Partial Alopecia Area Retains Bulge Hair Follicle Progenitor Cells in Indian Androgenetic Alopecia Patients

ABSTRACT

Background:

Androgenic alopecia (AGA) is a pathological condition characterized by the progressive decrease of scalp hair follicle density. Completely bald areas of AGA scalp exhibit a drastically decreased number of progenitor cells. However, it is unknown when the progenitor cells begin to diminish in number during the progression of AGA. Despite the prevalence of AGA in 58% of the Indian male population, no study to date has characterized the hair follicle stem and progenitor cell populations in AGA patients in this ethnic group.

Aims and Objective:

In this study, we tested the presence of progenitor cells marked by CD34 and Sox9 in the partially and fully bald scalp of Indian AGA patients. Material and Methods: We utilized punch biopsies from the scalp of Indian AGA patients and quantified the status of hair follicle progenitor cells via histological and immunofluorescence analysis.

Results:

We observed that the partially bald area retains progenitor cells expressing CD34 and Sox9, but they are not present in the hair follicles in the completely bald area.

Conclusion:

Therapeutic interventions based on maintaining and activating hair follicle progenitor cells would be most effective at the partially bald stage of AGA.

INTRODUCTION

Androgenic alopecia (AGA), or male pattern hair loss, begins after the onset of puberty and progresses through adulthood.[1] Studies report that AGA can affect 50% of men by the age of 50 and 50% of women by the age of 60 in the Caucasian population.[2] This can lead to decreased quality of life because of its significant impact on a person’s psychological well-being.

The hair follicle undergoes cyclical growth (anagen), regression (catagen), and resting (telogen) phases. In patients who are genetically predisposed to androgen-stimulated hair follicle miniaturization, the size of the scalp hair follicles is decreased with every cycle due to alterations in hair cycle dynamics. Eventually, patients start producing microscopic hair due to the transformation of terminal hair to vellus (dormant) hair follicles.[3] This results in the gradual replacement of large pigmented hair by depigmented hairs that are barely visible. Scalp skin biopsies from AGA patients exhibit decreased hair density, increase in the number of vellus hair, and altered hair follicle architecture.[4]

Hair follicle stem cells (HFSCs) reside in a niche in the hair follicle called the bulge. These HFSCs play a crucial role in the hair cycle by providing a continuous source of new cells for hair growth during the anagen phase. These stem cells are usually quiescent, but during the anagen phase, they divide and give rise to transit-amplifying progenitor cells,[5] which are responsible for the growth of the hair shaft.[6] Studies suggest that therapies aimed at treating AGA can utilize either stem cells to reactivate hair follicles or regulatory mechanisms controlling hair follicle activity to stimulate hair growth.[7] Realization of these therapeutic approaches is dependent upon understanding the status of the hair follicle stem and progenitor cells in the AGA scalp. Previous reports have shown that in the bald area of the scalp in AGA patients, HFSCs are retained, but progenitor cells are diminished, indicating that the conversion of stem cells into progenitor cells is affected.[8,9]

Recent studies conducted on Caucasian and Egyptian AGA patients have shown that, in the completely bald area of the AGA scalp, HFSCs expressing K15 are present,[8,10] but the progenitor cell population expressing CD200 and CD34 are diminished.[8] This suggests that AGA can potentially be reversed by re-activating the conversion of HFSCs into progenitor cells.[11]

Previous studies have mainly focused on the fully bald area, but the hair follicles of the partially bald area of AGA have not been characterized for the status of stem and progenitor cells. Moreover, most of the studies have been conducted on Caucasian patients, although the prevalence of AGA differs with ethnicity.[12,13,14] Although the prevalence is 58% in Indian males aged 30–50 years,[12] very few studies have been conducted on the Indian population. Thus, in this study, we tested the presence of progenitor cells in the partially and fully bald scalp of Indian AGA patients.

MATERIALS AND METHODS

Human subjects

Ten patients were included in the study. Diagnosis of AGA was established by the characteristic distribution of frontal and vertex hair. Patients more than 20 years old and diagnosed as male AGA, type III to VI, using the Hamilton–Norwood classification were included. Demographic details of all patients, including age, family history, and duration of disease, are summarized in Table 1.

Table 1.

Clinical characteristics of alopecia patients

Patient number	Age (years)	Family history of alopecia	Duration of disease
1	28	Present	4 years
2	24	Present	2 years
3	29	Absent	1 year
4	31	Present	1 year
5	35	Absent	6 months
6	30	Present	6 years
7	26	Absent	5 years
8	24	Absent	1 year
9	24	Absent	4 years
10	26	Present	5 years

Sample collection

Three mm punch biopsy specimens were obtained from all the patients from both the frontal area of the scalp with partial loss of hair and the occipital scalp skin that serves as a control. From 4 patients out of these, biopsies were also collected from completely bald frontal areas. The samples from the three different areas of biopsy have been denoted as occipital area, partially bald frontal area, and bald frontal area.

Tissue preparation and histology/immunofluorescence

Skin samples were fixed in 4% paraformaldehyde solution for 24 h at 40°C. Tissues were quenched in 125 mM glycine solution in Tris-buffered saline for 30 min and then incubated in 5%, 10%, and 20% sucrose solution for 6 h, 12 h, and 24 h, respectively, before embedding in tissue freezing medium (Leica). Sections that were 30 μm thick were sliced using a cryostat. H and E staining was used for viewing gross follicle histology. For immunofluorescence staining, the following primary antibodies and dilutions were used: Sox9 (Abcam, catalog number ab185230, 1:200), CD34 (Abcam, catalog number ab81289, 1:100). Alexa Fluor 488-labeled anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) was used at a dilution of 1:200. DAPI (4’,6-diamidino-2-phenylindole) stain was used to mark the nuclei.

Image collection and analysis

Imaging was performed with an Olympus IX73 microscope (Olympus), FV1000 or FV3000 confocal microscope (Olympus). Images were analyzed using the Fiji (ImageJ) software.

Statistical analysis

Quantification of the number of Sox9⁺ and CD34⁺ cells in the occipital and frontal areas of the scalp in 6 patient sample sets was done using the paired, nonparametric, one-tailed, Wilcoxon matched-pairs signed rank test. GraphPad Prism 5.02 (GraphPad Software) was used for all statistical analyses. P <0.05 was considered statistically significant.

RESULTS

Bald area of androgenic alopecia scalp lacks the complete structure of hair follicle and progenitor cells.

The hair follicle is comprised of the infundibulum, bulge, sub-bulge, lower outer root sheath (ORS), and bulb based on the anatomy and the stem cell positions.[15] To investigate the status of the hair follicle structure, we performed histological analysis of sections of hair follicles from the occipital area and bald frontal area of the AGA scalp [Figure 1a]. This analysis revealed that the hair follicle from the occipital area contains the complete hair follicle structure, whereas the bald frontal area lacks the bulge, sub-bulge, lower ORS, and bulb structures. The remnant structure resembles the follicular ostia [Figure 1b]. We further investigated the status of progenitor cells in these two regions from the AGA scalp. CD34⁺ progenitor cells are found in the ORS of the sub-bulge and suprabulbar region of the anagen hair follicle.[15,16,17] Another important population of cells expressing Sox9 is found in the ORS of the bulge, sub-bulge, and proximal bulb area in the anagen stage.[18] Immunostaining for these markers revealed that the occipital unaffected area contains CD34⁺ and Sox9⁺ progenitor cells, whereas the completely bald area lacks them [Figure 1c and d]. Our results are consistent with previous reports that the hair follicles of the bald area of AGA scalp from Caucasian patients lack progenitor cells derived from HFSCs[8] and also suggest that a cause of alopecia is due to a defect in the conversion of HFSCs to progenitor cells.[8,9]

Figure 1.

Bald frontal area of the androgenic alopecia scalp (AGA) lacks the complete structure of the hair follicle with the absence of hair follicle progenitor cells. (a) Schematic of the scalp of an AGA patient marking the sites of hair follicle biopsy collection: Bald frontal area with vellus hair (marked by asterisk); partially bald frontal area characterized by thinning/recession (marked by arrowhead); occipital unaffected area with terminal hair (marked by hash). (b) Hematoxylin and eosin staining of hair follicles from the occipital unaffected area and the bald frontal area of the scalp. Immunofluorescence staining (green) for hair follicle progenitor cells marked by CD34 and Sox9 in (c) occipital unaffected area and (d) bald frontal area, respectively. Nuclei are marked by DAPI (4’,6-diamidino-2-phenylindole) in blue. Scale bars: 100 μm. Images in (c) are composite images created by tiling several images together.

Partially bald area of androgenic alopecia scalp retains Sox9⁺ and CD34⁺ progenitors

An outstanding question in the field is when progenitor cells start to diminish. Thus, we tested the status of the progenitor cells in the partially bald frontal area of the AGA scalp. Immunostaining for CD34 and Sox9 markers revealed that the hair follicles of the bald frontal area of AGA scalp lack the progenitor cells [Figure 1d], but they are still retained in the hair follicles of the partially bald frontal area [Figure 2a and b]. Quantification of CD34⁺ and Sox9⁺ cells revealed that the number of progenitor cells is not significantly reduced in the partially bald area compared to the hair follicles of the occipital unaffected area [Figure 2a and b]. This suggests that loss of progenitor cells occurs after the partially bald stage of AGA.

Figure 2.

Partially bald area of AGA scalp retains CD34⁺ and Sox9⁺ progenitors. Immunofluorescence staining (green) for (a) CD34⁺ and (b) Sox9⁺ hair follicle progenitor cells in the unaffected occipital and partially bald frontal area of AGA scalp reveals that these progenitor cells are retained in the partially bald frontal area. Nuclei are marked by DAPI (4’,6-diamidino-2-phenylindole) in blue. Scale bars: 100 μm. Quantification indicates no significant difference in number of CD34⁺ or Sox9⁺ cells between the two areas. n = 6. Data are presented as mean ± standard error of the mean. Each dot represents the number of progenitor cells found per microscopic field. P values were calculated using nonparametric Wilcoxon matched-pairs signed-ranks test. P <0.05 was considered significant; “ns” indicates “not significant,” Scale bar: 100 μm

DISCUSSION

Recent studies have shed light on the defect in the stem cell to progenitor cell conversion in the AGA hair follicles,[8] highlighting the importance of characterizing various progenitor cells in the AGA scalp. Previous works have primarily focused on Caucasian patient samples, and since the prevalence of AGA differs with ethnicity, our study provides novel insights into the progenitor cell status of AGA in the Indian population. We found that the hair follicles in the completely bald area of AGA lack progenitor cells expressing CD34 and Sox9, but they are retained in the partially bald area. This indicates that progenitor cells likely diminish during the terminal stage, and there is a window of opportunity for therapeutic intervention.

AGA is one of the most common dermatological problems worldwide for which treatment is sought.[3] The cost of 5-year treatment for AGA ranges from hundreds to thousands of dollars depending on the type of treatment.[19] Although studies have shown that AGA can cause significant psychological distress in patients,[20] there are limited therapeutic options available due to the lack of a complete understanding of the cellular and signaling mechanisms underlying AGA progression. Previous work has reported that androgens, especially dihydrotestosterone (DHT) dysregulate the entry of hair follicles into the anagen phase by impairing HFSC differentiation via upregulating the production of DKK-1 which is an antagonist of WNT signaling.[21] Current treatment modalities often involve finasteride, which is used to inhibit the production of DHT. Minoxidil is another compound used in the treatment of AGA, which increases hair growth by shortening the telogen phase and causing premature entry of those hair follicles into the anagen phase.[22] Alternative therapies include hormonal and platelet-rich plasma therapies, but these have been shown to have limited effectiveness and are prone to several side effects.[19,23] While hair transplant is very effective in all stages of AGA, this method is costly and time-consuming.[19] Intradermal injection of autologous stem cells is an exciting new therapy.[24] Progenitor cell enriched micrografts are emerging as a novel therapy,[25] but the long-term effectiveness remains to be determined. Elucidating the underlying mechanisms of AGA would help to optimize these therapeutic approaches.

CONCLUSION

Therapies aimed at activating/maintaining stem and progenitor cells would theoretically be more effective at the stage when there is initiation of active hair loss and deterioration in hair quality (partial alopecia). On the other hand, therapies intended to stimulate new hair growth in completely bald areas need to focus on replenishing the source of stem and progenitor cells.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Grants from the Department of Biotechnology of the Government of India (BT/PR8738/AGR/36/770/2013 and BT/PR32539/BRB/10/1814/2019) and the Rajiv Gandhi University of Health Sciences (Bangalore, India) (RN003) and core funds from the Institute for Stem Cell Science and Regenerative Medicine (Bangalore, India).