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, 8 (9), e74174

Label Retaining Cells (LRCs) With Myoepithelial Characteristic From the Proximal Acinar Region Define Stem Cells in the Sweat Gland


Label Retaining Cells (LRCs) With Myoepithelial Characteristic From the Proximal Acinar Region Define Stem Cells in the Sweat Gland

Yvonne Leung et al. PLoS One.


Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating different skin appendages.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Sweat gland LRCs are localized in the acinar gland region of SGs.
(A,B) H2BGFP is expressed in the epidermis and sweat glands before doxycycline treatment. (C,D) Sweat gland LRCs are found in the acinar gland region after 4 weeks of chase with doxycycline. (E,F) Tissue histology on sections with H&E staining. Abbreviations: LRCs, label-retaining cells; H2BGFP, histone 2B conjugated with green fluorescent protein; H&E, hematoxylin and eosin staining.
Figure 2
Figure 2. Sweat gland LRCs are attached to the basement membrane and possess myoepithelial characteristics.
(A) Sweat gland LRCs are attached to the basement membrane positive for β4 integrin (red) and are (B) found in the basal layer co-localizing with K14. (C,D) Sweat gland LRCs do not express lumenal layer markers K8 and K18, respectively. (E) Within the basal layer, LRCs co-localize with myoepithelial cell marker p63, inset denotes p63 single channel, arrows and (F) with myoepithelial cell marker smooth muscle actin (SMA), arrows. (G,H) Whole mount staining of SGs with laminin with and without DAPI. Arrows denote co-localization of markers with H2BGFP marked LRCs. Abbreviations: LRCs, label-retaining cells; H2BGFP, histone 2B conjugated with green fluorescent protein; SMA, smooth muscle actin; K, keratin.
Figure 3
Figure 3. Sweat gland LRCs express Keratin 15 and contribute long term to the acinar SG structure.
(A) K15 staining of sweat gland LRCs indicate positive K15 expression. (B) Fluorescent photo of K15CrePR/R26eYFPRU palm containing YFP positive sweat glands after long term YFP activation. (C) Section of K15CrePR/R26tdTomRU crossed onto K5TetOff/TreH2BGFP sweat glands after 4 weeks of chase with doxycycline followed by 2 days of RU treatment. (D) K14 basal layer staining co-localizes with GFP expression in K15-GFP transgenic sweat glands. (E) K18 lumenal marker staining co-localizes with K15-GFP expression in sweat glands. (F) FACS analysis of K15-GFP sweat glands demonstrates that approximately half of the K15-GFP positive cells are localized to the basal layer expressing α6 integrin. (G) Histology of X-Gal-treated K15CrePR/R26LacZ transgenic mice, blue stain indicates transgene expression for more than 6 months after RU activation in sweat glands. (H) K15 expression co-localizes with LRCs in the acinar sweat gland region.
Figure 4
Figure 4. Isolation strategy for sweat gland LRCs.
(A) Whole K5TetOff/TreH2BGFP toe tip under the stereomicroscope, dotted yellow line marks sweat gland dissection. (B) Dissected sweat glands with surrounding sole’s epidermis. (C) Separation of sweat glands from attached sole’s epidermis after collagenase treatment. (D) A series of further enzymatic digestions result in a single cells suspension of sweat gland cells for FACS. (E) Sweat gland cells sorted for H2BGFP and α6 integrin-PE double positive cells as well as α6 integrin-PE single positive surrounding basal cells.
Figure 5
Figure 5. Molecular characteristics of sweat gland LRCs define BMP signaling as a requirement for SG formation.
(A) GFP+/α6+ sweat gland LRCs and GFP−/α6+ sweat gland non-LRCs basal cells were compared to the basal layer of the sole’s epidermis. The resulting gene expression profiles of these sweat gland LRCs and basal cells were then compared to each other. (B) Gene expression profiles consistently found in two independent microarray analyses from independent biological samples were categorized into ion and protein transport, signaling, transcription, extracellular matrix (ECM) and cell adhesion based on function. (C) Sodium Potassium ATPases were confirmed to be expressed in the sweat glands. (D) Gja1 is confirmed to be expressed in SG LRCs (arrows) as well as non-LRCs. (E) Phospho-Smad2 co-localizes with SG LRCs, arrows. (F) Corresponding phospho-Smad2 and K8 channels. Arrows indicate LRCs marked in panel “E”. (G) Positive phospho-smad1/5/8 staining indicates active BMP signaling in sweat glands. (H) Corresponding phospho-Smad1/5/8 and K8 channels with arrows indicating co-localization with some LRCs. (I) Downgrowth of sweat glands is observed in P1 control paws but is absent (J) in Bmpr1a/K14Cre/K14-H2BGFP KO paws. (K) Similarly, more developed sweat glands are observed in P8 control paws but are still absent (L) in KO mice. (M) The basal layer of the epidermis as well as the sweat glands is proliferative at P1. (N) Although sweat glands are absent in Bmpr1a/K14Cre/K14-H2BGFP KO paws, the epidermal basal layer is still capable of division.
Figure 6
Figure 6. Acinar sweat gland cells do not contribute to the epidermis during typical wound healing.
(A) K15CrePR/R26LacZRU marked sweat gland cells do not contribute to the epidermis at 24 h, (B) 48 h, and (C) 72 h after wounding. (D) BrdU pulse shows that a few SG cells are activated upon injury (inset, arrows) while most SG LRCs remain quiescent. (E) Ki67 staining confirms that the acinar sweat gland region is quiescent while the SG duct and epidermal basal layer is proliferative at 24 h and (F) 48 h. (G) Under normal homeostasis, cells of the SG duct and epidermal basal layer are active in the cell cycle. (H) Corresponding single Ki67 (red) channel. Abbreviations: du, sweat ducts.
Figure 7
Figure 7. Sweat gland LRCs can trans-differentiate into the epidermis under prolonged isolated wound healing conditions.
(A) DIC photo of the back skin area containing transplanted H2BGFP labeled sweat glands at 34 days. (B) Corresponding fluorescent image displaying the presence of H2BGFP positive cells in the grafted area. Arrows indicate regions with H2BGFP positive cells while “*” marks autofluorescence of the wounded area. (C) Biopsy was taken at 38 days where H2BGFP sweat glands were observed in the dermis, arrow. (D) Higher exposure time for the GFP channel on the same section from “C” detected marked cells with lower H2BGFP intensities in the epidermis. (E,I) K5 basal layer staining demonstrating the contribution of sweat gland cells to the newly formed epidermis at 38 and 46 days, respectively. (F,J) These H2BGFP labeled cells are still able to proliferate as indicated by Ki67 staining. (G,K) Sweat gland cells can differentiate into cells of the suprabasal layer marked by K1 at 38 and 46 days, respectively. (H,L) These sweat gland cells can also contribute to the granular layer as marked by loricrin. (M,N) When 4 weeks chased sweat glands are transplanted and kept on doxycycline treatment, the H2BGFP label gets diluted out of some sweat glands (arrows), as confirmed by K5 and K8 staining for sweat glands. (O) Ki67 staining show that these sweat glands lacking visible H2BGFP LRCs (green - nuclear) defined by K5 positive staining (green membrane staining) contains Ki67 positive dividing cells (arrows). (P) H2BGFP sweat gland LRCs can contribute to the newly formed epidermis as indicated by K5 basal layer staining (arrows). (Q) Some sweat glands are connected to the newly formed epidermis lacking visible H2BGFP LRCs. (R) Sweat gland LRCs contributing to the epidermis are proliferative near the CD104 marked epidermal basal layer as marked by Ki67, inset shows magnification of co-localization. (S) Sweat gland LRCs can contribute to the suprabasal layer marked by K1 as well as the (T) granular layer expressing loricrin. White dotted lines mark dermal-epidermal interfaces. Abbreviations: CD104, β4 integrin.
Figure 8
Figure 8. Dissociated sweat gland cells can regenerate sweat glands, hair follicles, and the epidermis.
(A) Chamber graft of K5TetOff/TreH2BGFP dissociated 4 weeks chased sweat gland cells mixed with unmarked newborn dermal fibroblasts yield GFP positive hair-like fibers at 29 days. (B) Section through graft confirms the presence of H2BGFP positive hair follicles. (C) K5 staining marks the outer root sheath of this H2BGFP+ hair follicle. (C’) AE15 stains the inner root sheath and medulla, arrows. (C”) AE13 stains the hair shaft. (D) 70 days after transplantation, H2BGFP positive sweat gland structures were found, as confirmed by Na+/K+ ATPase expression (inset), in addition to H2BGFP positive hair follicle. (E) Magnification of the hair follicle in “D” with (E’) GFP single channel. (F) The sweat gland structures found also expressed K5 basal and K8 lumenal layer markers. (G) H2BGFP positive cells were also found in the K5 basal layer, (H) K1 suprabasal layer, and (I) loricrin marked granular layer of the newly regenerated epidermis. (J,K) In an independent experiment, 39 days after subcutaneous injections of 4 weeks chased unsorted dissociated cells from H2BGFP labeled SGs with unmarked newborn dermal fibroblasts, a cluster of GFP positive cells containing hair follicles were observed. (L) Magnification of a GFP+ hair follicle from panel “K”. (M) Sections show the presence of H2BGFP labeled SG structures expressing K8 lumenal layer marker. (N) H2BGFP positive cells are again also found in the basal, suprabasal, and (O) granular layers of the epidermis. White dotted lines mark dermal-epidermal interfaces.

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