Human Eccrine Sweat Gland Cells Research Articles

Molecular Characterization of Aged Human Eccrine Sweat Gland Cells Using Single-Cell Transcriptomic Analysis

Molecular Characterization of Aged Human Eccrine Sweat Gland Cells Using Single-Cell Transcriptomic Analysis

Detection of fluid secretion of three-dimensional reconstructed eccrine sweat glands by magnetic resonance imaging.

We previously showed three-dimensional (3D) reconstructed eccrine sweat glands have similar structures as native eccrine sweat glands, but whether the 3D reconstructed sweat glands appropriately secrete fluid is still unknown. In this study, Matrigel-embedded human eccrine sweat gland cells or Matrigel alone were implanted into the groin subcutis of the nude mice. Ten weeks post-implantation, images of the subcutaneously formed plugs, as well as footpads of rats, pre- and post-pilocarpine/normal saline (NS) injection were acquired using a fat-suppressed proton density-weighted magnetic resonance imaging (MRI) sequence at 7.0T, and the regions of interest (ROIs) in plugs and rat footpads were analysed and graphed. A significant increase in the ROI mean proton intensity occurred in both 3D reconstructed and native eccrine sweat glands after pilocarpine injection. The mean proton intensity had no noticeable changes in ROIs of Matrigel plugs between pre- and post-pilocarpine injection, and in ROIs of rat footpads between pre- and post-NS injection. In conclusion, the 3D reconstructed sweat glands possess fluid secretion, which is detectable by fat-suppressed proton density-weighted MRI.

A novel organotypic 3D sweat gland model with physiological functionality.

Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.

Serum is an indispensable factor in the maintenance of the biological characteristics of sweat gland cells.

The tolerance of sweat gland cells for in vitro amplification and subcultivation is low as they are somatic cells. The present study aimed to formulate an optimal medium for the culture of human eccrine sweat gland cells (HESGCs) and to establish a method for induction of HESGCs proliferation, whilst maintaining the characteristics of sweat gland cells. HESGCs cultured in sweat gland (SG):keratinocyte growth medium-2 (KGM-2) (1:1) medium had a higher proliferation rate and a stable morphology compared with cells cultured in SG and KGM-2 medium only. Reverse transcription-quantitative polymerase chain reaction indicated that cells cultured in the SG:KGM-2 (1:1) medium exhibited higher expression levels of α-smooth muscle actin, keratin (K)77, carcinoembryonic antigen, K8, K18, ectodysplasin A receptor, c-Myc, Kruppel-like factor 4 and octamer-binding transcription factor 4 compared with cells cultured in SG only or KGM-2 only medium. Three-dimensional culture analysis revealed that HESGCs cultured in SG:KGM-2 1:1 medium differentiated into sweat gland-like structures, whereas cells cultured in KGM-2 only medium underwent cornification. The present study also determined that the maintenance of the biological characteristics of HESGCs occurred due to the presence of fetal bovine serum (FBS). Cells cultured in medium without FBS differentiated into keratinocytes. Therefore, the SG:KGM-2 (1:1) medium may be a suitable culture medium for HESGCs. In conclusion, this mixed medium is a valuable compound and should be considered to be a potential supplemental medium for HESGCs.

Human eccrine sweat gland cells reconstitute polarized spheroids when subcutaneously implanted with Matrigel in nude mice.

Increasing evidence indicates that maintenance of cell polarity plays a pivotal role in the regulation of glandular homeostasis and function. We examine the markers for polarity at different time points to investigate the formation of cell polarity during 3D reconstitution of eccrine sweat glands. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 2, 3, 4, 5 and 6weeks post-implantation, Matrigel plugs were removed and immunostained for basal collagen IV, lateral β-catenin, lateroapical ZO-1 and apical F-actin. The results showed that the cell polarity of the spheroids appeared in sequence. Formation of basal polarity was prior to lateral, apical and lateroapical polarity. Collagen IV was detected basally at 2weeks, β-catenin laterally and ZO-1 lateroapically at 3weeks, and F-actin apically at 4weeks post-implantation. At week 5 and week 6, the localization and the positive percentage of collagen IV, β-catenin, ZO-1 or F-actin in spheroids was similar to that in native eccrine sweat glands. We conclude that the reconstituted 3D eccrine sweat glands are functional or potentially functional.

Human Eccrine Sweat Gland Cells Turn into Melanin-Uptaking Keratinocytes in Dermo-Epidermal Skin Substitutes

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Human Eccrine Sweat Gland Cells Can Reconstitute a Stratified Epidermis

Eccrine sweat glands are generally considered to be a possible epidermal stem cell source. Here we compared the multilayered epithelia formed by epidermal keratinocytes and those formed by eccrine sweat gland cells. We demonstrated both in vitro and in vivo the capability of human eccrine sweat gland cells to form a stratified interfollicular epidermis substitute on collagen hydrogels. This is substantiated by the following findings: (1) a stratified epidermis consisting of 10-12 cell layers is formed by sweat gland cells; (2) a distinct stratum corneum develops and is maintained after transplantation onto immuno-incompetent rats; (3) proteins such as filaggrin, loricrin, involucrin, envoplakin, periplakin, and transglutaminases I and III match with the pattern of the normal human skin; (4) junctional complexes and hemidesmosomes are readily and regularly established; (5) cell proliferation in the basal layer reaches homeostatic levels; (6) the sweat gland-derived epidermis is anchored by hemidesmosomes within a well-developed basal lamina; and (7) palmo-plantar or mucosal markers are not expressed in the sweat gland-derived epidermis. These data suggest that human eccrine sweat glands are an additional source of keratinocytes that can generate a stratified epidermis. Our findings raise the question of the extent to which the human skin is repaired and/or permanently renewed by eccrine sweat gland cells.

PAR-2 receptor-induced effects on human eccrine sweat gland cells

Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells

Background and aims Steroid hormones target K + channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3. Results Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K + conductance, which is abolished by clotrimazole (30 μM), an inhibitor of the intermediate conductance calcium activated K + channel (IK Ca). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K + conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IK Ca channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IK Ca channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation. Conclusions Estradiol rapidly regulates a K + conductance through the IK Ca channel in an estrogen receptor independent manner. E2 stimulates the translocation of IK Ca to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.

PAR‐2 receptor‐induced intracellular signalling in isolated intact human eccrine sweat gland cells and primary tissue culture cells

PAR‐2 receptors can evoke intracellular signalling and anion transport in a cell line derived from human sweat glands, however, it is unknown whether PAR‐2 receptors are present and play a role in intact human sweat gland secretory cells. PAR‐2 receptors were investigated in isolated sweat glands and tissue culture cells derived from primary explants. Skin samples were obtained by informed consent and ethics approval from volunteers for immunohistochemistry and sweat gland isolation for; Western blot analysis; calcium imaging or explanted for tissue culture. Immunohistochemistry and Western blot using an anti human PAR‐2 antibody demonstrated the presence of these receptors in sweat gland secretory cells. Calcium imaging experiments using trypsin and the agonist peptide SLIGKV showed that both could increase intracellular calcium levels in intact glands and in the primary sweat gland cultures. Results also demonstrated that the effects of the agonist peptide were reversible upon washout, but not trypsin, and that the reverse peptide VKGILS failed to initiate changes in intracellular calcium. The results of this study demonstrate that PAR‐2 receptors are present in isolated human sweat gland secretory cells and in primary cultured cells and that these receptors are functionally active and can induce increases in intracellular calcium.The study was supported by Glasgow Caledonian University.

Nucleotide-evoked ion transport and [Ca 2+] i changes in normal and hyperhidrotic human sweat gland cells

Apical and basolateral application of ATP and UTP evoked [Ca 2+] i and short circuit current (Isc) increases in normal and hyperhidrotic human eccrine sweat gland cells grown into functionally polarised epithelia on permeable supports. Basolateral application to hyperhidrotic cells exhibited a markedly greater increase in Isc than in normal cells. Hyperhidrotic cells also demonstrated differences from the normal in [Ca 2+] i and Isc responses to ATP when pre-treated with thapsigargin. The data demonstrate the presence of apical and basolateral receptors that allow nucleotides to increase [Ca 2+] i and Isc. The results suggest that changes from the normal in transepithelial ion transport contribute to the characteristic excessive fluid production of hyperhidrotic sweat glands.

An outward-rectifying potassium channel in primary cultures of sweat glands from cystic fibrosis subjects

We have previously described a high conductance calcium-activated ‘maxi K’ channel in primary cultures of human eccrine sweat gland cells both from normal subjects and those with cystic fibrosis. In further studies we have now identified a potassium-selective channel of much lower conductance which shows outward-reactification and which is present in sweat glands isolated from cystic fibrosis subjects. In experiments with inside-out patches using symmetrical pipette and bath solutions containing 140 mM K + the channel showed an outward slope conductance (at +50 mV) of ≈ 26 pS and an inward conductance (at −50 mV) of ≈ 11 pS. When K + in the bath was replaced by Na + the reversal potential shifts to reveal a permeability ratio P K/ P Na ≈ 40 Unlike the maxi-K + channel, the outward-rectifying channel does not show sensitivity to Ca 2+. Channels were found in cells cultured from the glands of four out of five cystic fibrosis subjects. In cells cultured from 30 subjects who did not have cystic fibrosis, an outward-rectifying potassium channel was seen in only one out of ≈ 3000 patches.

A high-conductance Ca2+-activated K+ channel in cultured human eccrine sweat gland cells

Recordings have been made of a potassium-selective ion channel in primary cultures of cells derived from explants of human eccrine sweat glands obtained from normal subjects and from subjects suffering from cystic fibrosis. There appears to be no functional difference between potassium channels derived from normal subjects and those from cystic fibrosis subjects. The channel falls into the group generally known as "Maxi-K" channels, and has a slope conductance, with symmetrical solutions in bath and pipette containing 140 mM K+, of approximately 230 pS. It is calcium-activated, pH-sensitive and can be blocked by barium and quinine. The channel appears only very rarely in patches derived from confluent cells (in approximately 1 in 30 patches containing channels), but it is more frequently observed in younger cultures of dividing cells derived from recently explanted (within the previous 48-72 h) glands. It is possible that this channel is normally located on the basolateral membrane of the cell, and is responsible for the calcium-dependent secretory and absorptive events seen in the intact sweat gland.

Different physiological signatures of sweat gland secretory and duct cells in culture.

Human eccrine sweat gland cells grown in culture were found to lose their characteristic shape, becoming flattened and organized into multilayers. The resting membrane potentials of the cultured secretory cells (-35 +/- 2 mV, n = 36) were significantly higher than those measured for cultured duct cells (-22 +/- 1 mV, n = 58, P less than or equal to 0.01). When the cholinergic agonist methacholine (10(-5) or 10(-6) M) was administered, the cultured secretory cells could be distinguished unequivocally by their atropine-sensitive hyperpolarizing response (-20 +/- 2 mV, n = 43), whereas no cultured duct cells responded. When the sodium conductance antagonist amiloride (10(-5) or 10(-6) M) was administered, 44% of cultured secretory cells responded by hyperpolarization (-8 +/- 1 mV, n = 8), whereas 87% of cultured duct cells hyperpolarized (-15 +/- 1 mV, n = 46) and by a significantly greater margin (P less than or equal to 0.01). Substitution of chloride with gluconate in the bathing medium caused membrane potential depolarization in both cultured secretory and duct cell populations, which is consistent with the presence of a chloride conductance in the plasma membrane. The beta-adrenoceptor agonist isoproterenol induced a transient hyperpolarization of 5-10 mV in three out of six cultured secretory cells tested but had no effect on cultured duct cells.