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 (KRT15hi
) by flow cytometry, since KRT15 is expressed at the highest levels in bulge cells (3
). To further verify that KRT15hi
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
Small cell size has been associated with stem cells in multiple tissues (17
). 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
). Functional assays demonstrate that quiescent epidermal cells possess the greatest proliferative potential (9
). Label-retaining cell (4
), 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
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:
). Cells of increasing percentile of staining for KRT15 were measured to detect cell size by forward scatter (Figure A). 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 A).
KRT15 expression levels define a gradient of stem cell characteristics in isolated scalp keratinocytes.
To determine cell cycle attributes of cells expressing different levels of KRT15, we used flow cytometry to analyze Ki67 expression and DNA content (Figure , 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 B; 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 , 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 C; 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 D; 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 KRT15hi 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 ). We defined KRT15hi 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 KRT15hi cells was the same in bald and haired scalp (Figure , A–C; 4.6% ± 0.9% vs. 5.0% ± 0.02%, P = 0.3, n = 8).
Preservation of KRT15hi hair follicle stem cells in bald scalp, but depletion of CD200hiITGA6hi and CD34hi progenitor cells.
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 KRT15hi stem cells are depleted in bald scalp.
Recently, CD200 expression was identified in human bulge cells in haired scalp from women (13
). 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 , 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 CD200hiITGA6hi 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 CD200hiITGA6hi (Figure D), as well as cells gated as KRT15hiITGA6hi (Figure A), with an otherwise ungated population of all ITGA6hi cells (Supplemental Figure 1E). CD200hiITGA6hi cells expressed lower levels of KRT15 compared with KRT15hi cells (n = 3, P = 0.036), and higher levels of KRT15 (n = 3, P = 0.046) compared with ITGA6hi cells (Figure A). In line with this, we found almost no CD200 expression among the KRT15hi cells (Figure D; n = 3, P = 0.008), which indicates that these populations are distinct. Given the intermediate expression of KRT15 in the CD200hiITGA6hi cells, the gradient of stem cell characteristics (Figure ) predicts that these cells would be intermediate in cell size and cell cycle; indeed, this was the case (Figure , B and C). CD200hiITGA6hi cells were 75% ± 2% as large as all ITGA6+ cells (Figure B; n = 6, P = 3 × 10–5), but were significantly larger than the KRT15hi cells (n = 6, P = 0.007). Thus, CD200hiITGA6hi cells were of intermediate size compared with the KRT15hi cells.
CD200hiITGA6hi and CD34hi cells are distinct from KRT15hi stem cells and possess a progenitor cell phenotype.
To determine the level of quiescence of the CD200hiITGA6hi cells, we performed cell cycle analysis. The CD200hiITGA6hi population showed 69% ± 5% of cells in G0 (Figure C; n = 2), significantly higher than all basal cells (21% ± 1.9%, P = 0.02), but lower than the percentage of KRT15hi cells in G0 (98% ± 0.6%, P = 0.05). Thus, CD200hiITGA6hi cells were of intermediate size and quiescence compared with KRT15hi 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 , 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 KRT15hi
stem cells (Figure , E and F). These findings are consistent with a role for these cells as progenitors descendent from the bulge cells (14
Human CD200hiITGA6hi cells localize to the hair follicle bulge and to the secondary germ.
To better localize the CD200hi
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 ). In addition to KRT15, we used follistatin (FST) as a bulge cell marker (13
). The majority of CD200hi
cells were positive for both KRT15 and FST (Figure , B and C). However, approximately 15% were negative, which indicates that this portion resides outside of the bulge.
CD200hiITGA6hi cells localize to the hair follicle bulge and to the secondary germ.
To further define the location of the CD200hi
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 , D–F, Supplemental Figure 3, A and B, and ref. 24
). Of the CD200hi
cells, 16% were positive for Ber-EP4 (Figure F), 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 E 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 CD200hi
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
). We found LGR5
mRNA elevated 1,443-fold in CD200hi
cells (Figure G; n
= 3, P
As another test of the hypothesis that loss of the CD200hiITGA6hi 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 H). 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 H; n = 4, P < 0.01), indicative of a loss of LGR5 mRNA in bald scalp. The enrichment of LGR5 in the CD200hiITGA6hi population and the loss of LGR5 in bald scalp underscores that the decrease of CD200hiITGA6hi cells in bald scalp is not simply due to downregulation of CD200 expression, but rather to loss of these cells.
Mouse CD200hiItga6hi cells localize to the hair follicle bulge and secondary germ.
To enable functional studies of the CD200hi
cells, we sought to define an analogous cell population in the mouse hair follicle. In mice, CD200hi
cells accounted for approximately 8% of the total viable epithelial cell population from back skin (Figure C 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 , A and B, and Supplemental Figure 3C). By FACS analysis, approximately 82% of the CD200hi
cells were CD34+
and therefore localized to the bulge (Figure F). Together with the immunostaining data, these results indicate that roughly 18% of CD200hi
cells localized to the secondary germ. This corresponds closely to the 15% of human CD200hi
cells that localized to the secondary germ based on their Ber-EP4hi
status (Figure F). Thus, the mouse and human CD200hi
populations localize to both bulge and secondary germ in equivalent ratios.
Mouse CD200hiItga6hi cell location, cell cycle status, and gene expression are similar to those of human CD200hiITGA6hi cells.
To further compare the human CD200hiITGA6hi population with mouse CD200hiItga6hi cells, we performed cell cycle analysis of the mouse as we did on human CD200hiItga6hi cells (Figure ). Specifically, we compared cell cycle features in mouse bulge (CD200+CD34+) and secondary hair germ (CD200+CD34–) cells (Figure G) with the human CD200hiITGA6hi population (Figure ).
Consistent with previous studies demonstrating quiescence of the bulge cells (4
), the proportion of bulge cells in S phase (0.84% ± 0.1%) was significantly lower than in all cells (Figure G; 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 CD200hi
in the secondary hair germ (Figure G; 6.07% ± 0.3%, n
= 3, P
In an inverse pattern, the percentage of cells in G1/G0 was decreased in cells with elevated levels of G2/M. Therefore, CD200hiItga6hi 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 CD200hiItga6hiCD34– population compared with CD34+ bulge cells was similar to our cell cycle analysis demonstrating decreased G0 levels in human CD200hiItga6hi cells compared with KRT15hi bulge cells (Figure ). We conclude that both mouse and human CD200hiITGA6hi cells show evidence of cell cycle activation compared with cells of the bulge.
To further compare the mouse and human CD200hiItga6hi cells, we analyzed their global gene expression patterns using microarrays. Given that mouse CD200hiItga6hi cells were composed of both bulge and secondary hair germ cells (Figure , E and F), we compared the expression of human CD200hiItga6hi 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 H). 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 CD200hiITGA6hi 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 CD200hiITGA6hi 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 CD200hiItga6hi cells are multipotent and capable of regenerating hair follicles in a skin reconstitution assay.
We performed functional analysis on the CD200hi
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
We sorted CD200hiItga6hi or CD200–Itga6hi cells from ROSA26 reporter mice. Grafting of CD200hiItga6hi keratinocytes together with neonatal dermis successfully reconstituted hair follicles (Figure , A, B, and E). CD200–Itga6hi keratinocytes produced few hair follicles, despite injection of equal numbers of cells (Figure , C–E). Contaminating neonatal epidermis from neonatal dermal preparations contributed to some hair follicle formation in both samples (Figure , A–D), but could be distinguished by its lack of β-galactosidase activity. Histologic sectioning of reconstituted hair-bearing cysts demonstrated contribution of CD200hiItga6hi 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.
Mouse CD200hiItga6hi cells are multipotent and capable of reconstituting a hair follicle.