in ADX mice stayed in telogen for only about 2 weeks, and they repeatedly entered anagen in a synchronized fashion (FIG. 5J). Over an approximately 16-month period after adrenalectomy, ADX mice went through around 10 synchronized hair cycles in their back skin, whereas the hair follicles of sham mice entered anagen asynchronously and rarely, and only about 3 hair cycles were completed within 16 months (FIG. IE). Telogen became around 30 days long in 17- 18-month-old ADX mice, which is still significantly shorter than the length of the second telogen in young sham mice (which begins at around 1.5 months old), and is in sharp contrast to that of old sham mice at the same age16,17 (FIG. 5K). At 18-22 months of age, ADX mice had a hair coat density similar to that of young mice, and showed robust anagen entry (FIG. IF, FIGS. 5J, 5L, and FIG. 6A). These mice showed normal hair follicle morphology, the presence of different hair types, and an absence of aberrant overgrowth (FIGS. 6A-6C). Moreover, HFSC numbers were maintained despite repeated anagen entry (FIG. 6C). These data suggest that systemic factors secreted from the adrenal glands are key regulators of HFSC quiescence, and indicate that the ability of HFSCs to regenerate hair follicles in ADX mice does not decline after rounds of anagen entry.
Corticosterone regulates HFSC quiescence
The adrenal gland produces several hormones, including corticosterone, adrenaline (also known as epinephrine), noradrenaline (also known as norepinephrine) and aldosterone18 (FIG. 6D). The levels of each hormone were quantified and it was found that corticosterone showed the most substantial decrease — becoming barely detectable — in ADX mice compared with control mice (FIGS. 6D-6E). Supplementation with corticosterone effectively suppressed the aberrant activation of HFSCs in ADX mice (FIGS. 2A-2B, FIG. 6F). Together, these results suggest that corticosterone secreted from the adrenal glands acts as a key systemic regulator to suppress anagen entry under normal physiological conditions. Increased corticosterone levels inhibit anagen
To test if increased corticosterone levels inhibit HFSC activation, wild-type second-telogen mice were given supplementary corticosterone in their drinking water19, leading to enhanced levels of circulating corticosterone (FIG. 2C, FIG. 6G). Transient increases in corticosterone levels had a minimal effect on the hair cycle (FIG. 6H), but long-term supplementation prolonged the telogen phase (FIG. 2C, FIG. 61). Corticosterone-fed mice did not display obvious changes in weight, skin thickness or apoptosis (FIGS. 6J-6L). When corticosterone was removed, mice were able to enter anagen (FIG. 6M), which suggests that the effect of corticosterone on HFSCs is reversible.
To examine changes in HFSCs under physiological contexts in which corticosterone levels are altered, a chronic unpredictable stress model was adapted (see Methods)20,21. Stressed mice displayed increased corticosterone levels and significantly extended telogen (FIG. 2D, FIGS. 7A-7C). Consistent with this, adrenalectomy prevented prolonged telogen (FIG. 7D). Because aged mice — similar to stressed mice — display significantly extended telogen length16,17 (FIGS. 5J-5K), it was asked whether corticosterone is also responsible for the long telogen that is seen in aged mice. Consistent with this, older mice showed increased levels of corticosterone, and adrenalectomy in 17-18-month-old mice also led to rapid anagen entry (FIGS. 7E-7F). Collectively, the data show that increased corticosterone levels — whether physiological (for example, arising from chronic stress or ageing) or exogenously provided — extend the length of the telogen phase.
Corticosterone acts on dermal papillae
To identify the cell types on which corticosterone acts to regulate HFSC quiescence, its receptor, GR, was first depleted from HFSCs using the driver K15- CrePGR. Despite efficient depletion of the encoding gene GR — also known as Nr3cl — in HFSCs, the resulting K15-crePGR;GRfl/fl mice did not display significant differences in telogen length (FIGS. 8A-8B), which indicates that corticosterone does not act on HFSCs directly.
It was then asked whether corticosterone acts on cells in the niche to influence stem cell activity. Dermal fibroblasts are a heterogenous population that surrounds the HFSCs. Some fibroblast subpopulations, including the dermal papillae (DP) and adipocyte precursor cells, can regulate HFSC activity22,23. To test whether corticosterone regulates HFSC activity through fibroblasts, GR was depleted using Pdgfra-CreER, a driver expressed in the majority of fibroblast populations including DP and adipocyte precursor cells24 (FIG. 3A, FIG. 8C). Similar to ADX mice, Pdgfra- crcER;GRll/fl mice displayed precocious anagen entry, and their bulge and upper ORS continued to proliferate in late anagen (FIG. 3B, FIGS. 8D-8E). Pdgfra-crcER;GRn/l1 mice — like ADX mice — also entered anagen repeatedly, with short telogen phases in between (FIGS. 8F-8H).
Quantitative PCR with reverse transcription (RT-qPCR) data show that DP express a higher level of GR than do the rest of the fibroblasts (FIG. 9A). To determine whether DP mediate the effect of corticosterone, GR was deleted using Sox2-CreER, a driver expressed in a subset of the DP25,26. Analysis of Sox2- CreER;Rosa-YFP mice suggested that CreER is active in the DP of guard hairs and absent in other hair follicle types (FIG. 3C, FIG. 9B). Similarly, Sox2-CreER;GRfl/fl mice showed GR depletion only in the guard hair DP (2-3% of total hair follicles) (FIG. 9C). These mice displayed precocious anagen entry and entered more rounds of the hair cycle only in guard hair follicles, not in other hair follicles in which Sox2- CreER was not active (FIGS. 3D-3E, FIG. 9D). Late anagen (Anagen V-Anagen IV) Sox2-CreER;GRfl/fl guard hair follicles were also more proliferative in their bulge and upper ORS regions and grew longer hair shafts (FIGS. 9E-9G). Together, these results indicate that DP has a key role in mediating the effect of corticosterone on prolonging telogen.
Corticosterone alters the HFSC transcriptome
To explore the molecular changes in HFSCs that are influenced by corticosterone, RNA sequencing (RNA-seq) was performed on HFSCs purified by fluorescence-activated cell sorting (FACS) from sham, ADX, ADX + corticosterone, Pdgfra-CreER control and Pdgfra-CreER;GRfl/fl mice (all in telogen). First, pairwise comparisons were performed between samples (sham versus ADX; ADX versus ADX + corticosterone; control versus Pdgfra-CreER;GRfl/fl) to identify differentially expressed genes (1.5-fold change, Padj < 0.05), and overlap was looked for between these datasets (FIG. 3F, FIGS. 10A-10B). This analysis revealed 121 common differentially expressed genes, 54 of which are related to cell-cycle regulation or to cytokinesis machineries (FIG. 3G, FIG. IOC). By contrast, genes relating to signalling pathways and transcription factors that are known to regulate HFSC quiescence2-4,7,27- 30 were not significantly altered (FIG. 10D). RNA-seq showed that genes encoding cell-cycle and cell-division machineries are upregulated in the HFSCs of ADX mice despite their being phenotypically quiescent, which suggests that the HFSCs are primed for activation. Moreover, these cell-cycle-related genes were downregulated to levels similar to those in sham controls when the ADX mice were fed with corticosterone, indicating that these molecular changes are downstream of corticosterone (FIG. IOC). Given that these genes also overlapped with those that were differentially expressed in HFSCs purified from Pdgfra-CreER;GRfl/fl mice, the RNA-seq data further supported the idea that dermal fibroblasts relay the effect of corticosterone to HFSCs (FIG. IOC).
To validate these molecular changes, seven core genes (Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl and Cdkl) that represent different cell-cycle or cytokinesis- related categories were selected to verify with RT-qPCR, and all were confirmed using independent biological sample sets (FIGS. 10D-10F). Moreover, these seven genes became downregulated in HFSCs isolated from mice that were subjected to chronic unpredictable stress as well as from mice that were fed with corticosterone, but displayed no changes in the epidermis. This further indicates that these genes are related to the molecular changes in HFSCs that occur downstream of systemic corticosterone (FIGS. 11A-11D).
Gas6 is upregulated in the DP upon adrenalectomy
Next, it was desired to identify dermal genes that relay the effect of corticosterone to HFSCs. For this, RNA-seq was conducted using FACS-enriched DP cells from sham and ADX mice. In parallel, RNA-seq was also conducted of DP from control and Pdgfra-CreER;GRfl/fl mice (FIG. 11E). To avoid identifying changes that simply track with the different stages of the hair cycle, all samples were taken at telogen (FIGS. 11F-11H). From these, differentially expressed genes were identified (1.5-fold change, Padj < 0.05) in DP upon adrenalectomy or dermal GR depletion (FIGS. 11I-11J). Because DP probably exerts regulatory effects on HFSCs through secreted proteins, comparative secretome analyses was also conducted to identify differentially expressed secreted factors in ADX or dermal GR-knockout DP cells (see Methods). This approach identified a total of seven shared secreted factors for which expression levels were significantly altered in the DP of both ADX mice and Pdg fra-C cE R ; GR 11/11 mice compared with controls (FIG. 12A). Among the candidate secreted factors, Gas6 stood out as being abundantly expressed and significantly upregulated (FIGS. 12B-12C). Gas6 encodes a gamma-carboxyglutamic acid- containing secreted protein and predominantly binds to AXL, a member of the TYR03, AXL and MERTK (TAM) family of receptor tyrosine kinases31. It was confirmed by in situ hybridization that Gas6 is upregulated in the DP of ADX mice, and returns to baseline levels when ADX mice are fed with corticosterone (FIG. 4A). In late anagen, Gas6 is downregulated in the DP of control mice, but its expression levels remain high in the DP of ADX mice. In catagen, Gas6 is down-regulated both in control and in ADX mice (FIG. 12D). Moreover, HFSCs at the bulge and at the hair germ express Axl — the gene encoding the GAS6 receptor — whereas the matrix and epidermis have significantly lower levels of Axl (FIGS. 12E-12F). Of all three TAM receptors, Axl is most highly expressed in HFSCs (FIG. 12G).
GAS6 relays the effect of corticosterone
To examine the function of GAS6 in HFSC regulation, HFSCs were purified by FACS, plated in culture, and recombinant GAS6 was added to the media. HFSCs formed more colonies in the presence of GAS6 (FIG. 4B). Moreover, blocking AXL activity with the AXL- specific inhibitor R428 inhibited the ability of GAS 6 to promote HFSC proliferation in vitro (FIG. 12H). To evaluate the potency of GAS6 in promoting HFSC activation in vivo, CAG-GFP and CAG-Gas6 were generated and packaged into adeno-associated viruses (AAVs) and these AAVs were injected into the skin (FIG. 4C, FIG. 13A). RT-qPCR confirmed that Gas6 transcripts were upregulated in the dermal fibroblasts in the AAV-injected regions (FIG. 13B). AAV- CAG-Gas6 injection promoted precocious anagen entry at the site of injection (FIG. 4C). Similar to the results seen in ADX and Pdgfra-creER;GRfl/fl mice, HFSCs continued to proliferate at late anagen when Gas6 was overexpressed, but cells in no other compartment did (FIGS. 13C-13G). In addition, the seven shared core genes that were upregulated in ADX mice also became upregulated in HFSCs when Gas6 was overexpressed (FIG. 13H). Among the seven shared core genes, Cdkl, Ccnbl and Rad51 have been reported as downstream targets of the GAS6-AXL pathway32-34, which supports the idea that GAS6 relays the effects of corticosterone to HFSCs.
Then the effect of the AXL inhibitor R428 was tested in vivo. Topical application of R428 to ADX mice suppressed precocious anagen entry, suggesting that blocking the GAS6-AXL pathway can in part suppress the aberrant HFSC activity that is seen upon the loss of corticosterone (FIG. 131). On a molecular level, R428 also suppressed the expression of the seven core genes that were upregulated in HFSCs isolated from ADX mice (FIG. 13J). Together, these data confirm the role of the corticosterone-GAS6-AXL axis in regulating HFSC activity.
BMP signalling also suppresses Gas6
Notably, Gas6 expression in the DP is low in early (refractory) telogen and is upregulated in late (competent) telogen (FIG. 14A), in agreement with previously reported DP microarray data6. Because corticosterone levels are relatively constant at different telogen stages (FIG. 7E), it was reasoned that additional upstream signals — such as bone morphogenetic protein (BMP) signalling — might also regulate Gas6 levels. BMPs are secreted from fibroblasts and dermal adipocytes to promote HFSC quiescence, and are present at high levels in early telogen but low levels in late telogen4,6 — opposite to the trend that was observed for Gas6. To test whether BMPs regulate GAS6, AAV was used to overexpress Noggin, a secreted BMP inhibitor. Increased levels of Gas6 were observed, which suggests that BMP suppresses the expression of Gas6 (FIGS. 14A-14B). Moreover, suppressing BMP signalling with noggin in ADX mice led to significantly higher Gas6 expression than in the case of ADX or Noggin overexpression alone, and further reduced telogen length relative to that of ADX mice (FIGS. 14A-14C). These data suggest that corticosterone (a systemic hormone) and BMP (a niche signal) represent two upstream signals that both suppress Gas6 levels in DP.
Restoring Gas6 overcomes the effects of stress
Finally, it was asked whether the mechanisms identified here might be harnessed to counteract the effects of increased corticosterone levels and to promote HFSC activation under stress. To this end, it was first tested if extended telogen is shortened by depleting GR specifically in dermal fibroblasts in chronically stressed mice (FIG. 14D). Indeed, it was found that Pdgfra-CrcER;GRll/fl mice displayed significantly shorter telogen phases compared with control mice under chronic stress (FIG. 14D). Gas6 levels were then assessed in DP isolated from stressed mice and mice fed with corticosterone, and it was found that Gas6 levels were significantly downregulated in DP during stress or after an increase in corticosterone levels (FIGS. 14E-14F). To determine whether the restoration of Gas6 expression is sufficient to overcome the stress-induced inhibition of HFSCs, AAV-CAG-Gas6 were injected intradermally and the mice were subjected to chronic unpredictable stress or to long term corticosterone feeding (FIG. 4D). The overexpression of Gas6 effectively mitigated prolonged telogen caused by either condition (FIG. 4D), suggesting that restoration of Gas6 expression is sufficient to promote HFSC activation in a high- corticosterone environment. Together, the data suggest that corticosterone regulates HFSC activity by inhibiting the expression of Gas6 in the DP (FIG. 4E).
Discussion
Stem cells integrate both local and systemic inputs to couple tissue regeneration with the overall physiological state of the animal (Supplementary Discussion). Here, a mechanism was identified by which a systemic factor regulates a stem cell population by inhibiting a niche factor. Previous findings have suggested several mechanisms by which acute stress affects the biology of melanocyte stem cells or hair follicles35-37. The results now suggest that chronic stress delays anagen entry through a distinct mechanism. The findings not only reveal important regulators of HFSC quiescence and activation at both local and systemic levels, but also identify the cellular and molecular mechanisms by which chronic stress influences the hair cycle (FIG. 14G). Moreover, it was demonstrated that the tissue-regeneration capacity of HFSCs remains robust even after significantly increased rounds of anagen entry throughout life. It might therefore be possible to exploit the ability of HFSCs to promote hair-follicle regeneration by modulating the corticosterone-GAS6 axis.
Methods
Data reporting
Mice were randomly assigned to control or experimental groups whenever possible, except in experiments that required specific genotypes, for which littermate controls were used. For analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), RNA-seq library preparation and sequencing, experimenters were blinded to experimental conditions. Blinding was not possible in mouse studies when specific genotypes or surgical models had to be identified according to experimental designs. Mice
C57BL/6J, GR™8, K15-CrePGR39, Pdgfra-CreER40, Sox2-CreER41 and R26- lsl-YFP42 mice were obtained from The Jackson Laboratory. Given the inherent differences in the timing of the hair cycle in wild-type male and female mice — and that male mice start to fight after P70, which confounds hair cycle analyses — data was presented from female mice for most experiments. However, consistent trends were observed in both males and females. For ADX and corticosterone-fed mice, results from both male and female mice were included, and the data are presented separately (female data: FIGS. 1A, 2C; male data: FIG. 5B, FIG. 61). For GR-knockout experiments, sex-matched littermates without Cre or without GRM were used as controls. All the control mice received the same dose of tamoxifen (in the case of Pdgfra-CreER and Sox2-CreER) or RU486 (in the case of K15-CrePGR) as the experimental mice. The mice were maintained in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were approved by the Institutional Animal Care and Use Committee at Harvard University or the Icahn School of Medicine at Mount Sinai. Mice were housed in individually ventilated cages at a maximum density of five mice per cage with Nestlet bedding and a red hut for enrichment and kept on a 12 h-12 h light-dark cycle. Room temperature was maintained at 22 °C ± 1 °C with 30-70% humidity. Mice were fed ad libitum with rodent diet (Prolab IsoPro RMH 30005P75) and water. None of the mice were involved in any previous procedures before the study.
Adrenalectomy
C57BL/6J mice were anaesthetized and small incisions were made on the back skin directly above each adrenal gland. Both adrenal glands were removed with a pair of curved forceps. Sham mice underwent the same procedures as the ADX mice, except their adrenal glands were not removed. Because adrenal glands also secrete aldosterone to regulate salt balance, drinking water was supplemented with 1% w/v saline solution for both ADX and sham mice.
Chemical treatment and viral injection
RU486 (TCI America, M1732; 4%) in ethanol was used to induce K15- CrePGR. RU486 was applied topically once per day for 10 days to K15- CrcPGR;GRll/fl mice and sex-matched, littermate controls. Tamoxifen (Millipore Sigma, T5648) was dissolved in corn oil to a final concentration of 20 mg ml-1 and was used to induce CreER recombinase. Tamoxifen was injected into Pdgfra- CreER;GRfl/fl, Sox2-CreER;GRfl/fl, and sex-matched, littermate controls intraperitoneally once per day for 4-6 days. To inhibit AXL activity, R42843 (APExBIO, A8329; 2 mM in ethanol) was applied to ADX mice topically once a day. EdU (Lumiprobe Corporation, 10540; 25 mg kg-1) was administered by intraperitoneal injections. AAVs were produced as described previously1,44 and injected directly into the dermis through intradermal injections. Two-month-old C57BL/6J mice were injected with AAV-CAG-GFP, AAV-CAG-Gas6, or AAV- CAG-noggin (5 x 1010 genome copy numbers per mouse).
Hair cycle analysis
Hair cycle progression was documented by standardized photographs at the start of each experiment and weekly thereafter. Anagen was determined by darkening of the skin followed by hair growth as previously described5,45. The back skin of mice was shaved with an electric clipper to reveal skin colour changes and hair coat recovery. Once the hair coat recovery reached about 90% of the back skin, the mice were shaved again to monitor the entry into next anagen. To assess the length of each hair cycle phase (telogen, anagen, catagen) of sham and ADX mice (P43-P140), skin colour changes were documented every 2-3 days. The length of each hair cycle was quantified as described previously4 and was confirmed by histological section analysis.
Analysis of hair shaft length and type
Mice were shaved before ADX surgery or tamoxifen treatment to trim off the original hair coat. The mice were then followed through a complete anagen cycle and hair shafts (actual hairs) were plucked in the following telogen phase after anagen was completed. Individual hair shafts were sorted according to hair type (based on their unique banding patterns), and the shaft lengths were measured and analysed under a Keyence BX-700 microscope with 4x, 20x or 40x objectives. Chronic corticosterone feeding
Corticosterone (35 mg ml-1) (Millipore Sigma, C2505) was dissolved in 0.45% hydroxypropyl-P-cyclodextrin and added to the drinking water during the entire corticosterone feeding period19. Corticosterone water was changed every 3 days to prevent degradation. Control mice received vehicle water (0.45% hydroxypropyl-b- cyclodextrin).
Chronic unpredictable stress
Chronic unpredictable stress was adapted from protocols described previously20,21. C57BL/6 mice, sham, ADX, Pdgfra-CrcER;GRll/fl mice, and their littermate controls were exposed to diverse stressors for 9 weeks. Two of the following stressors were applied each day in a randomized fashion: cage tilt, isolation, crowding, damp bedding, rapid light-dark changes, restraining, empty cage, and three cage changes.
ELISA
Blood corticosterone levels were measured by ELISA (ARBOR assays, K014- Hl) according to the manufacturer’s instructions. Serum was collected by the tail clip bleeding method into heparinized tubes (Microvette CB 300 LH (16.443.100) or Microvette 300 LH (20.1309.100), Sarstedt) between 10:00 and 12:00.
Liquid chromatography-tandem mass spectrometry
Blood adrenaline and noradrenaline were measured by LC-MS/MS. A stable- isotope-labelled internal standard (d6-adrenaline, Cambridge Isotope Laboratories, E- 077) was used for absolute quantification. The standards for the HPLC system were prepared using a catecholamine mixture (adrenaline and noradrenaline) (Millipore Sigma, C-109). All samples were analysed on an Agilent 6460 Triple Quadrupole with an Agilent 1290 Infinity LC system.
Histology and immunohistochemistry
Skin samples were fixed in 4% paraformaldehyde (PLA, Electron Micros-copy Sciences, 15713) for 15 min at room temperature, washed in PBS, immersed in 30% sucrose solution overnight at 4 °C, and embedded in optimal cutting temperature compound (OCT, Sakura Finetek, 4583). Sections of approximately 35-50 pm were fixed in 4% PFA for 2 min and washed with PBS and 0.1% Triton X-100 in PBS. The slides were then blocked in blocking buffer (5% donkey serum, 1% bovine serum albumin, 2% cold-water fish gelatin in 0.3% Triton X-100 in PBS) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C, and incubated with secondary antibodies for 2-4 h at room temperature. The following antibodies and dilutions were used: CD34 (eBioscience, 14-0341-82, 1:100), CD140a (R&D Systems, AF1062, 1:100), P-Cadherin (R&D Systems, AF761, 1:400), GFP (Abeam, ab290, 1:5000), cleaved caspase-3 (Cell Signaling Technology, 9661S, 1:300), Sox9 (Millipore Sigma, AB5535, 1:500), phosphorylated histone H3 (Cell Signaling Technology, 3377S, 1:500) and glucocorticoid receptor (Cell Signaling Technology, 3660S, 1:100). DAPI was used as a counterstain for the nucleus. Cell proliferation assays were performed using a Click-It EdU Proliferation kit (Thermo Fisher Scientific, C10337) according to the manufacturer’s instructions. Haematoxylin and eosin (H&E) staining was performed according to standard protocols.
In situ hybridization
Unfixed dorsal skin samples were collected and embedded in OCT. In situ hybridization was performed using the RNAscope 2.5 HD detection kit (Red) (322360, Advanced Cell Diagnostics) with Gas6 probe (450941, Advanced Cell Diagnostics), Axl probe (450931, Advanced Cell Diagnostics), or negative control probe (bacterial gene DapB, 310043, Advanced Cell Diagnostics) according to the manufacturer’s protocol. Mean pixel intensities were measured using ImageJ (v.l.52h).
Fluorescence-activated cell sorting
Dermal cells were isolated as described6,24,44,46’47. Mouse dorsal skin was dissected and treated with collagenase in Hank’s Balanced Salt Solution for 20-30 min at 37 °C on an orbital shaker. The dermal fraction was collected by scraping followed by centrifugation at 300g for 10 min. Dermal single-cell suspensions were obtained after 0.25% trypsin treatment for 10-20 min at 37 °C followed by filtering and centrifugation. Samples were stained for 30 min on ice. The following antibodies were used: CD140a-biotin (eBioscience, 13-1401-82, 1:250), CD45-eFluor450 (eBioscience, 48-0451-82; 1:250), CD31-PE-Cy7 (eBioscience, 25-0311-81, 1:200), Sca-l-PerCP-Cy5.5 (eBioscience, 45-5981-82, 1:1000), CD24-FITC (eBioscience, 11-0242-82; 1:250), and Streptavidin-APC (eBioscience, 17-4317-82, 1:500). DAPI was used to exclude dead cells. DP cells were enriched as
CD45_CD31_CD140a+CD24_Sca-l_ cells, as described and validated previously24,44. The FACS strategy was further validated by the enrichment of DP signature genes46 48 (FIG. 11H).
For the isolation of HFSCs, mouse dorsal skin was dissected and the fat layer was removed using a surgical scalpel. The skin was incubated in trypsin-EDTA at 37 °C for 35-45 min on an orbital shaker. A single-cell suspension was obtained by scraping the epidermal side and filtering. Cells were centrifuged for 8 min at 350g at 4 °C, resuspended in 5% fetal bovine serum, and stained for 30-40 min. The following antibodies were used: CD49f (integrin alpha 6)-PE (eBioscience, 12-0495- 82, 1:500), CD34-eFluor660 (eBioscience, 50-0341-82, 1:100), Sca-l-PerCP-Cy5.5 (eBioscience, 45-5981-82, 1:1,000) and CD45-eFluor450 (eBioscience, 48-0451-82, 1:250). The HFSCs were isolated as CD45_integrin alpha 6+CD34+Sca-l_ cells, and the epidermal stem cells were isolated as CD45_integrin alpha 6+CD34_Sca-l+ cells, as described previouslyl,17. For example FACS plots, see Supplementary Figure 1. The data were analysed with FACSDiva (BD Biosciences, v.8.0.2) and FlowJo (FlowJo LLC, v.10.0.7).
RNA isolation
Cell populations, after isolation by FACS, were sorted directly into TRI-zol LS Reagent (Thermo Fisher Scientific, 10296028). RNA was isolated using an RNeasy Micro Kit (Qiagen, 74004), using QIAcube according to the manufacturer’s instructions. RNA concentration and RNA integrity were determined by Bioanalyzer (Agilent) using the RNA 6000 Pico kit (Agilent, 5067-1513). High-quality RNA samples with RNA integrity number > 8 were used as input for RT-qPCR and RNA- seq.
Complementary DNA synthesis and quantitative PCR
Complementary DNA was synthesized using the Superscript IV VILO Master Mix with ezDNase Enzyme (Thermo Fisher Scientific, 11766050). Quantitative PCR was performed using power SYBR Green dye (Thermo Fisher Scientific, 4368706) on a QuantStudio 6 Flex Real-Time PCR system. Ct values were normalized to an internal control (b-actin). RNA sequencing and computational analysis
RNA sequencing libraries were prepared using 1 ng of total RNA as input. A SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, 634888) was used for cDNA synthesis, with a 10-cycle PCR enrichment. Sequencing libraries were made using Illumina’s Nextera XT Library Prep kit (Illumina, FC-131-1024). Single read sequencing reads for DP samples were obtained using the Illumina NextSeq 500 platform, and aligned to the mouse reference genome (mmlO) using Salmon (v.1.55)49. Paired-end sequencing reads for HFSC samples were trimmed with Trim Galore! (v.0.4.1, bioinformatics.babraham.ac.uk/projects/tr-im_galore/) and aligned to mmlO using STAR (v.2.5.3)50. The reads were annotated using featurecounts51. Differential expression analysis was performed using the DESeq2 package (v.1.22.2) in R (v.3.5.1) and RStudio (v.1.1.453)52. Gene Ontology (GO) enrichment analysis was performed using the database for annotation, visualization and integrated discovery (DAVID) web-accessible tool (v.6.8)53,54. Transcripts per kilobase million (TPM), calculated from counts of control HFSC samples, were used to determine the expression levels of the AXL receptor shown in FIG. 12G. Signal peptide prediction and secretome analysis were performed on differentially expressed genes using the Phobius web-accessible tool55 (phobius.sbc.su.se/) and the secretome knowledge base available at the MetazSecKB web-accessible tool56.
Colony formation assay
FACS -purified HFSCs were plated on mitomycin C (P212121, M920 Re treated J2 fibroblast feeders at a density of 10,000 cells per well in 12-well plates in E media supplemented with 15% (v/v) serum and 0.3 mM calcium (calcium chloride; Millipore Sigma, C3881) as described in previous studies57. For GAS6 treatment, E medium was supplemented with recombinant mouse GAS6 protein (R&D systems, 8310-GS) at 500 ng ml-1. For R428-treatment experiments, E medium was supplemented with R428 (APExBIO, A8329) at 1 mM. Cells were fixed and stained with 1% (w/v) Rhodamine B (Millipore Sigma, R6626). Colony diameter was measured from scanned images of plates using ImageJ (v.l.52h). Imaging and image analysis
Images were obtained with a Zeiss LSM 880 confocal microscope with a 20x air objective or 40x oil-based objective (Carl Zeiss) or a Keyence BX-700 epifluorescence microscope with 4x, 20x or 40x objective (Keyence). Images are presented as maximum intensity projection images or a single Z stack. Images were further processed and assembled into panels using Adobe Photoshop (v.21.2.4) and Adobe Illustrator (v.24.3).
Statistical analyses
Statistical analyses were performed with GraphPad Prism v.8.4.2 (GraphPad Software) using an unpaired two-sided Student’s t-test, one-way ANOVA with Tukey’s two-sided multiple comparisons, two-way ANOVA with Bonferroni’s two- sided multiple comparisons, or two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. The DESeq2 package used the Wald test for hypothesis testing and the Benjamini-Hochberg method for multiple testing correction, and genes were considered differentially expressed if they had an adjusted P value of less than 0.05. DAVID used one-sided Fisher’s exact test with false- discovery rate (FDR) correction. The data are presented as mean ± s.e.m. No statistical methods were used to predetermine sample size.
Statistics and reproducibility
FIG. 1A, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. IB, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 1C, n = 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. ID, n = 5 mice per condition. The experiments were performed twice with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. IE, n = 7 (sham), n = 6 (ADX) mice. The experiments were performed twice with similar results. Two-way repeated measures ANOVA with Bonferroni’s two- sided multiple comparisons. FIG. IF, n = 5 mice per condition. The experiments were performed twice with similar results. FIG. 2A, n = 3 mice per condition. The experiments were performed three times with similar results. One-way ANOVA with Tukey’s two-sided multiple comparisons test. FIG. 2B, Left, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. Right, the experiments were performed three times with similar results. FIG. 2C, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 2D, n = 10 mice per condition. The experiments were performed three times with similar results. Two- way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons.
FIG. 3A, n = 3 biologically independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 3B, n = 6 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 3C, The experiments were performed three times with similar results. FIG. 3D, The experiments were performed three times with similar results. FIG. 3E, n = 5 mice per condition. The experiments were performed three times with similar results. FIG. 3G, n = 2 biological replicates from each condition. The experiments were performed once. One-sided Fisher’s exact test with FDR correction.
FIG. 4A, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. One-way ANOVA with Tukey’s two- sided multiple comparisons test. FIG. 4B, n = 3 biologically independent samples per condition. The experiments were performed three times with similar results. Two- sided unpaired t-test. FIG. 4C, Left, n = 5 mice per condition. The experiments were performed three times with similar results. Right, the experiments were performed three times with similar results. FIG. 4D, n = 5 mice per condition. The experiments were performed three times with similar results.
FIG. 5 A, The experiments were performed three times with similar results. FIG. 5B, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 5D, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t- test. FIG. 5E, n = 20 hair follicles (guard hairs), n = 30 hair follicles (awl/auchene hairs and zigzag hairs) from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 5F, The experiments were performed three times with similar results. FIG. 5G, The experiments were performed three times with similar results. FIG. 5H, n = 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two- sided unpaired t-test. FIG. 51, n = 30 skin regions from 5 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t- test. FIG. 5J, n = 7 (sham), n = 6 (ADX) mice. The experiments were performed twice with similar results. FIG. 5K, n = 5 mice per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 5L, n = 40 skin regions from 4 mice per condition. The experiments were performed twice with similar results. One-way ANOVA with Tukey’s two-sided multiple comparisons.
FIG. 6A, The experiments were performed three times with similar results. FIG. 6B, n = 114, 127, 100 hair follicles from 3 mice (sham), n = 100, 100, 119 hair follicles from 3 mice (ADX). The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 6C, Left, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Right, n = 2 biological independent samples per condition. The experiments were performed twice with similar results. FIG. 6D, n = 3 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 6E, n = 3 mice per condition. The experiments were performed twice with similar results. Two- sided unpaired t-test. FIG. 6F, n = 5 mice per condition. The experiments were performed twice with similar results. Two-way ANOVA repeated measures with Bonferroni’s two-sided multiple comparisons. FIG. 6G, n = 3 mice. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 6H, n = 5 mice per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 61, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA repeated measures with Bonferroni’s two-sided multiple comparisons. FIG. 6J, n = 5 mice per condition. The experiments were performed twice with similar results. Two-way ANOVA repeated measures with Bonferroni’s two-sided multiple comparisons. FIG. 6K, n = 30 skin regions from 3 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 6L, The experiments were performed three times with similar results. FIG. 6M, n = 4 mice (vehicle), n = 5 mice (corticosterone). The experiments were performed twice with similar results. Two- way ANOVA repeated measures with Bonferroni’s two-sided multiple comparisons.
FIG. 7A, n = 3 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 7B, The experiments were performed three times with similar results. FIG. 7C, The experiments were performed three times with similar results. FIG. 7D, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 7E, n = 4 mice per condition. The experiments were performed three times with similar results. One-way ANOVA with Tukey’s two-sided multiple comparisons. FIG. 7F, n = 5 mice (sham), n = 7 mice (ADX). The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons.
FIG. 8A, Top, n = 3 biologically independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. Bottom, the experiments were performed three times with similar results. FIG. 8B, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 8C, The experiments were performed three times with similar results. FIG. 8D, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 8E, n = 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 8F, n = 5 mice per condition. The experiments were performed twice with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 8G, n = 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 8H, n = 30 skin regions from 5 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
FIG. 9A, n = 2 biological independent samples per condition. The experiments were performed twice with similar results. FIG. 9B, n = 118, 139, 149 hair follicles per mouse from 3 mice. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 9C, The experiments were performed three times with similar results. FIG. 9D, n = 3 mice per condition. The experiments were performed twice with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons test. FIG. 9E, n = 20 guard hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 9F, n = 20 guard hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two- sided unpaired t-test. FIG. 9G, n = 20 guard hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
FIGS. 10A-10D, n = 2 biological independent samples for each condition. The experiments were performed once. FIG. 10E, n = 4 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 10F, n = 3 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test.
FIGS. 1 lA-1 IB, n = 3 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIGS. 1 lC-1 ID, n = 4 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIGS. 1 lF-11G, The experiments were performed three times with similar results. FIG. 11H, n = 3 biological independent samples. The experiments were performed once. FIGS lll- ll J, n = 2 biological independent samples for each condition. The experiments were performed once.
FIG. 12A, n = 2 biological independent samples for each condition. The experiments were performed once. FIGS. 12B-12C, n = 2 bio-logical independent samples for each condition. The experiments were performed once. Two-sided Wald test with multiple testing adjustments using the Benjamini-Hochberg method in DESeq2. FIG. 12D, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 12E, Top, the experiments were performed three times with similar results. Bottom, n = 4 biological independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 12F, The experiments were performed three times with similar results. FIG. 12G, n = 2 biological independent samples per condition. The experiments were performed once. FIG. 12H, n = 3 biological independent samples per condition. The experiments were performed three times with similar results. One way ANOVA with Tukey’s two-sided multiple comparisons.
FIG. 13A, The experiments were performed four times with similar results. FIG. 13B, n = 2 biologically independent samples per condition. The experiments were performed twice with similar results. FIG. 13C, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two- way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 13D, n = 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two-way ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 13E, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t- test. FIG. 13F, n = 30 hair follicles from 5 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 13G, n = 30 skin regions from 5 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test. FIG. 13H, n = 3 biologically independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 131, n = 5 mice per condition. The experiments were performed three times with similar results. Two-way repeated measures ANOVA with Bonferroni’s two-sided multiple comparisons. FIG. 13J, n = 3 (sham + EtOH), n = 4 (ADX + EtOH), and n = 4 biologically independent samples (ADX + EtOH). The experiments were performed twice with similar results. Two- sided unpaired t-test.
FIG. 14A, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. One-way ANOVA with Tukey’s two- sided multiple comparisons. FIG. 14B, n = 3 biologically independent samples per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 14C, n = 5 per condition. The experiments were performed three times with similar results. Two-way ANOVA repeated measures with Bonferroni’s two-sided multiple comparisons. FIG. 14D, n = 5 mice per condition. The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 14E, n = 3 biologically independent samples per condition (left), n = 4 biologically independent samples per condition (right). The experiments were performed twice with similar results. Two-sided unpaired t-test. FIG. 14F, n = 20 hair follicles from 4 mice per condition. The experiments were performed three times with similar results. Two-sided unpaired t-test.
References
1. Shwartz, Y. et al. Cell types promoting goosebumps form a niche to regulate hair follicle stem cells. Cell 182, 578-593.el9 (2020).
2. Lay, K., Kume, T. & Fuchs, E. FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential. Proc. Natl Acad. Sci. USA 113, E1506-E1515 (2016).
3. Wang, L., Siegenthaler, J. A., Dowell, R. D. & Yi, R. Foxcl reinforces quiescence in self-renewing hair follicle stem cells. Science 351, 613-617 (2016).
4. Plikus, M. V. et al. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature 451, 340-344 (2008).
5. Miiller-Rover, S. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Invest. Dermatol. 117, 3-15 (2001).
6. Greco, V. et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4, 155-169 (2009).
7. Hsu, Y. C., Li, L. & Fuchs, E. Transit-amplifying cells orchestrate stem cell activity and tissue regeneration. Cell 157, 935-949 (2014).
8. Sawaya, M. E. & Hordinsky, M. K. Glucocorticoid regulation of hair growth in alopecia areata. J. Invest. Dermatol. 104, 30S (1995).
9. Stenn, K. S., Paus, R., Dutton, T. & Sarba, B. Glucocorticoid effect on hair growth initiation: a reconsideration. Skin Pharmacol. 6, 125-134 (1993).
10. Perez, P. et al. Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor. FASEB J. 15, 2030-2032 (2001).
11. Rose, J. & Sterner, M. The role of the adrenal glands in regulating onset of winter fur growth in mink (Mustela vison). J. Exp. Zool. 262, 469-473 (1992). 12. Butcher, E. O. Hair growth in adrenalectomized, and, adrenalectomized thyroxin-treated rats. Am. J. Physiol. 120, 427-434 (1937).
13. Whiteley, H. J. The effect of adrenalectomy and adrenocortical hormones on the hair growth cycle in the rabbit and rat. J. Endocrinol. 17, 167-176 (1958).
14. Hsu, Y. C., Pasolli, H. A. & Fuchs, E. Dynamics between stem cells, niche, and progeny in the hair follicle. Cell 144, 92-105 (2011).
15. Rompolas, P., Mesa, K. R. & Greco, V. Spatial organization within a niche as a determinant of stem-cell fate. Nature 502, 513-518 (2013).
16. Chen, C. C. et al. Regenerative hair waves in aging mice and extra-follicular modulators follistatin, Dkkl, and Sfrp4. J. Invest. Dermatol. 134, 2086-2096 (2014).
17. Keyes, B. E. et al. Nfatcl orchestrates aging in hair follicle stem cells. Proc. Natl Acad. Sci. USA 110, E4950-E4959 (2013).
18. Walczak, E. M. & Hammer, G. D. Regulation of the adrenocortical stem cell niche: implications for disease. Nat. Rev. Endocrinol. 11, 14-28 (2015).
19. Besnard, A. et al. Targeting Kruppel-like factor 9 in excitatory neurons protects against chronic stress-induced impairments in dendritic spines and fear responses. Cell Rep. 23, 3183-3196 (2018).
20. Heidt, T. et al. Chronic variable stress activates hematopoietic stem cells. Nat. Med. 20, 754-758 (2014).
21. Tye, K. M. et al. Dopamine neurons modulate neural encoding and expression of depression-related behaviour. Nature 493, 537-541 (2013).
22. Enshell-Seijffers, D., Lindon, C., Kashiwagi, M. & Morgan, B. A. b-catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair. Dev. Cell 18, 633-642 (2010).
23. Festa, E. et al. Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling. Cell 146, 761-771 (2011).
24. Zhang, B. et al. Hair follicles’ transit-amplifying cells govern concurrent dermal adipocyte production through Sonic Hedgehog. Genes Dev. 30, 2325-2338 (2016). 25. Clavel, C. et al. Sox2 in the dermal papilla niche controls hair growth by fine- tuning BMP signaling in differentiating hair shaft progenitors. Dev. Cell 23, 981-994 (2012).
26. Driskell, R. R., Giangreco, A., Jensen, K. B., Mulder, K. W. & Watt, F. M. Sox2 -positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development 136, 2815-2823 (2009).
27. Horsley, V., Aliprantis, A. O., Polak, L., Glimcher, L. H. & Fuchs, E. NFATcl balances quiescence and proliferation of skin stem cells. Cell 132, 299-310 (2008).
28. Rhee, H., Polak, L. & Fuchs, E. Lhx2 maintains stem cell character in hair follicles. Science 312, 1946-1949 (2006).
29. Leishman, E. et al. Foxpl maintains hair follicle stem cell quiescence through regulation of Fgfl8. Development 140, 3809-3818 (2013).
30. Choi, Y. S. et al. Distinct functions for Wnt/p-catenin in hair follicle stem cell proliferation and survival and interfollicular epidermal homeostasis. Cell Stem Cell 13, 720-733 (2013).
31. Rothlin, C. V., Carrera-Silva, E. A., Bosurgi, L. & Ghosh, S. TAM receptor signaling in immune homeostasis. Annu. Rev. Immunol. 33, 355-391 (2015).
32. Wang, Y. et al. Axl-altered microRNAs regulate tumorigenicity and gefitinib resistance in lung cancer. Cell Death Dis. 5, el 227 (2014).
33. Asiedu, M. K. et al. AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells. Oncogene 33, 1316-1324 (2014).
34. Balaji, K. et al. AXL inhibition suppresses the DNA damage response and sensitizes cells to PARP inhibition in multiple cancers. Mol. Cancer Res. 15, 45-58 (2017).
35. Aoki, E., Shibasaki, T. & Kawana, S. Intermittent foot shock stress prolongs the telogen stage in the hair cycle of mice. Exp. Dermatol. 12, 371-377 (2003).
36. Zhang, B. et al. Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells. Nature 577, 676-681 (2020). 37. Arck, P. C. et al. Stress inhibits hair growth in mice by induction of premature catagen development and deleterious perifollicular inflammatory events via neuropeptide substance P-dependent pathways. Am. J. Pathol. 162, 803-814 (2003).
38. Mittelstadt, P. R., Monteiro, J. P. & Ashwell, J. D. Thymocyte responsiveness to endogenous glucocorticoids is required for immunological fitness. J. Clin. Invest. 122, 2384-2394 (2012).
39. Morris, R. J. et al. Capturing and profiling adult hair follicle stem cells. Nat. Biotechnol. 22, 411-417 (2004).
40. Kang, S. H., Fukaya, M., Yang, J. K., Rothstein, J. D. & Bergles, D. E. NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration. Neuron 68, 668-681 (2010).
41. Arnold, K. et al. Sox2+ adult stem and progenitor cells are important for tissue regeneration and survival of mice. Cell Stem Cell 9, 317-329 (2011).
42. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).
43. Holland, S. J. et al. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res. 70, 1544-1554 (2010).
44. Goldstein, J. M. et al. In situ modification of tissue stem and progenitor cell genomes. Cell Rep. 27, 1254-1264.e7 (2019).
45. Plikus, M. V. & Chuong, C. M. Complex hair cycle domain patterns and regenerative hair waves in living rodents. J. Invest. Dermatol. 128, 1071-1080 (2008).
46. Rezza, A. et al. Signaling networks among stem cell precursors, transit- amplifying progenitors, and their niche in developing hair follicles. Cell Rep. 14, 3001-3018 (2016).
47. Rendl, M., Lewis, L. & Fuchs, E. Molecular dissection of mesenchymal- epithelial interactions in the hair follicle. PLoS Biol. 3, e331 (2005).
48. Joost, S. et al. The molecular anatomy of mouse skin during hair growth and rest. Cell Stem Cell 26, 441-457.e7 (2020). 49. Patro, R., Duggal, G., Love, M. L, Mzarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417-419 (2017).
50. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013).
51. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923- 930 (2014).
52. Love, M. L, Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
53. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1-13 (2009).
54. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-
57 (2009).
55. Kail, L., Krogh, A. & Sonnhammer, E. L. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 338, 1027-1036 (2004).
56. Meinken, J., Walker, G., Cooper, C. R. & Min, X. J. MetazSecKB: the human and animal secretome and subcellular proteome knowledgebase. Database (Oxford)
2015, bav077 (2015).
57. Nowak, J. A. & Fuchs, E. Isolation and culture of epithelial stem cells. Methods Mol. Biol. 482, 215-232 (2009).

Claims

CLAIMS What is claimed is:
1. A method of promoting or increasing hair growth in an individual in need thereof comprising administering to the individual an effective amount of an agent that increases Gas6 expression.
2. The method of claim 1, wherein the agent increases the Gas6- Tyro3/Axl/Mertk (TAM) interaction or pathway.
3. The method of claim 2, wherein the TAM interaction or pathway is an AXL interaction or pathway.
4. The method of claim 2, wherein the TAM interaction or pathway is a Tyro3 interaction or pathway.
5. The method of claim 2, wherein the TAM interaction or pathway is a Mertk interaction or pathway.
6. The method of claim 1, wherein the agent suppresses BMP signaling.
7. The method of claim 6, wherein the agent is noggin.
8. The method of claim 1, wherein the agent increases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl.
9. The method of claim 1, wherein the agent increases expression of one or more genes selected from the group consisting of Ccnbl , Rad51, and Cdkl .
10. The method of any one of claims 1-9, wherein the agent is administered using an AAV vector.
11. The method of claim 10, wherein the AAV is AAV8.
12. The method of any one of claims 1-11, wherein the agent is administered through intradermal injection.
13. The method of any one of claims 1-12, wherein hair growth is increased by at least 10% relative to a suitable control.
14. The method of any one of claims 1-13, wherein hair growth is increased by at least 25% relative to a suitable control.
15. A method of decreasing hair growth in an individual in need thereof comprising administering to the individual an effective amount of an agent that decreases or inhibits Gas6 expression.
16. The method of claim 15, wherein the agent decreases the Gas6- Tyro3/Axl/Mertk (TAM) interaction or pathway.
17. The method of claim 16, wherein the TAM interaction or pathway is an AXL interaction or pathway.
18. The method of claim 16, wherein the TAM interaction or pathway is a Tyro3 interaction or pathway.
19. The method of claim 16, wherein the TAM interaction or pathway is a Mertk interaction or pathway.
20. The method of claim 15, wherein the agent comprises an AXL inhibitor.
21. The method of claim 20, wherein the agent comprises R428.
22. The method of claim 15, wherein the agent activates BMP signaling.
23. The method of claim 15, wherein the agent decreases expression of one or more genes selected from the group consisting of Aurkb, Ccnbl, Ccnb2, Cdca2, Rad51, Prcl, and Cdkl .
24. The method of claim 15, wherein the agent decreases expression of one or more genes selected from the group consisting of Ccnbl , Rad51, and Cdkl .
25. The method of any one of claims 15-24, wherein the agent is administered using an AAV vector.
26. The method of claim 25, wherein the AAV is AAV8.
27. The method of any one of claims 15-26, wherein the agent is administered through intradermal injection.
28. The method of any one of claims 15-27, wherein hair growth is decreased by at least 10% relative to a suitable control.
29. The method of any one of claims 15-28, wherein hair growth is decreased by at least 25% relative to a suitable control.
PCT/US2022/022956 2021-03-31 2022-03-31 Methods of modulating hair growth WO2022212790A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163169042P 2021-03-31 2021-03-31
US63/169,042 2021-03-31

Publications (1)

Publication Number Publication Date
WO2022212790A1 true WO2022212790A1 (en) 2022-10-06

Family

ID=83459814

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/022956 WO2022212790A1 (en) 2021-03-31 2022-03-31 Methods of modulating hair growth

Country Status (1)

Country Link
WO (1) WO2022212790A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080089873A1 (en) * 2004-06-14 2008-04-17 Shiseido Company, Ltd. Method for Regenerating Hair Follicles by Suppressing a Gene with Hair Follicle Formation-Inhibiting Ability or by Activating a Gene with Hair Follicle Formation-Inducing Ability
US20130158103A1 (en) * 2011-11-23 2013-06-20 The Curators Of The University Of Missouri Method of Tissue-Selective Targeted Gene Transfer
US20160046725A1 (en) * 2012-11-05 2016-02-18 Pierre Fabre Medicament Novel antigen binding proteins and their use as addressing product for the treatment of cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080089873A1 (en) * 2004-06-14 2008-04-17 Shiseido Company, Ltd. Method for Regenerating Hair Follicles by Suppressing a Gene with Hair Follicle Formation-Inhibiting Ability or by Activating a Gene with Hair Follicle Formation-Inducing Ability
US20130158103A1 (en) * 2011-11-23 2013-06-20 The Curators Of The University Of Missouri Method of Tissue-Selective Targeted Gene Transfer
US20160046725A1 (en) * 2012-11-05 2016-02-18 Pierre Fabre Medicament Novel antigen binding proteins and their use as addressing product for the treatment of cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PLIKUS MAKSIM V., MAYER JULIE ANN, DE LA CRUZ DAMON, BAKER RUTH E., MAINI PHILIP K., MAXSON ROBERT, CHUONG CHENG-MING: "Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 451, no. 7176, 1 January 2008 (2008-01-01), London, pages 340 - 344, XP055976141, ISSN: 0028-0836, DOI: 10.1038/nature06457 *
WANG ET AL.: "Identification and characterization of mouse Gas6 promoter", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION S, vol. 371, 5 May 2008 (2008-05-05), pages 567 - 572, XP022670743, DOI: 10.1016/j.bbrc.2008.04.130 *

Similar Documents

Publication Publication Date Title
Choi et al. Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence
Chang et al. The dystrophin glycoprotein complex regulates the epigenetic activation of muscle stem cell commitment
Zhao et al. Endocardial Notch signaling promotes cardiomyocyte proliferation in the regenerating zebrafish heart through Wnt pathway antagonism
Kamiya et al. Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts
Leishman et al. Foxp1 maintains hair follicle stem cell quiescence through regulation of Fgf18
Bär et al. Telomerase expression confers cardioprotection in the adult mouse heart after acute myocardial infarction
Shimomura et al. Biology and genetics of hair
Quarto et al. Exogenous activation of BMP-2 signaling overcomes TGFβ-mediated inhibition of osteogenesis in Marfan embryonic stem cells and Marfan patient-specific induced pluripotent stem cells
Mochizuki et al. Combinatorial CRISPR/Cas9 approach to elucidate a far-upstream enhancer complex for tissue-specific Sox9 expression
US11725033B2 (en) GDF11 variants and uses thereof
Escobedo et al. Syndecan 4 interacts genetically with Vangl2 to regulate neural tube closure and planar cell polarity
Du et al. A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration
Maioli et al. Hyaluronan esters drive Smad gene expression and signaling enhancing cardiogenesis in mouse embryonic and human mesenchymal stem cells
Mademtzoglou et al. Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells
Tripurani et al. Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development
Wang et al. A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells
Li et al. Downregulation of miR‑98 contributes to hypoxic pulmonary hypertension by targeting ALK1
Forouhan et al. AR cooperates with SMAD4 to maintain skeletal muscle homeostasis
Song et al. MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration
WO2022212790A1 (en) Methods of modulating hair growth
Dong et al. Wnt4 is crucial for cardiac repair by regulating mesenchymal-endothelial transition via the phospho-JNK/JNK
Bailey et al. MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states
Gouignard et al. Paracrine regulation of neural crest EMT by placodal MMP28
US20220387552A1 (en) Methods of modulating hair follicle stem cell quiescence by modulating dermal niche activator gas6
Iacono et al. The Hay Wells syndrome-derived TAp63αQ540L mutant has impaired transcriptional and cell growth regulatory activity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22782261

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22782261

Country of ref document: EP

Kind code of ref document: A1