Next Article in Journal
Stability of Poly[Ni(Salen)]-Based Electrodes in the Presence of Halide Impurities: Coordination and Redox Contributions
Previous Article in Journal
Synthesis and Antitumor Potency of 2E,21E-bis-(2-Pyridinylidene)-hollongdione in NCI-60 Panel and Zebrafish Model
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 27
Issue 4
10.3390/ijms27041815
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo

by
David Broadley
1,†,
Alizée Le Riche
1,†,
Ying Yu
2,
Helene El-Bacha
1,
Hanieh Erdmann
3,
Francisco Jimenez
4,5,
Mikhail Geyfman
2,
Neil Poloso
2,
Janin Edelkamp
1 and
Marta Bertolini
1,*
1
QIMA Life Sciences, QIMA Monasterium GmbH, Mendelstraße 17, 48149 Münster, Germany
2
AbbVie Inc., Irvine, CA 92612, USA
3
Praxis Dr. Pajouh, 22941 Bargteheide, Germany
4
Mediteknia Clinic, 35010 Las Palmas de Gran Canaria, Spain
5
Ciencias de la Salud, University Fernando Pessoa Canarias, 35010 Las Palmas de Gran Canaria, Spain
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2026, 27(4), 1815; https://doi.org/10.3390/ijms27041815
Submission received: 2 October 2025 / Revised: 20 January 2026 / Accepted: 26 January 2026 / Published: 13 February 2026
(This article belongs to the Section Molecular Biology)
Browse Figures
Versions Notes

Abstract

Androgen receptor (AR) signaling plays a key role in male pattern baldness. We investigated whether targeting Dickkopf 1 (DKK1) and Secreted frizzled-related protein 1 (SFRP1), two AR-regulated genes, offers a novel therapeutic strategy for hair loss. AR expression was validated in freshly frozen human scalp hair follicles (HFs). AR knockdown was induced in human HFs using AR spherical nucleic acid (SNA). DKK1 and SFRP1 siRNA treatment were performed in HEK293 cells, human dermal papilla cells (hDPC), and human HFs ex vivo. Functional effects of single and combined DKK1 and SFRP1 knockdown were analyzed in human HFs ex vivo by quantitative (immuno)histomorphology. AR knockdown decreased SFRP1 and DKK1 expression. We found reciprocal mRNA upregulation between DKK1 and SFRP1 following their siRNA knockdown in HEK293 and hDPC. We therefore applied a single and combined treatment of DKK1 and SFRP1 siRNA in HFs ex vivo. SFRP1 knockdown prolonged anagen, increased hair matrix keratinocyte proliferation, reduced apoptosis, and increased DKK1 levels in HFs ex vivo, whereas DKK1 knockdown had no effect, and combined knockdown did not enhance SFRP1’s benefits. The culture-dependent compensatory regulation of SFRP1 and DKK1 underscores Wnt-signaling complexity in hair growth and strengthens the rationale for SFRP1 based therapies in anagen maintenance and hair loss.

1. Introduction

Male pattern hair loss (MPHL), also known as male pattern androgenetic alopecia (mpAGA), is the most prevalent type of hair loss in men [1], affecting approximately 80% of individuals by the age of 70 [2,3]. The human hair cycle typically consists of three phases: anagen (growth), catagen (regression), and telogen (rest). In healthy individuals up to 90% of the scalp hair follicles (HFs) are in the anagen phase, during which they produce thick, pigmented terminal hair shafts [4]. In contrast, in HFs of mpAGA-affected scalp regions the anagen phase is significantly shortened and the telogen phase is prolonged, resulting in an increased lag period before the generation of a new hair shaft [5,6,7,8]. Furthermore, mpAGA-affected HFs undergo a miniaturization process, during which terminal HFs progressively develop into intermediate and/or vellus HFs that form thinner and less pigmented hair shafts [9,10]. Thus, therapeutic strategies to address mpAGA-affected HFs should prolong anagen/delay catagen, promote the telogen-to-anagen transition, or prevent HF miniaturization [11].
Excessive AR activation caused by elevated local concentrations of dihydrotestosterone (DHT) in human dermal papilla cells (hDPC) of androgen-sensitive HFs is one of the key mechanisms underlying mpAGA pathophysiology [5,12,13,14,15,16]. Accumulation of DHT is driven by the increased activity of 5α-reductase which catalyzes NADH-mediated double bond reduction of free testosterone [17,18,19]. In addition to direct production within HFs, androgens, including DHT, can also be synthesized locally in the skin indicating the HF could also be subjected to this external androgens present in the skin micromilieu [20]. However, their specific contribution in mpAGA has yet to be fully clarified. The 5α-reductase inhibitor Finasteride is one of the two FDA-approved drugs for mpAGA [2,5]. While this drug prevents disease progression, adverse effects are observed in about 2% of patients that can persist even after therapy discontinuation [21,22,23]. Thus, alternative strategies are currently being evaluated to target this pathway [24].
Increasing evidence indicates that AR activation modulates Wnt/β-catenin signaling, a key pathway involved in HF regeneration, growth, and anagen phase duration in healthy and mpAGA-affected human scalp HFs [25,26,27]. Indeed, AR activation downregulates β-catenin expression and activity in murine adult skin [28] and upregulates the Wnt/β-catenin pathway antagonist Dickkopf 1 (DKK1) in balding hDPCs [29]. Furthermore, activated AR signaling in hDPCs from mpAGA patients induces expression of the Wnt antagonist GSK-3β, resulting in reduced epithelial HF stem cell differentiation [30]. In line with these observations, AR stimulation in an androgen-responsive hDPC led to a decreased WNT5 and WNT10b expression and an increased DKK1 expression [31]. Moreover, cyclosporine, a drug promoting hair regrowth and hypertrichosis [32], promotes anagen maintenance also via suppression of the Wnt inhibitor Secreted frizzled-related protein 1 (SFRP1) [33]. Modulation of Wnt signaling by SFRP1 [34], which is expressed specifically in the dermal papilla (DP) [33], to promote hair regrowth has also been targeted with non-drug compounds [35]. Thus, AR- and Wnt signaling, and their interplay, play crucial roles in hair cycle regulation, highlighting their importance as therapeutic targets for the management of mpAGA.
Currently, RNA interference (RNAi)-based therapies, which can be delivered locally [36], are being developed for cosmeceutical and pharmacological applications, showing clinical benefit in several dermatological indications [37]. A DKK1-targeting siRNA, enhancing Wnt pathway activation, has recently been shown to significantly improve hair regeneration in mice in vivo [38], and two further RNAi-based therapeutics targeting the AR are currently being developed, namely asymmetric small interfering RNA (asiRNA) and self-assembled micelle inhibitory RNA (SAMiRNA). The asiRNA-based AR-targeting approach demonstrated effective gene knockdown in a murine mpAGA model in vivo as well as in human HFs ex vivo. AR knockdown by asiRNA induced hair growth promotion in mice, as well as anagen maintenance and an increase in hair bulb size in human HFs ex vivo [39]. The SAMiRNA was interrogated in a small 60-subject double-blind clinical study and although a positive efficacy signal was identified, the effect was not significant [40]. Thus, to achieve more satisfactory clinical results, RNAi-based approaches for promoting hair growth in mpAGA patients require further research and development.
Here, we investigated whether combinational strategies may be more effective in modulating hair growth. Specifically, by silencing AR in human HFs ex vivo, we identified SFRP1 as a Wnt pathway inhibitor, whose expression is directly modulated by AR signaling. Silencing DKK1 or SFRP1 in HEK293 or hDPCs in vitro resulted in the reciprocal up-regulation of the other molecule, depending on the cell type. Consequently, we applied both single and combined DKK1 and SFRP1 siRNA treatments to human HFs ex vivo and observed an increase in DKK1 transcript abundance upon SFRP1 knockdown. Finally, we investigated the effects of the dual silencing of DKK1 and SFRP1 on healthy human HF function ex vivo, without additional AR stimulation. We found that only SFRP1 siRNA treatment effectively prolonged anagen in HFs ex vivo, whereas DKK1 siRNA and combined DKK1 and SFRP1 siRNA treatments had no functional effects. These findings underscore the importance of fine-tuning targeted approaches to modulate Wnt signaling to achieve optimal benefits for hair growth in both healthy individuals and mpAGA patients.

2. Results

2.1. AR Knockdown Effectively Reduces mRNA Expression of the Wnt Pathway Modulators SFRP1 and DKK1 in Human HFs Ex Vivo

AR mRNA expression has been frequently reported for DP cells, whereas AR protein expression in other HF compartments, including the outer root sheath (ORS), is still controversial [41]. Thus, to be able to effectively evaluate AR silencing in our HF experiments, we initially investigated the localization of AR protein in organ cultured healthy HFs by in situ hybridization (ISH) and immunostaining after ex vivo culture for 24 h. Overall, low levels of AR mRNA were detected but a positive signal was seen mainly in the DP, DP stalk (DPst), and dermal cup (DC), and to a lesser extent in germinative and pre-cortical hair matrices (gHM and pcHM), whilst almost no signal was detected in the proximal ORS (Figure 1a). Qualitative immunofluorescence analysis demonstrated AR protein expression in the nucleus of DP fibroblasts from human HFs, obtained from three independent donors (Figure 1b), while AR protein levels were low in the proximal ORS, gHM and pcHM (Figure 1b). Thus, the AR is mainly expressed in the DP, and to a lesser extent also in the ORS and HM of human healthy HFs.
AR knockdown was validated by qPCR after application of 10 µM, 25 µM or 50 µM AR SNA or control SNA for six days, in the absence of additional testosterone or DHT stimulation, resulting in a significant reduction of more than 50% in AR mRNA levels when all tested concentrations were pooled (Figure 2a). Given the comparable results across the tested doses, all further experiments were performed with the lowest concentration of AR SNA (10 µM). Quantitative immunofluorescence confirmed the significant reduced AR expression in the DP and ORS, as well as a trend toward a lower percentage of AR+ cells in the DP (Figure 2b,c). Functionally, AR silencing in the absence of testosterone or DHT resulted in reduced mRNA levels of the Wnt inhibitor DKK1 (Figure 2d), confirming an AR-dependent modulation of Wnt signaling in human HFs ex vivo. In addition, lower mRNA expression of SFRP1 was also detected after AR silencing in human HFs (Figure 2d).
After confirming this, we proceeded to silence AR expression in human HFs ex vivo using spherical nucleic acids (SNAs), which consist of a nanoparticle core densely coated with nucleic acids, serving as an effective tool for delivering nucleic acids into cells [42].
Thus, our investigation confirmed that DKK1 expression is inhibited by AR also in human HFs ex vivo and identified SFRP1 as an additional AR target gene.

2.2. Downregulation of SFRP1 or DKK1 Following siRNA Treatment Induces Reciprocal Gene Expression in HEK293 and HDPCs In Vitro

We hypothesized that RNAi-mediated suppression of the Wnt pathway inhibitors DKK1 and SFRP1, resulting in disinhibition/activation of the Wnt signaling pathway, would represent a clinically safer strategy compared to the upregulation of a Wnt activator such as LEF1, which would directly activate the Wnt pathway. Therefore, we first confirmed the spatial expression patterns of SFRP1 and DKK1 in human HFs ex vivo. SFRP1 expression was in line with previously published results [32], with high SFRP1 mRNA levels detected in the DP, DPst, and DC and lower levels present in the HM, ORS, and pre-cortex of healthy HFs ex vivo (Figure S1a). SFRP1 protein expression was predominantly found in the DP, gHM and pcHM, but also in the ORS of three independent donors (Figure S1b). DKK1 mRNA was mainly detected in the CTS and distal ORS, with no transcripts being present in the DP of HFs ex vivo (Figure S1c). Analysis of DKK1 protein was consistent with the mRNA findings, as it was primarily detected in the CTS and distal ORS. However, low levels of DKK1 protein were also present in the DC, but not the DP of HFs of three independent donors (Figure S1d). These findings show different expression patterns for the two Wnt target genes DKK1 and SFRP1 in healthy human HFs ex vivo.
Next, we validated the knockdown efficacy of DKK1 and SFRP1 by siRNA in vitro in HEK293 cells and hDPCs. Treatment with DKK1 and SFRP1 siRNA reduced the respective mRNA levels to 68% and 41% in HEK293 cells and 20% and 0% in hDPCs, relative to treatment with scrambled siRNA (Figure 3a,b). Interestingly, downregulation of DKK1 caused an upregulation of SFRP1 mRNA in HEK293 cells (Figure 3a), whereas in hDPCs, downregulation of SFRP1 induced enhanced DKK1 mRNA expression (Figure 3b).
Thus, these data indicate a differential interplay between DKK1 and SFRP1 across various cell types and suggest that dual targeting of these molecules may produce synergistic effects.

2.3. Knockdown of SFRP1 Induces Upregulation of DKK1 mRNA and Protein in Healthy Human HFs Ex Vivo

Considering the different expression patterns of DKK1 and SFRP1 between distinct HF compartments, and their opposed compensatory regulation in two cell systems in vitro, we next investigated whether the simultaneous knockdown of DKK1 and SFRP1 might hold potential for additive or even synergetic effects on HF function. Sole applications of the respective siRNA as well as their combined application resulted in reduced DKK1 and SFRP1 mRNA levels. Consistent with and validating our results obtained in hDPCs (Figure 3b), DKK1 mRNA was upregulated by SFRP1 siRNA treatment, whereas there was no reciprocal effect of DKK1 knockdown on SFRP1 mRNA (Figure 3c). To confirm these findings at the protein level, ELISA was performed on the HF supernatant obtained at the end of the HF culture. Secretion of DKK1 and SFRP1 protein was significantly reduced by the respective siRNA treatment in male and female HFs (Figure 3d). Combined siRNA treatment induced a trend toward reduced DKK1 levels, and significantly decreased SFRP1 release into the medium, when compared to scrambled siRNA control (Figure 3d). However, SFRP1 siRNA treatment resulted in increased DKK1 protein secretion at the end of the HF cultures, and DKK1 siRNA significantly reduced SFRP1 protein release (Figure 3d). Analysis of the Wnt target genes LEF1 and AXIN2 following single and combined DKK1 and SFRP1 siRNA treatments revealed that LEF1 mRNA expression tended to increase with single SFRP1 siRNA treatment and with combined SFRP1 and DKK1 siRNA treatment, whereas AXIN2 expression was only increased by DKK1 siRNA treatment in one out of three donors (Figure S2). This differential regulation of two additional Wnt target genes following SFRP1 and DKK1 knockdown further underscores the complex interplay of Wnt signaling in human HFs ex vivo.
These findings provide additional evidence of a delicate and complex interaction in the regulation of SFRP1 and DKK1 expression at the transcriptional and post-transcriptional levels.

2.4. Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair-Growth-Promoting Effect of SFRP1 in Healthy Human Hair Follicles Ex Vivo

Finally, we investigated the consequences of DKK1 and SFRP1 knockdown on HF function ex vivo. Neither single treatment with DKK1 or SFRP1 siRNA, nor their combined treatment, affected hair shaft elongation (Figure 4a). While DKK1 siRNA had only a minor positive effect on anagen prolongation, SFRP1 siRNA effectively increased the number of female and male HFs in anagen, which was also accompanied by a decreased hair cycle score, significantly increased hair matrix keratinocyte proliferation, and a trend towards a reduced apoptosis (Figure 4b–f). Although a modest anagen-prolonging effect was observed, HFs treated with dual silencing of DKK1 and SFRP1 exhibited a higher number of catagen-phase follicles compared to those treated with scramble control (Figure 4c), which is also reflected by a slight increase in the hair cycle score (Figure 4d).
Taken together, these data reveal that dual targeting of two, apparently synergetic, Wnt modulators does not result in an enhanced beneficial effect on hair growth promotion ex vivo. In addition, SFRP1 is further confirmed as an interesting, and more therapeutically compelling target for anagen maintenance.

3. Discussion

Elevated AR signaling is a molecular driver of mpAGA and reduced AR signaling via Finasteride-mediated inhibition of 5-alpha reductase is one of the two FDA-approved treatments for mpAGA [2]. Direct AR targeting with pharmacological antagonists [2] has shown clinically meaningful impact on improving the appearance of patients. Yet, these therapeutics are associated with side effects [2], that could be avoided by developing non-systemic therapeutic strategies. To this purpose, AR silencing techniques have been tested, which did not yet result in the expected outcome [40]. While suboptimal delivery may have contributed to the lack of efficacy so far, further understanding of the downstream biology of AR signaling is required to develop alternative, more effective therapeutics for mpAGA.
In our study, we pursued this goal by identifying novel AR target genes, notably the Wnt inhibitor SFRP1, which may play a role in modulating hair growth, using an ex vivo HF culture system. We also examined the interplay between SFRP1 and DKK1, another AR-target gene known to modulate Wnt signaling, in both in vitro and ex vivo HF models. Lastly, we assessed whether dual targeting of these two AR target genes, in conjunction with Wnt signaling inhibitors, could potentiate their individual hair-growth–promoting effects in HF organ culture.
Our data demonstrates that AR silencing in human HFs ex vivo leads to decreased transcription of the Wnt modulators DKK1 and SFRP1. Interestingly, when one of these genes was silenced in vitro, a compensatory expression mechanism was observed, which led to the reciprocal up-regulation of the other molecules, in a cell-type dependent manner. Indeed, also in human HFs ex vivo, silencing of SFRP1 led to increased DKK1 expression. Additionally, knockdown of SFRP1 prolonged anagen and significantly increased hair matrix keratinocyte proliferation, while neither knockdown of DKK1 nor double knockdown of the two genes had any effect on the hair cycle or HF function. These findings indicate that, by targeting the compensatory expression mechanisms between DKK1 and SFRP1, the beneficial effects of SFRP1 on hair growth promotion are not further enhanced.
Based on those findings, along with the fact that AR activation mediates the suppression of Wnt/β-catenin signaling in mpAGA [26,28,29,30,31], the potential of targeting Wnt signaling as an alternative or in addition to targeting AR signaling for mpAGA treatment is becoming increasingly evident. This strategy is reinforced by genetic and transcriptional evidence linking Wnt signaling components to the condition [43,44]. Additionally, several Wnt signaling modulators are being investigated preclinically or are already marketed as hair growth promoting agents, including the SFRP1 antagonist WAY-316606 [32] and the cosmetic SFRP1 inhibitors Bioscalin or KY19382. KY19382 acts as a Wnt/β-catenin signaling activator via inhibition of the interaction between CXXC-type zinc finger protein 5 (CXXC5) and Dishevelled (Dsh) [45]. Moreover, several studies have demonstrated the efficiency of natural or synthetic Wnt-activating compounds as an adjuvant strategy against hair loss [46,47], further supporting the specific therapeutic targeting of Wnt signaling for mpAGA treatment, also facilitated in our study.
Therefore, we focused on the Wnt inhibitors DKK1 and SFRP1, which were also reduced upon AR knockdown. While DKK1 is a known AR target gene [29], this has not been described for SFRP1. Instead, SFRP1 is known to inhibit AR expression [12,27]. The clinical significance of DKK1 and SFRP1 in hair loss progression in mpAGA patients is supported by the findings that both genes are upregulated in mpAGA-affected hDPC [29,48,49,50], and that they trigger the anagen-to-catagen transition in human HFs [32,35,51]. Furthermore, DKK1 reduces the size of the HF bulb, which directly affects the width of the hair shaft [52]. Notably, the role of SFRP1 in hair growth has been extensively described in the literature, in human and murine models with its therapeutic inhibition shown to promote hair growth [32,34,53]. These studies underscore the relevance of targeting DKK1 and SFRP1 for hair growth promotion, and prompted us to assess whether their combined knockdown could accelerate the effect of SFRP1 inhibition alone
Silencing of DKK1 resulted in upregulation of SFRP1 in HEK293 cells, while silencing of SFRP1 resulted in upregulation of DKK1 in hDPCs and human HFs ex vivo. It is known that Wnt signaling can be up- or downregulated in response to the same stressor depending on the model system [54], which could explain the differences between HEK293 cells, hDPCs and human HFs ex vivo.
To our knowledge, no compensatory expression mechanism or direct reciprocal regulatory loop between DKK1 and SFRP1 has been previously reported that could explain the observed differences in expression patterns or on the distinct functional outcomes between the three treatments. However, SFRP1 inhibition enhances Wnt/β-catenin signaling and, therefore, it is plausible that Wnt antagonists such as DKK1 are transcriptionally induced as part of a compensatory negative feedback mechanism [55]. This would serve to buffer the increased Wnt activity resulting from SFRP1 loss and thereby contribute to the restoration of pathway homeostasis. Additionally, previous data have shown that SFRP1 knockdown also increased LEF1 expression, which, unlike DKK1, functions as an anagen-promoting factor [56]. In line with this, we found increased LEF1 mRNA expression following SFRP1 knockdown, and thus most likely also enhanced β-catenin–LEF/TCF transcriptional activity. This, in turn, could drive the upregulation of DKK1, and explain the observed prolongation of anagen. Furthermore, SFRPs also interact with Wnt-unrelated molecules that play a role in the hair cycle, including fibronectin, receptor activator for nuclear factor kappa B (NF-κB) ligand (RANKL), bone morphogenetic protein (BMP)/Tolloid and Unc5H3 [57]. In summary, our data suggest that SFRP1 may play a more diverse role than DKK1 in modulating the hair cycle, as reflected in our findings. Nevertheless, these interpretations require experimental confirmation. Particularly the pronounced effects of single SFRP1 knockdown on the hair cycle and hair matrix keratinocyte proliferation, in contrast to the minimal effects observed with single DKK1 knockdown or combined SFRP1/DKK1 knockdown, remain difficult to explain.
In human HFs ex vivo, only the knockdown of SFRP1 substantially prolonged anagen and significantly increased or reduced hair matrix proliferation or apoptosis, respectively. Although we hypothesized that the double knockdown of both genes would result in a more profound synergistic effect on HF function, it did not augment the observed effects and even reduced the positive effect of SFRP1 knockdown. One provocative explanation for these observations could be drawn from a recent article describing a role of SFRP proteins as Wnt ligand carriers or inhibitors, depending on their abundance relative to Wnt ligands [58]. Only at a high concentration of SFRPs, Wnt molecules become sequestered and thus unavailable for receptor binding. We can, therefore, hypothesize that knocking down SFRP1 results in an expression level that is sufficient for its role as a Wnt carrier, thereby further increasing Wnt pathway activity. This provides a plausible explanation for the enhanced ability of SFRP1 to promote anagen.
Contrary to SFRP1 silencing, the single knockdown of DKK1, as well as the combined knockdown of DKK1 and SFRP1 had only a minor effect on anagen maintenance and did not affect hair matrix keratinocyte proliferation or apoptosis. Of note, mouse data demonstrate that treatment with a DKK1-targeting siRNA during anagen induction significantly accelerated the hair cycle but did not prolong anagen [38]. Since DKK1 siRNA did not prolong anagen in our model either, it is possible that its knockdown does not maintain anagen but rather accelerates re-entry into the hair cycle. In line with this, Wnt/β-catenin signaling is a potent driver of the telogen-to-anagen transition [59].
Moreover, the discrepancy between results obtained from murine and human HFs could also be explained by the low expression of DKK1 in healthy human HFs ex vivo demonstrated here, suggesting that DKK1 plays a less significant role in clinically healthy anagen VI HFs from the occipital scalp region than in mpAGA-affected HFs, where its expression is upregulated [49,51,55]. This aligns with the fact that DKK1 is an androgen-responsive gene, but occipital HFs are androgen-independent [3,5].
We acknowledge several limitations in our study, including the small number of human donors and the lack of in-depth mechanistic investigations into how different Wnt modulators influence each other’s function and expression. Although this interaction is highly interesting and of broad biological relevance, it was beyond the scope of the present work. Additionally, we analyzed only clinically healthy occipital HFs, which may mechanistically differ from HFs affected by mpAGA. Thus, our data warrants further investigation, particularly in the context of mpAGA management and the development of RNAi-based therapeutic strategies for hair growth promotion. Additional research on the relationship of Wnt regulators in the context of androgen sensitive versus androgen insensitive human HFs is needed to advance our understanding of the molecular pathophysiology of mpAGA. Specifically, it remains to be determined whether the compensatory mechanism between DKK1 and SFRP1 reported here also occurs in fronto-temporal, androgen-sensitive mpAGA HFs.
In conclusion, our findings reveal intricate regulatory mechanisms governing AR and Wnt signaling in healthy human HFs. The insights from this study underscore the complexity behind therapeutic approaches targeting both AR, and Wnt signaling inhibition for anagen maintenance and hair growth promotion. This compensatory behavior between DKK1 and SFRP1 transcripts has, to our knowledge, not been described before and may have wider implications in biology. Furthermore, our results confirmed previous observations by underscoring the potential of SFRP1 as a therapeutic target for hair loss management.

4. Materials and Methods

4.1. Hair Follicle Donor Information

This study was conducted in accordance with the Declaration of Helsinki principles. Scalp anagen VI HFs were obtained from 12 healthy donors (Table 1) after informed written patient consent and Ethics Committee approval (Monasterium Laboratory Biobank approval 2019-297-f-S; study plan 2020-954-f-S, University of Münster 2015-602-f-S, and Comité de Bioética de la Universidad Fernando Pessoa Canarias (03 (22 June 2020))).

4.2. HEK293 Cell Culture and siRNA Treatment

HEK293 STF cells (CRL-3249™, ATCC, Manassas, VA, USA) were cultivated at 37 °C and 5% CO2 in assay medium comprised of 80% DMEM F12 Medium, (30-2006™, ATCC, Manassas, VA, USA) and, 20% FBS (ThermoFisher, Carlsbad, CA, USA) supplemented with 200 µg/mL G-418 (ThermoFisher, Carlsbad, CA, USA; 2 mL in 500 mL media). Then, 6.0 × 105 HEK293 cells/well were seeded in a 6-well plate and transfected with 25 nM DKK1 targeting, SFRP1 targeting and NTC siRNA oligos using Dharmafect 2 reagent (Horizon Discovery, Lafayette, CO, USA) as described by the manufacturer. The cells were incubated at 37 °C for 48 h. Then the cells were harvested to test the target gene mRNA expressions. All siRNAs were purchased from Dharmacon (DKK1 siRNA target: 5’-CUGUGAUUGCAGUAAAUUA-3’, SFRP1 siRNA target: 5’-AGAAGAUGGUGCUGCCCAA-3’, or NTC siRNA: 5’-UAGCGACUAAACACAUCAA-3’).

4.3. Dermal Papilla Fibroblast Culture and siRNA Treatment

Primary hDPCs and cell culture media were obtained from PromoCell (Heidelberg, Germany). In compliance with the Human Tissue Act (2004), all PromoCell cells were isolated with an explicit informed consent of the donors and/or their next-of-kin. Second to sixth passages of subcultures were seeded in a 6-well plate and then cultivated at 37 °C and 5% CO2. DharmaFECT 2 (Horizon Discovery, Lafayette, CO) was used to transfect cells, as per the manufacturer’s protocol (DharmaconTM). Briefly, 4 μL of DharmaFECT 2 was combined with 25 nM of siRNA in a volume of 400 μL Opti-mem media (GIBCO, Waltham, MA, USA) and incubated for 20 min. The complexes of DharmaFECT 2 and siRNAs were then added directly to each well with a total volume of 2 mL. The cells were incubated at 37 °C for 48 h. Then the cells were harvested to test the target gene mRNA expressions. All siRNAs were purchased from Dharmacon (DKK1 siRNA target: 5’-CUGUGAUUGCAGUAAAUUA-3’, SFRP1 siRNA target: 5’-AGAAGAUGGUGCUGCCCAA-3’, or NTC siRNA: 5’-UAGCGACUAAACACAUCAA-3’).

4.4. Hair Follicle Organ Culture and Treatment

Human amputated HFs were micro-dissected from follicular unit extraction (FUE) or from scalp skin samples obtained from facelift surgery (Table 1) as previously described [60,61,62]. In short, HFs were cultured in a minimal medium consisting of William’s E medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 2 mM of L-Glutamine (Gibco), 10 ng/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL insulin (Sigma-Aldrich), and 1% penicillin/streptomycin (Gibco) at 37 °C and 5% CO2 [63,64]. After quality control, anagen VI HFs were randomly assigned to the following experimental groups for up to 5–8 days of culture: (1) control spheric nucleic acid (SNA; 10, 25 and 50 µM) or AR SNA (10, 25 and 50 µM) or (2) scrambled/NTC (non-template control) siRNA (10 µM), DKK1 siRNA (10 µM), SFRP1 siRNA (10 µM) or DKK1 (10 µM) + SFRP1 (10 µM) siRNA. At the end of the culture, HFs were embedded in an OCT cryomatrix (Fisher Scientific, Waltham, MA, USA) and sectioned with a cryostat (Leica, Wetzlar, Germany), and consecutive 6-μm sections of each amputated HF were collected and stored at −80 °C. All siRNAs were purchased from Dharmacon (DKK1 siRNA target: 5’-CUGUGAUUGCAGUAAAUUA-3’, SFRP1 siRNA target: 5’-AGAAGAUGGUGCUGCCCAA-3’, or NTC siRNA: 5’-UAGCGACUAAACACAUCAA-3’).

4.5. Hair Follicle Elongation/Hair Shaft Production

To determine hair shaft growth, each HF was measured from the end of the connective tissue sheath (CTS) to the end of the distal ORS at days 0, 4, 5, 6, and 8 in culture with a digital light microscope at 50× magnification (VHX900; Keyence Corporation, Osaka, Japan), and affiliated software [60,61,62].

4.6. Immunohistochemistry, Immunofluorescence Microscopy, and Quantitative (Immu-No)histomorphometry

Characterization of AR, SFRP1, and DKK1 expression was performed on 6 µM cryosections, and AR and SFRP1 expression were evaluated by immunofluorescence. Sections were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.1–0.5% Triton X-100, and blocked in 10% normal goat serum (NGS) prior to overnight incubation at 4 °C with primary antibodies against AR (1:50; monoclonal rabbit-anti-AR; clone: ERP1535(2), abcam, Cambridge, UK) [41] or SFRP1 (1:200; polyclonal rabbit-anti-SFRP1, abcam) [32]. Secondary antibody incubation employed either a rhodamine-labeled goat anti-rabbit antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) or an Alexa Fluor 488-labeled goat anti-rabbit antibody (1:400, Invitrogen, Waltham, MA, USA) and was performed at room temperature (RT) for 45 min.
Immunohistochemistry was performed to detect DKK1 expression. Sections were fixed in 4% PFA and endogenous peroxidase activity was quenched by incubation with 3% H2O2 for 15 min at RT. To reduce background staining, sections were blocked with 10% NGS for 1 h at RT, followed by sequential incubation with avidin and biotin blocking solutions (15 min each). Sections were then incubated overnight at 4 °C with the primary antibody against DKK1 (1:200; monoclonal mouse anti-DKK1, clone 2 A5, Novus Biologicals). After washing, sections were incubated with a biotinylated goat anti-mouse secondary antibody (1:200, Jackson ImmunoResearch) for 45 min at RT. Antigen–antibody complexes were visualized using the Avidin–Biotin Complex–Peroxidase (VEC-PK-6100, Vector) for 30 min at RT, followed by development with the AEC substrate kit (SK-4200, Vector) according to the manufacturer’s instructions.
To stain apoptotic and proliferating cells, a Ki-67/TUNEL double staining was performed using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Burlington, MA, USA) combined with a Ki-67 staining, as described previously [60,62,65]. Briefly, after fixation with 4% PFA and pre-treatment with the ApopTag solutions for the TdT-Enzyme, cryosections were blocked with goat normal serum and incubated over night at 4 °C with a mouse anti-Ki-67 antibody (1:800 in PBS; Cell Signalling Technology, Danvers, MA, USA). Ki-67 antibody was detected with a goat anti-mouse IgG rhodamine secondary antibody (1:200; Jackson ImmunoResearch) and apoptotic cells were detected with a Fluorescent-labeled Anti-Digoxigenin antibody.
Counterstaining with DAPI was performed during every immunofluorescence staining protocol to visualize nuclei, while with Hematoxylin and Eosin staining during the histochemistry protocol according to routine histochemical methods. Images were taken using a Keyence fluorescence microscope (BZ9100; Osaka, Japan), maintaining a constant set exposure time throughout imaging for further analysis [35]. Staining immunoreactivity or the number of positive cells were counted in the corresponding reference areas depicted in the different figures. Analyses were carried out with ImageJ 1.54p software (National Institutes of Health, Bethesda, MD, USA).
For the histochemical visualization of melanin, the Masson–Fontana staining was performed as previously described [65,66,67].

4.7. Hair Cycle Staging and Scoring

Microscopic hair cycle staging was performed at the end of the culture based on morphology, Masson–Fontana histochemistry and Ki-67/TUNEL immunostaining, as previously described [60,61]. The hair cycle score was calculated using a standardized, arbitrary score (anagen = 100; catagen = 200; early catagen = 300; mid-catagen = 400) [68]. Thus, a lower score means that the HFs are more established in anagen, and a higher score means that the HFs are more progressed to catagen.

4.8. In Situ Hybridization (ISH)

ISH was performed with the RNAscope® multiplex Fluorescent Reagent Kit V2 following the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA, USA) using the following RNAscope™ Probes: Hs-AR-C2 (NM_000044.3, target region 5604–6660 bp), Hs-SFRP1-C2 (NM_003012.4, target region 401–1971 bp), Hs-DKK1-C3 (NM_012242.2, target region 229–1523 bp), PPIB (positive control; NM_000942.4, target region 139–989 bp), and DapB (negative control, EF191515, target region 414–862 bp) [69,70].

4.9. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

For HEK293 and DP qPCR experiments, RNA was purified from cell pellets using TRIzol (Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer’s suggested protocol. cDNA synthesis was performed with 400 ng total RNA according to the manufacturer’s suggested protocol (Superscript kit 11752–250, ThermoFisher, Carlsbad, CA, USA). qPCR reactions were set up according to the TaqMan gene expression master mix protocol (4369016, ThermoFisher, Carlsbad, CA) with 2 µL cDNA and 10 µL total reaction volume using TaqMan probes (ThermoFisher, Carlsbad, CA, USA) run on the QuantStudio 12K Flex Real Time PCR System (Thermo Fisher, Carlsbad, CA, USA) using standard TaqMan settings in a 384-well format. For HFs, total RNA was extracted from 2–3 amputated HFs using the Arcturus® PicoPure® RNA Extraction Kit following the manufacturer’s instructions (Thermo Fisher), then RNA purity and concentration were determined using the Nanodrop ND-1000 assay (Fisher Scientific). For the qPCR protocol, 200–300 ng of mRNA was reverse-transcribed to cDNA using the Tetro cDNA synthesis kit (Bioline, Heidelberg, Germany) according to the manufacturer’s protocols. Quantitative PCR was run in triplicate using TaqMan Fast Advanced Master Mix Product Insert and gene Expression Assay transcripts on the Quantstudio3 Real-Time PCR system, plus associated software (Applied Biosystems; [69]). The following gene TaqMan Expression Assay probes were used: Id: Hs00171172_m1 (AR), Hs00610060_m1 (SFRP1), HS00183740_m1 (DKK1), Hs99999905_m1 (GAPDH), and Hs99999907_m1 (B2M) (Applied Biosystem). The amount of the transcripts was normalized to those of the housekeeping gene (GAPDH or B2M) using the ΔΔCT method.

4.10. ELISA

Detection of secreted DKK1 and SFRP1 was measured on pooled culture medium (duplicate) collected at the end of the culture medium, using the Human Dkk-1 Quantikine ELISA kit (R&D; DKK100B) and the Human SFRP1 ELISA kit (Abcam; ab277082) respectively, following the manufacturer’s instructions. For the ELISAs, the culture medium was diluted in a 1:1 ratio in culture medium and absorbances were measured at 450 nm on a plate reader (GloMax® Discover System, Promega, Madison, WI, USA). The concentrations were determined based on internal standards. Standard curves were generated using linear regression in GraphPad Prism 9, and the unknown DKK1 or SFRP1 concentrations in the samples were interpolated accordingly.

4.11. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Normal distribution of the data was assessed using the D’Agostino and Pearson omnibus normality test. For normally distributed data, multiple group comparison was performed by one-way ANOVA, followed by Tukey’s multiple comparison test, while two-group comparisons were performed using an unpaired Student’s t-test. If the data did not follow a normal distribution, multiple group comparison was carried out using the Kruskal–Wallis test with Dunn’s multiple comparison test, and two-group comparisons were performed using the Mann–Whitney U-test. Data are presented as mean ± SEM. p-values < 0.05 were considered statistically significant.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041815/s1.

Author Contributions

Conceptualization, M.B., N.P., M.G., and J.E.; methodology, M.B., J.E., A.L.R., N.P., M.G., and Y.Y.; validation, A.L.R., D.B., J.E., M.G., and M.B.; formal analysis, A.L.R., D.B., and Y.Y.; investigation, A.L.R., D.B., Y.Y., and H.E.-B.; resources, F.J., H.E.; data curation, A.L.R., D.B., and Y.Y.; writing—original draft preparation, A.L.R., D.B., and M.B.; writing—review and editing, all authors; visualization, A.L.R. and D.B.; supervision, M.B. and N.P.; project administration, M.B.; funding acquisition, M.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee (Monasterium Laboratory Biobank approval 2019-297-f-S, study plan 2020-954-f-S, University of Münster 2015-602-f-S, and Comité de Bioética de la Universidad Fernando Pessoa Canarias (03, 22 June 2020)).

Informed Consent Statement

Informed written consent was obtained from all subjects involved in the study.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Kayumba Ng‘andu for technical support, Lisa Epping for editorial input, and Max van Lessen and Tamas Biro for scientific discussion and input.

Conflicts of Interest

Authors D.B., A.L.R., H.E.-B., J.E. and M.B. are employed by the company QIMA Life Sciences, QIMA Monasterium GmbH. Authors M.G. and N.P. are employed by the company AbbVie Inc., while Y.Y. was employed by the company AbbVie Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
mpAGAMale pattern androgenetic alopecia
ARAndrogen receptor
CTSConnective tissue sheath
DCDermal cup
DPDermal papilla
DPst
hDPC		Dermal papilla stalk
Human dermal papilla cells
DKK1Dickkopf 1
gHMGerminative hair matrix
pcHMPrecortical hair matrix
HFHair follicle
MPHLMale pattern hair loss
NTCNon-template control
ORSOuter root sheath
asiRNAAsymmetric small interfering RNA
SAMiRNASelf-assembled micelle inhibitory RNA
RNAiRNA interference
SFRP1Secreted frizzled related protein 1
SNASpherical nucleic acid

References

  1. Sinclair, R. Male pattern androgenetic alopecia. BMJ 1998, 317, 865–869. [Google Scholar] [CrossRef] [PubMed]
  2. Devjani, S.; Ezemma, O.; Kelley, K.J.; Stratton, E.; Senna, M. Androgenetic Alopecia: Therapy Update. Drugs 2023, 83, 701–715. [Google Scholar] [CrossRef] [PubMed]
  3. Redmond, L.C.; Limbu, S.; Farjo, B.; Messenger, A.G.; Higgins, C.A. Male pattern hair loss: Can developmental origins explain the pattern? Exp. Dermatol. 2023, 32, 1174–1181. [Google Scholar] [CrossRef] [PubMed]
  4. Schneider, M.R.; Schmidt-Ullrich, R.; Paus, R. The Hair Follicle as a Dynamic Miniorgan. Curr. Biol. 2009, 19, R132–R142. [Google Scholar] [CrossRef]
  5. Altendorf, S.; Bertolini, M.; Le Riche, A.; Tosti, A.; Paus, R. Frontiers in the physiology of male pattern androgenetic alopecia: Beyond the androgen horizon. Physiol. Rev. 2025, 106, 121–195. [Google Scholar] [CrossRef]
  6. Ellis, J.A.; Sinclair, R.; Harrap, S.B. Androgenetic alopecia: Pathogenesis and potential for therapy. Expert Rev. Mol. Med. 2002, 4, 1–11. [Google Scholar] [CrossRef]
  7. Guarrera, M.; Rebora, A. The Higher Number and Longer Duration of Kenogen Hairs Are the Main Cause of the Hair Rarefaction in Androgenetic Alopecia. Ski. Appendage Disord. 2018, 5, 152–154. [Google Scholar] [CrossRef]
  8. Kaufman, K.D. Androgens and alopecia. Mol. Cell. Endocrinol. 2002, 198, 89–95. [Google Scholar] [CrossRef]
  9. Gilhar, A.; Keren, A.; Paus, R. Vellus-to-terminal Hair Follicle Reconversion Occurs in Male Pattern Balding and is Promoted by Minoxidil and Platelet-rich Plasma: In Vivo Evidence from a New Humanized Mouse Model of Androgenetic Alopecia. Acta Dermato-Venereologica 2023, 103, adv12320. [Google Scholar] [CrossRef]
  10. Miranda, B.; Tobin, D.; Sharpe, D.; Randall, V. Intermediate hair follicles: A new more clinically relevant model for hair growth investigations. Br. J. Dermatol. 2010, 163, 287–295. [Google Scholar] [CrossRef]
  11. Vasserot, A.P.; Geyfman, M.; Poloso, N.J. Androgenetic alopecia: Combing the hair follicle signaling pathways for new therapeutic targets and more effective treatment options. Expert Opin. Ther. Targets 2019, 23, 755–771. [Google Scholar] [CrossRef]
  12. Ceruti, J.M.; Leirós, G.J.; Balañá, M.E. Androgens and androgen receptor action in skin and hair follicles. Mol. Cell. Endocrinol. 2018, 465, 122–133. [Google Scholar] [CrossRef]
  13. Hamilton, J.B. Patterned loss of hair in man: Types and incidence. Ann. New York Acad. Sci. 1951, 53, 708–728. [Google Scholar] [CrossRef] [PubMed]
  14. Itami, S.; Kurata, S.; Sonoda, T.; Takayasu, S. Characterization of 5α-Reductase in Cultured Human Dermal Papilla Cells from Beard and Occipital Scalp Hair. J. Investig. Dermatol. 1991, 96, 57–60. [Google Scholar] [CrossRef] [PubMed]
  15. Itami, S.; Kurata, S.; Takayasu, S. 5α-Reductase Activity in Cultured Human Dermal Papilla Cells from Beard Compared with Reticular Dermal Fibroblasts. J. Investig. Dermatol. 1990, 94, 150–152. [Google Scholar] [CrossRef] [PubMed]
  16. Trüeb, R.M. Molecular mechanisms of androgenetic alopecia. Exp. Gerontol. 2002, 37, 981–990. [Google Scholar] [CrossRef]
  17. Irwig, M.S. Testosterone therapy for transgender men. Lancet Diabetes Endocrinol. 2017, 5, 301–311, Correction in Lancet Diabetes Endocrinol. 2017, 5, e2. https://doi.org/10.1016/S2213-8587(16)30143-7. [Google Scholar] [CrossRef]
  18. Gu, Y.; Tang, G.T.; Cheung, A.S.; Sebaratnam, D.F. Dermatological considerations for transgender and gender diverse patients: An Australian perspective. Australas. J. Dermatol. 2023, 65, 24–36. [Google Scholar] [CrossRef]
  19. Taillefer, V.; Kelley, J.; Marsolais, S.; Chiniara, L.; Chadi, N. Expected vs. perceived effects of gender-affirming hormone therapy among transmasculine adolescents. J. Pediatr. Endocrinol. Metab. 2023, 36, 1072–1078. [Google Scholar] [CrossRef]
  20. Slominski, R.M.; Raman, C.; Jetten, A.M.; Slominski, A.T. Neuro–immuno–endocrinology of the skin: How environment regulates body homeostasis. Nat. Rev. Endocrinol. 2025, 21, 495–509, Correction in Nat. Rev. Endocrinol. 2025, 21, 513. https://doi.org/10.1038/s41574-025-01126-8.. [Google Scholar] [CrossRef]
  21. Andy, G.; John, M.; Mirna, S.; Rachita, D.; Michael, K.; Maja, K.; Aseem, S.; Zeljana, B. Controversies in the treatment of androgenetic alopecia: The history of finasteride. Dermatol. Ther. 2018, 32, e12647. [Google Scholar] [CrossRef]
  22. Cilio, S.; Tsampoukas, G.; Morgado, A.; Ramos, P.; Minhas, S. Post-finasteride syndrome—a true clinical entity? Int. J. Impot. Res. 2025, 37, 426–435. [Google Scholar] [CrossRef]
  23. Leliefeld, H.H.J.; Debruyne, F.M.J.; Reisman, Y. The post-finasteride syndrome: Possible etiological mechanisms and symptoms. Int. J. Impot. Res. 2023, 37, 414–421. [Google Scholar] [CrossRef] [PubMed]
  24. Gupta, A.K.; Talukder, M.; Williams, G. Emerging and traditional 5-α reductase inhibitors and androgen receptor antagonists for male androgenetic alopecia. Expert Opin. Emerg. Drugs 2024, 29, 251–261. [Google Scholar] [CrossRef] [PubMed]
  25. Bellani, D.; Patil, R.; Prabhughate, A.; Shahare, R.; Gold, M.; Kapoor, R.; Shome, D. Pathophysiological mechanisms of hair follicle regeneration and potential therapeutic strategies. Stem Cell Res. Ther. 2025, 16, 1–12. [Google Scholar] [CrossRef] [PubMed]
  26. Horton, C.; Liu, Y.; Wang, J.; Green, J.; Tsyporin, J.; Chen, B.; Wang, Z.A. Modulation of the canonical Wnt activity by androgen signaling in prostate epithelial basal stem cells. Stem Cell Rep. 2023, 18, 1355–1370. [Google Scholar] [CrossRef]
  27. Premanand, A.; Rajkumari, B.R. Androgen modulation of Wnt/β-catenin signaling in androgenetic alopecia. Arch. Dermatol. Res. 2018, 310, 391–399. [Google Scholar] [CrossRef]
  28. Kretzschmar, K.; Cottle, D.L.; Schweiger, P.J.; Watt, F.M. The Androgen Receptor Antagonizes Wnt/β-Catenin Signaling in Epidermal Stem Cells. J. Investig. Dermatol. 2015, 135, 2753–2763. [Google Scholar] [CrossRef]
  29. Kwack, M.H.; Sung, Y.K.; Chung, E.J.; Im, S.U.; Ahn, J.S.; Kim, M.K.; Kim, J.C. Dihydrotestosterone-Inducible Dickkopf 1 from Balding Dermal Papilla Cells Causes Apoptosis in Follicular Keratinocytes. J. Investig. Dermatol. 2008, 128, 262–269. [Google Scholar] [CrossRef]
  30. Leirós, G.; Attorresi, A.; Balañá, M. Hair follicle stem cell differentiation is inhibited through cross-talk between Wnt/β-catenin and androgen signalling in dermal papilla cells from patients with androgenetic alopecia. Br. J. Dermatol. 2012, 166, 1035–1042. [Google Scholar] [CrossRef]
  31. Leirós, G.J.; Ceruti, J.M.; Castellanos, M.L.; Kusinsky, A.G.; Balañá, M.E. Androgens modify Wnt agonists/antagonists expression balance in dermal papilla cells preventing hair follicle stem cell differentiation in androgenetic alopecia. Mol. Cell. Endocrinol. 2017, 439, 26–34. [Google Scholar] [CrossRef] [PubMed]
  32. Hawkshaw, N.J.; Haslam, I.S.; Ansell, D.M.; Shamalak, A.; Paus, R. Re-Evaluating Cyclosporine A as a Hair Growth–Promoting Agent in Human Scalp Hair Follicles. J. Investig. Dermatol. 2015, 135, 2129–2132. [Google Scholar] [CrossRef] [PubMed]
  33. Hawkshaw, N.J.; A Hardman, J.; Haslam, I.S.; Shahmalak, A.; Gilhar, A.; Lim, X.; Paus, R. Identifying novel strategies for treating human hair loss disorders: Cyclosporine A suppresses the Wnt inhibitor, SFRP1, in the dermal papilla of human scalp hair follicles. PLOS Biol. 2018, 16, e2003705. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, C.; Du, Y.; Lu, C.; Bi, L.; Ding, Y.; Fan, W. The role of SFRP1 in human dermal papilla cell growth and its potential molecular mechanisms as a target in regenerative therapy. Regen. Ther. 2024, 28, 161–168. [Google Scholar] [CrossRef]
  35. Bertolini, M.; Chéret, J.; Pinto, D.; Hawkshaw, N.; Ponce, L.; Erdmann, H.; Jimenez, F.; Funk, W.; Paus, R. A novel nondrug SFRP1 antagonist inhibits catagen development in human hair follicles ex vivo. Br. J. Dermatol. 2020, 184, 371–373. [Google Scholar] [CrossRef]
  36. Tang, Q.; Khvorova, A. RNAi-based drug design: Considerations and future directions. Nat. Rev. Drug Discov. 2024, 23, 341–364. [Google Scholar] [CrossRef]
  37. Chang, Y.-T.; Huang, T.-H.; Alalaiwe, A.; Hwang, E.; Fang, J.-Y. Small interfering RNA-based nanotherapeutics for treating skin-related diseases. Expert Opin. Drug Deliv. 2023, 20, 757–772. [Google Scholar] [CrossRef]
  38. Papukashvili, D.; Liu, C.; Rcheulishvili, N.; Xie, F.; Wang, X.; Feng, S.; Sun, X.; Zhang, C.; Li, Y.; He, Y.; et al. DKK1-targeting cholesterol-modified siRNA implication in hair growth regulation. Biochem. Biophys. Res. Commun. 2023, 668, 55–61. [Google Scholar] [CrossRef]
  39. Moon, I.J.; Yoon, H.K.; Kim, D.; Choi, M.E.; Han, S.H.; Park, J.H.; Hong, S.W.; Cho, H.; Lee, D.-K.; Won, C.H. Efficacy of Asymmetric siRNA Targeting Androgen Receptors for the Treatment of Androgenetic Alopecia. Mol. Pharm. 2022, 20, 128–135. [Google Scholar] [CrossRef]
  40. Yun, S.-I.; Lee, S.-K.; Goh, E.-A.; Kwon, O.S.; Choi, W.; Kim, J.; Lee, M.S.; Choi, S.J.; Lim, S.S.; Moon, T.K.; et al. Weekly treatment with SAMiRNA targeting the androgen receptor ameliorates androgenetic alopecia. Sci. Rep. 2022, 12, 1–15, Correction in Sci. Rep. 2022, 12, 5675. https://doi.org/10.1038/s41598-022-10024-2.. [Google Scholar] [CrossRef]
  41. Miranda, B.H.; Charlesworth, M.R.; Tobin, D.J.; Sharpe, D.T.; Randall, V.A. Androgens trigger different growth responses in genetically identical human hair follicles in organ culture that reflect their epigenetic diversity in life. FASEB J. 2018, 32, 795–806. [Google Scholar] [CrossRef] [PubMed]
  42. Kapadia, C.H.; Melamed, J.R.; Day, E.S. Spherical Nucleic Acid Nanoparticles: Therapeutic Potential. BioDrugs 2018, 32, 297–309. [Google Scholar] [CrossRef] [PubMed]
  43. Ober-Reynolds, B.; Wang, C.; Ko, J.M.; Rios, E.J.; Aasi, S.Z.; Davis, M.M.; Oro, A.E.; Greenleaf, W.J. Integrated single-cell chromatin and transcriptomic analyses of human scalp identify gene-regulatory programs and critical cell types for hair and skin diseases. Nat. Genet. 2023, 55, 1288–1300. [Google Scholar] [CrossRef] [PubMed]
  44. Pirastu, N.; Joshi, P.K.; de Vries, P.S.; Cornelis, M.C.; McKeigue, P.M.; Keum, N.; Franceschini, N.; Colombo, M.; Giovannucci, E.L.; Spiliopoulou, A.; et al. GWAS for male-pattern baldness identifies 71 susceptibility loci explaining 38% of the risk. Nat. Commun. 2017, 8, 1–10, Correction in Nat. Commun. 2018, 9, 2536. https://doi.org/10.1038/s41467-018-04857-7.. [Google Scholar] [CrossRef]
  45. Ryu, Y.C.; Lee, D.; Shim, J.; Park, J.; Kim, Y.; Choi, S.; Bak, S.S.; Sung, Y.K.; Lee, S.; Choi, K. KY19382, a novel activator of Wnt/β-catenin signalling, promotes hair regrowth and hair follicle neogenesis. Br. J. Pharmacol. 2021, 178, 2533–2546. [Google Scholar] [CrossRef]
  46. Choi, B.Y. Targeting Wnt/β-Catenin Pathway for Developing Therapies for Hair Loss. Int. J. Mol. Sci. 2020, 21, 4915. [Google Scholar] [CrossRef]
  47. Shin, D.W. The Molecular Mechanism of Natural Products Activating Wnt/β-Catenin Signaling Pathway for Improving Hair Loss. Life 2022, 12, 1856. [Google Scholar] [CrossRef]
  48. Fawzi, M.M.T.; Mahmoud, S.B.; Shaker, O.G.; A Saleh, M. Assessment of tissue levels of dickkopf-1 in androgenetic alopecia and alopecia areata. J. Cosmet. Dermatol. 2015, 15, 10–15. [Google Scholar] [CrossRef]
  49. Mahmoud, E.A.; Elgarhy, L.H.; Hasby, E.A.; Mohammad, L. Dickkopf-1 Expression in Androgenetic Alopecia and Alopecia Areata in Male Patients. Am. J. Dermatopathol. 2019, 41, 122–127. [Google Scholar] [CrossRef]
  50. Zhou, L.-B.; Cao, Q.; Ding, Q.; Sun, W.-L.; Li, Z.-Y.; Zhao, M.; Lin, X.-W.; Zhou, G.-P.; Fan, W.-X. Transcription factor FOXC1 positively regulates SFRP1 expression in androgenetic alopecia. Exp. Cell Res. 2021, 404, 112618. [Google Scholar] [CrossRef]
  51. Kwack, M.H.; Kim, M.K.; Kim, J.C.; Sung, Y.K. Dickkopf 1 Promotes Regression of Hair Follicles. J. Investig. Dermatol. 2012, 132, 1554–1560. [Google Scholar] [CrossRef] [PubMed]
  52. Lei, M.; Guo, H.; Qiu, W.; Lai, X.; Yang, T.; Widelitz, R.B.; Chuong, C.; Lian, X.; Yang, L. Modulating hair follicle size with Wnt10b/DKK1 during hair regeneration. Exp. Dermatol. 2014, 23, 407–413. [Google Scholar] [CrossRef] [PubMed]
  53. Sunkara, R.R.; Mehta, D.; Sarate, R.M.; Waghmare, S.K. BMP-AKT-GSK3β Signaling Restores Hair Follicle Stem Cells Decrease Associated with Loss of Sfrp1. Stem Cells 2022, 40, 802–817. [Google Scholar] [CrossRef] [PubMed]
  54. Sambo, D.; Gohel, C.; Yuan, Q.; Sukumar, G.; Alba, C.; Dalgard, C.L.; Goldman, D. Cell type-specific changes in Wnt signaling and neuronal differentiation in the developing mouse cortex after prenatal alcohol exposure during neurogenesis. Front. Cell Dev. Biol. 2022, 10, 1011974. [Google Scholar] [CrossRef]
  55. Papukashvili, D.; Rcheulishvili, N.; Liu, C.; Xie, F.; Tyagi, D.; He, Y.; Wang, P.G. Perspectives on miRNAs Targeting DKK1 for Developing Hair Regeneration Therapy. Cells 2021, 10, 2957. [Google Scholar] [CrossRef]
  56. DasGupta, R.; Fuchs, E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 1999, 126, 4557–4568. [Google Scholar] [CrossRef]
  57. Bovolenta, P.; Esteve, P.; Ruiz, J.M.; Cisneros, E.; Lopez-Rios, J. Beyond Wnt inhibition: New functions of secreted Frizzled-related proteins in development and disease. J. Cell Sci. 2008, 121, 737–746. [Google Scholar] [CrossRef]
  58. Magalhaes, T.d.A.; Liu, J.; Chan, C.; Borges, K.S.; Zhang, J.; Kane, A.J.; Wierbowski, B.M.; Ge, Y.; Liu, Z.; Mannam, P.; et al. Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens. Dev. Cell 2023, 59, 244–261.e6. [Google Scholar] [CrossRef]
  59. Hawkshaw, N.; Hardman, J.; Alam, M.; Jimenez, F.; Paus, R. Deciphering the molecular morphology of the human hair cycle: Wnt signalling during the telogen–anagen transformation. Br. J. Dermatol. 2019, 182, 1184–1193. [Google Scholar] [CrossRef]
  60. Langan, E.A.; Philpott, M.P.; Kloepper, J.E.; Paus, R. Human hair follicle organ culture: Theory, application and perspectives. Exp. Dermatol. 2015, 24, 903–911. [Google Scholar] [CrossRef]
  61. Edelkamp, J.; Gherardini, J.; Bertolini, M. Methods to Study Human Hair Follicle Growth Ex Vivo: Human Microdissected Hair Follicle and Human Full Thickness Skin Organ Culture. In Molecular Dermatology; Botchkareva, N.V., Westgate, G.E., Eds.; Methods in Molecular Biology; Springer: New York, NY, USA, 2020; Volume 2154, pp. 105–119. ISBN 978-1-0716-0647-6. [Google Scholar]
  62. Campiche, R.; Le Riche, A.; Edelkamp, J.; Botello, A.F.; Martin, E.; Gempeler, M.; Bertolini, M. An extract of Leontopodium alpinum inhibits catagen development ex vivo and increases hair density in vivo. Int. J. Cosmet. Sci. 2022, 44, 363–376. [Google Scholar] [CrossRef]
  63. Philpott, M.P.; Sanders, D.; Westgate, G.E.; Kealey, T. Human hair growth in vitro: A model for the study of hair follicle biology. J. Dermatol. Sci. 1994, 7, S55–S72. [Google Scholar] [CrossRef]
  64. Philpott, M.P.; Green, M.R.; Kealey, T. Human hair growth in vitro. J. Cell Sci. 1990, 97, 463–471. [Google Scholar] [CrossRef]
  65. Bertolini, M.; Gherardini, J.; Chéret, J.; Alam, M.; Sulk, M.; Botchkareva, N.V.; Biro, T.; Funk, W.; Grieshaber, F.; Paus, R. Mechanical epilation exerts complex biological effects on human hair follicles and perifollicular skin: An ex vivo study approach. Int. J. Cosmet. Sci. 2023, 46, 175–198. [Google Scholar] [CrossRef] [PubMed]
  66. Chéret, J.; Gherardini, J.; Bertolini, M.; Paus, R. Fluoxetine promotes human hair follicle pigmentation ex vivo: Serotonin reuptake inhibition as a new antigreying strategy? Br. J. Dermatol. 2019, 182, 1492–1494. [Google Scholar] [CrossRef] [PubMed]
  67. Gherardini, J.; Wegner, J.; Chéret, J.; Ghatak, S.; Lehmann, J.; Alam, M.; Jimenez, F.; Funk, W.; Böhm, M.; Botchkareva, N.V.; et al. Transepidermal UV radiation of scalp skin ex vivo induces hair follicle damage that is alleviated by the topical treatment with caffeine. Int. J. Cosmet. Sci. 2019, 41, 164–182. [Google Scholar] [CrossRef] [PubMed]
  68. Kloepper, J.E.; Sugawara, K.; Al-Nuaimi, Y.; Gáspár, E.; Van Beek, N.; Paus, R. Methods in hair research: How to objectively distinguish between anagen and catagen in human hair follicle organ culture. Exp. Dermatol. 2010, 19, 305–312. [Google Scholar] [CrossRef]
  69. Edelkamp, J.; Lousada, M.B.; Pinto, D.; Chéret, J.; Calabrese, F.M.; Jiménez, F.; Erdmann, H.; Wessel, J.; Phillip, B.; De Angelis, M.; et al. Management of the human hair follicle microbiome by a synthetic odorant. J. Dermatol. Sci. 2023, 112, 99–108. [Google Scholar] [CrossRef]
  70. Lousada, M.B.; Edelkamp, J.; Lachnit, T.; Fehrholz, M.; Jimenez, F.; Paus, R. Laser capture microdissection as a method for investigating the human hair follicle microbiome reveals region-specific differences in the bacteriome profile. BMC Res. Notes 2023, 16, 1–6. [Google Scholar] [CrossRef]
Ijms 27 01815 g001
Figure 1. Characterization of androgen receptor (AR) expression in healthy human HFs ex vivo. (a) Representative images showing AR mRNA expression in a healthy human HF from n = 1 donor after 24 h of ex vivo culture, visualized by ISH. Qualitative observation was performed in n = 3 independent donors. White arrows indicate AR transcript expression. PPIB and DapB serve as positive and negative controls, respectively. Scale bars = 50 µM. (b) Representative images showing AR protein expression in anagen VI HFs of n = 3 independent donors after 24 h of ex vivo culture, visualized by immunofluorescence staining. Scale bars = 100 µM and 50 µM, in the original images and zoom-ins, respectively. AR: androgen receptor, DP: dermal papilla, DPst: dermal papilla stalk, DC: dermal cup, gHM: germinative hair matrix, HF: hair follicle, ORS: outer root sheath, pcHM: pre-cortical hair matrix.
Figure 1. Characterization of androgen receptor (AR) expression in healthy human HFs ex vivo. (a) Representative images showing AR mRNA expression in a healthy human HF from n = 1 donor after 24 h of ex vivo culture, visualized by ISH. Qualitative observation was performed in n = 3 independent donors. White arrows indicate AR transcript expression. PPIB and DapB serve as positive and negative controls, respectively. Scale bars = 50 µM. (b) Representative images showing AR protein expression in anagen VI HFs of n = 3 independent donors after 24 h of ex vivo culture, visualized by immunofluorescence staining. Scale bars = 100 µM and 50 µM, in the original images and zoom-ins, respectively. AR: androgen receptor, DP: dermal papilla, DPst: dermal papilla stalk, DC: dermal cup, gHM: germinative hair matrix, HF: hair follicle, ORS: outer root sheath, pcHM: pre-cortical hair matrix.
Ijms 27 01815 g001
Ijms 27 01815 g002
Figure 2. AR SNA efficiently downregulates AR mRNA and protein, as well as AR target gene expression in healthy human HFs. (a) Pooled quantification of AR mRNA expression assessed by qRT-PCR in healthy human HFs treated with 10 µM, 25 µM or 50 µM control or 10 µM, 25 µM or 50 µM AR SNA for 6 days, n = 4 independent donors. (b,c) Quantification (b) and representative images (c) of AR protein expression assessed by immunofluorescence staining in healthy human VI HFs treated with 10 µM control or 10 µM AR SNA for 6 days, in n = 7–8 HFs from 2 independent donors. Scale bars = 50 µM, in the original images and 25 µM in the zoom-ins. (d) Quantification of DKK1 and SFRP1 mRNA expression assessed by qRT-PCR in healthy human HFs treated with 10 µM control or 10 µM AR SNA for 6 days, n = 3 technical replicates from 1 donor. Dots of different shapes represent different donors. All data are presented as mean ± SEM. D’Agostino & Pearson normality test followed by Mann–Whitney test, * p < 0.05, ** p < 0.01. AR: androgen receptor, DKK1: dickkopf 1, DP: dermal papilla, DPst: dermal papilla stalk, DC: dermal cup, gHM: germinative hair matrix, HF: hair follicle, ORS: outer root sheath, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.
Figure 2. AR SNA efficiently downregulates AR mRNA and protein, as well as AR target gene expression in healthy human HFs. (a) Pooled quantification of AR mRNA expression assessed by qRT-PCR in healthy human HFs treated with 10 µM, 25 µM or 50 µM control or 10 µM, 25 µM or 50 µM AR SNA for 6 days, n = 4 independent donors. (b,c) Quantification (b) and representative images (c) of AR protein expression assessed by immunofluorescence staining in healthy human VI HFs treated with 10 µM control or 10 µM AR SNA for 6 days, in n = 7–8 HFs from 2 independent donors. Scale bars = 50 µM, in the original images and 25 µM in the zoom-ins. (d) Quantification of DKK1 and SFRP1 mRNA expression assessed by qRT-PCR in healthy human HFs treated with 10 µM control or 10 µM AR SNA for 6 days, n = 3 technical replicates from 1 donor. Dots of different shapes represent different donors. All data are presented as mean ± SEM. D’Agostino & Pearson normality test followed by Mann–Whitney test, * p < 0.05, ** p < 0.01. AR: androgen receptor, DKK1: dickkopf 1, DP: dermal papilla, DPst: dermal papilla stalk, DC: dermal cup, gHM: germinative hair matrix, HF: hair follicle, ORS: outer root sheath, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.
Ijms 27 01815 g002
Ijms 27 01815 g003
Figure 3. Reciprocal modulation of DKK1 and SFRP1 after siRNA in HEK293 cells and DP fibroblasts in vitro and healthy human HFs ex vivo. (a) Quantification of DKK1 and SFRP1 mRNA expression assessed by qRT-PCR in HEK293 cells treated with 25 nM DKK1 or SFRP1 siRNA or 25 nM non-template control (NTC) siRNA for 48 h, n = 3 independent experiments, (b) or in human DP fibroblasts treated with 25 nM NTC siRNA, DKK1 siRNA or SFRP1 siRNA for 48 h, n = 2 technical replicates from 1 donor. (c) Quantification of DKK1 and SFRP1 mRNA expression in healthy human HFs, treated with 10 µM NTC siRNA, DKK1 siRNA or SFRP1 siRNA for 6 days, n = 4 independent donors. (d) Quantification of DKK1 and SFRP1 secretion from healthy human HFs, treated with each 10 µM NTC siRNA, DKK1 siRNA or SFRP1 siRNA. Medium was collected after 4 days of culture and following termination of the culture. n = 4 independent donors. All data are presented as mean ± SEM. Kruskal–Wallis test followed by Dunn’s multiple comparison test, # p < 0.05, or Mann–Whitney test, * p < 0.05. Dots of different shapes indicate different donors (filled dots) or experiments (empty dots). Data points reflecting the female donor are indicated by circles in c and d. DKK1: dickkopf 1, DP: dermal papilla, NTC: non template control, SFRP1: secreted frizzled receptor 1.
Figure 3. Reciprocal modulation of DKK1 and SFRP1 after siRNA in HEK293 cells and DP fibroblasts in vitro and healthy human HFs ex vivo. (a) Quantification of DKK1 and SFRP1 mRNA expression assessed by qRT-PCR in HEK293 cells treated with 25 nM DKK1 or SFRP1 siRNA or 25 nM non-template control (NTC) siRNA for 48 h, n = 3 independent experiments, (b) or in human DP fibroblasts treated with 25 nM NTC siRNA, DKK1 siRNA or SFRP1 siRNA for 48 h, n = 2 technical replicates from 1 donor. (c) Quantification of DKK1 and SFRP1 mRNA expression in healthy human HFs, treated with 10 µM NTC siRNA, DKK1 siRNA or SFRP1 siRNA for 6 days, n = 4 independent donors. (d) Quantification of DKK1 and SFRP1 secretion from healthy human HFs, treated with each 10 µM NTC siRNA, DKK1 siRNA or SFRP1 siRNA. Medium was collected after 4 days of culture and following termination of the culture. n = 4 independent donors. All data are presented as mean ± SEM. Kruskal–Wallis test followed by Dunn’s multiple comparison test, # p < 0.05, or Mann–Whitney test, * p < 0.05. Dots of different shapes indicate different donors (filled dots) or experiments (empty dots). Data points reflecting the female donor are indicated by circles in c and d. DKK1: dickkopf 1, DP: dermal papilla, NTC: non template control, SFRP1: secreted frizzled receptor 1.
Ijms 27 01815 g003
Ijms 27 01815 g004
Figure 4. Silencing of SFRP1 prolongs anagen in healthy human HFs. Healthy human HFs were treated with 10 µM NTC siRNA, DKK1 siRNA, or SFRP1 siRNA for 5–8 days ex vivo. (a) Quantification of hair shaft production on days 1, 4, 5, 6, and 8 in culture with n = 24 (D1, D4), n = 6 (D5), n = 12–13 (D6), and n = 6 (D8) HFs from 1–4 independent donors. (b) Representative images of Ki-67/TUNEL immunofluorescence and Masson–Fontana histochemistry. (c) Quantitative analysis of hair cycle staging, n = 4 donors. (d) Quantitative analysis of the hair cycle score. Arbitrary units (au) were assigned to anagen (100), early catagen (200), mid catagen (300), and dystrophic (500) HFs. n = 23–24 HFs from 4 independent donors. (e,f) Quantitative analysis of hair matrix keratinocyte proliferation (e) and apoptosis (f). n = 23–24 HFs from 4 independent donors. All data are presented as mean ± SEM. Mann–Whitney test or unpaired t-test, * p < 0.05. Dots of different shapes indicate different donors. Data points reflecting the female donor are indicated by circles. DKK1: dickkopf 1, DP: dermal papilla, gHM: germinative hair matrix, HF: hair follicle, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.
Figure 4. Silencing of SFRP1 prolongs anagen in healthy human HFs. Healthy human HFs were treated with 10 µM NTC siRNA, DKK1 siRNA, or SFRP1 siRNA for 5–8 days ex vivo. (a) Quantification of hair shaft production on days 1, 4, 5, 6, and 8 in culture with n = 24 (D1, D4), n = 6 (D5), n = 12–13 (D6), and n = 6 (D8) HFs from 1–4 independent donors. (b) Representative images of Ki-67/TUNEL immunofluorescence and Masson–Fontana histochemistry. (c) Quantitative analysis of hair cycle staging, n = 4 donors. (d) Quantitative analysis of the hair cycle score. Arbitrary units (au) were assigned to anagen (100), early catagen (200), mid catagen (300), and dystrophic (500) HFs. n = 23–24 HFs from 4 independent donors. (e,f) Quantitative analysis of hair matrix keratinocyte proliferation (e) and apoptosis (f). n = 23–24 HFs from 4 independent donors. All data are presented as mean ± SEM. Mann–Whitney test or unpaired t-test, * p < 0.05. Dots of different shapes indicate different donors. Data points reflecting the female donor are indicated by circles. DKK1: dickkopf 1, DP: dermal papilla, gHM: germinative hair matrix, HF: hair follicle, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.
Ijms 27 01815 g004
Table 1. Donor information.
Table 1. Donor information.
DonorGenderAgeScalp RegionFUE/Scalp SkinExperiment(s)
1male58occipitalFUEAR, SFRP1, DKK1 ISH analysis
2male25occipitalFUEAR, SFRP1, DKK1 protein analysis
3male54occipitalFUEAR, SFRP1, DKK1 ISH and protein analysis
4male27temporalscalp skinAR, SFRP1, DKK1 ISH and protein analysis
5male55occipitalFUEAR, SFRP1, DKK1 ISH analysis
6male18occipitalFUEAR, SFRP1, DKK1 protein analysis
7male31occipitalFUEAR SNA
8male23occipitalFUEAR SNA
9female24temporalscalp skinSFRP1 siRNA, DKK1 siRNA
10male57temporal
and occipital		scalp skinSFRP1 siRNA, DKK1 siRNA
11male38occipitalscalp skinSFRP1 siRNA, DKK1 siRNA
12male59temporalFUESFRP1 siRNA, DKK1 siRNA
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Broadley, D.; Le Riche, A.; Yu, Y.; El-Bacha, H.; Erdmann, H.; Jimenez, F.; Geyfman, M.; Poloso, N.; Edelkamp, J.; Bertolini, M. Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo. Int. J. Mol. Sci. 2026, 27, 1815. https://doi.org/10.3390/ijms27041815

AMA Style

Broadley D, Le Riche A, Yu Y, El-Bacha H, Erdmann H, Jimenez F, Geyfman M, Poloso N, Edelkamp J, Bertolini M. Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo. International Journal of Molecular Sciences. 2026; 27(4):1815. https://doi.org/10.3390/ijms27041815

Chicago/Turabian Style

Broadley, David, Alizée Le Riche, Ying Yu, Helene El-Bacha, Hanieh Erdmann, Francisco Jimenez, Mikhail Geyfman, Neil Poloso, Janin Edelkamp, and Marta Bertolini. 2026. "Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo" International Journal of Molecular Sciences 27, no. 4: 1815. https://doi.org/10.3390/ijms27041815

APA Style

Broadley, D., Le Riche, A., Yu, Y., El-Bacha, H., Erdmann, H., Jimenez, F., Geyfman, M., Poloso, N., Edelkamp, J., & Bertolini, M. (2026). Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo. International Journal of Molecular Sciences, 27(4), 1815. https://doi.org/10.3390/ijms27041815

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop