JCI - Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells

KRT15 expression levels correlate with epithelial stem cell characteristics of small cell size and cell cycle quiescence. To evaluate changes in hair follicle stem cell numbers in human scalp, we used KRT15 expression as a stem cell marker. We focused on cells in the top 5% of KRT15 expression (KRT15^hi) by flow cytometry, since KRT15 is expressed at the highest levels in bulge cells (3, 14). To further verify that KRT15^hi cells possess epithelial stem cell properties, we analyzed the relationship of KRT15 expression to the known stem cell characteristics of quiescence and cell size (12, 17).

Small cell size has been associated with stem cells in multiple tissues (17–19). In epithelia, early studies indicated that small human epidermal keratinocytes were clonogenic and had the greatest proliferative potential (17). More recent analysis confirmed that cells isolated from the bulge are small in size and are highly proliferative in vitro, consistent with their role as stem cells (14). Small corneal epithelial cells also exhibit the highest proliferative potential (19). Finally, cell size may regulate cell cycle progression, since large cell size triggers proliferation (20).

Quiescence remains a defining characteristic of epithelial stem cells in the skin and other tissues (11, 21, 22). Functional assays demonstrate that quiescent epidermal cells possess the greatest proliferative potential (9, 10). Label-retaining cell (4, 7), lineage (9), and cell ablation studies (2) confirm that quiescent keratinocytes in the bulge are responsible for constant regeneration of the hair follicle. Human basal bulge cells also retain label and have a quiescent proliferative profile (3, 23).

To assess the relationship between KRT15 expression and the stem cell properties of quiescence and small cell size, we measured the cell size and cell cycle characteristics of KRT15-expressing keratinocytes from human adult scalp. We analyzed viable keratinocytes for expression of ITGA6, KRT15, and Ki67 by flow cytometry. The gating strategy is listed in Supplemental Figure 1, A–G (supplemental material available online with this article; doi: 10.1172/JCI44478DS1). Cells of increasing percentile of staining for KRT15 were measured to detect cell size by forward scatter (Figure 1A). Cells at the 50th percentile and higher of KRT15 staining were significantly smaller than cells at the 20th percentile (n = 5, P = 0.002 to P = 1.58 × 10^–5). KRT15 expression positively correlated with small cell size: cells expressing the highest levels of KRT15 were smallest (Figure 1A).

To determine cell cycle attributes of cells expressing different levels of KRT15, we used flow cytometry to analyze Ki67 expression and DNA content (Figure 1, B–D, and Supplemental Figure 1, H–J). We compared cells at increasing percentiles of KRT15 staining with those at the 20th percentile. The percentage of cells in G₀ significantly increased at the 40th and higher percentiles of KRT15 staining (Figure 1B; n = 5, P = 0.05 to P = 3.85 × 10^–5). The percentage of proliferative, Ki67⁺ cells was lowest for cells at the 95th percentile of KRT15 expression: 82% ± 3% of these cells were in G₀, with 12% ± 3% in G₁, 5% ± 2% in G₂/M, and 0.3% ± 0.1% in S (Figure 1, B–D). Coincident with the increase in the percentage of cells in G₀, there was a decrease in cells in G₁, which indicated that most cells not in G₀ were instead in G₁. The percentage of cells in G₁ was significantly decreased at the higher percentiles (Figure 1C; n = 5, P = 0.05 to P = 3.1 × 10^–5). Similarly, cells expressing high levels of KRT15 were less likely to be in S phase (Figure 1D; n = 5, P = 0.04 to P = 0.0003). G₂/M changes were more variable according to KRT15 levels, as has been published previously (9), but were significantly decreased at the 80th and 90th percentiles (Supplemental Figure 1K; n = 5, P = 0.04 and P = 0.05). Thus, high levels of KRT15 expression correlate well with a quiescent stem cell phenotype.

Bald scalp retains KRT15^hi stem cells. Having verified that cells with the highest level of KRT15 expression possess properties of epithelial stem cells, we next addressed whether hair follicle stem cell numbers decrease in bald versus haired scalp from men with AGA. We isolated single-cell suspensions of epithelial cells from bald and haired scalp from the same individuals. These cells were stained with antibodies against ITGA6 and KRT15 and then analyzed by flow cytometry (Figure 2). We defined KRT15^hi cells as those in the top 5% of staining in haired scalp. In each paired sample from the same individual, an identical gate defining the top 5% of cells in haired scalp was applied to the cells from bald scalp. The flow cytometry was performed on the same day with identical instrument settings (see Methods for details). On average, the percentage of KRT15^hi cells was the same in bald and haired scalp (Figure 2, A–C; 4.6% ± 0.9% vs. 5.0% ± 0.02%, P = 0.3, n = 8).

Immunohistochemical staining for KRT15 also showed many strongly positive cells in miniaturized follicles from scalp with androgenetic alopecia (Supplemental Figure 2, A and B), which supported the notion that hair follicle stem cells are maintained in bald scalp.

Progenitor cell populations distinct from KRT15^hi stem cells are depleted in bald scalp. Recently, CD200 expression was identified in human bulge cells in haired scalp from women (13, 14). In these studies, the CD200⁺ population overlapped substantially with the K15⁺ population. To define changes in CD200⁺ cells in men with AGA, we analyzed CD200 expression together with expression of the epithelial basal cell marker ITGA6 by flow cytometry in matched bald and haired scalp. We excluded CD45⁺ hematopoietic cells and CD117⁺ melanocytes from the starting population and confirmed that the CD200⁺ cells were negative for these nonepithelial markers (Supplemental Figure 1, L and M).

Surprisingly, we found that a well-demarcated population of cells expressing high levels of both CD200 and ITGA6 was markedly decreased in haired versus bald scalp (Figure 2, D–F; 2.3% ± 0.7% vs. 0.28% ± 0.1%, P = 0.005, n = 9). This population represented 10.0% ± 0.1% (n = 9) of the entire CD200⁺ population; to our knowledge, it has not been studied previously.

To better characterize CD200^hiITGA6^hi cells with respect to their stem cell properties, we determined their level of KRT15 expression, cell size, and degree of quiescence. We compared cells gated as CD200^hiITGA6^hi (Figure 2D), as well as cells gated as KRT15^hiITGA6^hi (Figure 2A), with an otherwise ungated population of all ITGA6^hi cells (Supplemental Figure 1E). CD200^hiITGA6^hi cells expressed lower levels of KRT15 compared with KRT15^hi cells (n = 3, P = 0.036), and higher levels of KRT15 (n = 3, P = 0.046) compared with ITGA6^hi cells (Figure 3A). In line with this, we found almost no CD200 expression among the KRT15^hi cells (Figure 3D; n = 3, P = 0.008), which indicates that these populations are distinct. Given the intermediate expression of KRT15 in the CD200^hiITGA6^hi cells, the gradient of stem cell characteristics (Figure 1) predicts that these cells would be intermediate in cell size and cell cycle; indeed, this was the case (Figure 3, B and C). CD200^hiITGA6^hi cells were 75% ± 2% as large as all ITGA6⁺ cells (Figure 3B; n = 6, P = 3 × 10^–5), but were significantly larger than the KRT15^hi cells (n = 6, P = 0.007). Thus, CD200^hiITGA6^hi cells were of intermediate size compared with the KRT15^hi cells.

To determine the level of quiescence of the CD200^hiITGA6^hi cells, we performed cell cycle analysis. The CD200^hiITGA6^hi population showed 69% ± 5% of cells in G₀ (Figure 3C; n = 2), significantly higher than all basal cells (21% ± 1.9%, P = 0.02), but lower than the percentage of KRT15^hi cells in G₀ (98% ± 0.6%, P = 0.05). Thus, CD200^hiITGA6^hi cells were of intermediate size and quiescence compared with KRT15^hi cells.

To assess whether other progenitor cell populations were depleted in bald scalp, we quantitated the number of CD34⁺ cells, which juxtapose the bulge and localize below it in the outer root sheath. We found that CD34⁺ cells were diminished roughly 10-fold in bald versus haired scalp (Figure 2, G–I; 1.9% ± 1% vs. 10.5% ± 0.3%, P = 0.01, n = 3). CD34⁺ cells expressed low levels of KRT15 and were larger than the KRT15^hi stem cells (Figure 3, E and F). These findings are consistent with a role for these cells as progenitors descendent from the bulge cells (14).

Human CD200^hiITGA6^hi cells localize to the hair follicle bulge and to the secondary germ. To better localize the CD200^hiITGA6^hi cells that were depleted in bald scalp, we isolated these cells from haired scalp and analyzed them for expression of markers from different compartments of the hair follicle (Figure 4). In addition to KRT15, we used follistatin (FST) as a bulge cell marker (13). The majority of CD200^hiITGA6^hi cells were positive for both KRT15 and FST (Figure 4, B and C). However, approximately 15% were negative, which indicates that this portion resides outside of the bulge.

To further define the location of the CD200^hiITGA6^hi cells that did not localize to the bulge, we used the Ber-EP4 antibody, which detects epithelial cell adhesion molecule (EPCAM), to stain for secondary germ cells (Figure 4, D–F, Supplemental Figure 3, A and B, and ref. 24). Of the CD200^hiITGA6^hi cells, 16% were positive for Ber-EP4 (Figure 4F), indicative of their localization to the secondary germ. By immunohistochemistry, we detected CD200 expression in the bulge region and in secondary germ cells in telogen human hair follicles from haired scalp (Figure 4E and Supplemental Figure 4, D and E). In agreement with our fluorescence-activated cell sorting (FACS) analysis, we found a qualitative decrease in staining for CD200⁺ cells in bald scalp (Supplemental Figure 2, C and D).

To further investigate the relationship of CD200^hiITGA6^hi cells to the secondary germ cells, we evaluated expression of LGR5 by quantitative PCR (qPCR). LGR5 recently has been touted as a marker of hair follicle progenitor cells in the lower bulge and secondary germ (11, 25). We found LGR5 mRNA elevated 1,443-fold in CD200^hiITGA6^hi versus CD200^–ITGA6^hi cells (Figure 4G; n = 3, P < 0.01).

As another test of the hypothesis that loss of the CD200^hiITGA6^hi population in AGA represents a loss of activated bulge cells, but not quiescent bulge cells, we compared changes in LGR5 and KRT15 mRNA levels in haired and bald scalp using qPCR (Figure 4H). Loss of epithelial cells expressing LGR5 and KRT15 in bald scalp would result in a haired/bald ratio of gene expression greater than 1. The haired/bald ratio of KRT15 mRNA was 0.58 ± 0.14, indicating at least proportional, if not absolute, maintenance of signal in miniaturized hair follicles (Supplemental Figure 5). However, the ratio of haired to bald scalp mRNA for LGR5 was significantly elevated at 3.3 ± 0.87 (Figure 4H; n = 4, P < 0.01), indicative of a loss of LGR5 mRNA in bald scalp. The enrichment of LGR5 in the CD200^hiITGA6^hi population and the loss of LGR5 in bald scalp underscores that the decrease of CD200^hiITGA6^hi cells in bald scalp is not simply due to downregulation of CD200 expression, but rather to loss of these cells.

Mouse CD200^hiItga6^hi cells localize to the hair follicle bulge and secondary germ. To enable functional studies of the CD200^hiITGA6^hi cells, we sought to define an analogous cell population in the mouse hair follicle. In mice, CD200^hiItga6^hi cells accounted for approximately 8% of the total viable epithelial cell population from back skin (Figure 5C and Supplemental Figure 6). To localize these cells, we took advantage of the known specific expression of CD34 by hair follicle bulge cells in the mouse epithelium (15) and compared CD34 and CD200 staining patterns. Immunostaining demonstrated overlap of their expression in the bulge, but extension of CD200 staining into the CD34^– secondary germ (Figure 5, A and B, and Supplemental Figure 3C). By FACS analysis, approximately 82% of the CD200^hiItga6^hi cells were CD34⁺ and therefore localized to the bulge (Figure 5F). Together with the immunostaining data, these results indicate that roughly 18% of CD200^hiItga6^hi cells localized to the secondary germ. This corresponds closely to the 15% of human CD200^hiITGA6^hi cells that localized to the secondary germ based on their Ber-EP4^hi status (Figure 4F). Thus, the mouse and human CD200^hiITGA6^hi populations localize to both bulge and secondary germ in equivalent ratios.

To further compare the human CD200^hiITGA6^hi population with mouse CD200^hiItga6^hi cells, we performed cell cycle analysis of the mouse as we did on human CD200^hiItga6^hi cells (Figure 3). Specifically, we compared cell cycle features in mouse bulge (CD200⁺CD34⁺) and secondary hair germ (CD200⁺CD34^–) cells (Figure 5G) with the human CD200^hiITGA6^hi population (Figure 3).

Consistent with previous studies demonstrating quiescence of the bulge cells (4, 9, 15), the proportion of bulge cells in S phase (0.84% ± 0.1%) was significantly lower than in all cells (Figure 5G; 1.44% ± 0.004%, n = 3, P = 0.02). The mouse bulge did show increased numbers of cells in G₂/M (all cells 1.46% ± 0.5%, bulge cells 2.84% ± 0.5%, P = 0.02), as described previously (9). The 4.44% ± 1.5% of secondary germ cells (CD34^–) in G₂/M was higher than that of the bulge (26). The cells demonstrating the highest percentage of G₂/M were CD200^hiItga6^hi in the secondary hair germ (Figure 5G; 6.07% ± 0.3%, n = 3, P = 0.01).

In an inverse pattern, the percentage of cells in G₁/G₀ was decreased in cells with elevated levels of G₂/M. Therefore, CD200^hiItga6^hi cells of the secondary hair germ showed significantly lower levels of cells in G₁/G₀ (89.5% ± 0.4%, n = 3) than did bulge cells (95.7% ± 0.2%, n = 3, P = 0.02). In summary, the decreased proportion of cells in G₁/G₀ in the mouse CD200^hiItga6^hiCD34^– population compared with CD34⁺ bulge cells was similar to our cell cycle analysis demonstrating decreased G₀ levels in human CD200^hiItga6^hi cells compared with KRT15^hi bulge cells (Figure 3). We conclude that both mouse and human CD200^hiITGA6^hi cells show evidence of cell cycle activation compared with cells of the bulge.

To further compare the mouse and human CD200^hiItga6^hi cells, we analyzed their global gene expression patterns using microarrays. Given that mouse CD200^hiItga6^hi cells were composed of both bulge and secondary hair germ cells (Figure 5, E and F), we compared the expression of human CD200^hiItga6^hi cells with those of mouse bulge and mouse secondary hair germ in a cross-species comparison. Gene lists of enriched genes for each population were created (see Methods) and compared for overlap (Figure 5H). Although mouse bulge and mouse secondary hair germ gene expression patterns showed the most overlap (499 genes), it is likely that this is explained by species homogeneity. All 3 populations shared 178 genes. Interestingly, of the populations uniquely shared between the human CD200^hiITGA6^hi cells and the mouse cell populations, more genes were shared with mouse bulge (151 genes) than with mouse secondary hair germ (39 genes). The greater overlap with the bulge compared with the secondary hair germ matches the cellular composition of the CD200^hiITGA6^hi populations in both mice and humans. Further analysis of these gene lists (Supplemental Figure 9) showed that shared human and mouse genes present in the bulge were enriched in biologic adhesion proteins, whereas transcripts of the secondary hair germ were enriched in genes regulating death and apoptosis. These results are consistent with the concept that substrate attachment maintains the quiescent phenotype of the bulge cells and that the loss of these adhesions is associated with differentiation to secondary hair germ cells.

Mouse CD200^hiItga6^hi cells are multipotent and capable of regenerating hair follicles in a skin reconstitution assay. We performed functional analysis on the CD200^hiItga6^hi population using a reconstitution assay, which tests the ability of isolated cell populations to regenerate hair follicles. Isolated single cells from epithelium are combined with neonatal dermal cells and injected intradermally into an immunodeficient mouse host. After 4 weeks, the injected tissue is examined for the presence of newly formed hair follicles, epidermis, and sebaceous glands (9, 27).

We sorted CD200^hiItga6^hi or CD200^–Itga6^hi cells from ROSA26 reporter mice. Grafting of CD200^hiItga6^hi keratinocytes together with neonatal dermis successfully reconstituted hair follicles (Figure 6, A, B, and E). CD200^–Itga6^hi keratinocytes produced few hair follicles, despite injection of equal numbers of cells (Figure 6, C–E). Contaminating neonatal epidermis from neonatal dermal preparations contributed to some hair follicle formation in both samples (Figure 6, A–D), but could be distinguished by its lack of β-galactosidase activity. Histologic sectioning of reconstituted hair-bearing cysts demonstrated contribution of CD200^hiItga6^hi cells to all hair follicle lineages, including outer root sheath, inner root sheath, and sebaceous gland (Supplemental Figure 7, A–C), indicative of the multipotency of these cells.