Mature Epithelial Cells Research Articles

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro

ABSTRACT Purpose Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). Methods ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin’s effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. Results Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. Conclusions This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin’s viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

Glucuronyl C5-epimerase is crucial for epithelial cell maturation during embryonic lung development.

Glucuronyl C5-epimerase (Hsepi) is a key enzyme in the biosynthesis of heparan sulfate that is a sulfated polysaccharide expressed on the cell surface and in the extracellular matrix of alveolar walls and blood vessels. Targeted interruption of the Hsepi gene, Glce, in mice resulted in neonatal lethality, which is most likely due to lung atelectasis. In this study, we examined the potential mechanisms behind the defect in lung development. Histological analysis of the lungs from embryos revealed no difference in the morphology between wild-type and mutant animals up to E16.5. This suggests that the initial events leading to formation of the lung primordium and branching morphogenesis are not disturbed. However, the distal lung of E17.5-18.5 mutants is still populated by epithelial tubules, lacking the typical saccular structural characteristic of a normal E17.5 lung. Immunostaining revealed strong signals of surfactant protein-C, but a weaker signal of T1α in the mutant lungs in comparison to WT littermates, suggesting differentiation of type I alveolar epithelial cells (AT1) is impaired. One of the parameters contributed to the failure of AT1 maturation is reduced vascularization in the developing lungs.

Abstract A30: Single-cell RNA-sequencing analysis of estrogen and the endocrine-disrupting chemical induced mouse mammary gland reorganization after surgical menopause

Abstract Menopause is considered a critical window of susceptibility (WOS) for its sensitivity to endocrine-disrupting chemicals due to decline of endogenous estrogen. Using a surgical menopausal (ovariectomized) mouse model, we assessed how mammary gland tissue was affected by both 17β-estradiol (E2) and polybrominated diphenyl ethers (PBDEs). These endocrine-disrupting chemicals have been used as flame retardants in common household products since the 1970s and are vastly detected in human serum. During physiologically relevant exposure to E2, PBDEs enhanced E2-mediated regrowth of mammary glands with terminal end budlike structures. To better elucidate the effects of E2 and PBDEs, single-cell RNA sequencing (scRNAseq) analysis was performed. In a definitive manner, E2 was found to induce Pgr expression in both Esr1+ and Esr1- cells. We identified PBDE-impacted cell populations, Esr1+/Pgr+ and Esr1-/Pgr+ cells, in both mature luminal epithelial and progenitor cells, as well as the mammary gland population of M2 macrophages. Significantly, scRNAseq data show gene expression differences among cells with differential expression of Esr1 and Pgr. The results help clarify how exposure to both E2 and endocrine-disrupting chemicals like PBDEs after menopause impacts mammary gland structure. Ultimately, the findings advance understanding of how such exposure can increase the risk of developing breast cancer through the expansion of estrogen-responsive cells. (Supported by NIH U01ES026137.) Citation Format: Shiuan Chen, Noriko Kanaya, Gregory Chang, Xiwei Wu, Kohei Saeki, Lauren Bernal, Timothy Dynold, Susan L. Neuhausen. Single-cell RNA-sequencing analysis of estrogen and the endocrine-disrupting chemical induced mouse mammary gland reorganization after surgical menopause [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A30.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER‐Grem1 flx/flx) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7–13 days). Post‐mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Augmentation of the Conversion of 25-hydroxyvitamin D into 1,25-Dihydroxyvitamin D Specifically in Inflamed Intestines for the Treatment of Inflammatory Bowel Disease

Abstract An important pathogenic mechanism of inflammatory bowel disease (IBD) is the penetration of intestinal epithelium barrier by luminal microbes, which does not have a clinical solution. In this respect, previous data have shown that Lgr5+ intestinal stem cells (ISCs) can differentiate into all intestinal epithelial cells and that 1,25(OH)2D, i.e. the active vitamin D metabolite, is essential for the development of Lgr5+ ISCs. However, supplementation of native vitamin D, though effectively raising blood 25(OH)D levels, did not reach the expected outcomes in IBD patients. The data suggest that the conversion of 25(OH)D into 1,25(OH)2D cannot meet the demand in intestines. In addition, our recent findings support that direct systemic delivery of the requied 1,25(OH)2D levels to circumvent the insufficient conversion is impossible because of attending hyercalcemia. Here, we propose to augment such conversion specifically in inflamed intestines using engineered macrophages (MAC-CYP cells) that specifically home into inflamed intestines and overexpress 1alpha-hydroxylase (i.e. the physiological enzyme that converts 25[OH]D into 1,25[OH]2D). We showed that intraperitoneally administered MAC-CYP cells homed specifically into inflamed intestines, significantly suppressed ongoing experimental colitis without causing hypercalcemia, and augmented the migration of Lgr5+ ISCs which differentiated into mature intestinal epithelial cells under physiological and colitis conditions. In vitro, we showed that 1,25(OH)2D enhanced the survival of multipotent Lgr5+ ISCs. In conclusion, our data suggest that specific augmentation of 1alpha-hydroxylase activity in inflamed intestines is a promising therapeutic strategy for IBD.

The aged thymus is impaired in supporting T cell central tolerance

Abstract During aging, the thymus undergoes involution, decreasing dramatically in size and mass, and becoming increasingly disorganized. Notably, thymic epithelial cells decline with age, especially within the compartment of mature medullary thymic epithelial cells (mTECs) that express and present tissue-restricted antigens to thymocytes. Age-associated changes in the composition of hematopoietic antigen-presenting cells (APCs) have also been observed, but are not well characterized. Given that both mTECs and APCs present diverse self-antigens to thymocytes, we hypothesized that central tolerance is impaired in the aged thymus. Using live-cell two-photon microscopy, we determined that CD4-single positive (CD4SP) thymocytes, regardless of thymocyte age, had reduced motility and accumulation within the medulla of an aged thymus. We report age-associated changes in both the localization and composition of thymic APC subsets. To test whether this altered thymic environment functionally impacted central tolerance, we used thymic slice assays to measure tolerance induction of CD8− and CD4SP thymocytes encountering a range of model self-antigens. These assays indicated that central tolerance in the aged thymus was impaired, particularly against low-avidity auto-antigens. Our findings indicate that newly generated T cells selected in an aged thymic environment have not undergone a rigorous screening process against autoimmunity.

Thymic epithelial miR-155 promotes regulatory T cell development through safeguarding medullary thymic epithelial cell maturation

Abstract During thymocyte development, medullary thymic epithelial cells (mTECs) provide appropriate instructive cues in the thymic microenvironment for not only negative selection but also the generation of regulatory T (Treg) cells. Here, we identify that miR-155, a microRNA whose expression in Treg cells has previously been shown to be crucial for their development and homeostasis, also contributes to thymic Treg (tTreg) cell differentiation by promoting mTEC maturation. Mechanistically, we show that RANKL stimulation induces expression of miR-155 to safeguard the thymic medulla through targeting multiple known and previously uncharacterized molecules within the TGFβ signaling pathway, which is recognized for its role in restricting the maturation and expansion of mTECs. Our work uncovers a miR-155-TGFβ axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tTreg cells and suggests that miR-155 ensures proper tTreg cell development in both cell-intrinsic and -extrinsic manners.

Comparisons between Animal Models and Intestinal Crypt Cultures when Investigating the Impact of Fatty Acids on Gene Expression in Intestinal Epithelial Cells

Developmental delay of UDP‐glucuronosyltransferase 1A1 (UGT1A1) expression is central toward the development of neonatal hyperbilirubinemia, observed clinically as increased total serum bilirubin (TSB) in newborns. UGT1A1 is the sole enzyme responsible for the glucuronidation and detoxification of bilirubin. We have developed a murine model that resembles neonatal hyperbilirubinemia. In humanized UGT1 (hUGT1) mice, the expression of intestinal but not hepatic UGT1A1 is inversely associated with TSB levels. In addition, the expression of intestinal UGT1A1 is closely associated with proliferation and maturation of intestinal epithelial cells (IECs). It is known that fatty acids (FAs), the major component of breast milk, plays an important role in intestinal development. We have treated hUGT1 neonatal mice with oleic acid (OA, 16:1), a monounsaturated fatty acid. Following OA exposure, TSB levels in hUGT1 neonatal mice were reduced significantly, and RT‐qPCR analysis demonstrated that intestinal human UGT1A1 was induced, along with increased expression of Cyp4a10, a target gene of peroxisome proliferator‐activated receptor alpha (PPARα). In addition, IEC maturation markers including sucrase‐isomaltase (Sis) and alkaline phosphatase 3 (Akp3) are also induced. To examine the mechanism of OA induction, intestinal crypt cells were isolated from hUGT1 mice. The organoids were exposed to different fatty acids, including OA, palmitoleic acid (PA), arachidonic acid (AA), linoleic acid (LA), and docosahexaenoic acid (DHA). Gene expression parameters were monitored 24 hours after exposure. We found that OA‐ and PA‐treated organoids have similar survival rates in comparison to controls, but at similar concentrations of LA, DHA or AA cell death and decreased organoid viability was observed. At lower FA concentrations, RT‐qPCR analysis demonstrated that the UGT1A1 gene was not induced in any of the samples, including the IEC maturation markers Sis and Akp3. However, increased expression of the Cyp4a10 gene resulted in DHA, AA, and LA‐treated organoids. By comparing the in vivo and in vitro experiments, it suggests that unlike Cyp4a10, the regulation of UGT1A1 by fatty acids is associated with the development of the intestinal tract. Under current culture conditions, the application of intestinal organoid cultures in the study of developmental‐associated events is limited.Support or Funding InformationSupported in part by NIH grants GM126074 and ES010337

Lysophosphatidic acid enhances intestinal tuft cell differentiation in Myo5b KO mice independent of Pou2f3

We have reported that adult tamoxifen‐induced, intestine‐specific, Myosin Vb knockout (Myo5b KO) mice recapitulate malabsorption syndrome in a congenital diarrheal disease, microvillus inclusion disease, and that daily treatment with lysophosphatidic acid (LPA) stimulated brush border maturation and sodium/glucose absorption in the Myo5b KO mice. Influence of Myo5b loss and LPA in sensory cell differentiation had not been studied. The present study evaluated the global changes in gene expression following Myo5b loss and LPA treatment, focusing on the differentiation of a nutrient sensor cell lineage, tuft cell. VillinCreERT2;Myo5bflox/flox mice were treated with LPA or vehicle for 4 days after a single tamoxifen injection. Immunostaining on small intestinal sections and whole‐mounted enteroids was used to quantify tuft cell density. RNA sequencing was performed on isolated jejunal epithelial cells. The sections of entire small intestine were analyzed by whole‐slide scanning and digital analysis with Cell Profiler. The frequency of DCLK1+ tuft cell was decreased by 80% with Myo5b loss, whereas enteroendocrine cells showed no difference between Myo5b KO mice and littermate controls. Paneth cell numbers were significantly increased in Myo5b KO, correlating with hyperproliferation in crypts. LPA did not alter the increased Paneth cells or Ki67+ cells. Intraperitoneal LPA treatment re‐established the frequency of tuft cells in Myo5b KO mice, indicating that a tuft cell‐specific differentiation mechanism was activated by LPA. In contrast, enteroids that were generated from VillinCreERT2;Myo5Bflox/flox jejunum had no difference in tuft cell number between hydroxytamoxifen‐treated (iKO) and control enteroids. The disrupted tuft cell differentiation by Myo5b loss was not represented in in vitro system, although microvillus formation was immature in iKO enteroids. RNA‐seq revealed that 844 genes in jejunal epithelial cells were significantly down‐regulated, and 657 genes were up‐regulated by tamoxifen‐induced loss of Myo5b compared to control. Three stem cell transcription factors were significantly increased in KO, whereas two tuft cell‐specific transcription factors (Spib and Pou2f3) were decreased. Epithelial Myo5b loss demonstrated a broad impact on epithelial cell differentiation and maturation. Treatment of Myo5b KO mice with ip LPA up‐regulated only 9 genes and had no effect on the expression of transcription factors, suggesting that LPA may modulate transporter trafficking to stimulate nutrient absorption rather than change the transcription. Myo5b loss abolished the differentiation of a sensory tuft cell, and in vivo treatment with LPA re‐established the tuft cell population independent of Pou2f3 expression. These findings suggest unexplored functions for Myo5b and LPA signaling in cell lineage choice in the intestinal epithelial cells, interacting with non‐epithelial cells.Support or Funding InformationR01 DK48370 and R01 DK70856 and a gift from the Christine Volpe Fund to J.R.G. This work was supported by core resources of the Vanderbilt Digestive Disease Center (P30 DK058404), the Vanderbilt‐Ingram Cancer Center (P30 CA68485), VUMC Digital Histology Shared Resource (supported by a VA Shared Equipment Grant 1IS1BX003097).

Multifunctional synthetic Bowman's membrane-stromal biomimetic for corneal reconstruction

As the outermost layer of the eye, the cornea is vulnerable to physical and chemical trauma, which can result in loss of transparency and lead to corneal blindness. Given the global corneal donor shortage, there is an unmet need for biocompatible corneal substitutes that have high transparency, mechanical integrity and regenerative potentials. Herein we engineered a dual-layered collagen vitrigel containing biomimetic synthetic Bowman's membrane (sBM) and stromal layer (sSL). The sBM supported rapid epithelial cell migration, maturation and multilayer formation, and the sSL containing tissue-derived extracellular matrix (ECM) microparticles presented a biomimetic lamellar ultrastructure mimicking the native corneal stroma. The incorporation of tissue-derived microparticles in sSL layer significantly enhanced the mechanical properties and suturability of the implant without compromising the transparency after vitrification. In vivo performance of the vitrigel in a rabbit anterior lamellar keratoplasty model showed full re-epithelialization within 14 days and integration of the vitrigel with the host tissue stroma by day 30. The migrated epithelial cells formed functional multilayer with limbal stem cell marker p63 K14 expressed in the lower layer, epithelial marker K3 and K12 expressed through the layers and tight junction protein ZO-1 expressed by the multilayers. Corneal fibroblasts migrated into the implants to facilitate host/implant integration and corneal stromal regeneration. In summary, these results suggest that the multi-functional layers of this novel collagen vitrigel exhibited significantly improved biological performance as corneal substitute by harnessing a fast re-epithelialization and stromal regeneration potential.

Epidermal Delivery of Retinyl Palmitate Loaded Transfersomes: Penetration and Biodistribution Studies.

The alteration of retinoids levels in the skin can cause different disorders in the maturation of epithelial skin cells. Topical administration of these lipophilic molecules is a challenge that can be addressed by encapsulation into drug delivery systems. In this study, retinyl palmitate transferosomes formulated in cream were developed and the increases in the penetration of the active ingredients as well as the biodistribution were evaluated in vitro and in vivo. Transfersomes demonstrated a significant increase in the administration of retinyl palmitate to the epidermis by quantification of the active ingredients in the different layers of the skin, as well as by fluorescence microscopy of biopsies of non-dermatomized pig-ear skin. These results suggest that transfersomes may be an efficient vehicle for the delivery of retinoids to inner layers of the skin, such as the epidermis.

No Zones Left Behind: Democratic Hepatocytes Contribute to Liver Homeostasis and Repair.

Despite minimal turnover, liver cells possess immense regenerative capacity. Some studies suggest existence of a hepatocyte subset with such unique capabilities. However, in the current issue of Cell Stem Cell, three independent studies (Chen etal., 2020; Matsumoto etal., 2020, and Sun etal., 2020) demonstrate an equitable homeostatic and reparative potential of all hepatocytes, irrespective of their lobular location or ploidy status.

Aldosterone controls primary cilium length and cell size in renal collecting duct principal cells.

Primary cilia are nonmotile sensory organelles found on the surface of almost all kidney tubule epithelial cells. Being exposed to the tubular lumen, primary cilia are thought to be chemo- and mechanosensors of luminal composition and flux, respectively. We hypothesized that, Na+ transport and primary cilia exist in a sensory functional connection in mature renal tubule epithelial cells. Our results demonstrate that primary cilium length is reduced in mineralocorticoid receptor (MR) knockout (KO) mice in a cell autonomous manner along the aldosterone-sensitive distal nephron (ADSN) compared with wild type (as µm ± SEM; 3.1 ± 0.2 vs 4.0 ± 0.1). In mouse cortical collecting duct (mCCD)cl1 cells, which are a model of collecting duct (CD) principal cells, changes in Na+ transport intensity were found to mediate primary cilium length in response to aldosterone (as µm ± SEM: control: 2.7 ± 0.9 vs aldosterone treated: 3.8 ± 0.8). Cilium length was positively correlated with the availability of IFT88, a major intraflagellar anterograde transport complex B component, which is stabilized in response to exposure to aldosterone treatment. This suggests that the abundance of IFT88 is a regulated, rate limiting factor in the elongation of primary cilia. As previously observed in vivo, aldosterone treatment increased cell volume of cultured CD principal cells. Knockdown of IFT88 prevents ciliogenesis and inhibits the adaptive increase in cell size that was observed in response to aldosterone treatment. In conclusion, our results reveal a functional connection between Na+ transport, primary cilia, and cell size, which may play a key role in the morphological and functional adaptation of the CD to sustained changes in active Na+ reabsorption due to variations in aldosterone secretion.

Lactobacillus plantarum IS-10506 promotes renal tubular regeneration in pyelonephritic rats.

Lactobacillus plantarum IS-10506 is a novel probiotic isolated from Indonesian traditional fermented buffalo milk dadih. Probiotics are clinically proven to be effective in accelerating recovery after urinary tract infection (UTI) and in decreasing recurrent UTI in children with or without structural disorders of the urinary tract. This study aimed to investigate the role of the probiotic L. plantarum IS-10506 in activating and regenerating renal epithelial stem cells in pyelonephritic rats. Fifty-five 20-weeks-old male Sprague-Dawley rats were randomised into control, lipopolysaccharide (LPS) and treatment (LPS and probiotic) groups. Pyelonephritis was induced with Escherichia coli ATCC 25922 LPS administered via a urethral catheter on the first experimental day to the LPS and treatment groups. Microencapsulated L. plantarum IS-10506 was orally administered once daily for up to 14 days to the treatment group. On days 3, 5, 7, 10 and 14, five rats per group were sacrificed and their kidneys were immunohistochemically analysed for production of interleukin (IL)-10, lipocalin-2 and Ki-67 in the renal tubular cells. IL-10 production was upregulated starting from day 3 through day 14 in the treatment group (P<0.05) compared with that in the control and LPS groups. Moreover, treatment with the probiotic led to increased activation of renal tubular stem cell, as indicated by a higher lipocalin-2 production, and further proliferation of renal tubular stem cells was documented by a higher Ki-67 production. Taken together, these results indicate that the anti-inflammatory probiotic L. plantarum IS-10506 improves renal injury in pyelonephritic rats by activating endogenous renal tubular stem cells to proliferate into mature renal tubular epithelial cells.

Infantile colic from the perspective of evidence-based medicine: prevalence, principles of differential diagnosis and diet therapy algorithm

The article presents data on the prevalence of infant colic, etiological factors, diagnostic criteria, differential diagnosis and therapy with justification of approaches to the choice of formula in children on artificial feeding. When choosing a formula, it is necessary to take into account the compositional features aimed at overcoming the functional immaturity of the gastrointestinal tract, enteric nervous system, and the ongoing formation of the intestinal microbiome – the presence of a partially hydrolyzed protein, a reduced lactose content, the presence of prebiotics (fructooligosaccharides) and probiotics. The formula Similac Comfort is fully compliant. This formula additionally contains oligosaccharide of the breast milk 2 fucosillactose (2’-FL), 0.2 g / l – identical to that found in breast milk, which has proven positive effects in the form of an immunomodulating effect, anti-infectious effect, is involved in the regulation of growth and maturation of intestinal epithelial cells, in the development of the nervous system.

Notch Signaling in Kidney Development, Maintenance, and Disease.

Kidney development involves formation of nephrons intricately aligned with the vasculature and connected to a branched network of collecting ducts. Notch signaling plays multiple roles during kidney development involving the formation of nephrons composed of diverse epithelial cell types arranged into tubular segments, all the while maintaining a nephron progenitor niche. Here, we review the roles of Notch signaling identified from rodent kidney development and injury studies, while discussing human kidney diseases associated with aberrant Notch signaling. We also review Notch signaling requirement in maintenance of mature kidney epithelial cell states and speculate that Notch activity regulation mediates certain renal physiologic adaptations.

Temporal Regulation of Notch Signaling and Its Influence on the Differentiation of Ex Vivo Cultured Limbal Epithelial Cells

ABSTRACTPurpose: Notch signaling plays a vital role in the differentiation and proliferation of corneal epithelial cells from limbal stem cells. The temporal regulation of Notch signaling during this differentiation remains unknown. Hence, we investigated the importance of temporal activation/blockage of Notch signaling during corneal differentiation.Methods: Human limbal epithelial cultures were established with and without Notch activators (rec-Human Jagged1 Fc chimera) and pharmacological blockers (LY-411575). The modulation of Notch signaling was done at different time points during cell differentiation, which were collected on Day 14 for further analysis of differentiation, proliferation, maturation and apoptosis using RT-qPCR and immunofluorescence staining.Results: The activation of Notch signaling at Day 8 resulted in the highest number of mature corneal epithelial cells (p = .008) and pro-apoptosis marker BAX (p = .0001) with no increase in the number of corneal progenitors, and proliferation marker Ki67 compared to untreated controls. Cultures grown in the presence of Notch signaling blockers showed a significantly higher number of corneal progenitors (p = .0001) and proliferation marker Ki67 (p = .02) but lower corneal epithelial marker CK3/CK12 (p = .0007) and no difference in the pro-apoptotic marker BAX compared to untreated controls.Conclusion: During the differentiation of limbal epithelial cells to the corneal epithelial cell type, Day 8 seems to be a crucial window to modulate Notch signaling for a customized outcome.

617 Generation of immortalized sweat gland myoepithelial cells and eccrine sweat gland research model

The increased incidence of heat stroke is caused by reduced sweating as a result of failures in tissue homeostasis that occur with aging. Specifically, tissue homeostasis is disrupted by the exhaustion of stem cells. We previously revealed that myoepithelial cells are the stem cells of sweat glands, thereby heat stroke can be avoided by preventing myoepithelial cell loss. Although myoepithelial cells serve as a useful tool for revealing their depletion, only a single strain of sweat gland cells, NCL-SG3, can differentiate into mature cells in adherent culture conditions. Recently, mammary gland cells were shown to maintain a stem cell phenotype in floating spheroid culture. Similarly, we reported the culture of primary undifferentiated myoepithelial cells in spheroid culture conditions. However, these myoepithelial cells differentiated into mature epithelial cells after a few passages. Therefore, we attempted to generate immortalized undifferentiated myoepithelial cells in spheroid culture conditions. Although we first tried to transduce single sweat gland cells with lentiviral vectors carrying immortalized genes hTERT and SV40Tt, they were hardly transduced. Given that the surface (virus-cell contact) area of single cells is narrow, we directly transduced immortalized genes into spheroid architectures covered by myoepithelial cells, which expanded their surface area. Compared with primary sweat gland cells, immortalized sweat gland cells continued to form spheroids beyond the 20th passage. Furthermore, immortalized sweat gland cells co-expressed α-smooth muscle actin and keratin 14, indicating a myoepithelial phenotype. Thus, we generated immortalized myoepithelial cells, which will help to establish sweat gland evaluation systems that mimic sweat gland organs.

Role of the Microenvironment in Regulating Normal and Cancer Stem Cell Activity: Implications for Breast Cancer Progression and Therapy Response.

The epithelial cells in an adult woman’s breast tissue are continuously replaced throughout their reproductive life during pregnancy and estrus cycles. Such extensive epithelial cell turnover is governed by the primitive mammary stem cells (MaSCs) that proliferate and differentiate into bipotential and lineage-restricted progenitors that ultimately generate the mature breast epithelial cells. These cellular processes are orchestrated by tightly-regulated paracrine signals and crosstalk between breast epithelial cells and their tissue microenvironment. However, current evidence suggests that alterations to the communication between MaSCs, epithelial progenitors and their microenvironment plays an important role in breast carcinogenesis. In this article, we review the current knowledge regarding the role of the breast tissue microenvironment in regulating the special functions of normal and cancer stem cells. Understanding the crosstalk between MaSCs and their microenvironment will provide new insights into how an altered breast tissue microenvironment could contribute to breast cancer development, progression and therapy response and the implications of this for the development of novel therapeutic strategies to target cancer stem cells.