Single-cell transcriptomics reveals hair growth retardation mediated by aberrant connective tissue sheath contraction in male androgenetic alopecia

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
Androgenetic alopecia (AGA) manifests as progressive hair follicle (HF) miniaturization; however, its drivers remain poorly elucidated. Combining spatial and single-cell transcriptomics, we generate a concise single-cell atlas of anagen HFs in male AGA, revealing early changes in cell subpopulations, altered HF stem cell fate determination, and disrupted cell-cell communications. Through ex vivo HF organ culture and humanized mouse models, we demonstrate that hypercontractility of connective tissue sheath (CTS) activates the mechanosensitive channel PIEZOS1 in anagen HFs. This mechanotransduction induces ectopic apoptosis of HF progenitor cells and suppresses matrix/ORS cell proliferation, depleting progenitoPr pools and impairing HF growth, thereby driving progressive miniaturization. CritNically, pharmacological inhibition of CTS contraction via ML-7, a selective myosin light chain kinase (MLCK) inhibitor, improves HF growth in both male AGA patient-dLerived ex vivo models and humanized mice. Our study delineates the cellular dynamics underlying male AGA pathogenesis and identifies mechanopathologically activated CTS as a key driver of HF miniaturization, positioning the peri-follicular CTAS as a promising therapeutic target for AGA intervention.

Introduction
Androgenetic alopecia (AGA), also known as male pattern baldness, is the most common type of hair loss, affecting both men and women. Although existing evidence suggests that androgen signaling pathways may play important roles in AGA pathogenesis1, 2, 3, 4, 5, the precise mechanisms underlying its development remain largely unknown. AGA incidence increases with age, with over 50% of Caucasian males affected by age 506, 7. Its profound psychosocial impact drives strong treatment-seeking behaviors, particularly among females and young males8, 9, 10, 11, 12. Despite this clinical urgency, therapeutic development remains constrained by limited pathophysiological understanding. Currently, only two therapies (oral finasteride and topical minoxidil) have been approved by the Food and Drug Administration (FDA), both carrying persistent safety concerns13, 14, 15, 16, 17, 18. This underscores the critical need to systematically characterize the pathogenic regulatory networks underlying AGA for developing mechanism-driven therapeutic interventions.

AGA pathogenesis is characterized by progressive and patterned conversion of large terminal hair follicles (HFs) into small vellus-like structures (termed as HF miniaturization) through dysregulated cycling dynamics19, 20. All HFs undergo periodic cycles of growth (anagen), regression (catagen), and rest (telogen) stageSs throughout the lifetime21, 22, 23. While normal human HFs maintain prolonged anagen phases (2-6 years) to sustain robust hair growth, AGA-affected HFs exhibit truPncated anagen duration with premature catagen entry and prolonged telogen. TheNse perturbations lead to diminished HF size, reduced hair shaft diameter and de creased anagen/telogen ratios, culminating in microscopic hairs characteristic ofL balding scalps as the HF cycles24, 25, 26. In HF cycle, the growth of HFs primarily relies on interactions between the rapidly proliferating hair matrix cells and the components of niche (including dermal papilla (DP), dermal sheath (DS), fibroblasts, adiApocytes and blood vessels etc.)19, 27, 28, 29, 30, 31, 32. Crucially, although HF stem cells (HFSCs) persist in AGA-affected bulges, their activated progenitor cell progeny—essential for fueling matrix cell pool—become depleted33, 34. This creates an unresolved paradox: preserved stem cell reservoirs coexisting with insufficient regenerative capacity.

Current AGA research remains constrained by methodological limitations. Histomorphological analyses and bulk transcriptomic approaches fail to capture cellular heterogeneity, while existing animal models inadequately recapitulate human AGA pathology6, 13, 35. Emerging single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics technologies offer unprecedented resolution for deconstructing cellular dynamics, lineage trajectories, and niche interactions in complex tissues36, 37, 38, 39, 40. Nevertheless, these cutting-edge tools have yet to be applied to systematically investigate AGA pathogenesis.

In this study, we integrate scRNA-seq with spatial transcriptomics to construct the first cellular atlas of human anagen HFs in male AGA, revealing early pathogenic alterations in cellular hierarchies and communication networks. Through synergistic use of ex vivo HF organ culture models and humanized mouse models, we identify hypercontractility of the connective tissue sheath (CTS) as a mechanopathological driver of HF miniaturization. Our findings not only elucidate CTS-mediated mechanotransduction pathways impairing HF growth, but also demonstrate therapeutic efficacy thrSough targeted CTS relaxation— providing a paradigm-shifting strategy for AGA treatment

Results
Single-cell and spatial transcripto mics reveals cell heterogeneity of male androgenetic alopecia and healtLhy anagen HF units

To dissect the cellular heterogeneity and explore the early changes in AGA development, we obtained anagen HF units of both frontal balding (B) scalps and occipital non-balding (NB) scalps from the same male AGA patients (n = 4), and frontal normal scalps of healthy male volunteers (HS, n = 4), to perform scRNA-seq; and acquired a normal frontal scalp containing anagen HFs from one healthy male volunteer to perform spatial transcriptomics (Stereo-seq) (Fig. 1a). Among these, anagen HFs of balding scalps were at the early stage of hair miniaturization, isolated from the edge of frontal hairline of male AGA patients. Prior to scRNA-seq and Stereo-seq profiling, we implemented a multimodal verification strategy to confirm anagen phase status in human HFs, adapting established methodologies for hair cycle staging41, 42, 43. Our results showed that compared to catagen HFs, anagen HFs exhibited hair matrix (HM) with larger volume, more onion-shaped DP and maximal melanin content; besides, the HM cells displayed obvious proliferative activity and no apoptotic signals (Fig. S1). After sequencing and stringent cell filtration of scRNA-seq data, 76368 cells were retained for subsequent analyses (Supplementary Data 1). Following a standard scRNA-seq data analysis procedure, totally 28 clusters (C0-C27) were retained, which were sufficiently separated from each other (Fig. S2a), and these clusters could be reproduced using cells from each of the 12 samples, suggesting that they are robustly present across different samples and conditions (including HS, NB and B) (Fig. S2b, c).

To dissect the cell types, we first defined the expression pattern of SOX9 (Fig. S2d), a reported marker for hair follicle cells44. By immunohistochemistry, we confirmed that SOX9 is expressed in most cells of hair follicle anSd almost none in other cells, distinguishing hair follicle cells from other epidermal and dermal cells (Fig. S2e). Based on the expression of SOX9 and known lineaPge markers, the above clusters were preliminarily identified as the followinNg different cell types, including Bulge (KRT15+/CXCL14+), DP (VCAN+/CFD +), DS (ACTA2+/CCN2+), outer root sheat suprabasal (ORS SB; KRT6A+/SOLX9+), matrix (MKI67+/SOX9+), endothelial cells (Endo; PECAM1+/VWF+), immune cells (CD3D+), Langerhans (CD207+/CD1A+), Melanocytes (DCT+/MLANA+), Vascular smooth muscle cells/pericytes (vSMCs; ACTA2+/MYH11+/TAAGLN+), sebaceous gland cells (DCD+/SCD+), sweet gland cells (SwGs; KRT8+/KRT18+), sweet gland duct cells (SGDCs; KRT77+), interfollicular epidermis spinous (IFE spinous; KRT1+/KRT10+), IFE granular (FLG+/KRT2+)，IFE proliferative (KRT10+/MKI67+); cells highly expressing KRT14 but not SOX9 were defined as IFE basal45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 (Fig. 1b, c; Supplementary Data 2; Fig. S2f-m).

To resolve the ambiguities of cell clustering in scRNA-seq analyses, we projected single-cell annotations onto Stereo-seq spatial maps and refined cluster classifications using spatial localization patterns. This integrative analysis not only confirmed the accuracy of our original single-cell clustering but also enabled definitive identification of previously ambiguous populations: ORS basal (S100A2⁺), inner infundibulum/isthmus cells (Inner II; S100A8⁺/S100A9⁺), and Outer II (SEMA5A⁺/PTN⁺) (Fig. 1b-e; Supplementary Data 2, 3; Fig. S3a, b). Significantly, spatial transcriptomics uncovered the inner root sheath (IRS) population with high TCHH expression—undetectable in single-cell datasets (Fig. S3b, c). Consistent with the scRNA-seq and Stereo-seq data, immunostaining analysis showed that KRT15 was highly expressed in bulge HFSCs and moderately expressed in IFE basal cells (Fig.1f), similar to previous observations33, 46. As an ORS marker, KRT6A was exactly expressed mainly in the ORS SB and some of the inner layer cells of the infundibulum and isthmus, which are the upward continuation of ORS (Fig.1g and Fig. S4a). Moreover, we verified IFE spinous cells with KRT10, and DS cells with α-SMA and CCN2 (Fig. 1h, i). We determined the relative proportion of each HF ceSll type in all samples, showing increased outer II cells, and slightly decreased ORS basal cells in B anagen HF units compared with NB or HS (Fig. S4b; SupplemPentary Data 4). We next explored the molecular changes in each cell type of baldNing HF unit compared to that of NB/HS group (including differentially expressed gene s (DEGs), and enriched pathways). Our results uncovered a landscape of transcripLtional dysregulation across all major cell types in AGA anagen HFs (Fig. S4c; Supplementary Data 5, 6). Notably, HFSCs exhibited a pronounced inflammatory signature, characterized by upregulation of pro-inflammatory pathways such as ATNF signaling, concomitant with a downregulation of pathway governing organ growth. DP cells, the signaling orchestrators of HF regeneration, showed a marked suppression of angiogenesis-related pathways, suggesting a compromised microvascular niche. Matrix cells, which are directly responsible for hair shaft production, were ensnared in a pro-inflammatory milieu, as evidenced by the significant upregulation of cytokine-cytokine receptor interaction. The complete datasets of differential gene expression, and upregulated or downregulated pathways for all cell types is available in Supplementary Data 5 and 6.

To further explore the potential heterogeneity, HFSCs (bulge cells) were subclustered into three distinct subclusters (Fig. 2a), in which the percentage of subcluster 1 was reduced in the balding bulge of AGA patients (Fig. 2b). Wilcoxon rank-sum test was used to characterize the marker genes specifically enriched in each subpopulation versus other subpopulations. Top marker genes with avg_log2FC>0.25, pct (minimum fraction in either of the two populations)>0.25 and P value<0.05 of each subcluster were showed in Supplementary Data 7. Among these, KRT15 was commonly highly expressed (Fig. S2f); CD200 was visibly enriched in subcluster 0 (CD200+ HFSCs), while CD34 was mainly enriched in subcluster 1 (CD34+ HFSCs), and subcluster 2 was enriched with DSG1 (DSG1+ HFSCs) (Fig. 2c). Consistently, CD34+ HFSCs were decreased in balding anagen HFs (Fig. 2d). By immunostaining, we defined that CD200+ HFSCs are located in the upper bulge with high stemness, while CD34 is mainly exSpressed in the lower bulge cells referred to as HF progenitor cells (HFPCs), which are differentiated from the upper bulge HFSCs (Fig. 2e, f). Via RNA Velocyto analysis, wPe further confirmed that CD200+ HFSCs have the ability to differentiate into CD34+ HNFSCs (namely HFPCs) (Fig. 2g). As progeny of HFPCs, matrix cells were also foun d to be decreased in the balding HFs of AGA patients (Fig. S5a).

Subclustering of DP cells, the signaling center for hair growth28, revealed 5 subclusters (Fig. 2h, Fig. S5b and Supplementary Data 8). The percentage and distribution of subclusters were comAparable across HS, NB and B, suggesting that DP cell identity may not alter in AGA anagen HFs (Fig. 2i and Supplementary Data 9). Gene ontology (GO) analysis showed that pathways involving collagen trimer, appendage morphogenesis and epidermis development were upregulated in B DP cells (Fig. 2j); while angiogenesis-related pathway was downregulated (Fig. 2j), consistent with our previous findings from bulk RNA sequencing19.

DS cells were divided into 4 subclusters (Fig. 2k, Fig. S5c and Supplementary Data 10), which showed no significant changes in the balding anagen HFs of AGA patients (Fig. 2l and Supplementary Data 11). Pathway enrichment analysis of differential genes revealed muscle contraction was obviously upregulated in balding anagen HFs compared to non-balding or healthy groups (Fig. 2m).

In human hair follicle, the DS is tightly surrounded by a network of blood vessels, composed of vSMCs and endothelial cells57. Among them, vSMCs were separated into 4 subclusters, and the percentage and distribution of subclusters were comparable across three groups (Fig. 2n, o; Fig. S5d; Supplementary Data 12 and 13). Pathway enrichment analysis showed that smooth muscle contraction and multiple pathways involved in inflammatory regulation (such as IL-17 signaling pathway) were upregulated in vSMCs of balding anagen HFs (Fig. 2p). Endothelial cells were divided into 4 subclusters, which exhibited no obvious alterations (Fig. S5e-g; Supplementary Data 14 and 15), and IL-17 signaling pathway was also upregulated in endothelial ceSlls of balding anagen HFs (Fig. S5h).

Immune cells expressing CD3D were further separated into 6 subclusters, including Th17 cells (IL17A+/IL17F+), naive CD4+ T Ncells (IL7R+), CD8+ T cells (GZMK+/CD8A+), natural killer T (NKT) cells (ZNF683 +) and two unknown subclusters (Fig. S5i-k; Supplementary Data 16). Cell comLposition analysis and co-immunostaining of CD4 and IL17A showed significant increase of Th17 cells in balding HF units (Fig. S5l-n; Supplementary Data 17), which provides evidence for inflammatory infiltration in AGA.

Identification of Aearly changes in HFSC fate determination and cell-cell communications in AGA

Epidermal and sebaceous differentiation of HFSCs has been reported to be associated with HF miniaturization during ageing-induced and obesity-induced hair loss in mice58, 59, but it was unclear whether there existed HFSC fate determination alterations in AGA development. To address this question, we conducted cell fate analysis among potential cell types: HFSCs, HF lineage (including ORS basal and matrix cells), IFE lineage (including Outer II and IFE basal cells) or sebaceous gland cells. As expected, more HFSCs showed a pronounced tendency to differentiate into IFE lineage and sebaceous gland cells in balding HFs compared with non-balding and healthy HFs, and obviously decreased HFSCs differentiated into HF lineage cells (Fig. S6a, d). To explore the underlying mechanisms, branched expression analysis modeling (BAEM) was performed, and identified three common cell fate determination genes, COL17A1, PPP1R1C and SGK1 (Fig. S6b, c, e, f).

To further characterize the cell-cell communications between different cell types in HF units. Cellchat40 was employed to compare the cell-cell communications between balding and non-balding/healthy HFs. Although the interaction strength showed that most cell types had no obvious changes in incoming and outgoing interaction strength, Th17 cells, SwGs and SGDCs exhibited increased incoming interaction strength in balding and non-balding HFs compared to healthy HFs, while HFSCSs and outer II cells displayed decreased outgoing interaction strength in balding anagen HFs in AGA (Fig. S6g).

Apoptotic loss of HF progenitor cells is correlated with HF miniaturization in AGA

AGA is characterized by the progressive cNonversion of the terminal (t) scalp HFs into intermediate/miniaturized (i/m) HFs, wh ich we called the process of HF miniaturization and may be attributed to the deficLiency of HFSCs and their descendant cells (such as progenitor cells and matrix cells) based on our results as mentioned above (Fig. 2b, d; Fig. S5a) and the early study33. To verify changes in HFSC and progenitor cell numbers, we performed immuAnostaining of CD200 and CD34 on anagen HFs from frontal balding (B) and occipital non-balding scalps (NB) of AGA patients, and frontal normal scalps of healthy individuals (HS). HFs retain CD200+ HFSCs with the same percentage in AGA, despite a decrease in cell numbers in B scalps (Fig. S7a-c). However, CD34+ progenitor cells were gradually diminished during the process of HF miniaturization (Fig. 3a-c). Further analysis indicated a strong positive correlation between the number/ proportion of CD34+ progenitor cells and the hair follicle diameter in balding scalps of AGA patients (Fig. 3d-g). Collectively, these observations suggest that loss of progenitor cells is associated with HF miniaturization in AGA.

Next, investigation into the causes of the progenitor cell loss via signaling pathway enrichment analysis revealed upregulated apoptotic signals in progenitor cells of balding anagen HFs (Fig. 3h). A significant increase in apoptosis was observed in the CD34+ progenitor cells of balding anagen HFs according to Cleaved Caspase-3 staining (Fig. 3i, j). These results support the notion that an apoptosis-induced gradual loss of CD34+ progenitor cells may be involved in the development of AGA.

Hyperactive CTS contraction suppresses hair follicle growth via inducing progenitor cell apoptosis and inhibiting ORS and matrix cell proliferation in AGA

Intimate crosstalk between stem cells and the surrounding microenvironment is vital for stem cell fate decisions. On the outside of the progenitor cells is the structure of DS (mainly composed of a layer of elastic DS cells) in muSrine pelage HF, while in human scalp HF is the multi-layer of connective tissue sheath (CTS, the homolog of murine DS) mainly composed of DS cells, collagens anPd vasculature (including vSMCs and endothelial cells) 57(Fig. 4a, b; Fig. S8Na). Pathway enrichment analysis revealed increased contractile activity in both DS cells and vSMCs of balding anagen HFs compared to non-balding or healthLy groups (Fig. 2m, p), which can be supported by the immunostaining of phosphorylated myosin light chain 2 (p-MLC2), a marker of myosin active state60 (Fig. 4c, d; Fig. S8b, c). To further confirm whether DS cells in balding anagen HFs exhibitA enhanced contractility or acquire hypercontractility under specific stimuli, we first profiled expression of mechanocontractile receptors. CHRM3 and EDNRA emerged as the two most abundantly expressed receptors in DS cells (Fig. S8d). Subsequent co-immunostaining analysis confirmed that CHRM3 is specifically expressed within the CTS compartments (encompassing DS cells and vSMCs), with comparable expression levels in NB and B anagen HFs (Fig. S8e). Although EDNRA was detectable in human HFs, its expression localized primarily to the ORS cells with minimal CTS presence (Fig. S8f). Notably, serum concentrations of acetylcholine (ACH) — a canonical agonist of CHRM3 — were significantly elevated in AGA patients compared to healthy controls. Moreover, ACH levels were higher within B HFs relative to NB HFs (Fig. S8g). We also observed that stimulation with methacholine (MCH, a synthetic ACH derivative61, 62, 63) induced greater Young's modulus and more pronounced contractility in isolated primary DS cells derived from B HFs of AGA patients versus NB controls (Fig. S8h-k). Crucially, at the organ-culture level, MCH stimulation provoked significantly stronger contraction in B HFs compared to NB HFs, recapitulating the cellular phenotype (Fig. 4e, f). Collectively, these results suggest that CTS contraction is enhanced in anagen HFs of balding scalps in AGA patients.

Given that the contractile force of DS is a key driver of HF regression in mice29, we wondered whether excessive contraction of the CTS, the analogous structure of murine DS in human, might affect hair follicle growth in AGA. TSo this end, we utilized MCH to activate CTS contraction in the in vitro cultured human anagen HF organs. The efficiency of MCH on CTS contraction in this model wPas evaluated and verified by p-MLC2 immunostaining and real-time confocal imaNging (Fig. S9a-c). Strikingly, MCH treatment resulted in a dose-dependent negative effect on hair growth and anagen maintenance (Fig. 4g-i, and Fig. S9d). Given the marked depletion of progenitor cells in AGA-affected balding HFs, we sought to investigate whether hyperactive CTS contraction could potentially mediate pathological effects on these progenitor populations. Our results showed that MCH Apromoted the loss and apoptosis of CD34+ progenitor cells in a concentration-dependent manner. Of note, Cleaved Caspase-3+CD34+ cells are barely detectable under 100 μM MCH treatment due to the fact that CD34+ progenitor cells were almost depleted under this condition (Fig. 4j-m). Given the crucial role of epidermal cell proliferation—particularly matrix cells—in HF growth, we examined the proliferative activity of the HF compartments, and found that MCH suppressed proliferation in both matrix and ORS cells in a dose-dependent manner (Fig. 4n, o). Notably, blockade of CTS contraction with ML-7 (Fig. S9b, c, e), a specific myosin activation inhibitor64, not only improved the growth retardation and catagen premature induction (Fig. 4p-r, and Fig. S9f), but also reversed the apoptotic loss of progenitor cells and the inhibited proliferation of matrix and ORS cells in MCH-treated human HFs (Fig. 4s-x), confirming the critical role of CTS contraction in MCH effects.

Prompted by a recent study demonstrating dihydrotestosterone (DHT, converted from testosterone via 5α-reductase) promotes muscle cell contraction via the membrane receptor GPR13365, we wondered whether DHT might similarly stimulate CTS contraction through GPR133 in AGA pathogenesis. We first examined GPR133 expression and found that it is indeed expressed in human HF CTS cells (Fig. S10a). Via immunoblotting, we showed that primary DS cells treated with DHT exhibited significantly upregulated MLC2 phosphorylation levels (Fig. S10b, c), while atomic force microscopy (AFM) confirmed enhanced cellular contractility (Fig. S10d); these DHT-inSduced effects were reversed by ML-7 treatment (Fig. S10c, d). Moreover, real-time confocal imaging directly validated DHT-induced contraction enhancement in both PDS cells and intact human hair follicles (Fig. S10e-h). Further HF organ culture exNperiments showed that DHT not only induced CTS hypercontraction, but also trigger ed progenitor cell apoptosis, suppressed matrix and ORS cell proliferation, and Lultimately inhibited HF growth—all of which were significantly reversed by either GPR133 knockdown (Fig. S10i-r) or ML-7 treatment (Fig. S11a-i). To be mentioned, while AR knockdown alleviated DHT’s inhibitory effects on HF growth, it did not alteAr CTS contraction (Fig. S11j-m). Consistently, we observed markedly reduced proliferative activity in matrix and ORS cells within balding HFs of AGA patients, accompanied by a significant increase in the proportion of HFs in the catagen phase (Fig. 4y, z; Fig. S12a-c). These findings significantly strengthen the link between enhanced CTS contraction and AGA pathogenesis.

Subsequently, to determine whether apoptosis is necessary, we applied Z-VAD-FMK to block cell apoptosis66. Our results showed that the observed phenotypes caused by MCH, including HF growth inhibition (Fig. S13a-c), premature catagen induction (Fig. S13d) and depletion of CD34+ progenitor cells (Fig. S13e-h), were partially improved after Z-VAD-FMK administration.

Taken together, these data suggest that over-activated CTS contraction compresses HF growth via induction of progenitor cell apoptosis and suppression of matrix and ORS cell proliferation.

CTS contraction induces progenitor cell apoptosis and inhibits ORS and matrix cell proliferation via PIEZO1 signaling

To pinpoint the specific molecular mechanisms by which the contractility of CTS mediates this effect, we first examined the expression levels of the known mechanosensitive ion channel proteins, which have been found to be expressed in HFs in mice67. Our results showed that of the eight mechanosensitive ion channel genes investigated, PIEZO1 was abundantly expressed across HFSCs (including progeniStor cells), ORS and matrix cells (Fig. 5a, b) and was expressed at higher levels in balding anagen HFs compared to non-balding group (Fig. S14a, b). We then used intraPcellular Ca2+ as a downstream indicator of PIEZO1 signaling68, 69. As quantified by NCal520 (a Calcium influx indicator70) relative fluorescent intensity (RFI), there wa s a significant increase in intracellular Ca2+ concentration in ORS (including prLogenitor cells) and matrix cells of balding anagen HFs (Fig. 5c, d). These results suggest that PIEZO1 signaling is activated in ORS and matrix cells of balding anagen HFs.

We next investigaAted whether activation of PIEZO1 is sufficient to induce HF growth retardation. To this end, we applied YODA1, a PIEZO1-specific activator to cultured anagen HFs (Fig. S14c, d). Intriguingly, anagen HFs treated with YODA1 displayed a phenotype similar to MCH, including growth retardation, progenitor cell apoptosis, and inhibited proliferation of ORS and matrix cells (Fig. 5e-m and Fig. S14e). In addition, the Ca2+ concentration in MCH-treated HF ORS and matrix cells was elevated and could be reversed by the specific PIEZO1 inhibitor GsMTX4 and the myosin activation inhibitor ML-7 (Fig. S14f, g), indicating the activation of PIEZO1 signaling by CTS contraction. Not surprisingly, inhibition of PIEZO1 signaling by GsMTX4 rescued the phenotypes induced by MCH (Fig. 5n-v and Fig. S14h). To further ascertain whether PIEZO1 activation directly suppresses proliferation and induces apoptosis in matrix and ORS cells, we treated primary human ORS cells in vitro with YODA1. Our results demonstrated that activation of PIEZO1 by YODA1 not only significantly suppressed ORS cell proliferation but also potently induced apoptosis (Fig. S14i, j). Furthermore, PIEZO1 activation directly elevated intracellular calcium, compromised mitochondrial function, and upregulated the apoptotic executioner molecule cleaved caspase-3 in ORS cells (Fig. S14k-m). Given that TRPV4 was also abundantly expressed in HFSCs and ORS cells (Fig. 5a), we sought to investigate whether it also mediates CTS hypercontraction-induced hair growth retardation. Consequently, treating MCH-challenged human anagen HFs with the selective TRPV4 inhibitor RN1734771, demonstrated tShat TRPV4 blockade failed to rescue the MCH-mediated suppression of HF growth (Fig. S14n, o).

Targeting CTS contraction by ML-7 improvePs the growth of HFs from the balding scalps of AGA patients

All the above results inspired us to w onder whether targeting CTS contraction is a promising therapy for AGA. We firsLt obtained anagen HFs from the balding scalps of AGA patients, which had been experiencing the process of miniaturization. As expected, the balding anagen HFs exhibited slower growth compared with those from occipital non-balding scalps of the same AGA patients in ex vivo organ culture system (Fig. S15a-c). We then treated the balding HFs with ML-7, or minoxidil (MNX), the FDA-approved drug for AGA treatment. Immunostaining of p-MLC2 confirmed that ML-7 abolished muscle contraction activity in CTS, but MNX did not (Fig. S15d). Excitingly, ML-7 could significantly improve the growth and premature regression of balding HFs of AGA patients, even better than MNX (Fig. S15e-h). Consistently, ML-7 showed a better effect on decreasing the apoptotic loss of progenitor cells compared with MNX (Fig. S15i, j). Furthermore, we demonstrated ML-7 did not affect the growth and anagen-to-catagen progression of cultured anagen HFs from occipital non-balding scalps of AGA patients, suggesting a specific therapeutic effect for balding anagen HFs with enhanced CTS contraction (Fig. S15k-m).

Given the recent development of an excellent humanized mouse model for preclinical AGA research26, 72, 73, we wondered whether targeting CTS contraction via ML-7 demonstrates therapeutic efficacy in this system. We first established the humanized mouse model by transplanting human scalps from non-balding and balding regions of AGA patients on SCID mice according to previous studies26, 72. Using this humanized mouse model, we demonstrated that administration of MCH enhanced CTS contraction, induced apoptosis in the ORS cells (including progenitor cells), suppressed the proliferation of matrix and ORS cells, and inhibited HF growth; conversely, application of ML-7 effectively countered these effects, markedly improSving the phenotypes induced by MCH within xenografted hair follicles derived from the non-balding scalps of AGA patients (Fig. 6a-f, and Fig. S16a-d). Similarly, in this huPmanized mouse model, DHT treatment phenocopied MCH-induced pathological mNanifestations within xenografted non-balding HFs of AGA patients, while ML-7 markedly ameliorated these DHT-triggered phenotypes—manifesting as HF Lgrowth retardation, progenitor cell apoptosis, and suppressed proliferation in both ORS and matrix cells (Fig. 6g-l, and Fig. S16e-i). We further compared the therapeutic efficacy of ML-7 versus minoxidil and their combination in this humanized Amouse models. Our results showed that both ML-7 and MNX significantly rescued DHT-induced hair growth suppression, while their combination demonstrated superior therapeutic outcomes (Fig. 6m-r). However, no statistically significant difference was observed between ML-7 and MNX monotherapies in vivo (Fig. 6m-r), contrasting with our prior ex vivo human HF organ culture data where ML-7 outperformed MNX (Fig. S15e-j). Most importantly, our longitudinal observations revealed that ML-7, administered for over one month, prominently improved hair growth even within xenografted HFs derived from the balding scalps of AGA patients (Fig. 6s-x).

Taken together, we propose that hyperactive CTS contraction in balding anagen HFs activates PIEZO1 signaling in ORS and matrix cells via mechano-transduction, thereby inducing cell apoptosis and suppressing cell proliferation, ultimately resulting in HF growth retardation (Fig. S17), which strongly suggests that relaxing HFs via targeting CTS contraction is a hopeful strategy for AGA therapy.

Methods
Human scalp and HF samples

Human scalp skin biopsies and HF units were collectedS from non-balding occipital and balding frontal sites of male AGA patients undergoing hair transplantation surgery, and normal frontal sites of male healthy volunteers Pfrom the Department of Dermatology in Xiangya Hospital, Central South UniversityN. The detailed information for all specimens I used in this study is listed in Supplem entary Data 18, including patient/donor sources, E biopsy sites, and respective methLodologies. The procedures were in accordance with C protocols set out in the WMA Declaration of Helsinki and the Department of Health and I Human Services Belmont Report and approved by the ethical committee of the Xiangya T Hospital of Central SAouth University (No. 202203076) and written informed consent was R acquired from all participants.

Identification of hair cycle phases in human HFs

Hair cycle phases of human HFs were determined by morphological analysis, HE staining, co-immunostaining of Ki67 and TUNEL, immunostaining of Cleaved Caspase-3 (C-Caspase-3), and Hematoxylin staining for melanin content as previously described41, 42, 43. With these identification methods, or among the representative ones, all human HFs used in this study (including scRNA-seq, Stetro-seq, staining, in vitro organ culture and humanized mouse model) had been confirmed in the typical anagen phase before experimentation unless otherwise stated.

Preparation of single-cell suspensions of HF units

To obtain single-cell suspension of entire HF units, DPs were isolated first by micro-dissection from HFs and were chopped. The remaining hair follicle tissue samples were cut with scissors and digested with the chopped DPs in 4mg/ml Dispase II and Collagenase IV for 40 min at 37℃, centrifuged at 200g for 5min. The pellet was resuspended and digested in 0.25% trypsin for 10 min at 37℃, washed in PBS+5%FBS and filtered through a 70 μm cell strainer. The Dead Cell Removal Kit (Miltenyi Biotec) was used in accordance with the manufacturer’s instruction to obtain live cells. 10 μL of the cell suspension were mixed with the same volume of Trypan blue for cell counting. The loading volume for sequencing was verified by the cSell concentration

ScRNA-seq

scRNA-seq mainly includes GEM (gel bead-in-Pemulsion) generation, barcoding, cDNA amplification, library construction and sNequencing. These steps were completed according to the user’s instructions of C hromium Single Cell 3ʹ Reagent Kits v3.1 (10× Genomics, product cLode: 1000268, 1000215, 1000120) (https://www.10xgenomics.com/support/single-cell-gene-expression). Libraries were sequenced by an Illumina NovaSeq6000 System. Approximately 10,000 cells (targeting 5000–12,000) per saAmple were used for single-cell RNA sequencing.

ScRNA-seq data processing and cell type identification

We used the default parameters of Cell Ranger software suite (v6.0.2) to align and quantify the raw reads data from 10x Genomics. After the initial Cell Ranger metric assessment, Seurat (v4.3.0)86 in R version 4.3.1 was used to exclude cells with fewer than 200 genes or more than 6000 genes detected, and more than 20% mitochondrial reads for downstream analysis.

After quality control, 76368 cells remained and were used for downstream bioinformatic analysis. The “NormalizeData” function was used to normalize the feature expression measurements for each cell by the total expression. The “ScaleData” function was used to scale and center the expression of each gene for dimensional reduction. To avoid batch effects among samples and experiments, we integrated data from all samples using harmony (v0.1.1). Total cell clustering was performed by “FindClusters”function with Louvain algorithm at a resolution of 0.2. Non-linear dimensional reduction was performed by “RunUMAP” function and visualized by Uniform Manifold Approximation and Projection (UMAP). Markers genes of each cell cluster were determined by “FindAllMarkers” function with Wilcoxon rank-sum test. Only those with |‘avg_logFC’| ≥0.25 and ‘adjusted P value’≤0.05 were considered as marker genes.

For subgroup cell clustering, cells of each cell type were extracted separately and clustered by their first 20 PCs and appropriate resolution. To further approximate the low-dimensional data manifold representing the differentiation trajectory for HFSCs, spliced status of mRNAs in single-cell RNA sequencing data were used to estimations of RNA velocities through velocyto87. Markers geNnes of each subcluster were identified by “FindAllMarkers” function with the defau lt parameters. “DoHeatmap” function were used to show top marker genes in heatmLap.

Spatial transcriptomics analysis

Spatial transcriptomic profiling was performed using the Stereo-seq platform (BGI Genomics). Raw sAequencing data were processed with the SAW pipeline (v8.1, https://github.com/STOmics/SAW) under default settings to align reads to the human reference genome. Gene expression matrix was subsequently normalized using the Stereopy package (Version 1.6.1, https://github.com/STOmics/stereopy). Cell-type identities were initially inferred by applying Cell2location88, using single-cell RNA-seq–based annotations as a reference. We then performed unsupervised clustering using the ‘tl.spatial_neighbors’ and the ‘tl.leiden’ functions in Stereopy package with the default parameter and refined the clusters by integrating spatial localization patterns and the expression of canonical marker genes. The final annotated dataset was converted into a Seurat object via the sceasy package (v0.0.7, https://github.com/cellgeni/sceasy), and marker gene expression across cell types was visualized using the DotPlot function.

Identification of balding-associated DEGs

We used the function of “FindMakers” in Seurat to identify balding-vs-nonbalding and balding-vs-healthy differentially expressed genes (DEGs) for each cell type and sub-cell type. The adjusted P values of each DEG were calculated by non-parametric two-sided Wilcoxon rank-sum test and only those with |‘avg_logFC’| > 0.25 and ‘adjusted P value’ < 0.05 were to generate ranked gene lists for downstream Gene Set Enrichment Analysis (GSEA).

Function enrichment analysis

Gene Set Enrichment Analysis (GSEA) of GO anSd KEGG was performed by clusterProfiler R package (v4.5.1.902) and visualized with ggplot2 R package (v3.4.1)89. Representative terms selected from the top rankPed GO terms and KEGG pathways from MsigDB were displayed.

Pseudotime analysis

To reconstruct the differentiation trajectory and do “branched expression analysis modeling” (BAEM) for HFSC fate cell determination related cell types, such as HFSCs to HF lineage and IFE lineage/sebaceous gland cells, Monocle290 was used to reconstruct the differentiation trAajectory for small set of cell types and “BAEM” function were performed to identify cell determination related molecules.

Cell-cell communication analysis

To assess cell-cell communications among different cell types, we used cellchat (v1.1.3) to infer the intercellular communication network from single-cell RNA-seq data. Only cell types with more than 10 cells were considered in the analysis. The “trimean” method is used for calculating the average gene expression per cell group in “computeCommunProb” function. Pairwise comparison and visualization are performed by cellchat build-in function, only interactions with P value lower than 0.05 are considered to be real.

HF organ culture

Human HFs were obtained from the non-balding occipital sites (unless stated otherwise) of male AGA patients undergoing hair transplantation surgery. After microdissection, the HFs were first incubated in William’E medium (ThermoFisher) for 24 hours to re-equilibrate. HFs in anagen VI phase were randomly assigned to the different experimental groups. HFs were cultured at 37℃ with 5% CO in WEM supplemented with 2 mM of L-2 glutamine, 10 ng/ml hydrocortisone, 10 ug/ml insulin and 1% penicillin/streptomycin mixure, and photographed under a stereoscope every two days to count hair shaft length and hair cycle stage. The hair cycle stage (anagen/early-catagen/mid-catagen) of HFs was determined according to the identification methods dSescribed above41, 42.

Humanized mouse model

All animals used in this study were bred and hPoused in individually ventilated cages in specific pathogen‐free holdings with tempNerature and light control (12 h light and dark cycle). All animal experiments were car ried out under the approval of and guidelines of the ethical committee of the XLiangya Hospital of Central South University (No. 2022020197). The humanized mouse model was established with slight modifications to existing protocols26, 72. Anagen-stage HFs from the occipital (non-balding) and frontal (balding) scalp regioAns of male AGA patients were implanted into dorsal incisions of male SCID mice (n=10-20 HFs per mouse) under isoflurane anesthesia. Starting on post-grafting day 30, the mice received treatments of MCH (1 mM), ML-7 (100 μM), DHT (0.625 mg/mL), or Minoxidil (30 mg/mL), as indicated. Mice were sacrificed at day 30/32 post-treatment. HF morphology and hair shaft dimensions (length/diameter) were assessed using photomicroscopy and ImageJ analysis.

Chemical stimulation of HFs

All the chemicals were purchased from Selleck. After 24 hours, medium was replaced and HFs were treated with vehicle (0.1% DMSO), MCH (0.1,1 10,100 μM as indicated), ML-7(0.3 μM), Z-VAD-FMK (10 μM), YODA1(15 μM), DHT (10 μM), RN17347 (3 μM), or GsMTX4(1 μM) according to the design of different experiments and the medium was changed daily with the indicated treatment. HFs were cultured for 2-10 days for analysis of hair growth and immunostaining.

Immunostaining and TUNEL staining

Immunostaining was performed as previously described91. Briefly, OCT-embedded samples were sectioned (10 μm) with a Leica cryostat. The sections were fixed for 10 min with 4% PFA, and washed with PBS, then blocked for 60 min with blocking buffer (5% NDS, 1% BSA, 0.3% Triton X-100). Primary antibodies were incubated overnight at 4℃. Secondary antibody was incubated for 60 min at room temperature. After washed, sections were counterstained with DAPI. TUENL stainiSng was performed with TUNEL assay kit (Roche, USA) according to manufacturer’s instructions, and then stained with CD34 following the immunostaining protocol. For the co-immunostaining of two antibodies from the same host species, double immuNnohistochemistry for paraffin sections (8 μm) was conducted by using the Opal 4 color manual immunohistochemistry (IHC) kit (NEL810001KT, PerkinELlmer). The signal of p-MLC2 was amplified by the Opal 4 color manual IHC kit (NEL810001KT, PerkinElmer). All pictures were taken with a Zeiss Axioplan 2 microscope. The fluorescence intensity was evaluated with ImageJ. The primary antibodies wAere used in this study: Rabbit anti-SOX9 (1:100, Cell Signaling), Mouse anti-KRT15 (1:2000, Lab vision), Mouse anti-KRT14 (1:4000, Abcam), Rabbit anti-KRT6A (1:500, Atlas Antibodies), Rabbit anti-KRT10 (1:1000, Abcam), Mouse anti-α-SMA (1:4000, Abcam), Rabbit anti-CD200 (1:200, Cell Signaling), Rabbit anti-CD34 (1:250, Abcam), Rabbit anti-Cleaved Caspase-3 (1:400, Cell Signaling), Rabbit anti-p-MLC2 (1:100, Cell Signaling), Rabbit anti-Ki67 (1:500, Abcam), Rabbit anti-PIEZO1 (1:100, Proteintech), Rabbit anti-EDNRA (1:500, Abcam), Rabbit anti-GPR133 (1:100, Novus), Rabbit anti-CCN2 (1:100, Proteintech), Rabbit anti-AR(1:100, Cell Signaling). Staining intensity was evaluated in well-defined reference by quantitative (immuno-) histomorphometry using NIH ImageJ software.

Isolation and culture of primary human DS cells

Non-balding and balding human HFs from the same AGA patient were micro-dissected under sterile conditions using fine forceps and microscissors. Adipose tissue and supra-sebaceous segments were meticulously removed under microscopic guidance. Isolated HFs were enzymatically treated with 1 mg/mL dispase II in Hanks' balanced salt solution for 1 hour at 37°C to separate the dermal sheath from the epithelial component. The liberated dermal sheath tissue was then transferred to a solution containing 2 mg/mL collagenase type IV and incubated for 1 hour at 37°C with periodic agitation. The digested suspension was dissociated by gentle pipetting and immediately quenched with DMEM supplemented with 5% fetal bovine serum. The cell suspSension was sequentially filtered through a 40-μm cell strainer and centrifuged at 500 g for 10 minutes at 4°C. The pelleted cells were resuspended in growth medium consPisting of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin. Cells were seedNed onto tissue culture plates pre-coated with 50 μg/mL rat tail collagen type I and mai ntained at 37°C in a humidified 5% CO₂ incubator. Medium changes were performed Levery 72 hours until 80% confluency, with subsequent passages using 0.25% trypsin-EDTA. Primary cells between passages 2-4 were utilized for experiments.

In vitro Ca2+ imaginAg

HFs were incubated with Calbryte™ 520 AM fluorescent calcium indicator (AAT Bioquest) in culture medium at 37℃ for 1 hour. Afterward HFs were washed with HBSS and cultured with HBSS supplemented with 1mM calcium. For different treatments, MCH, ML-7, YODA1 or GsMTX4 was added to the culture medium for 30 min. The HFs were embedded in OCT and were sectioned with a Leica cryostat. Images were taken immediately to record the fluorescence intensity to indicate Ca2+ concentration and the adjacent section was used for CD34 staining to indicate progenitor cells.

Atomic force microscopy (AFM) for cell stiffness measurement

Primary human DS cells were seeded at a density of 5 × 10³ cells/cm² on sterile 35-mm Petri dishes pre-coated with collagen type I (5 μg/cm²) and allowed to adhere for 24 h before AFM analysis. Stiffness measurements were performed using a JPK Nanowizard IV AFM system coupled with an inverted optical microscope (Nikon Eclipse Ti2) as previously described83. Cells were maintained in 1× phosphate-buffered saline (PBS) at 37°C during measurements. A cylindrical nitride cantilever (Bruker SAA-SPH-5UM; tip radius: 5 μm, spring constant: 0.20–0.30 N/m) was used for all experiments. The cantilever’s spring constant was calibrated before each experiment via the thermal fluctuation method. Indentations were performed at a constant approach rate of 5 μm/s with a maximum cantilever deflection set point of 2.0 nN to ensure elastic-dominant deformation. Force-indentation curves were recorded aSt 5 points over the perinuclear region of individual cells (≥6 cells per group).

Young’s modulus (E) was derived by fitting theP approaching force curve to the Hertzian contact model for a spherical indenter (Eq. N1):

F(x) = 4 ∙ Ε √rx3⁄2
E3 1-υ 2

where F = applied force, r = tip radius (5 μm), δ = indentation depth, and υ = Poisson’s ratio (fixed at 0.5 based on bioIlogical soft materials). Baseline correction and contact point estimation were automRated using JPK Data Processing Software (v6.3). Cells with excessive drift or incomplete retraction curves were excluded.

Ex vivo hair follicle and DS cell contraction assays

Human HFs were micro-dissected from scalp tissue, approved by the ethical committee of the Xiangya Hospital of Central South University (No. 202203076) and written informed consent was acquired from all participants. HFs underwent 60 min equilibration at 37°C in glucose-supplemented HBSS ± 1 µM ML-7. Real-time imaging used a Leica TCS SP8 confocal system (10× air objective, 37°C, 5% CO₂). Immediately prior to time-lapse imaging, the buffer was replaced with HBSS containing 1 mM CaCl₂ supplemented with or without contraction inducers (10 µM DHT or MCH). Brightfield images were acquired at 5-min intervals for 15 min. Primary DS cells isolated from matched follicles were cultured on collagen I-coated 24-well plate. After identical pretreatment, induction buffers were replaced simultaneously with HF experiments. Phase-contrast imaging proceeded for 30 min at 10-min intervals. HF diameter was measured perpendicular to the axis at five locations (200-400 µm superior to dermal papilla, 50 µm intervals) using Fiji/ImageJ v.2.3.0. Cell area was quantified via automated thresholding.

ACH content quantification

ACH levels in serum and HFs were measured using an ACH assay kit (Catalog No. A105-1-1 for HFs, A105-2-1 for serum, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

Detection of cell apoptosis using Annexin V-FITC assSay kit

Cell apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (ThermoFisher). Cells were resuspended in bPinding buffer (1 × 10^6 cells/mL) and incubated with 5 μL Annexin V-FITC and 5 μNL propidium iodide (PI) for 15 minutes at room temperature in the dark. After adding 40 0 μL of binding buffer, cells were analyzed by flow cytometry. Early apoptotic cells wLere Annexin V-FITC-positive, PI-negative, while late apoptotic or necrotic cells were both Annexin V-FITC and PI-positive.

Isolation and culture of primary human ORS cells

Outer root sheath (OARS) cells were isolated from hair follicles (HFs) of AGA patients using previously described methods (PMID: 40776528). HFs were harvested and digested with 0.1% dispase (Sigma, USA) at 37°C for 1 hour. The connective tissue sheath (CTS) was carefully removed under a stereomicroscope to expose the ORS. The ORS was then dissociated into single cells by incubation with 0.05% trypsin (Gibco, USA) at 37°C for 30 minutes. The resulting cell suspension was vortexed and filtered through a 40 μm cell strainer (Falcon, USA) to remove debris. Cells were then centrifuged at 300 × g for 5 minutes, resuspended in culture medium, and seeded onto culture plates pre-coated with 10 μg/mL human fibronectin (Sigma, USA). The plates were incubated at 37°C for 1 hour, and non-adherent cells were removed by changing the culture medium. ORS cells were cultured in CNT-07 medium (CELLNTEC, Switzerland) at 37°C with 5% CO2. The culture medium was replaced every 2 days.

Detection of mitochondrial function

Mitochondrial function in ORS cells was assessed using the JC-1 Mitochondrial Membrane Potential Kit (Beyotime). Cells were incubated with 2 μM JC-1 at 37°C for 20 minutes, then washed and analyzed by fluorescence microscopy. Red fluorescence indicates high mitochondrial membrane potential, while green fluorescence indicates depolarization. An increase in the green-to-red fluorescence ratio suggests increased mitochondrial permeability, a sign of mitochondrial dysfunction.

Immunoblotting

Cells were lysed in RIPA buffer (ThermoFisher) containing protease inhibitors (ThermoFisher) after washed with cold PBPS. The proteins were quantified via bicinchoninic acid assay (ThermoFisher) aNnd separated on SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat milk for 1h at room temperature and incubated with Lprimary antibodies overnight at 4℃. The secondary antibodies HRP-conjugated Goat anti-Mouse IgG (Santa Cruz) and HRP-conjugated Goat anti-Rabbit IgG secondary antibody (Santa Cruz) were incubated for 1h at room temperature. The imAmunoreactive bands were visualized by the HRP substrate (Sigma) on ChemiDoc XRS+ system (Bio-Rad). Data were analyzed by GE Healthcare ImageQuant LAS 4000 Mini and images have been cropped for presentation. The corresponding uncropped images of immunoblotting results were provided in Resource data file. The primary antibodies used in this study were: Rabbit anti-p-MLC2 (1:1000, Cell Signaling), Rabbit anti MLC2 (1:2000, Proteintech), Rabbit anti-GPR133 (1:1000, Novus), Rabbit anti cleaved-CASPASE3(1:1000, Cell Signaling), Mouse anti GAPDH (1:10000, Proteintech).

RNA interference

Small interferring RNA (siRNA) were purchased from GenePharma (China). The sequences for siRNA: sihGPR133: CCAGGCCAAGTGTTATGAGAA; sihAR: CACCAATGTCAACTCCAGGAT; ORS cells were transfected with siRNA at 0.06 nM packaged by Lipofectamine 3000 (Invitrogen) in 6-well plates (5 × 105cells/well). Hair follicles were transfected with siRNA at 0.06 nM packaged by Lipofectamine 3000 (Invitrogen) in 24-well plates (1 hair follicle/well).

EdU proliferation assay

Cell proliferation was evaluated using the EdU Cell Proliferation Assay kit (RiboBio). ORS cells were cultured in medium with YODA1 as indicated. After 24 hours of incubation in medium containing 10 µM EdU, cells were fixed with 4% paraformaldehyde. Subsequently, cell nuclei were stained with DAPI to facilitate visualizatioSn. EdU-positive cells, indicative of DNA synthesis, were quantified using fluorescence microscopy. The proportion of EdU-incorporating cells was calculated relative to the total cell population.

RNA extraction, real-time PCR (qPCR)

RNA was extracted from the bulbs of human hair follicles using TRIzol Reagent (ThermoFisher) and was reverse-Ltranscribed to cDNA by PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). qPCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme). The relative gene expression was measured by delta-delta CT relative to GAPDH, aAnd the fold change was normalized to the control group. The primer sequences used in this study were presented in Supplementary Data 19.

Statistical analysis

All data were expressed as mean±SEM and were analyzed by ANOVA or Kruskall-Wallis test when more than two groups were compared or Student’s t-test when only two groups were compared. Graphpad Prism 10, R (v4.1.3) and Excel (Microsoft) were used to assess statistical significance. Statistical significance was set at a P < 0.05. *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively. All corresponding P values are listed in the Source data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a very intriguing study that shifts attention in human AGA research from the role of androgens to the CTS by proposing that continuous, hyperactive CTS contraction eventually causes the PIEZO1-dependent loss of epithelial HF progenitor cells, mainly through excessive & ectopic apoptosis. This seems to be somewhat in line with previous work from the lab of M Rendl on the importance of CTS contraction in murine hair follicle (HF) biology, whose clinical relevance has remained obscure, and as such enriches the human hair research literature substantially. However, in its current form, the study has many shortcomings.\n1. Some statements in the Introduction & Discussion question how familiar the authors really are with human hair follicle biology and AGA, which (avoidably) mitigates enthusiasm for this study. For example: \"In AGA, the growth phase decreases to only days or weeks, followed by premature entry into hair regression; this leads to an increase in the percentage of resting HFs, generating microscopic hairs in the balding scalp as the HF cycles(8, 9). In HF cycle, the growth of HFs relies on the proliferation and differentiation of HF stem/progenitor cells, whose activities are mainly regulated by the components of HF niche (including dermal papilla (DP), dermal sheath (DS), adipocytes and blood vessels etc.)\". These statements require correction, and need to be better supported by optimally chosen citations.\na. Even in AGA, anagen typically lasts much longer than days or weeks.\nb. Premature catagen entry does not cause HF miniaturization, but telogen effluvium. In fact, HF miniaturization can only occur during anagen, and can happen even within a single ongoing anagen phase (Whiting JAAD 2001). Instead, HF miniaturization is driven mainly by a declining size, cell number and inductive potential of the dermal papilla, while a reduction in the pool of epithelial progenitor cells seems to be only one of several additional relevant signals leading to HF miniaturization.\nc. HF growth, or more precisely: the duration of anagen and the speed of hair shaft production, primarily relies on interactions between the rapidly proliferating cells of the of anagen hair matrix and inductive signals arising from/communication with the fibroblasts of the dermal papilla ( epithelial stem cells and extrafollicular signals all prominently play into this, but are not the recognized driving forces).\nd. In the Results, it is stated \"Our data demonstrate that bulge cells first differentiate into ORS and IRS cells...\" Yet, it is common knowledge that only hair matrix cells can terminally differentiate into IRS keratinocytes.\ne. Keratin 6A is not a specific marker for the IRS, but is also expressed prominently throughout the ORS.\nThese selected examples, of which one could cite many more, suggest strongly that this study would profit from recruiting an expert hair biologist and AGA researcher as additional co-authors for professional text editing and literature synthesis and to assist with data interpretation.\n2. The statement (Introduction) \"To date, the pathogenetic study of AGA has been scant and limited to histomorphology, global gene analysis and immunohistochemistry due to the fact that most laboratory models are not fully representive of AGA(15, 16)\" ignores that there is now an excellent humanized mouse model for preclinical AGA research, in which the authors' main working hypotheses could and should be experimentally probed: human AGA scalp xenotransplants on SCID mice (e.g., Zeltzer AA et al. Acta Derm Venereol. 2024, Gilhar et al. Acta Derm Venereol. 2024). This model permits exactly those functional long-term studies directly in human scalp skin that are required to convincingly support the authors' main claims.\n3. The Introduction fails to clearly state the specific research questions addressed, and does not sufficiently explain the rationale for the chosen research design. For example, since scRNAseq analyses always struggle with gene expression profiling artifacts caused by enzymatic digestion and cell dispersion (which are not even discussed here) the use of spatial transcriptomics, combined with CellChat analysis, would overcome the limitations of scRNAseq. This would also have provided better insights into spatiotemporal interactions of unmanipulated HF cell populations within the native tissue habitat of AGA-affected versus AGA-non-affected scalp HFs (occipital versus frontotemporal).\n4. That the gene expression profile of all investigated cell types \"had differences in various degree (Fig. S2N, Supplementary Table 2)\" is entirely expected, but does not characterize (and much less so explain) important transcriptional changes the precede the early stages of hair miniaturization in AGA - not the least since these early stages (=intermediate HFs, see Miranda et al. FASEB J 2018) do not seem to have been specifically studied. The latter, however, is mandatory if one wishes to make any plausible claims reg. the molecular controls that drive the early stages of HF miniaturization in AGA.\n5. May be I just missed the points the authors tried to make here, but I fail to recognize in which way exactly this study advances the field fundamentally over what has already been published on HF stem cells and their immediate progeny, their heterogeneity, and corresponding AGA-associated abnormalities in the Garza/Cotsarelis study and later work (e.g., Purba et al. Sci Rep 2017). It is essential to clarify this.\n6. The subclusters of DP cells and related changes in AGA reported here are interesting and potentially important - if the transcriptomic data are backed up on the decisive protein expression level by.\n7. It is a general shortcoming of this study that too few transcriptional changes reported here that are critical for supporting the authors' main working hypothesis have been confirmed by IF/IHC and quantitatively analyzed at the protein level. It is strongly recommended that the authors expand these protein data sets, including but not restricted to verifying the reported changes in immune cell populations by quantitative immunohistomorphometry, following published standard protocols.\n8. Measurements of changes in HF cycling seem to largely rely on macroscopic assessment. This is so unreliable as to be unacceptable. For hair cycle staging of organ-cultured human HFs, standard hair cycle histomorphometry needs to be employed (Kloepper et al. Exp Dermatol 2010), analyzing a sufficient number of HFs from at least 3 different donors.\n9. It remains too unclear on which data exactly the differences between murine pelage and human scalp HF CTS that are depicted in the cartoon (Fig. 4A) are based. What exactly is the take home message from this cartoon, and how does this relate to the authors' main hypothesis? Please explain this more lucidly and corroborate the main claims contained in this cartoon by convincing evidence.\n10. While the postulate that \"hyperactive CTS contraction drives premature hair regression via inducing apoptosis of progenitor cells in AGA\" is very intriguing, I am unconvinced by the currenly presented evidence. In human terminal anagen scalp HFs, the early stages of catagen development occur in the anagen hair matrix, i.e. very far distant from the bulge stem cells and their immediate progeny. While - in line with the Garza/Cotsarelis AGA study - it is theoretically conceivable that excessive progenitor apoptosis might result from hyperactive CTS contraction and may somehow contribute to HF miniaturization, it is therefore highly implausible that this should also induce premature catagen induction. To support this claim, much more direct evidence would have to be provided, such as the demonstration that key anagen-maintaining growth factors (IGF-1, KGF, HGF, Wnt activity) are reduced, while key drivers of catagen (TGFb1/2, SFRP1 etc.) are increased.\n11. I am also intrigued by the reported hair growth promotion by ML-7, which would represent a welcome novel AGA treatment principle. However, convincing evidence that ML-7 is indeed more effective than minoxidil is best generfated in the humanized AGA model after long-term appliucation in vivo - especially since human HF organ culture is a notoriously unreliable assay for evaluating minoxidil activities (Magerl et al. Exp Dermatol 2004).\nMinor issues:\n-- The main concepts emanating from this study should be synthesized in an instructive cartoon (presented as part of the last core figure or as a supplementary fig.).\n-- Complex statements like \"To investigate the trajectories among all 20 cell types, monocle3(36) was employed to perform the pseudotime analysis with the root node setting at start point of bulge on the SOX9+ HF branch and start point of IFE basal on the SOX9- IFE branch separately....\" (Results) will not be easily digestible by many non-expert readers and should be rephrased to be more instructive & self-explanatory.\n-- \"For the SOX9- IFE branch, IFE basal cells can directly differentiate into SOX9- PR and IFE spinous cells which finally develop into IFE granular cells (Fig. 1E). These patterns are consistent with the knowledge from previous studies(26, 35).\"\nPlease explain what exactly these results add to the published literature, how they advance the field, and why they are even relevant in the context of AGA and CTS contraction.\n-- What exactly do the authors feel to be the role of DHT and AR in promoting the presumed hyperactive CTS contraction?\nCan they generate direct evidence to support or exclude such a role, e.g. in HF organ culture?", "reviewer_2": "The authors Li et al provide a single cell analysis of androgenic alopecia human skin. Androgenic alopecia is still largely unexplored and, although being a “cosmetic” disease, harbors a high burden on many affected patients concerning their quality of life. Thus, high throughput investigations of this conditions are urgently mandated. Comparing occipital with frontal skin (affected and unaffected) is a reasonable approach.\nMajor\nIn the beginning and in the abstract, the authors need to more clearly state their sample size, which seems quite low. Also, it needs to be clearer whether investigations have been performed in same patients or independent cohorts.\nThe authors need to make clear in abstract, title and throughout the manuscript that they are investigating male patients, as balding patterns strongly differ between males and females.\nThe authors might want to add a table with QC parameters such as genes per cell, cells per sample obtained, as well as a table showing how many cells were filtered out during QC, and how many cells are present for each cluster and each sample. Also, a baseline table with patients age etc needs to be provided to assess whether the biopsies are appropriately matched.\nA cutoff for DEGs of avg_log2FC>0.25 is very low, I would recommend to increase to 0.5 or 0.75 if feasible.\nThe authors need to better label their figures, the figures are very difficult to follow. Particularly also in supplements, the authors need to write what cells have been subclustered etc, right now plots are too sparsely labelled and partly incomprehensible.\nWhen sublcustering, authors need to explain (or label in figure) what they obtained. For example for endothelial cells, they perform subclustering and obtain 4 clusters, but do not explain what they find. Arteriolar or venular or capillary? If they perform subclustering, they should elaborate on them. This needs to be done for all figures.\nIn lymphocyte subclustering, the “unknown” clusters seem keratinocyte contaminations.", "reviewer_3": "The manuscript entitled Single-cell transcriptomics reveals cellular hierarchies and aberrant CTS contraction-mediated premature hair regression in androgenetic alopecia revealed aberrant CTS contraction in AGA patients, which is the crucial factor that causes premature hair regression. Focusing on intracellular contraction force in HF is quite interesting. However, this work missed critical evidence to confirm the enhanced mechanical stress, and lacked further exploration in the detailed molecular mechanism of how contraction force led to hair regression.\nMajor concerns\n1. The authors suggested enhanced connective tissue sheath as the significant driver of premature hair regression. However, bioinformatic analysis and immunostaining of p-MLC2 couldn’t fully support this hypothesis. Atomic force microscope is appreciated to examine and confirm the hyperactive contraction force in CTS.\n2. Piezo1 is one of the most investigated mechanosensitive ion channel proteins in recent years. How did the authors come to consider exploring Piezo1, but not other proteins? Is the expression pattern of Piezo1 upregulated, or other mechanosensitive pathways functionally enriched, in the single cell sequence metadata of frontal balding, especially the CD200 positive subset?\n3. The results indicated that Piezo1 inhibitor GsMTX4 could ameliorate hair regeneration. While in the next section, the authors also showed the positive effect of myosin activation inhibitor ML-7. From the authors’ perspective, inhibiting mechanosensitive ion channels via GsMTX4, or hindering cytoskeleton contraction via ML-7, which had a more significant regulatory effect on CTS contraction, and possibly be a therapeutic target. Please compare, verify, and illustrate.\n4. The authors showed that ML-7 had a better effect on treating androgenetic alopecia. I would also prefer to know the therapeutic effect of ML-7 comparing with finasteride. And could the combination of ML-7 with minoxidil/finasteride acquire a better clinical outcome?\n5. The authors didn’t explain the molecular mechanism of how Piezo1/myosin activation-induced CTS contraction induces apoptosis and thus causes premature hair regression, which is critical for an in-depth understanding of AGA progression.\nMinor concerns\n1. Before conducting scRNA-seq, the HFs were confirmed in anagen phase (line 117). Please describe the biomarker and briefly illustrate the sorting protocol for preparing single-cell suspension in the main body.\n2. The results showed that all cell types had differences in various degree (Fig. S2N, Supplementary Table 2), suggesting that anagen HFs undergo significant changes at the early stage of hair miniaturization in AGA (line 163-165). Please explain the change in gene expression pattern and the corresponding biological meanings."}, "author_response": "Dear reviewers:\nWe would like to express our deepest appreciation for your time and effort in\nreviewing our manuscript. Please find our point to point responses to the\ncomments as detailed below. We are hopeful that all of the additional data,\nanalyses and information we have provided satisfy your concerns.\nReviewer #1\nOverall comments:\nThis is a very intriguing study that shifts attention in human AGA research from\nthe role of androgens to the CTS by proposing that continuous, hyperactive\nCTS contraction eventually causes the PIEZO1-dependent loss of epithelial HF\nprogenitor cells, mainly through excessive & ectopic apoptosis. This seems to\nbe somewhat in line with previous work from the lab of M Rendl on the\nimportance of CTS contraction in murine hair follicle (HF) biology, whose clinical\nrelevance has remained obscure, and as such enriches the human hair\nresearch literature substantially. However, in its current form, the study has\nmany shortcomings.\nOverall response:\nWe thank the reviewer for the recognition of our work, and the helpful and\nconstructive comments, all of which have been addressed, as detailed below.\nComment 1:\n1. Some statements in the Introduction & Discussion question how familiar the\nauthors really are with human hair follicle biology and AGA, which (avoidably)\nmitigates enthusiasm for this study. For example: \"In AGA, the growth phase\ndecreases to only days or weeks, followed by premature entry into hair\nregression; this leads to an increase in the percentage of resting HFs, generating microscopic hairs in the balding scalp as the HF cycles (8, 9). In HF\ncycle, the growth of HFs relies on the proliferation and differentiation of HF\nstem/progenitor cells, whose activities are mainly regulated by the components\nof HF niche (including dermal papilla (DP), dermal sheath (DS), adipocytes and\nblood vessels etc.)\". These statements require correction, and need to be better\nsupported by optimally chosen citations.\nResponse 1:\nWe thank the reviewer for the kind suggestions, and apologize for the unprecise\nstatements. As suggested, in the revised manuscript we have made the\ncorresponding corrections according to optimally established literatures (J Am\nAcad Dermatol. 2001 Sep;45(3 Suppl):S81-6; PMID: 25905192; Exp Dermatol.\nworld-renowned experts in AGA research, as detailed below.\na. Even in AGA, anagen typically lasts much longer than days or weeks.\nResponse 1a:\nAs suggested, we have corrected the unprecise statement.\nb. Premature catagen entry does not cause HF miniaturization, but telogen\neffluvium. In fact, HF miniaturization can only occur during anagen, and can\nhappen even within a single ongoing anagen phase (Whiting JAAD 2001).\nInstead, HF miniaturization is driven mainly by a declining size, cell number and\ninductive potential of the dermal papilla, while a reduction in the pool of\nepithelial progenitor cells seems to be only one of several additional relevant\nsignals leading to HF miniaturization.\nResponse 1b:\nWe sincerely thank the reviewer for the constructive and helpful comments.\nBased on these comments and the established literatures, we have carefully\nre-analyzed and reconsidered our results. We agree that our initial\ninterpretation may have introduced confusion or inaccuracies, particularly\nregarding the direct causality between premature catagen entry and hair follicle\n(HF) miniaturization, and we acknowledge that premature catagen entry does\nnot directly cause HF miniaturization.\nFollowing the reviewer's suggestions, we performed a series of additional\nexperiments to examine the proliferation of HF cells enveloped by CTS,\nincluding the outer root sheath (ORS) cells and matrix cells surrounding the\ndermal papilla. Our results demonstrated that excessive CTS contraction not\nonly induces apoptosis in progenitor cells, but also significantly suppresses the\nproliferation of matrix cells and ORS cells (including progenitor cells) (Shown\nin new Figure 4n, o; new Figure 4w-z; new Figure 5l, m; new Figure 5u, v).\nMoreover, we have provided additional evidence showing that activating\nPIEZO1 in human hair follicle ORS cells directly with YODA1 in vitro not only\nsignificantly suppressed cell proliferation, but also induced apoptosis in a\nsubset of cells (Shown in new Supplementary Figure 14i-m)—consistent with\nour observations from ex vivo organ-cultured human hair follicles. These\nalterations, especially the significant suppression of matrix cell proliferation,\nlead to hair growth retardation. We propose that this retardation may\nprogressively contribute to HF miniaturization over time. Furthermore, in a\nsubset of follicles, the combined effects of increased progenitor cell apoptosis\nand significantly reduced proliferation of both the matrix and progenitor cells\nappear to culminate in premature entry into catagen. With additional\nexperiments and analyses, we observed that this premature catagen entry is\nreflected by the onset of apoptosis in matrix cells. Critically, this matrix cell\napoptosis occurs after the observed increase in progenitor cell apoptosis and\ndecrease in matrix/ORS cell proliferation (Shown in new Supplementary\nFigure 9g-i). This temporal sequence suggests that premature catagen entry\nis likely a consequence of these preceding events (apoptosis and proliferation\ndefects), rather than a direct cause of HF miniaturization.\nTherefore, consistent with these insights and new data, we have revised the\ndescription of the impact of excessive CTS contraction throughout the\nmanuscript. References to \"premature catagen entry\" have been replaced with\n\"hair follicle growth retardation\".\nNotably, our study provides no mechanistic evidence elucidating why\naberrant CTS contraction exerts differential effects on spatially distinct ORS\nand matrix cells within human hair follicles. Specifically, aberrant CTS\ncontraction simultaneously induces apoptosis and inhibits proliferation in ORS\ncells, whereas matrix cells exhibit only proliferation suppression at early stage.\nWe postulate that this heterogeneity may correlate with spatially varying\nmagnitudes of mechanical forces generated by CTS contraction along the hair\nfollicle. However, validation of this hypothesis will require more sophisticated\nbiomechanical experimentation in future studies. This limitation has been\nexplicitly addressed in the revised discussion section of our manuscript.\nc. HF growth, or more precisely: the duration of anagen and the speed of hair\nshaft production, primarily relies on interactions between the rapidly\nproliferating cells of the of anagen hair matrix and inductive signals arising\nfrom/communication with the fibroblasts of the dermal papilla (epithelial stem\ncells and extrafollicular signals all prominently play into this, but are not the\nrecognized driving forces).\nResponse 1c:\nAs suggested, we have corrected the unprecise statement.\nd. In the Results, it is stated \"Our data demonstrate that bulge cells first\ndifferentiate into ORS and IRS cells...\" Yet, it is common knowledge that only\nhair matrix cells can terminally differentiate into IRS keratinocytes.\nResponse 1d:\nWe apologize for the inaccurate result interpretation. To more accurately define\nhair follicle cell populations in our single-cell dataset, we performed spatial\ntranscriptomics analysis using non-balding occipital scalp tissue from AGA\npatients. Based on these spatial transcriptomics results, we re-analyzed the cell\nclustering within our single-cell data. While the re-analysis largely confirmed\nour original findings, it revealed that cells previously identified as IRS cells\nactually corresponded to ORS suprabasal cells (Shown in new Figure 1b-e;\nnew Supplementary Figure 3). However, IRS cells were not detected in our\nsingle-cell dataset. We attribute this absence of IRS cells to limitations during\nthe single-cell suspension preparation phase of our workflow, where these\nparticular cells may not have been captured or preserved. Since this specific\nfinding (shown in former Figure 1E) was not novel but consistent with existing\nliterature (PMID: 35606346), a point also noted by another reviewer, we have\nremoved this specific result from the revised manuscript.\nConsequently, we have corrected the corresponding interpretations\nthroughout the revised manuscript and ensured alignment with these updated\nfindings.\ne. Keratin 6A is not a specific marker for the IRS, but is also expressed\nprominently throughout the ORS.\nResponse 1e:\nWe thank the reviewer for the helpful comments, and apologize for the\ninaccurate result interpretation. Indeed, keratin 6A is not a specific marker for\nthe IRS, but is expressed prominently throughout the ORS, which is comfirmed\nby our spatial transcriptomics analysis results (Shown in new Figure 1b-e;\nnew Supplementary Figure 3). Therefore, we have corrected the\ncorresponding interpretations throughout the revised manuscript.\nThese selected examples, of which one could cite many more, suggest strongly\nthat this study would profit from recruiting an expert hair biologist and AGA\nresearcher as additional co-authors for professional text editing and literature\nsynthesis and to assist with data interpretation.\nResponse:\nWe thank the reviewer for the kind and helpful suggestion. As suggested, we\nhave recruited professor Mingxing Lei (Chongqing University), an experted hair\nbiologist and AGA researcher as an additional co-author for professional text\nediting and literature synthesis and to assist with data interpretation. With the\nhelp from professor Mingxing Lei, we have tried our best to improve the\nmanuscript.\nComment 2:\n2. The statement (Introduction) \"To date, the pathogenetic study of AGA has\nbeen scant and limited to histomorphology, global gene analysis and\nimmunohistochemistry due to the fact that most laboratory models are not fully\nrepresentive of AGA(15, 16)\" ignores that there is now an excellent humanized\nmouse model for preclinical AGA research, in which the authors' main working\nhypotheses could and should be experimentally probed: human AGA scalp\nxenotransplants on SCID mice (e.g., Zeltzer AA et al. Acta Derm Venereol. 2024,\nGilhar et al. Acta Derm Venereol. 2024). This model permits exactly those\nfunctional long-term studies directly in human scalp skin that are required to\nconvincingly support the authors' main claims.\nResponse 2:\nWe appreciate the reviewer highlighting the utility of the humanized mouse\nmodel for preclinical AGA research. As suggested, we first established the\nhumanized AGA mouse model by transplanting human scalps from non-balding\nand balding regions of AGA patients on SCID mice according to previous\nstudies (e.g., Zeltzer AA et al. Acta Derm Venereol. 2024; Gilhar et al. Acta\nDerm Venereol. 2023; Gilhar et al. Exp Dermatol. 2022). Using this humanized\nAGA mouse model, we demonstrated that administration of MCH or DHT: (1)\nenhances CTS contraction, (2) induces apoptosis in the ORS cells (including\nprogenitor cells), (3) significantly suppresses the proliferation of matrix cells and\nORS cells, and (4) over extended observation periods, inhibits hair growth.\nConversely, application of ML-7 effectively countered these effects, markedly\nimproving the phenotypes induced by both MCH and DHT within xenografted\nhair follicles derived from the non-balding scalps of AGA patients (Shown in\nnew Figure 6a-r; new Supplementary Figure 16). Most importantly, our\nlongitudinal observations revealed that ML-7, administered for over one month,\nprominently improved hair growth even within xenografted hair follicles derived\nfrom the balding scalps of AGA patients (Shown in new Figure 6s-x). These\ncompelling in vivo findings are in agreement with the results we previously\nobtained in ex vivo HF organ culture assays.\nWe wish to clarify that the use of DHT and its relationship with CTS\ncontraction have been specifically addressed in both our response to Minor\nissue 4 below and the corresponding Results section of the revised manuscript\n(Shown in new Supplementary Figure 10 and 11; new Figure 6g-r).\nComment 3:\n3. The Introduction fails to clearly state the specific research questions\naddressed, and does not sufficiently explain the rationale for the chosen\nresearch design. For example, since scRNAseq analyses always struggle with\ngene expression profiling artifacts caused by enzymatic digestion and cell\ndispersion (which are not even discussed here) the use of spatial\ntranscriptomics, combined with CellChat analysis, would overcome the\nlimitations of scRNAseq. This would also have provided better insights into\nspatiotemporal interactions of unmanipulated HF cell populations within the\nnative tissue habitat of AGA-affected versus AGA-non-affected scalp HFs\n(occipital versus frontotemporal).\nResponse 3:\nWe appreciate the reviewer's insightful comments regarding the need for\nclearer research questions and a stronger rationale for the research design in\nthe Introduction which have been improved in the revised manuscript, as well\nas the inherent limitations of scRNA-seq analysis, including potential artifacts\nintroduced by enzymatic digestion and cell dispersion, which we regrettably did\nnot explicitly discuss previously. Due to significant financial constraints,\nconducting large-scale spatial transcriptomics on multiple AGA-affected\nsamples was unfortunately not feasible for this study. However, we directly\nfollowed the reviewer's advice and performed spatial transcriptomics analysis\non scalp tissue from the non-balding occipital region of a single AGA patient.\nBased on this spatial transcriptomics data, published cell markers, and known\nhair follicle spatial organization, we meticulously re-analyzed our scRNA-seq\ncell clusters. This re-analysis largely corroborated our initial clustering results.\nNonetheless, it also revealed a specific error: we had previously misclassified\nORS suprabasal cells as IRS cells (Shown in new Figure 1b-e;\nnew Supplementary Figure 3). We have corrected this misclassification throughout\nthe revised manuscript based on the spatial transcriptomics evidence.\nImportantly, this revision does not alter the core conclusions of our study.\nFurthermore, we recognize the limitations of the scRNA-seq approach more\nbroadly. We have therefore added a detailed discussion of these limitations\n(including sample preparation artifacts and spatial context loss) within the\nrevised manuscript. To definitively address the spatiotemporal interactions\ncrucial for AGA pathogenesis, as strongly suggested by the reviewer, we plan\nfuture studies utilizing spatial transcriptomics on intact scalp tissue specimens.\nThis will specifically compare balding (frontotemporal) versus non-balding\n(occipital) regions from AGA patients, enabling a direct investigation within the\nnative tissue habitat.\nComment 4:\n4. That the gene expression profile of all investigated cell types \"had differences\nin various degree (Fig. S2N, Supplementary Table 2)\" is entirely expected, but\ndoes not characterize (and much less so explain) important transcriptional\nchanges the precede the early stages of hair miniaturization in AGA - not the\nleast since these early stages (=intermediate HFs, see Miranda et al. FASEB J\n2018) do not seem to have been specifically studied. The latter, however, is\nmandatory if one wishes to make any plausible claims reg. the molecular\ncontrols that drive the early stages of HF miniaturization in AGA.\nResponse 4:\nWe appreciate the reviewer for the constructive comments. We also sincerely\napologize for our previous inappropriate wording and fully acknowledge that our\ncurrent results indeed cannot make any plausible claims regarding the\nmolecular controls that drive the early stages of hair follicle miniaturization in\nAGA. Therefore, we have revised the relevant descriptions in the revised\nmanuscript. Additionally, we have performed an in-depth analysis of\ntranscriptional changes within our single-cell RNA sequencing dataset. This\ncomprehensive re-analysis specifically included conducting differentially\nexpressed gene (DEG) analyses and pathway enrichment analyses for the two\ncritical comparisons: B (Balding) vs. NB (Non-balding), and B (Balding) vs. HS\n(Healthy Scalp), aiming to better characterize the specific transcriptional\nalterations in balding anagen HFs of AGA patients. To reflect these new\nfindings, we have incorporated the full results of this in-depth transcriptional\nanalysis into the revised manuscript. A detailed description of these results,\nincluding the newly identified DEGs and enriched pathways relevant to AGA\ndevelopment, now appears in the Results section (Shown in new\nSupplementary Figure 4b, c; new Supplementary Table 5 and 6).\nComment 5:\n5. May be I just missed the points the authors tried to make here, but I fail to\nrecognize in which way exactly this study advances the field fundamentally over\nwhat has already been published on HF stem cells and their immediate progeny,\ntheir heterogeneity, and corresponding AGA-associated abnormalities in the\nGarza/Cotsarelis study and later work (e.g., Purba et al. Sci Rep 2017). It is\nessential to clarify this.\nResponse 5:\nWe acknowledge that our finding of reduced HF stem cells (HFSCs) and their\nimmediate progeny (progenitor cells) in the balding scalp of AGA patients\nindeed aligns with and is not unprecedented in the field, as consistently\nreported in previous seminal studies (J Clin Invest. 2011 Feb;121(2):613-22;\nSci Rep. 2017 Nov 9;7(1):15197). However, our study provides mechanistic\ninsights and novel observations. Firstly, we demonstrate that CTS contraction\n-mediated cellular apoptosis might be a contributing mechanism to this\nprogenitor cell reduction, a point not previously confirmed. Secondly, we\nprovide evidence that aberrant CTS contraction suppresses the proliferation of\nmatrix cells and ORS cells (Shown in new Figure 4n, o; new Figure 4w-z;\nnew Figure 5l, m; new Figure 5u, v),\npotentially representing a key driver\nunderlying HF miniaturization in AGA. Critically, as suggested by the reviewer,\nwe rigorously tested our main findings using the humanized AGA mouse model\n(human AGA scalp xenografts on SCID mice). This in vivo validation offers\nrobust, clinically oriented preclinical evidence supporting the targeting of CTS\ncontraction as a potential therapeutic strategy for AGA (Shown in new Figure\n6; new Supplementary Figure 16). Furthermore, our preliminary analysis via\nsingle-cell trajectory modeling suggests that HFSCs in AGA exhibit a\ndifferentiation bias towards epidermal and sebaceous gland lineages (Shown\nin new Supplementary Figure 6a-f). While this potential shift in fate\ncommitment requires further functional and mechanistic investigation in future\nstudies, it adds another layer to our understanding of HFSC dysregulation in\nAGA pathogenesis.\nWe have now included a direct comparison and discussion of our findings\nrelative to the aforementioned foundational work within the revised Discussion\nsection to clarify the specific advancements made by this study.\nComment 6:\n6. The subclusters of DP cells and related changes in AGA reported here are\ninteresting and potentially important - if the transcriptomic data are backed up\non the decisive protein expression level by.\nResponse 6:\nWe sincerely appreciate the reviewer’s constructive and insightful comments\nregarding the potential significance of DP cell subclusters and their changes in\nAGA. Following your suggestion, we carefully reanalyzed the DP data. While\nour analysis identified five distinct DP subclusters, no statistically significant\ndifferences in their proportion were observed between B vs. HS or B vs. NB\ncomparisons. Moreover, due to the very limited cell counts within each\nindividual DP subcluster, conducting reliable subcluster-specific differential\ngene expression (DEG) analysis risked substantial technical bias.\nConsequently, we performed DEG analysis for total DP cells in B vs. HS and B\nvs. NB. This yielded 19 DEGs (Padj < 0.05) for B vs. HS and 101 DEGs (Padj <\n0.05) for B vs. NB, with no overlap between the two lists (Shown in new\nSupplementary Table 5). Collectively, our results (subcluster proportions and\naggregate DEGs) reveal no robust transcriptional signature differentiating AGA\nbalding DP cells from either HS or NB controls within this dataset, suggesting\nno strong candidate pathway for immediate protein-level validation. We\nspeculate this may result from the inherently low abundance of captured DP\ncells, potentially exacerbated by transcriptomic perturbations during single-cell\nsuspension preparation. To address this limitation, we plan future studies\nutilizing multiple samples (B, NB, HS) through spatial transcriptomics or\noptimized suspension protocols to rigorously explore DP heterogeneity in AGA.\nWe have explicitly acknowledged these limitations in the revised Discussion,\nadvising readers to interpret our DP-related findings with caution.\nComment 7:\n7. It is a general shortcoming of this study that too few transcriptional changes\nreported here that are critical for supporting the authors' main working\nhypothesis have been confirmed by IF/IHC and quantitatively analyzed at the\nprotein level. It is strongly recommended that the authors expand these protein\ndata sets, including but not restricted to verifying the reported changes in\nimmune cell populations by quantitative immunohistomorphometry, following\npublished standard protocols.\nResponse 7:\nWe appreciate the reviewer’s kind suggestion regarding the need for greater\nprotein-level validation. As suggested, we conducted additional co-\nimmunostaining for CD4 and IL-17A, and performed quantitative analysis of\nboth the number of CD4-positive cells and the proportion of IL-17A-positive\ncells within the total CD4-positive population across the study groups (Shown\nin new Supplementary Figure 5n, o), which is consistent with our\ntranscriptomic findings. Furthermore, we have provided immunostaining of\nCleaved-Caspase 3 with quantitative analysis of apoptotic cell number in\nprogenitor/matrix cells, and p-MLC2 and α-SMA co-immunostaining with\nquantitative assessment of p-MLC2 intensity validating CTS hypercontraction—\nall critical evidence strengthening our mechanistic conclusions (Shown in\nrevised Figure 4c, d).\nComment 8:\n8. Measurements of changes in HF cycling seem to largely rely on macroscopic\nassessment. This is so unreliable as to be unacceptable. For hair cycle staging\nof organ-cultured human HFs, standard hair cycle histomorphometry needs to\nbe employed (Kloepper et al. Exp Dermatol 2010), analyzing a sufficient\nnumber of HFs from at least 3 different donors.\nResponse 8:\nWe thank the reviewer for this constructive critique and clarify that our hair\nfollicle cycling assessments were not solely based on macroscopic evaluation,\nbut rather adopted histomorphometric methods detailed in the previous\npublication (Exp Dermatol. 2015 Dec;24(12):903-11)—a methodology\nconcurrent with the approach mentioned by the reviewer (Kloepper et al. Exp\nDermatol 2010), both originating from the Ralf Paus (a world-renowned expert\nin AGA research) research group. Our original Supplementary Figure 1\nalready demonstrated cycle staging for HS, NB, and B groups using this\nstandardized framework, showing that compared to catagen HFs, anagen HFs\nexhibited hair matrix (HM) with larger volume, more onion-shaped DP and\nmaximal melanin content; besides, the HM cells displayed obvious proliferative\nactivity and no apoptotic signals. As suggested, to further validate our findings,\nwe performed additional analyses on organ-cultured HFs treated with MCH ±\nML-7, including: Cleaved-Caspase 3 immunostaining in matrix cells,\nHematoxylin staining for melanin content, and RT-qPCR quantification of\nanagen-maintaining factors (IGF-1, KGF) and catagen drivers (TGFβ1/2,\nSFRP1)—characterized across HFs from ≥3 donors per condition. These\ncomprehensive datasets reinforce our conclusions (Shown in revised\nSupplementary Figure 8b, c; new Supplementary Figure 9g-i; new\nSupplementary Figure 10r; new Supplementary Figure 11h, i; new\nSupplementary Figure 16d), with full methodological details now explicitly\nciting the literature (Kloepper et al. Exp Dermatol 2010) in our revised Methods\nsection and experimental specifics integrated throughout Results, Figures, and\nFigure legends.\nComment 9:\n9. It remains too unclear on which data exactly the differences between murine\npelage and human scalp HF CTS that are depicted in the cartoon (Fig. 4A) are\nbased. What exactly is the take home message from this cartoon, and how does\nthis relate to the authors' main hypothesis? Please explain this more lucidly and\ncorroborate the main claims contained in this cartoon by convincing evidence.\nResponse 9:\nWe regret any confusion caused by Fig. 4A, and clarify that this cartoon—\nmodified from prior literature (Exp Dermatol. 2021 Apr;30(4):512-521.)—\nvisually summarizes the key structural differences of CTS between murine\npelage and human scalp HF, specifically illustrating that ORS (including\nprogenitor cells) in mice is enveloped by DS structure (mainly composed of a\nlayer of elastic DS cells), whereas the human scalp CTS (the homolog of murine\nDS) forms a multilayer structure comprising DS cells, collagens, and vascular\nelements (including vSMCs and endothelial cells), as detailed in our Results\nsection. To avoid any confusion, we have described it more clearly in the result\nsection, and explicitly noted the cartoon’s origin in the revised figure legend.\nCrucially, this cartoon contextualizes our functional findings that both DS cells\nand vSMCs exhibit augmented contractility in balding HFs (Shown in revised\nFigure 2m, p)—directly relevant as these cells constitute the primary contractile\nunits of the human CTS, thereby underscoring how CTS hypercontraction\nmechanistically contributes to AGA pathogenesis.\nComment 10:\n10. While the postulate that \"hyperactive CTS contraction drives premature hair\nregression via inducing apoptosis of progenitor cells in AGA\" is very intriguing,\nI am unconvinced by the currenly presented evidence. In human terminal\nanagen scalp HFs, the early stages of catagen development occur in the\nanagen hair matrix, i.e. very far distant from the bulge stem cells and their\nimmediate progeny. While - in line with the Garza/Cotsarelis AGA study - it is\ntheoretically conceivable that excessive progenitor apoptosis might result from\nhyperactive CTS contraction and may somehow contribute to HF\nminiaturization, it is therefore highly implausible that this should also induce\npremature catagen induction. To support this claim, much more direct evidence\nwould have to be provided, such as the demonstration that key anagen-\nmaintaining growth factors (IGF-1, KGF, HGF, Wnt activity) are reduced, while\nkey drivers of catagen (TGFb1/2, SFRP1 etc.) are increased.\nResponse 10:\nWe sincerely thank the reviewer for the insightful comments. Based on these\ncomments, comment 1 also provided by the reviewer and the established\nliteratures, we have carefully re-analyzed and reconsidered our results. We\nacknowledge that our initial interpretation may have introduced confusion or\ninaccuracies, particularly regarding the direct causality between excessive\nprogenitor apoptosis resulting from hyperactive CTS contraction and hair follicle\n(HF) miniaturization. In order to provide a clearer explanation, we performed a\nseries of additional experiments to examine the proliferation of HF cells\nenveloped by CTS, including the outer root sheath cells and matrix cells\nsurrounding the dermal papilla. Our results demonstrated that excessive CTS\ncontraction not only induces apoptosis in progenitor cells, but also significantly\nsuppresses the proliferation of matrix cells and ORS cells (including progenitor\ncells) (Shown in new Figure 4n, o; new Figure 4w-z; new Figure 5l, m; new\nFigure 5u, v). Moreover, we have provided additional evidence showing that\nactivating PIEZO1 in human hair follicle ORS cells directly with YODA1 in vitro\nnot only significantly suppressed cell proliferation, but also induced apoptosis\nin a subset of cells (Shown in new Supplementary Figure 14i-m)—consistent\nwith our observations from ex vivo organ-cultured human hair follicles. These\nalterations, especially the significant suppression of matrix cell proliferation,\nlead to hair growth retardation. We propose that this retardation may\nprogressively contribute to HF miniaturization over time. Furthermore, in a\nsubset of follicles, the combined effects of increased progenitor cell apoptosis\nand significantly reduced proliferation of both the matrix cells and ORS cells\n(including progenitor cells) appear to culminate in premature entry into catagen.\nWith additional experiments and analyses suggested by the reviewer, including\nCleaved-Caspase 3 immunostaining in matrix cells, Hematoxylin staining for\nmelanin content, and RT-qPCR quantification of anagen-maintaining factors\n(IGF-1, KGF) and catagen drivers (TGFβ1/2, SFRP1), we observed that a\nportion of HFs treated with MCH or DHT indeed prematurely entered the\ncatagen phase (Shown in new Supplementary Figure 9g-i; new\nSupplementary Figure 10r; new Supplementary Figure 11h, i; new\nSupplementary Figure 16d). Critically, this matrix cell apoptosis\noccurs after the observed increase in progenitor cell apoptosis and decrease in\nmatrix/ORS cell proliferation (Shown in new Supplementary Figure 9g-i).\nThis temporal sequence suggests that premature catagen entry is likely a\nconsequence of these preceding events (apoptosis and proliferation defects),\nrather than a direct cause of HF miniaturization.\nTherefore, consistent with these insights and new data, we have revised the\ndescription of the impact of excessive CTS contraction throughout the\nmanuscript. References to \"premature catagen entry\" have been replaced with\n\"hair follicle growth retardation\".\nNotably, our study provides no mechanistic evidence elucidating why\naberrant CTS contraction exerts differential effects on spatially distinct ORS\nand matrix cells within human hair follicles. Specifically, aberrant CTS\ncontraction simultaneously induces apoptosis and inhibits proliferation in ORS\ncells, whereas matrix cells exhibit only proliferation suppression at early stage.\nWe postulate that this heterogeneity may correlate with spatially varying\nmagnitudes of mechanical forces generated by CTS contraction along the hair\nfollicle. However, validation of this hypothesis will require more sophisticated\nbiomechanical experimentation in future studies. This limitation has been\nexplicitly addressed in the revised discussion section of our manuscript.\nComment 11:\n11. I am also intrigued by the reported hair growth promotion by ML-7, which\nwould represent a welcome novel AGA treatment principle. However,\nconvincing evidence that ML-7 is indeed more effective than minoxidil is best\ngenerfated in the humanized AGA model after long-term appliucation in vivo -\nespecially since human HF organ culture is a notoriously unreliable assay for\nevaluating minoxidil activities (Magerl et al. Exp Dermatol 2004).\nResponse 11:\nWe thank the reviewer for the positive feedback and insightful suggestions\nregarding ML-7's therapeutic potential for AGA. As suggested, we extended our\nexperiments in the humanized AGA mouse model, demonstrating that both ML-\n7 and minoxidil (MNX) significantly counteracted DHT-induced growth\nsuppression in transplanted non-balding AGA hair follicles, with no statistically\nsignificant efficacy difference observed between them. Notably, combination\ntreatment yielded superior improvement (Shown in new Figure 6m-r). More\nstrikingly, ML-7 administration prominently stimulated hair growth in xenografts\nderived from balding AGA scalp regions (Shown in new Figure 6s-x),\nfurther\nunderscoring its translational relevance.\nMinor issue 1:\nThe main concepts emanating from this study should be synthesized in an\ninstructive cartoon (presented as part of the last core figure or as a\nsupplementary fig.).\nResponse 1:\nWe thank the reviewer for this excellent suggestion. As recommended, we have\nnow synthesized our main findings into an instructive cartoon (Presented as\nnew Supplementary Figure 17).\nMinor issue 2:\nComplex statements like \"To investigate the trajectories among all 20 cell types,\nmonocle3 (36) was employed to perform the pseudotime analysis with the root\nnode setting at start point of bulge on the SOX9+ HF branch and start point of\nIFE basal on the SOX9- IFE branch separately....\" (Results) will not be easily\ndigestible by many non-expert readers and should be rephrased to be more\ninstructive & self-explanatory.\nResponse 2:\nWe sincerely appreciate the reviewer’s constructive suggestion regarding the\naccessibility of our pseudotime analysis description. Considering that this\nparticular finding (former Figure 1E) lacked novelty and similar with previously\nreported studies—a point also noted by another reviewer—we have removed\nthis result from the revised manuscript. Concurrently, we have diligently refined\nsimilar technical descriptions throughout the manuscript to enhance clarity and\nensure self-contained explanatory value for non-specialist readers.\nMinor issue 3:\n\"For the SOX9- IFE branch, IFE basal cells can directly differentiate into SOX9-\nPR and IFE spinous cells which finally develop into IFE granular cells (Fig. 1E).\nThese patterns are consistent with the knowledge from previous studies(26,\n35).\" Please explain what exactly these results add to the published literature,\nhow they advance the field, and why they are even relevant in the context of\nAGA and CTS contraction.\nResponse 3:\nWe appreciate the reviewer’s constructive critique regarding the significance of\nour SOX9– IFE trajectory findings and their relevance to AGA pathogenesis. In\nreconsidering this result, we recognized it confirmed known epidermal\ndifferentiation paradigms without introducing conceptual novelty, was\nperipherally related to our core focus on CTS contraction mechanisms in AGA,\nand was rendered obsolete by subsequent analysis—after integrating spatial\ntranscriptomics data, all cell clustering was redefined without SOX9-based\nguidance. Thus, aligning with another reviewer’s assessment, we have\nremoved this analysis from the revised manuscript.\nMinor issue 4:\nWhat exactly do the authors feel to be the role of DHT and AR in promoting the\npresumed hyperactive CTS contraction? Can they generate direct evidence to\nsupport or exclude such a role, e.g. in HF organ culture?\nResponse 4:\nWe thank the reviewer for the interest in the potential role of DHT and AR in\nhyperactive CTS contraction, which indeed raises an intriguing question.\nPrompted by a recent Cell study demonstrating DHT promotes muscle cell\ncontraction via the membrane receptor GPR133 (Cell. 2025 Mar\n20;188(6):1589-1604.e24), we investigated whether DHT might similarly\nstimulate CTS contraction through GPR133 in AGA pathogenesis. Our new\ndata revealed that GPR133 is indeed expressed in human HF CTS cells\n(Shown in new Supplementary Figure 10a). In vitro assays using isolated\ndermal sheath (DS) cells—a key CTS component—demonstrated that DHT\npotently enhances DS cell contraction; this effect was markedly attenuated by\nGPR133 knockdown with siRNA (Shown in new Supplementary Figure 10b-\nf). Further HF organ culture experiments showed DHT not only induces CTS\nhypercontraction but also triggers progenitor cell apoptosis, suppresses matrix\nand ORS cell proliferation, and ultimately inhibits hair growth—all of which were\nsignificantly reversed by either GPR133 knockdown or ML-7-mediated\ncontractility inhibition (Shown in new Supplementary Figure 10g-r; new\nSupplementary Figure 11a-i). Importantly, while AR knockdown alleviated\nDHT’s inhibitory effects on hair follicle growth, it did not alter CTS contraction\n(Shown in new Supplementary Figure 11k-m). These findings suggest that\nDHT contributes to AGA pathogenesis through two distinct mechanisms: (1)\ncanonical AR signaling in follicular cells (e.g., DP and ORS cells), and (2)\nGPR133-mediated hypercontraction in CTS components (e.g., DS cells). This\nDHT-GPR133-dependent contractile mechanism might represent a key\nconsideration for AGA therapeutic development, particularly regarding AR-\ntargeting therapies since AR blockade alone may not address this pathway. We\nhave incorporated these mechanistic insights in the revised manuscript's\nDiscussion section.\nReviewer #2\nOverall comments:\nThe authors Li et al provide a single cell analysis of androgenic alopecia human\nskin. Androgenic alopecia is still largely unexplored and, although being a\n“cosmetic” disease, harbors a high burden on many affected patients\nconcerning their quality of life. Thus, high throughput investigations of this\nconditions are urgently mandated. Comparing occipital with frontal skin\n(affected and unaffected) is a reasonable approach.\nOverall response:\nWe thank the reviewer for the recognition of our work, and the insightful and\nhelpful comments, all of which have been addressed, as detailed below.\nComment 1:\nIn the beginning and in the abstract, the authors need to more clearly state their\nsample size, which seems quite low. Also, it needs to be clearer whether\ninvestigations have been performed in same patients or independent cohorts.\nResponse 1:\nWe sincerely appreciate the reviewer’s kind suggestion. Accordingly, we have\nclarified the sample size in the revised manuscript. Due to word constraints in\nthe abstract, we now explicitly detail sample information in the beginning of\nResults section, Figure legends and \"Human scalp and HF samples\"\nsubsection of Methods. Specifically, we have stated that scRNA-seq analysis\nutilized matched frontal balding/occipital non-balding HF units from 4 AGA\npatients and frontal HF units from 4 healthy donors, while HF organ culture and\nhumanized mouse model experiments employed specimens from distinct\ncohorts. To enhance transparency, we provide a supplementary table\ncataloging all specimens used across experiments, including patient/donor\nsources, biopsy sites, and respective methodologies (Shown in new\nSupplementary Table 18).\nComment 2:\nThe authors need to make clear in abstract, title and throughout the manuscript\nthat they are investigating male patients, as balding patterns strongly differ\nbetween males and females.\nResponse 2:\nWe sincerely appreciate the reviewer's constructive suggestion. As suggested,\nwe have clarified throughout the revised manuscript—including the abstract,\ntitle, and main text—that this study exclusively investigates male patients,\nthereby addressing potential ambiguities regarding sex-specific balding\npatterns. Also, we have discussed this limitation in the Discussion section of the\nrevised manuscript.\nComment 3:\nThe authors might want to add a table with QC parameters such as genes per\ncell, cells per sample obtained, as well as a table showing how many cells were\nfiltered out during QC, and how many cells are present for each cluster and\neach sample. Also, a baseline table with patients age etc needs to be provided\nto assess whether the biopsies are appropriately matched.\nResponse 3:\nWe thank the reviewer for this valuable suggestion. As suggested, we have\nadded two comprehensive tables in the revised manuscript: (1) a QC metrics\ntable detailing genes per cell, cells per sample, and the number of cells filtered\nduring quality control (Shown in new Supplementary Table 1); and (2) a\ncluster distribution table specifying cell counts per cluster and sample (Shown\nin new Supplementary Table 4). Additionally, a baseline characteristics table\nincluding patient age, biopsy site, and clinical parameters has been provided to\nconfirm appropriate cohort matching across all specimens (Shown in new\nSupplementary Table 18).\nComment 4:\nA cutoff for DEGs of avg_log2FC>0.25 is very low, I would recommend to\nincrease to 0.5 or 0.75 if feasible.\nResponse 4:\nWe appreciate the reviewer's suggestion regarding the DEG cutoff threshold.\nFor clarity, the selection criterion (avg_log2FC > 0.25) aligns with Seurat's\ndefault parameters, reflecting that scRNA-seq datasets typically yield fewer\nDEGs than bulk RNA-seq (where avg_log2FC > 1 is common). When testing\nstricter thresholds (avg_log2FC > 0.5/0.75), we observed a dramatic reduction\nin detected DEGs, with many cell clusters exhibiting minimal or no significant\nDEGs. This may reflect our use of early-stage AGA follicular specimens—\ncollected prior to marked miniaturization—where transcriptomic alterations are\ninherently subtle. Notwithstanding this lower threshold, we exclusively\nprioritized the top 20 DEGs ranked by avg_log2FC when identifying\ncell/subpopulation markers to ensure biological relevance.\nComment 5:\nThe authors need to better label their figures, the figures are very difficult to\nfollow. Particularly also in supplements, the authors need to write what cells\nhave been subclustered etc, right now plots are too sparsely labelled and partly\nincomprehensible.\nResponse 5:\nWe apologize for the insufficient labeling in our original figures and sincerely\nappreciate the reviewer’s constructive suggestion. Accordingly, we have\ncomprehensively relabeled all figures throughout the manuscript with detailed\nannotations, particularly highlighting cell-type specifications and subclustering\ninformation to ensure clarity and interpretability.\nComment 6:\nWhen sublcustering, authors need to explain (or label in figure) what they\nobtained. For example for endothelial cells, they perform subclustering and\nobtain 4 clusters, but do not explain what they find. Arteriolar or venular or\ncapillary? If they perform subclustering, they should elaborate on them. This\nneeds to be done for all figures.\nResponse 6:\nWe thank the reviewer for this insightful suggestion. Accordingly, in the revised\nmanuscript, we have provided definitive labeling and textual interpretation of all\ncellular subclustering results across figures, specifically delineating subtypes of\neach cell type in relevant annotations, legends, and results sections to ensure\ncomprehensive biological clarity.\nComment 7:\nIn lymphocyte subclustering, the “unknown” clusters seem keratinocyte\ncontaminations.\nResponse 7:\nWe fully agree with the reviewer’s insightful observation. The \"unknown\" cluster\nexpresses high levels of keratinocyte marker genes\n(e.g., KRT14, KRT15, KRT1, KRT10), suggesting these likely represent dying\nkeratinocyte-derived debris (or other dying cell-derived debris containing a\nlarge amount of mRNA from dead keratinocytes) inadvertently encapsulated\ninto droplets during single-cell processing. Although we cannot definitively\nverify this contamination source, these data points are retained in UMAP\nprojections to ensure data transparency.\nReviewer #3\nOverall comments:\nThe manuscript entitled Single-cell transcriptomics reveals cellular hierarchies\nand aberrant CTS contraction-mediated premature hair regression in\nandrogenetic alopecia revealed aberrant CTS contraction in AGA patients,\nwhich is the crucial factor that causes premature hair regression. Focusing on\nintracellular contraction force in HF is quite interesting. However, this work\nmissed critical evidence to confirm the enhanced mechanical stress, and lacked\nfurther exploration in the detailed molecular mechanism of how contraction\nforce led to hair regression.\nOverall response:\nWe thank the reviewer for the recognition of our work, and the insightful and\nhelpful comments, all of which have been addressed, as detailed below.\nMajor concern 1:\n1. The authors suggested enhanced connective tissue sheath as the significant\ndriver of premature hair regression. However, bioinformatic analysis and\nimmunostaining of p-MLC2 couldn’t fully support this hypothesis. Atomic force\nmicroscope is appreciated to examine and confirm the hyperactive contraction\nforce in CTS.\nResponse 1:\nWe greatly appreciate the reviewer’s constructive suggestion. As suggested,\nwe performed atomic force microscopy (AFM) measurements directly\ncomparing the contraction force of primary dermal sheath (DS) cells—key\ncellular components of the CTS—isolated from balding versus non-balding hair\nfollicles (HFs) of AGA patients. These experiments demonstrated that\nstimulators (e.g., MCH or DHT) significantly enhanced DS cell contractility, with\nDS cells from balding HFs exhibiting stronger stimulator-induced contraction\nforces versus non-balding counterparts (Shown in new Supplementary\nFigure 8h, i; new Supplementary Figure 10d; new Supplementary Figure\n8d-g; new Supplementary Figure 10a-c). Complementarily, real-time\nconfocal imaging of primary DS cells and human hair follicle organ cultures\nconsistently revealed that stimulators robustly promote contraction in both\nsystems; notably, DS cells and hair follicles derived from balding scalp of AGA\npatients showed markedly amplified contractile responses upon stimulation\n(Shown in new Figure 4e, f; new Supplementary Figure 8j, k; new\nSupplementary Figure 9b, c; new Supplementary Figure 10e-h). These\ndata directly validate hyperactive CTS contraction in AGA pathophysiology,\nstrengthening our initial mechanistic hypothesis.\nMajor concern 2:\n2. Piezo1 is one of the most investigated mechanosensitive ion channel\nproteins in recent years. How did the authors come to consider exploring Piezo1,\nbut not other proteins? Is the expression pattern of Piezo1 upregulated, or other\nmechanosensitive pathways functionally enriched, in the single cell sequence\nmetadata of frontal balding, especially the CD200 positive subset?\nResponse 2:\nWe thank the reviewer for the insightful comments and constructive\nsuggestions. In fact, our prior analysis of scRNA sequencing data had already\nassessed the expression profiles of multiple mechanosensitive ion channels\n(including PIEZO1, PIEZO2, TRPV4, KCNK2, KCNK4, KCNK10, STOML3 and\nTRPA1), revealing PIEZO1 and TRPV4 as the predominant channels in hair\nfollicle stem cells (HFSCs). However, since published evidence (PMID:\n34624205) established PIEZO1—but not TRPV4—as essential for murine\nHFSC function, we prioritized PIEZO1 for mechanistic validation in human AGA.\nThis rationale led us to initially verify PIEZO1 upregulation via\nimmunofluorescence in balding HFSCs (including progenitor cells) of AGA\npatients, rather than presenting our scRNA-seq data. In direct response to this\ncomment, we have now: (1) added these scRNA-seq analyses to the revised\nmanuscript, confirming PIEZO1’s enrichment in the HFSC subset and other\nmechanosensitive cell populations (e.g., ORS, matrix) in human hair follicles;\nand (2) performed enhanced immunofluorescence using tyramide signal\namplification (TSA) on balding versus non-balding AGA hair follicles, and our\nquantitative results directly validate PIEZO1 upregulation within ORS (including\nprogenitor cells), and matrix of balding HFs (Shown in new Figure 5a-d; new\nSupplementary Figure 14a, b). Simultaneously, we have provided additional\nevidence showing that inhibiting TRPV4 by its specific inhibitor RN1734 fails to\nrescue the MCH-induced growth suppression of human hair follicles (Shown in\nnew Supplementary Figure 14n, o). These results firmly establish the\npathological relevance of PIEZO1-mediated mechanotransduction in AGA.\nMajor concern 3:\n3. The results indicated that Piezo1 inhibitor GsMTX4 could ameliorate hair\nregeneration. While in the next section, the authors also showed the positive\neffect of myosin activation inhibitor ML-7. From the authors’ perspective,\ninhibiting mechanosensitive ion channels via GsMTX4, or hindering\ncytoskeleton contraction via ML-7, which had a more significant regulatory\neffect on CTS contraction, and possibly be a therapeutic target. Please\ncompare, verify, and illustrate.\nResponse 3:\nWe are grateful for the reviewer’s insightful comments on therapeutic target\nprioritization. To directly answer this question, we conducted additional\ncomparative functional assays using MCH-treated human hair follicles, and\ndemonstrated that both GsMTX4 and ML-7 effectively rescued growth inhibition,\nwith ML-7 exhibiting a modest but non-significant advantage in efficacy (Data\npresented below, for review only). Crucially, our mechanistic model indicates\nthat pathological CTS hypercontraction in AGA balding follicles (inhibited by\nML-7) drives downstream PIEZO1 activation in HF cells (e.g., progenitor cells,\nmatrix; inhibited by GsMTX4), thereby inducing apoptosis, suppressing\nproliferation, and ultimately impairing HF growth. Thus, ML-7’s blockade of the\nproximal mechanical trigger provides broader therapeutic leverage by targeting\nthe root cause, whereas GsMTX4 cannot rectify the primary CTS defect despite\nameliorating its consequences. This premise is strongly supported by\ncompelling preclinical validation: in humanized mouse models, ML-7 exhibited\nrobust efficacy (Shown in new Figure 6), underscoring its enhanced\ntranslational promise. We maintain that topical/follicle-targeted delivery of either\nagent—rather than systemic administration—represents the optimal strategy to\nmitigate potential adverse effects in clinical settings.\nMajor concern 4:\n4. The authors showed that ML-7 had a better effect on treating androgenetic\nalopecia. I would also prefer to know the therapeutic effect of ML-7 comparing\nwith finasteride. And could the combination of ML-7 with minoxidil/finasteride\nacquire a better clinical outcome?\nResponse 4:\nWe appreciate the reviewer's constructive suggestions. To address this, we\ncompared the therapeutic efficacy of ML-7 versus minoxidil (MNX) and their\ncombination in humanized mouse models. Our new results showed that both\nML-7 and MNX significantly rescued DHT-induced hair growth suppression,\nwhile their combination demonstrated superior therapeutic outcomes (Shown\nin new Figure 6m-r). Notably, no statistically significant difference was\nobserved between ML-7 and MNX monotherapies in vivo, contrasting with our\nprior ex vivo human hair follicle organ culture data where ML-7 outperformed\nMNX (Shown in revised Supplementary Figure 15e-j). This discrepancy may\nstem from MNX’s potential pro-angiogenic/vasodilatory effects within the\ncutaneous microenvironment—benefits not fully realized in isolated HF\ncultures. Moreover, given that finasteride primarily functions by inhibiting 5α-\nreductase to suppress DHT synthesis—and our current HF organ\nculture/humanized AGA mouse models (established by using exogenous DHT\nor MCH) do not target 5α-reductase activity or upstream regulatory pathways—\nwe have not performed direct efficacy assessments of finasteride in these\nsystems.\nMajor concern 5:\n5. The authors didn’t explain the molecular mechanism of how Piezo1/myosin\nactivation-induced CTS contraction induces apoptosis and thus causes\npremature hair regression, which is critical for an in-depth understanding of\nAGA progression.\nResponse 5:\nWe appreciate the reviewer's constructive comments. Previous studies have\nestablished that PIEZO1 activation in murine hair follicle stem cells elevates\nintracellular calcium, triggering mitochondrial membrane permeabilization to\nrelease pro-apoptotic factors and ultimately induce apoptosis (Cell Stem Cell.\n2022;29:70-85); concurrently, PIEZO1-mediated calcium signaling suppresses\nmurine HFSC activation and impedes hair regeneration (Sci Adv.\n2025;11:eadt2771). To elucidate whether this mechanism underlies AGA, we\nperformed calcium imaging using Cal520, a more specific calcium influx\nindicator than Fluo-4, in hair follicles from AGA patients (balding vs. non-balding\nregions) and MCH-treated human HF organs. This revealed significantly\nintensified calcium flux in the ORS (including progenitor cells) and matrix cells\nof balding HFs and MCH-treated HF organs (Shown in new Figure 5c, d; and\nnew Supplementary Figure 14c, d, f, g). Crucially, experiments on isolated\nprimary human ORS cells demonstrated that the PIEZO1 agonist YODA1\ndirectly elevated intracellular calcium, inhibited proliferation, compromised\nmitochondrial function, and upregulated the apoptotic executioner molecule\ncleaved caspase-3, culminating in apoptosis (Shown in new Supplementary\nFigure 14i-m). Thus, pathological CTS contraction—or the consequential\nPIEZO1 activation in ORS/matrix cells—drives aberrant calcium signaling that\nsimultaneously induces apoptosis (via mitochondrial-caspase axis) and\nsuppresses proliferation, collectively propagating AGA pathogenesis. Although\nour preliminary evidence suggests that PIEZO1-mediated dysregulated calcium\nsignaling may play a contributory role in this process, , the detailed molecular\nmechanisms by which PIEZO1 hyperactivation induces progenitor cell\napoptosis and suppresses proliferation of ORS/matrix cells in AGA need further\nstudy to clarify. These new experimental findings and supporting literature\ncitations have been incorporated into the revised manuscript and discussed in\ndepth in the Discussion section.\nMinor concern 1:\n1. Before conducting scRNA-seq, the HFs were confirmed in anagen phase\n(line 117). Please describe the biomarker and briefly illustrate the sorting\nprotocol for preparing single-cell suspension in the main body.\nResponse 1:\nWe thank the reviewer for this kind suggestion. As suggested, we have\ndescribed the relevant biomarker for confirming the anagen phase in the Result\nsection, and briefly illustrated the sorting protocol for preparing single-cell\nsuspensions in the “Preparation of single-cell suspensions of HF units” of\nMethods section of the revised manuscript.\nMinor concern 2:\n2. The results showed that all cell types had differences in various degree (Fig.\nS2N, Supplementary Table 2), suggesting that anagen HFs undergo significant\nchanges at the early stage of hair miniaturization in AGA (line 163-165). Please\nexplain the change in gene expression pattern and the corresponding biological\nmeanings.\nResponse 2:\nWe sincerely appreciate the reviewer's valuable suggestion. As suggested,\nwe have provided explanations for the change in gene expression pattern and the\ncorresponding biological meanings in the Results section (Detailed data was\nalso Shown in Supplementary Table 5 and 6).\nAgain, we appreciate the constructive and insightful comments, and helpful\nsuggestions from the reviewers. We hope that with the revisions the manuscript\nwill be acceptable for publication.\nBest wishes.\nSincerely\nJi li, PhD, Professor\nDepartment of Dermatology, Xiangya Hospital,\nHunan Key Laboratary of Aging Biology, Xiangya Hospital, Central South\nUniversity, Changsha, Hunan, China\nE-mail: liji_xy@csu.edu.cn\nTel: +86-731-8432-7472"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I wholeheartedly commend the authors for their extensive and very satisfactory efforts to greatly improve their study on multiple levels. In its current form, this study has matured into a major contribution that significantly advances the field: congratulations!\nI have only minor suggestions for some additional improvements the authors may wish to consider.\nResponse 1a\nThe authors are still not quoting the currently most relevant literature and should read and cite at least Altendorf et al. Physiol Rev 2025.\nResponse 1b\nThe additional data generated are most welcome. Yet, as the authors appear to recognize, they do not provide sufficient evidence for explaining HF miniaturization, which is primarily driven by a reduction in the volume, cell number and inductive properties of the dermal papilla and secondarily by a reduction in the capacity of bulge epithelial stem cells to generate progeny (as explained in Altendorf et al. 2025). Therefore, to convincingly explain how CTS-related events may lead to HF miniaturization, it would have to be documented that these CTS events reduce the volume, cell number and inductive properties of the dermal papilla and/or the capacity of K15+ epithelial stem cells to generate CD34+ progeny. This should be stated more clearly in the Discussion.\nThough I find the authors’ working hypothesis how aberrant CTS contraction may exert differential effects on spatially distinct ORS and matrix cells plausible, in the Discussion, the authors should 1) openly acknowledge that the study provides no mechanistic evidence in support of this hypothesis and 2) very briefly suggest some key experiments to probe it.\nThe revised Discussion would profit from dissecting even more clearly in which respects exactly the current study a) is in line with, b) conflicts with, and c) advances the field compared to the previous CTS work published by the Rendl Lab in mice.", "reviewer_2": "The authors have answered all the reviewers questions. However, sample size and DEG cutoffs are very small, raising the question of reproducibility. Also, their reasoning that most of their DEGs disappeared with higher threshold indicates the weakness of their data. They need to reproduce their findings in independent sample sets.\nParticularly, their reasoning to choose a FCH cutoff of 0.25 as it is the \"default\" in Seurat is a weak argument. They need to choose a threshold that is biologically meaningful. With such a small sample size and such a very low threshold, they need to more convincingly show that their detected genes are found in a reproducible fashion, which is impossible at n=4 and at their threshold.", "reviewer_3": "I appreciate the extensive amount of work the authors have undertaken in this revision, including new in vitro and ex vivo experiments, re-analysis of scRNA-seq data, AFM-based mechanical measurements, calcium imaging, and the humanized AGA mouse model. The revised manuscript shows substantial improvements in mechanistic depth, experimental rigor, and conceptual clarity. Most of the concerns I raised in the first round have been addressed thoroughly. However, several important issues still require clarification or further refinement to ensure that the manuscript reaches the level of completeness and scientific soundness expected for publication.\nBelow are my detailed second-round comments.\nMajor Comments\n1. Further clarification needed regarding evidence for hyperactive CTS contraction\nThe authors have added AFM measurements of dermal sheath (DS) cell contractility and provided real-time imaging data indicating stronger contraction of DS cells from balding scalp under stimulation. These additions largely address my earlier concerns.\nRemaining issues requiring clarification:\n- Were DS cells from balding and non-balding follicles obtained from the same individual donors in the AFM measurements?\n- Are the baseline (unstimulated) contraction forces also altered in DS cells from balding scalp?\n2. Justification for prioritizing PIEZO1 over other mechanotransducers\nThe authors have added scRNA-seq evidence, TSA-amplified immunofluorescence data, and YODA1 functional assays.\nRemaining suggestion:\n- Briefly explain why other mechanotransducers were not further explored in the Discussion.\n3. Comparison between ML-7 and GsMTX4 as therapeutic strategies\nThe new data show both agents rescue MCH-induced inhibition, with ML-7 showing stronger evidence in vivo.\n4. Clarification regarding comparison with finasteride\nPlease add a clear explanation in the Discussion section regarding why finasteride cannot be meaningfully compared within the current model system.\n5. Recommendation to include a schematic summarizing the proposed mechanistic pathway\nA visual summary would enhance conceptual clarity.\nOverall Assessment\nThe manuscript has improved significantly. I recommend minor revision before acceptance."}, "author_response": "Dear reviewers:\nWe would like to express our deepest appreciation for your time and effort in\nhandling our manuscript. Please find our point-to-point responses to the\nreviewers’ comments as detailed below. We are hopeful that all of the additional\ndata, analyses and information we have provided satisfy the reviewers’\nconcerns.\nREVIEWERS' COMMENTS\nReviewer #1:\nI wholeheartedly commend the authors for their extensive and very satisfactory\nefforts to greatly improve their study on multiple levels. In its current form, this\nstudy has matured into a major contribution that significantly advances the field:\ncongratulations!\nI have only minor suggestions for some additional improvements the authors\nmay wish to consider.\nComment#1a:\nThe authors are still not quoting the currently most relevant literature and should\nread and cite at least Altendorf et al. Physiol Rev 2025.\nResponse:\nWe thank the reviewer for this valuable suggestion. We have now cited\nAltendorf et al., Physiological Reviews, 2025, together with several recent high-\nimpact studies published in the past two years that are highly relevant to\nandrogenetic alopecia pathogenesis, and have incorporated these references\ninto the Introduction and Discussion.\nComment#1b:\nThe additional data generated are most welcome. Yet, as the authors appear\nto recognize, they do not provide sufficient evidence for explaining HF\nminiaturization, which is primarily driven by a reduction in the volume, cell\nnumber and inductive properties of the dermal papilla and secondarily by a\nreduction in the capacity of bulge epithelial stem cells to generate progeny (as\nexplained in Altendorf et al. 2025). Therefore, to convincingly explain how CTS-\nrelated events may lead to HF miniaturization, it would have to be documented\nthat these CTS events reduce the volume, cell number and inductive properties\nof the dermal papilla and/or the capacity of K15+ epithelial stem cells to\ngenerate CD34+ progeny. This should be stated more clearly in the Discussion.\nThough I find the authors’ working hypothesis how aberrant CTS contraction\nmay exert differential effects on spatially distinct ORS and matrix cells plausible,\nin the Discussion, the authors should 1) openly acknowledge that the study\nprovides no mechanistic evidence in support of this hypothesis and 2) very\nbriefly suggest some key experiments to probe it.\nThe revised Discussion would profit from dissecting even more clearly in which\nrespects exactly the current study a) is in line with, b) conflicts with, and c)\nadvances the field compared to the previous CTS work published by the Rendl\nLab in mice.\nResponse:\nWe thank the reviewer for these insightful comments. We have revised the\nDiscussion to more clearly situate our findings within the established dermal\npapilla–centric framework of hair follicle miniaturization, explicitly acknowledge\nthe mechanistic limitations of the present study, briefly outline key future\nexperimental directions, and clarify how our work relates to and extends\nprevious CTS studies from the Rendl laboratory. We thank the reviewer again\nfor these important and constructive comments.\nReviewer #2\nComment:\nThe authors have answered all the reviewers questions. However, sample size\nand DEG cutoffs are very small, raising the question of reproducibility. Also,\ntheir reasoning that most of their DEGs disappeared with higher threshold\nindicates the weakness of their data. They need to reproduce their findings in\nindependent sample sets.\nParticularly, their reasoning to choose a FCH cutoff of 0.25 as it is the \"default\"\nin Seurat is a weak argument. They need to choose a threshold that is\nbiologically meaningful. With such a small sample size and such a very low\nthreshold, they need to more convincingly show that their detected genes are\nfound in a reproducible fashion, which is impossible at n=4 and at their\nthreshold.\nResponse:\nWe thank the reviewer for raising these important concerns regarding sample\nsize, DEG thresholds, and reproducibility. We have clarified in the revised\nmanuscript that the avg_log2FC cutoff of 0.25 was used as an inclusive\nscreening threshold to generate ranked gene lists for downstream gene set\nenrichment analyses, which are designed to detect coordinated transcriptional\nshifts rather than strong individual gene effects. Accordingly, we have limited\ngene-level interpretations and emphasize that our biological conclusions are\nsupported by pathway-level enrichment, cross-cell-type consistency, spatial\ntranscriptomic validation, and multiple orthogonal functional assays, rather than\nreliance on individual DEGs alone.\nReviewer #3:\nI appreciate the extensive amount of work the authors have undertaken in this\nrevision, including new in vitro and ex vivo experiments, re-analysis of scRNA-\nseq data, AFM-based mechanical measurements, calcium imaging, and the\nhumanized AGA mouse model. The revised manuscript shows substantial\nimprovements in mechanistic depth, experimental rigor, and conceptual clarity.\nMost of the concerns I raised in the first round have been addressed thoroughly.\nHowever, several important issues still require clarification or further refinement\nto ensure that the manuscript reaches the level of completeness and scientific\nsoundness expected for publication.\nBelow are my detailed second-round comments.\nMajor Comments:\n1. Further clarification needed regarding evidence for hyperactive CTS\ncontraction\nThe authors have added AFM measurements of dermal sheath (DS) cell\ncontractility and provided real-time imaging data indicating stronger contraction\nof DS cells from balding scalp under stimulation. These additions largely\naddress my earlier concerns.\nRemaining issues requiring clarification:\n- Were DS cells from balding and non-balding follicles obtained from the same\nindividual donors in the AFM measurements?\n- Are the baseline (unstimulated) contraction forces also altered in DS cells from\nbalding scalp?\nResponse 1:\nWe thank the reviewer for this important clarification request. We have now\nexplicitly clarified in the Methods that DS cells used for AFM measurements\nwere derived from matched balding and non-balding follicles from the same\nindividual donors. In addition, we have clarified in the Results and Discussion\nthat baseline (unstimulated) DS contractility did not show a consistent\ndifference, whereas hypercontractility became evident under defined contractile\nstimulation, supporting a stimulus-dependent pathological sensitization rather\nthan constitutive activation.\n2. Justification for prioritizing PIEZO1 over other mechanotransducers\nThe authors have added scRNA-seq evidence, TSA-amplified\nimmunofluorescence data, and YODA1 functional assays.\nRemaining suggestion:\n- Briefly explain why other mechanotransducers were not further explored in the\nDiscussion.\nResponse 2:\nWe thank the reviewer for this helpful suggestion. We have revised the\nDiscussion to briefly clarify that PIEZO1 was prioritized based on its relatively\nhigher expression, consistent activation signature, and functional\nresponsiveness in our system, while other mechano-transducers were not\nexcluded but were beyond the scope of the current study.\n3. Comparison between ML-7 and GsMTX4 as therapeutic strategies\nThe new data show both agents rescue MCH-induced inhibition, with ML-7\nshowing stronger evidence in vivo.\nResponse 3:\nWe thank the reviewer for the positive assessment and for recognizing the\nstrength of the comparative evidence presented.\n4. Clarification regarding comparison with finasteride\nPlease add a clear explanation in the Discussion section regarding why\nfinasteride cannot be meaningfully compared within the current model system.\nResponse 4:\nWe thank the reviewer for this important point. We have added a clarification in\nthe Discussion explaining that finasteride acts upstream by inhibiting\ndihydrotestosterone production, whereas the hair follicle organ culture and\nhumanized androgenetic alopecia models used in this study rely on exogenous\ndihydrotestosterone or MCH to induce AGA-like phenotypes. As a result, these\nmodels do not engage 5α-reductase activity, precluding a meaningful or\nmechanistically informative comparison of finasteride within the current\nexperimental framework.\n5. Recommendation to include a schematic summarizing the proposed\nmechanistic pathway\nA visual summary would enhance conceptual clarity.\nResponse 5:\nWe thank the reviewer for this helpful suggestion. We have now added a\nschematic model summarizing the proposed CTS contraction–PIEZO1\nmechano-transduction pathway and its impact on progenitor cell survival and\nhair follicle growth in Fig S17.\nOverall Assessment:\nThe manuscript has improved significantly. I recommend minor revision before\nacceptance.\nResponse:\nWe thank the reviewer for this positive overall assessment and\nrecommendation.\nThank you again for your time and insightful comments and suggestions.\nYours sincerely,\nJi li, PhD, Professor\nDepartment of Dermatology, Xiangya Hospital,\nHunan Key Laboratary of Aging Biology, Xiangya Hospital, Central South\nUniversity, Changsha, Hunan, China\nE-mail: liji_xy@csu.edu.cn\nTel: +86-731-8432-7472"}]

Decision letter