03-12-2010, 07:59 PM
03-13-2010, 10:19 AM
In the adult skin, interfollicular epidermis (IFE) and sebaceous glands (SGs) are subject to constant self-renewal, whereas hair follicles (HFs) cycle between growth, involution, and resting phases (fig. S1) (1). Under normal conditions, these three skin cell populations are each believed to be maintained by their own discrete stem cells (2). When tissue homeostasis is disrupted, however, any of the three stem cell populations is capable of producing all three structures (2, 3). The IFE can be maintained without the recruitment of stem cells from the HF bulge (4–8), yet the exact identification of IFE stem cells has remained elusive. Within the SG, progenitors reportedly maintain this structure independent of the HF (5, 9). HF stem cells reside in the bulge, express CD34 and cytokeratin 15 (10–12), and retain DNA or histone labels (13–15). However, stem cells may reside in other areas of the HF as well (16–19).
We recently identified Lgr5 [leucine-rich repeat–containing G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor 5] as a marker of cycling stem cells in the intestine (20). Subsequently, we demonstrated that Lgr5 marks HF stem cells, which over very long periods of time contribute to all hair lineages but not to the SG or IFE (21). A closely related gene exists in the mammalian genome, Lgr6 (22). To evaluate a potential involvement of Lgr6 in stem cell biology, we obtained LacZ- and EGFP-Ires-CreERT2 (where EGFP is enhanced green fluorescent protein and Ires is internal ribosomal entry site) knock-in alleles (23) (figs. S1 and S2). Both integrations create null alleles. Homozygous mice of both strains were healthy and fertile. In adult Lgr6LacZ and EGFP-Ires-CreERT2 knock-in mice, we noticed prominent expression in rare cells in brain, mammary gland, lung, and skin. In the latter tissue, in situ hybridization confirmed the pattern observed with the knock-in alleles (Fig. 1 and figs. S1 to S3). Lgr6 was first observed around embryonic day 14.5 (E14.5) (Fig. 1A). Expression was evident throughout the epithelial compartment of placodes, whereas the epidermis was entirely negative (Fig. 1B). Lgr6 is thus one of the earliest placode markers, resembling Sonic Hedgehog (24) and Sox9 (25). Lgr6 expression persisted during hair peg development (Fig. 1C and fig. S2C). The resulting hair breaks through the overlying epidermis postnatally. Lgr6+ cells appeared in the IFE coincident with the emergence of hair (Fig. 1D and fig. S2D), suggesting an origin in the developing follicles. Epidermal Lgr6 expression peaked around postnatal day 7 to 15 (P7 to P15) and then became gradually more restricted, with expression persisting within adult HFs on the back and tail throughout life (Fig. 1, E and F, and fig. S2, E to I).
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[I] Fig. 1. Lgr6 is expressed in early hair progenitor cells and becomes restricted to a limited number of cells at the central isthmus. (A) Whole-mount picture of a Lgr6-LacZ embryo at E14.5. Scale bar indicates 500 Ám. (B to F) Cross sections of dorsal skin from Lgr6-LacZ knock-in mice obtained at various developmental stages (E14.5, P1, P7, P20, and P37, respectively) reveal restricted Lgr6 expression (blue) above the bulge. Scale bars, 50 Ám. Confocal microscopy reveals limited overlap with known hair follicle stem cell markers (in red) CD34 (G), Mts24 (H), and Lrig1 (I) in Lgr6-EGFP-Ires-CreERT2 mice analyzed at telogen stages. Scale bars, 25 Ám. Bu, bulge; Sg, sebaceous gland; and UI, upper isthmus.
Detailed analysis in the first (P20) and second (P56) resting states (telogen) revealed that Lgr6 marked a unique population, located directly above the CD34 and keratin 15–positive bulge (Figs. 1G and 2A and fig. S1). Lgr6 cells did not retain the DNA label 5-bromo-2'-deoxyuridine (BrdU) (fig. S4). MTS24 and Lrig1 (upper-isthmus markers) (17, 19) and Blimp1 (SG) (9) showed limited overlap with the tight Lgr6 cell cluster (Figs. 1, H and I, and 2A and fig. S1). Analysis of LacZ staining in telogen follicles of Lgr4 (26), Lgr5 (20), and Lgr6 LacZ knock-in mice confirmed that Lgr6 marked the central isthmus directly above the bulge, whereas Lgr4 expression was present in both the Lgr5+ and the Lgr6+ domains (Fig. 2B).
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[I]Fig. 2. Lgr6 marks a different stem cell population than Lgr5/CD34+ HF stem cells. (A) Fluorescence-activated cell sorting (FACS) analysis at first telogen reveals that Lgr6+ cells are largely distinct from CD34+ cells and MTS24+ cells. WT, wild type. (B) Expression analysis of Lgr family members illustrates that Lgr5 HF stem cells are located at the bulge (21), whereas Lgr4 has a wider expression pattern, including the tight cluster of Lgr6+ stem cells at the central isthmus. Scale bar, 50 Ám. CI, central isthmus; HG, hair germ; and DP, dermal papilla. (C) Gene expression analysis of Lgr5+ HF stem cells and Lgr6+ stem cells further indicates that Lgr6 marks a separate population with no overlap of bulge HF stem cells. Color scale bar represents log2 differences.
In agreement with our findings, gene expression profiles of late embryonic (E17.5) HF stem cells revealed Lgr5 and Lgr6 at the top of the enriched-gene list (27). We directly compared gene expression profiles of sorted Lgr5high and Lgr6high cells isolated from P20 dorsal skin. As expected, the Lgr5 population was strongly enriched for bulge markers such as CD34 (Fig. 2C). The only gene in the Lgr6 profile implicated in stem cell biology and HF development was Tnfrsf19/Troy (28, 29). Another gene, Il1r2, marks cells at a corresponding position below the SGs in human HFs (30). Thus, Lgr6 marked a unique, tight cell cluster at the central isthmus of the HF (Fig. 2B). Of note, although embryonic expression in nascent whiskers resembled that of other hair follicle types, no Lgr6+ zone was established postnatally at the equivalent location (fig. S9).
To study lineage relationships of Lgr6+ cells, we intercrossed Lgr6-EGFP-Ires-CreERT2 with the Cre reporter R26R-LacZ mice. Without tamoxifen, we essentially noted no leakiness of Cre activity. Single tamoxifen injections facilitated genetic tracing of Lgr6+ cells and their offspring. We first genetically marked Lgr6+ cells at E17.5, when Lgr6 expression is restricted to hair pegs (fig. S5A). Subsequent postnatal LacZ stainings were performed at various phases of the hair cycle. In all cases, widespread labeling of all three skin compartments was observed (Fig. 3B and fig. S5).
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[I]Fig. 3. After hair morphogenesis, Lgr6+ stem cells predominantly generate SGs and epidermis. Scale bars histochemistry (HC), 50 Ám. (A) LacZ staining (arrow) in dorsal skin, first visible after 3 days of tracing. (B) Quantification of lineage tracing from Lgr6 stem cells initiated at E17.5, P20, and P56, respectively. (Left) In postnatal mice, the vast majority of lineage tracings (~90%) originate in the isthmus. (Right) Tracing events remain constant over time and Lgr6 stem cells persistently generate IFE and SG, whereas HF potential diminishes with age of the mice. Error bars indicate standard deviation. (C to E) LacZ analysis of dorsal skin from Lgr6-EGFP-Ires-CreERT2/R26R-LacZ mice after CreERT2 induction at P20. Analysis during anagen at P38 [(C) and (D)] or after >1 year (E) with whole-mount microscopy or HC, respectively. Lgr6+ stem cells persistently trace toward epidermal [(D) and (E), upper left images], SG lineages [(D) and (E), lower left images], and occasional HF [(D) and (E), right images]. (F) HC analysis of transplanted Lgr6+/LacZ+ stem cells onto backs of nude mice confirmed multipotency.
When lineage tracing was induced at P20, sporadic single LacZ-labeled cells first became visible at P23 (Fig. 3A). The overwhelming majority of labeled cells still appeared at the isthmus, implying limited mobility in the intervening 3-day period (Fig. 3B). When analyzed 18 days after induction, blue clones were observed in SGs, the IFE, and, to a lesser extent, in the hair (Fig. 3, C and D). Even after >1 year, extensive lineage tracing was readily observed (Fig. 3E and fig. S6). Tracing induced at P56, the second telogen phase, yielded identical observations, albeit HF potential was further diminished (Fig. 3B and fig. S7). Quantification of lineage tracing initiated at E17.5, P20, or P56 underscored that, in virtually all cases, labeling was restricted to single cells in the isthmus 3 days after induction (Fig. 3B). Contribution to SG and IFE was relatively constant between E17.5, P20, and P56, whereas the contribution to the hair decreased with age (Fig. 3B).
In order to further document the stemness potential, we transplanted Lgr6+ stem cells, isolated at first telogen, onto the backs of nude mice. As expected, Lgr6+ cells reconstituted fully formed HFs. Multipotency of donor stem cells was confirmed by activating the R26R-LacZ locus in vivo 4 days before isolation. A small subset of Lgr6+ stem cells became LacZ-positive and contributed, once transplanted, to all skin lineages (Fig. 3F and fig. S6F).
The contribution of Lgr6+ cells to wound repair was assessed by inducing lineage tracing at first telogen (P20), followed by excision of 1 cm2 of full-thickness back skin 5 days later. Lgr6 progeny was traced over >3 months after wounding. As observed previously when bulge stem cells were LacZ-labeled (6), convergent bands of blue cells emanated from the border of the wound and migrated toward its center (Fig. 4, A to D). Such bands originating from HF bulge stem cells disappear by 20 days postwounding (6). The blue clones derived from Lgr6+ cells involved cells in the basal layer of the wound epithelium (Fig. 4, E and F), whereas the clones persisted for >3 months within the newly formed epidermis. As reported by Cotsarelis and colleagues (31), HF growth occurred de novo within the wound epithelium. When scored in a 60- and 100-days postwounding mouse, about 10% of these new HFs were derived from LacZ-marked Lgr6+ stem cells (3 in 34 and 4 in 31, respectively), comparable to the estimated percentage of surface area comprising LacZ-marked keratinocytes in the same wounds (7% and 11%, respectively) (Fig. 4, G and H, and fig. S8).
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[I]Fig. 4. Lgr6+ stem cells permanently contribute to wound healing, including hair neogenesis. (A) Top view of a wound in dorsal skin 2 days postwounding (dpw). White dashed line marks edge of the wound. Incision was made at day P25, 5 days after tracing initiation. Right image is magnification of the area marked by the white box. (B) Cross section of the wound reveals marked progeny migrating into the wound. Black arrowhead points to the edge of the wound. Scale bars IHC, 50 Ám; Ow, open wound. (C and D) As in (A) and (B), dorsal wound 7 dpw. (E and F) As in (A) and (B), 49 dpw Lgr6+ stem cells made persistent contributions. Ki67+ basal layer of scar tissue is Lgr6-derived (black arrows). (G and H) As in (A) and (B). After >100 dpw, Lgr6 progeny is still present within the wound. Moreover, newly formed hairs within the wound are occasionally LacZ positive.
Our study identifies Lgr6 as a marker for a distinct population of stem cells giving rise to all lineages of the skin. Unlike the Lgr5 gene, we found no evidence that Lgr6 is controlled by Wnt signaling. This is in agreement with the notion that the active hair lineage in the lower bulge requires Wnt signaling, whereas the sebaceous and epidermal lineages are Wnt-independent (2). A picture thus emerges in which a Wnt-independent Lgr6 stem cell pool can renew sebaceous cells and seed the epidermis throughout life, whereas a Wnt-dependent Lgr5 stem cell pool derives from the Lgr6 pool early in life but then becomes relatively independent.
03-13-2010, 09:05 PM
This is great... but, the timeline of "discovering" the stem cell to actual fruition of a commercially viable product would be decades, in the least.