Stem cell plasticity enables hair regeneration following Lgr5+ cell loss

Abstract

Under injury conditions, dedicated stem cell populations govern tissue regeneration. However, the molecular mechanisms that induce stem cell regeneration and enable plasticity are poorly understood. Here, we investigate stem cell recovery in the context of the hair follicle to understand how two molecularly distinct stem cell populations are integrated. Utilizing diphtheria-toxin-mediated cell ablation of Lgr5+ (leucine-rich repeat-containing G-protein-coupled receptor 5) stem cells, we show that killing of Lgr5+ cells in mice abrogates hair regeneration but this is reversible. During recovery, CD34+ (CD34 antigen) stem cells activate inflammatory response programs and start dividing. Pharmacological attenuation of inflammation inhibits CD34+ cell proliferation. Subsequently, the Wnt pathway controls the recovery of Lgr5+ cells and inhibition of Wnt signalling prevents Lgr5+ cell and hair germ recovery. Thus, our study uncovers a compensatory relationship between two stem cell populations and the underlying molecular mechanisms that enable hair follicle regeneration.

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Figure 1: Labelling and deletion of Lgr5+ bulge and hair germ stem cells.
Figure 2: Ablation of Lgr5+ cells inhibits telogen-to-anagen transition and hair growth.
Figure 3: Discontinuation of Lgr5+ cell ablation restores hair growth.
Figure 4: CD34+ stem cell activation and inflammation signature.
Figure 5: Attenuation of inflammation inhibits CD34+ stem cell activation.
Figure 6: CD34+ stem cells give rise to Lgr5+ cells.
Figure 7: BMP and Wnt signalling play opposite roles during Lgr5+ cell recovery.
Figure 8: Wnt signalling is required for Lgr5+ cell recovery.

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Acknowledgements

We are grateful to the Animal Unit, particularly W. Ortiz, the Center for Advanced Light Microscopy, especially L. Komuves, and the Fluorescence Activated Cell Sorting (FACS) laboratory at Genentech for technical help and advice. We thank P. Sotiropoulou at the Universite libre de Bruxelles for her advice on CD34+ cell FACS sorting. We thank H. Sharpe and C. Metcalfe for critical reading of the manuscript.

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Contributions

J.D.H. designed and conducted the majority of the experiments, performed the data analyses and wrote the manuscript. B.B. carried out the ISH and DAB stainings and co-wrote the manuscript. A.V.K., N.M.K., F.d.S.e.M. and B.A. helped with the in vivo experiments. H.K. analysed the ISH results. Z.M. managed the library generation and RNA-sequencing. R.P. analysed the RNA-sequencing data. F.J.d.S. supervised the project and co-wrote the manuscript.

Corresponding author

Correspondence to Frederic J. de Sauvage.

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Competing interests

All authors are employees of Genentech, Inc. and some own shares of Roche.

Integrated supplementary information

Supplementary Figure 1 Lgr5+ cell ablation in the skin of the Lgr5DTR/+ mouse model.

(a) Schematic representation of the Lgr5DTR/+ mouse model expressing a diphtheria toxin receptor (DTR)/enhanced green fluorescent protein (eGFP) fusion construct in the endogenous Lgr5 locus. Lgr5+ cells are depicted in green and are ablated upon diphtheria toxin treatment (DT). (b) Immunofluorescence co-stainings for GFP and the apoptosis marker cleaved Caspase 3 (Casp3) in Lgr5+/+ (wt) and Lgr5DTR/+ mice 24 h after administration of one dose of DT (100 μg kg−1 bodyweight) or 0.9% NaCl. Scale bars, 20 μm. (c) H&E staining on skin from untreated 3.5 week old Lgr5DTR/+ mice. Rectangle marks region shown below in high magnification. Scale bars, 50 μm. (d) Immunofluorescence staining for the hair germ stem cell marker P-Cad on skin from untreated 3.5 week old Lgr5DTR/+ mice. Asterisks mark autofluorescence from hair shafts. Scale bars, 50 μm. Where visible, the dermal papilla is separated from the hair follicle epithelium by a dotted line.

Supplementary Figure 2 Deletion of Lgr5+ cells during anagen results in the collapse of the bulb.

(a) Immunofluorescence staining for CD34 and GFP on Lgr5DTR/+ skin during anagen phase of the hair cycle. (b) Schematic depicting the dosing regimen of 5 week old Lgr5DTR/+ mice treated every 48 h with DT (100 μg kg−1 bodyweight) or 0.9% NaCl. DT/NaCl treatment was discontinued after three doses and tissue was harvested at d1 or d11. Rectangular bars show hair cycle stages of control mice at the indicated age. Ana, Anagen; Cat, Catagen; Tel, Telogen. (c,e) H&E staining on (c) d1 and (e) d11 skin from Lgr5DTR/+ mice treated with DT or NaCl. Rectangles mark regions shown below in high magnification. (d,f) Immunofluorescence staining for CD34 on (d) d1 and (f) d11 skin from Lgr5DTR/+ mice treated with DT or NaCl. Where visible, the dermal papilla is separated from the hair follicle epithelium by a dotted line. Bu, bulge. Scale bars, 50 μm.

Supplementary Figure 3 Lgr5+ cells are not required for wound healing.

(a) Schematic depicting the dosing regimen of 3.5 week old Lgr5+/+ and Lgr5DTR/+ mice treated every 48 h with DT (100 μg kg−1 bodyweight) or 0.9% NaCl. Two full-thickness wounds (6 mm diameter) were induced on d1 of the experiment and wound healing was analyzed at d8. (b) Brightfield images and H&E stainings on d8 skin from wounded Lgr5+/+ and Lgr5DTR/+ mice treated with DT or NaCl. Scale bars, 50 μm. (c) Quantification of the wound area (in cm2) at d8 on the back of Lgr5+/+ and Lgr5DTR/+ mice treated with DT or NaCl. Each data point represents the results from one mouse. n = 4 mice. Center values, mean; Error bars, s.e.m.; n.s., not significant (unpaired t-test).

Supplementary Figure 4 Lgr5+ cell deletion delays immediate but not long-term anagen induction.

(a) Schematic depicting the dosing regimen of 8 week old Lgr5+/+ and Lgr5DTR/+ mice in telogen treated every 48 h with DT (100 μg kg−1 bodyweight) or 0.9% NaCl. Hair plucking was performed 24 h after the last DT or saline injection. DT and NaCl treatment was discontinued after three doses and tissue was harvested at d25. Rectangular bar shows hair cycle stage of control mice at the indicated age. Tel, Telogen. (b) H&E staining on d25 skin from Lgr5+/+ and Lgr5DTR/+ mice treated with 3 doses of DT or NaCl. (c) Schematic showing the dosing regimen of 8 week old Lgr5+/+ and Lgr5DTR/+ mice in telogen treated every 48 h with DT (100 μg kg−1 bodyweight) or 0.9% NaCl. DT and NaCl treatment was discontinued after three doses and tissue was harvested at d14 and at d31. Rectangular bars show hair cycle stages of control mice at the indicated age. Ana, Anagen; Tel, Telogen. (d) H&E staining on d14 skin of Lgr5+/+ and Lgr5DTR/+ mice treated with 3 doses of DT or NaCl. (e) Immunofluorescence staining for CD34 and P-Cad on d14 skin from Lgr5+/+ and Lgr5DTR/+ mice treated with 3 doses of DT or NaCl. (f) H&E staining on d31 skin from Lgr5+/+ and Lgr5DTR/+ mice treated with 3 doses of DT or NaCl. Where visible, the dermal papilla is separated from the hair follicle epithelium by a dotted line. In (b,d,f), scale bars, 50 μm. In (e), scale bars, 20 μm.

Supplementary Figure 5 CD34+ cell divisions during Lgr5+ cell recovery.

(af) Immunofluorescence co-staining for CD34 and the cell division marker phospho-Histone H3 (pH3) on skin from Lgr5DTR/+ mice treated with 3 doses of (a) NaCl or (bf) DT at (a,b) d1, (c) d2, (d) d4, (e) d7 and (f) d11. Yellow arrows point out pH3+ cells. Asterisks mark autofluorescence from hair shafts. Where visible, the dermal papilla is separated from the hair follicle epithelium by a dotted line. Scale bars, 20 μm.

Supplementary Figure 6 CD34+ cell proliferation does not depend on developmental pathways.

(a) Gene set activity in DT-treated versus saline treated samples for selected gene sets associated with developmental pathways BMP, Wnt and Hh, unchanged in CD34+ hair follicle stem cells of DT-treated Lgr5DTR/+ mice at d1. For each gene set the mean and 95% confidence intervals are plotted and coloured according to their false discovery rate (FDR)-adjusted P-values when compared to zero. n = 3 mice. (b) Schematic showing the dosing regimen of 8 week old Lgr5DTR/+ mice treated with three doses of DT (100 μg kg−1 bodyweight) every 48 h. Starting 24 h before the first DT administration, mice were treated with either vehicle control (MCT), vismodegib (Vismo, 75 mg kg−1 bodyweight), LDN-193189 (LDN, 35 mg kg−1 bodyweight) or LGK-974 (LGK, 5 mg kg−1 bodyweight) twice daily (b.i.d.). Rectangular bars show hair cycle stage of control mice at the indicated age. Tel, Telogen. (c) Immunofluorescence co-staining for CD34 and the proliferation marker Ki67 on d1 skin from DT-treated Lgr5DTR/+ mice that received either MCT (upper left), Vismo (upper right), LDN (lower left) or LGK (lower right). (d) Histogram depicting the percentage of Ki67+ cells among CD34+ stem cells at d1 of MCT, Vismo, LDN or LGK treated Lgr5DTR/+ mice (DT-treated). n = 3 mice. Center values, mean; Error bars, s.e.m. (e) Gli1 in situ hybridisation on d1 hair follicles of DT treated Lgr5DTR/+ mice that received either MCT or Vismo. (f) 3,3′-Diaminobenzidine (DAB) staining for phospho-SMAD1/5 (p-SMAD1/5) on d1 hair follicles from DT treated Lgr5DTR/+ mice that received either MCT or LDN. (g) Axin2 in situ hybridisation on d1 hair follicles of DT treated Lgr5DTR/+ mice that received either MCT or LGK. Scale bars, 20 μm.

Supplementary Figure 7 Wnt inhibition blocks hair germ recovery, but has no effect on the physiological hair germ.

(a,b) DAB staining for (a) p-SMAD1/5, (b) β-Catenin (β-Cat) on d7 skin of Lgr5DTR/+ mice during the Lgr5GFP cell recovery phase. In (b), counterstain: haematoxylin. HG, Hair germ. Scale bars, 20 μm. (c) Wnt5a (upper panels), Wnt10b (lower panels) in situ hybridisation on hair follicles of saline or DT treated Lgr5DTR/+ mice at d7 of Lgr5GFP cell recovery. Scale bars, 10 μm. (d) Dosing regimen of 8 week old Lgr5DTR/+ mice treated with DT (100 μg kg−1 bodyweight, every 48 h), discontinued after three doses and starting 24 h later, mice were treated twice daily (b.i.d.) periorally with MCT (control), vismodegib (Vismo, 75 mg kg−1 bodyweight), LDN-193189 (LDN, 35 mg kg−1 bodyweight) or LGK-974 (LGK, 5 mg kg−1 bodyweight). Skin harvest: d11. Rectangular bar: hair cycle stage of control mice at indicated age. Tel, Telogen. (e) GFP staining on d11 skin from Lgr5DTR/+ mice treated with DT and MCT, Vismo, LDN or LGK. Scale bars, 20 μm. (f) Number of GFP+ cells per hair follicle at d11 in Lgr5DTR/+ mice treated with DT and MCT (n = 4 mice), Vismo (n = 5 mice), LDN (n = 3 mice) or LGK (n = 3 mice). Red circles, individual data points; Center values, mean; Error bars, s.e.m.; n.s., not significant, P = 0.0285 (unpaired t-test). (g) DAB staining for p-SMAD1/5 on skin from DT treated Lgr5DTR/+ mice at d11 that received either MCT or LGK. Scale bars, 20 μm. (h) P-Cad and GFP co-staining on skin from non-DT administered Lgr5DTR/+ mice treated with MCT or LGK-974 (5 mg kg−1 bodyweight) for 11 days b.i.d. Scale bars, 20 μm. (i) Taqman qRT-PCR analysis for Lgr5 and GFP transcript levels (relative to Actb) in skin from non-DT administered Lgr5DTR/+ mice treated with MCT (control) or LGK-974 b.i.d. for 11 days. Histogram: Fold change normalised to mRNA levels in MCT treated Lgr5DTR/+ control mice. n = 4 mice. Center values, mean; Error bars, s.e.m; n.s., not significant (unpaired t-test). Where visible, the dermal papilla is separated from the hair follicle epithelium by a dotted line.

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Hoeck, J., Biehs, B., Kurtova, A. et al. Stem cell plasticity enables hair regeneration following Lgr5+ cell loss. Nat Cell Biol 19, 666–676 (2017). https://doi.org/10.1038/ncb3535

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