Transient-amplifying Cells Research Articles

The Potential Reversible Transition between Stem Cells and Transient-Amplifying Cells: The Limbal Epithelial Stem Cell Perspective.

Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs.

The Hippo-YAP/TAZ Signaling Pathway in Intestinal Self-Renewal and Regeneration After Injury.

The Hippo pathway and its downstream effectors, the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), control stem cell fate and cell proliferation and differentiation and are essential for tissue self-renewal and regeneration. YAP/TAZ are the core components of the Hippo pathway and they coregulate transcription when localized in the nucleus. The intestinal epithelium undergoes well-regulated self-renewal and regeneration programs to maintain the structural and functional integrity of the epithelial barrier. This prevents luminal pathogen attack, and facilitates daily nutrient absorption and immune balance. Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation of the entire digestive tract. Impaired mucosal healing is a prominent biological feature of IBD. Intestinal self-renewal is primarily dependent on functional intestinal stem cells (ISCs), especially Lgr5+ crypt base columnar (CBC) cells and transient-amplifying (TA) cells in the crypt base. However, intestinal wound healing is a complicated process that is often associated with epithelial cells, and mesenchymal and immune cells in the mucosal microenvironment. Upon intestinal injury, nonproliferative cells rapidly migrate towards the wound bed to reseal the damaged epithelium, which is followed by cell proliferation and differentiation. YAP is generally localized in the nucleus of Lgr5+ CBC cells, where it transcriptionally regulates the expression of the ISC marker Lgr5 and plays an important role in intestinal self-renewal. YAP/TAZ are the primary mechanical sensors of the cellular microenvironment. Their functions include expanding progenitor and stem cell populations, reprogramming differentiated cells into a primitive state, and mediating the regenerative function of reserve stem cells. Thus, YAP/TAZ play extremely crucial roles in epithelial repair after damage. This review provides an overview of the Hippo–YAP/TAZ signaling pathway and the processes of intestinal self-renewal and regeneration. In particular, we summarize the roles of YAP/TAZ in the phases of intestinal self-renewal and regeneration to suggest a potential strategy for IBD treatment.

A2 MUCUS DEFICIENCY IMPACTS INTESTINAL EPITHELIAL CELL PROLIFERATION INDEPENDENT OF THE MICROBIOTA

BackgroundIntestinal homeostasis is highly dependent on the proliferation and differentiation of intestinal epithelial cells (IEC). IEC arise from intestinal stem cells (ISCs) that reside at the bottom of intestinal crypts. Following proliferation, the IEC migrate up as transient amplifying (TA) cells, and differentiate into mature IEC subtypes. When this process is disrupted, it can lead to aberrant IEC proliferation and differentiation. Mucus production by secretory goblet cells is also crucial for intestinal homeostasis, as mucus separates the IEC from luminal microbiota. Surprisingly, mice lacking Muc2, the main protein component of mucus, display increased distal colonic IEC proliferation and crypt hyperplasia at baseline, suggesting a relationship between Muc2 production and IEC proliferation.AimsWe investigated how mucus production impacts IEC proliferation and differentiation in the intestinal crypt.MethodsWe used wildtype (Muc2+/+) and Muc2 deficient (Muc2-/-) littermates to measure distal colon crypt length and IEC proliferation pattern via microscopy. Organoids were also derived from distal colons of Muc2+/+ and Muc2-/- mice, and quantified for size, density and proliferation for 7 days to test whether the hyper-proliferation phenotype was also seen in vitro, thus epithelial-intrinsic. Crypts and organoids were collected for RNA sequencing to examine changes in IEC proliferation pathways. Proliferation assessments were repeated in germ-free (GF) Muc2+/+ and Muc2-/- mice. Muc2-/- mice were also cross-bred with Lgr5-EGFP-IRES-CreERT2 mice to investigate the contribution of ISCs to IEC hyperproliferation resulting from Muc2 deficiency.ResultsSignificant crypt hyperplasia was observed in the distal colons of Muc2-/- mice in concert with a > twofold increase in Ki67+ TA cells as compared to Muc2+/+ mice. Similarly, Muc2-/- organoids also displayed significantly greater size, density and an increased number of Ki67+ cells than Muc2+/+ organoids. Hyperproliferation was also seen in GF mice and organoids, suggesting that mucus impacts IEC proliferation independent of the microbiome. Muc2-/- Lgr5-EGFP+ mice showed no significant increase in numbers of Lgr5+ cells, indicating that Muc2 deficiency does not directly impact ISC number, but rather their proliferation and differentiation reflected in increased numbers of TA cells. RNA-sequencing results suggested that changes in lipid metabolism may underlie the increased IEC proliferation seen in Muc2-/- mice.ConclusionsMucus not only promotes IEC homeostasis by separating luminal bacteria from the intestinal epithelium, but also intrinsically modifies IEC proliferation independent of the microbiota. Taken together, our results emphasize the importance of mucus in controlling gut health through mechanisms independent of its role in barrier function.Funding AgenciesCCC, CIHRC.H.I.L.D Foundation

An Anhydrous Sodium Chloride Skin Preservation Model for Studies on Keratinocytes Grafting into the Wounds.

Background. Human skin is needed for covering large body areas lost by trauma. The shortcomings of contemporary methods of skin storage are limited preservation time and high immunogenicity if allogeneic. Methods. We investigated whether long-lasting skin preservation in anhydrous sodium chloride (NaCl) may be the source of keratinocytes (KCs) for transplantation. Dehydrated skin fragments were preserved for a time frame from 1 week to 12 months. Then, skin fragments were rehydrated, and KCs were isolated. The viability of KCs was assessed in viability/cytotoxicity test. NaCl-preserved KCs were cultured for 7 days and transplanted to the dorsum of SCID mice. Results. The morphology of NaCl-preserved KCs was unaltered. KCs from all epidermal layers could be identified. All grafts were accepted by the recipients. Transplanted KCs: synthesized keratins 10 and 16 expressed antigens specific for stem cells and transient-amplifying cells, and remained HLA-I-positive. Moreover, they expressed the proliferative marker PCNA. Cells isolated from transplants remained viable and produced enzymes. Conclusions. Transplantation of KCs obtained from human skin and stored in anhydrous NaCl may be considered for the closure of extensive skin wounds. The originality of this method consists of an effective storage procedure and easy preparation of keratinocytes for transplantation.

Integrin αEβ7+ T cells direct intestinal stem cell fate decisions via adhesion signaling.

Intestinal stem cell (ISC) differentiation is regulated precisely by a niche in the crypt, where lymphocytes may interact with stem and transient amplifying (TA) cells. However, whether and how lymphocyte-stem/TA cell contact affects ISC differentiation is largely unknown. Here, we uncover a novel role of T cell-stem/TA cell contact in ISC fate decisions. We show that intestinal lymphocyte depletion results in skewed ISC differentiation in mice, which can be rescued by T cell transfer. Mechanistically, integrin αEβ7 expressed on T cells binds to E-cadherin on ISCs and TA cells, triggering E-cadherin endocytosis and the consequent Wnt and Notch signaling alterations. Blocking αEβ7-E-cadherin adhesion suppresses Wnt signaling and promotes Notch signaling in ISCs and TA cells, leading to defective ISC differentiation. Thus, αEβ7+ T cells regulate ISC differentiation at single-cell level through cell-cell contact-mediated αEβ7-E-cadherin adhesion signaling, highlighting a critical role of the T cell-stem/TA cell contact in maintaining intestinal homeostasis.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Senescent Progenitor Cells in the Skin of Patients with Cutaneous Lupus Erythematosus

Senescent Progenitor Cells in the Skin of Patients with Cutaneous Lupus Erythematosus

Single-cell transcriptome analysis reveals differential nutrient absorption functions in human intestine.

The intestine plays an important role in nutrient digestion and absorption, microbe defense, and hormone secretion. Although major cell types have been identified in the mouse intestinal epithelium, cell type-specific markers and functional assignments are largely unavailable for human intestine. Here, our single-cell RNA-seq analyses of 14,537 epithelial cells from human ileum, colon, and rectum reveal different nutrient absorption preferences in the small and large intestine, suggest the existence of Paneth-like cells in the large intestine, and identify potential new marker genes for human transient-amplifying cells and goblet cells. We have validated some of these insights by quantitative PCR, immunofluorescence, and functional analyses. Furthermore, we show both common and differential features of the cellular landscapes between the human and mouse ilea. Therefore, our data provide the basis for detailed characterization of human intestine cell constitution and functions, which would be helpful for a better understanding of human intestine disorders, such as inflammatory bowel disease and intestinal tumorigenesis.

In vitro endothelial cell migration from limbal edge-modified Quarter-DMEK grafts

Endothelial cell migration plays a crucial role in achieving corneal clearance after corneal transplantation when using smaller-sized endothelial grafts to increase the donor pool. In this study we investigated how different strategies of Quarter-Descemet Membrane Endothelial Keratoplasty (Quarter-DMEK) limbal graft edge modification influence peripheral endothelial cell migration in an in vitro culture environment. For this study, 15 Quarter-DMEK grafts, prepared from 7 corneas deemed ineligible for transplantation but with intact and viable endothelial cells, were embedded in a cooled biocompatible, thermoresponsive matrix for culture. The limbal edge of ten Quarter-DMEK grafts were modified, either by using a small diameter punch or by peripheral radial cuts. All Quarter-DMEK grafts showed substantial collective endothelial cell migration from the radial cut graft edges, as observed by light microscopy at standardized time intervals. Grafts were retrieved from the polymer matrix after the two-week culture for immunohistochemistry analyses of the newly formed cell monolayers; this demonstrated the presence of tightly packed and viable cells that showed higher migratory ability at the leading edge. Peripheral endothelial cell migration, however, was not triggered by increasing cell exposure to free space through surgical modifications of the far periphery. Our data suggest that alterations in the far peripheral area of Quarter-DMEK grafts were insufficient to triggering cell migration from the limbal graft edge. This may be due to transient-amplifying cells that reside in the far periphery and which lack cytokinetic directional cues. Understanding the migration capacity of the peripheral endothelium could unlock cells’ therapeutic potential which are, at present, routinely discarded from transplantation. Encouraging peripheral cell migration may also improve clinical outcomes from Quarter-DMEK, but a more effective solution is required prior to clinical implementation of modified grafts.

Chromatin-Remodelling Complex NURF Is Essential for Differentiation of Adult Melanocyte Stem Cells.

MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. We show that MITF associates the NURF chromatin-remodelling factor in melanoma cells. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf-mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes.

The EGFR-HER2 module: a stem cell approach to understanding a prime target and driver of solid tumors.

The epidermal growth factor receptor (EGFR) and a co-receptor denoted HER2/ERBB2, are frequently overexpressed or mutated in solid tumors, such as carcinomas and gliomas. In line with driver roles, cancer drugs intercepting EGFR or HER2 currently outnumber therapies targeting other hubs of signal transduction. To explain the roles for EGFR and HER2 as prime drivers and targets, we take lessons from invertebrates and refer to homeostatic regulation of several mammalian tissues. The model we infer ascribes to the EGFR-HER2 module pivotal functions in rapid clonal expansion of progenitors called transient amplifying cells (TACs). Accordingly, TACs of tumors suffer from replication stress, hence accumulate mutations. In addition, several lines of evidence propose that in response to EGF and related mitogens, TACs might undergo de-differentiation into tissue stem cells, which might enable entry of oncogenic mutations into the stem cell compartment. According to this view, antibodies or kinase inhibitors targeting EGFR-HER2 effectively retard some solid tumors because they arrest mutation-enriched TACs and possibly inhibit their dedifferentiation. Deeper understanding of the EGFR-HER2 module and relations between cancer stem cells and TACs will enhance our ability to control a broad spectrum of human malignancies.

Tic-TACs: Refreshing Hair Growth

Although stem cells are subject to niche control, evidence is emerging that they also contribute to generating the niche through their offspring. Using the hair follicle as a model, Hsu at al. demonstrate that the transient-amplifying cells, downstream of stem cells and well-known cell producers, signal back to stem cells to maintain long-term regenerative capacity.

Expression of α-taxilin in the murine gastrointestinal tract: potential implication in cell proliferation

α-Taxilin, a binding partner of the syntaxin family, is a candidate tumor marker. To gain insight into the physiological role of α-taxilin in normal tissues, we examined α-taxilin expression by Western blot and performed immunochemical analysis in the murine gastrointestinal tract where cell renewal vigorously occurs. α-Taxilin was expressed in the majority of the gastrointestinal tract and was prominently expressed in epithelial cells positive for Ki-67, a marker of actively proliferating cells. In the small intestine, α-taxilin was expressed in transient-amplifying cells and crypt base columnar cells intercalated among Paneth cells. In the corpus and antrum of the stomach, α-taxilin was expressed in cells localized in the lower pit and at the gland, respectively, but not in parietal or zymogenic cells. During development of the small intestine, α-taxilin was expressed in Ki-67-positive regions. Inhibition of cell proliferation by suppression of the Notch cascade using a γ-secretase inhibitor led to a decrease in α-taxilin- and Ki-67-positive cells in the stomach. These results suggest that expression of α-taxilin is regulated in parallel with cell proliferation in the murine gastrointestinal tract.

A stem cell proliferation burst forms new layers of P63 expressing suprabasal cells during zebrafish postembryonic epidermal development

SummaryOrgan growth during development is a highly regulated process with both temporal and spatial constraints. Epidermal stratification is essential for skin growth and development. Although the zebrafish has been well studied, it is not known when and how epidermal stratification occurs. This is because beyond the first five days of development our knowledge is currently limited. We found that epidermal stratification in zebrafish begins when the larvae reach a standard length (SL) of 6 mm at approximately 25 days of age. Over the next four days (from a SL of 6 to 9 mm), epidermis thickness increases almost four-fold. This represents a sudden increase in organ size, since for the previous 20 days of development, the epidermis has been only two layers thick. This pattern is different from that observed in mammals that undergo continuous stratification from E14.5–E18.5. To study how stem cell proliferation gives rise to the new epidermal layers, we used a combination of markers: one for cell proliferation (proliferating cell nuclear-antigen PCNA) and one for epidermal stem cells (P63 transcription factor). We identified, throughout the stratification process, two different waves of cell division. Initially, the most basal epidermal cells divided and generated a subset of suprabasal cells (possibly transient-amplifying cells); within the next several days, the basal cells stopped dividing, and the suprabasal cells began proliferation, giving rise to most of the cell types in the new layers. This part of the process is similar to what has been recently found during epidermal stratification in mammals.

Polarized distribution of AMPA, but not GABAA, receptors in radial glia‐like cells of the adult dentate gyrus

Glial fibrillary acidic protein (GFAP)-positive astrocytes with radial processes [radial glia (RG)-like cells] in the postnatal dentate gyrus share many of the characteristics of embryonic radial glia and appear to act as precursor cells for adult dentate neurogenesis, a process important for pattern separation and hippocampus-dependent learning. Although much work has delineated the mechanisms underlying activity-neurogenesis coupling via gamma-amino butyric acid (GABA)ergic neurotransmission on GFAP-negative transient-amplifying cells and neuroblasts, little is known regarding the effects of neurotransmitters on RG-like cells. Conflicting evidence exists for both GABA and glutamate receptors on these cells. Here, using GFAP reporter mice, we show that the somatic membrane of RG-like cells carries GABAA receptors and glutamate transporters but not ionotropic glutamate receptors, whereas 2-amino-3-(hydroxyl-5-methylisoxazole-4-yl) propionic acid (AMPA) and GABAA receptors are expressed on the processes of these cells. Almost all RG-like cells expressed the GluA2 subunit, which restricts the Ca(2+) permeability of AMPA receptors. The glial GABAA receptors mainly comprised α2/α4, β1, and γ1/γ3. The selective presence of AMPA receptors on the radial processes may be important for sensing and responding to local activity-driven glutamate release and supports the concept that RG-like astrocytes are composed of functional and structural domains.

Tu1733 MicroRNA-322/503 Repress Translation of Smurf1 and Smurf2 to Regulate TGFβ/SMAD Signaling and Gut Epithelial Homeostasis

Background and aims: The intestinal epithelium is a vigorously self-renewing tissue. Stem cells and transient-amplifying cells are located at the base of the crypts and give rise to terminally differentiated cells which migrate up the villous tip. A tightly regulated balance between proliferation and cell death is of utmost importance. Survivin is a unique member of the inhibitor of apoptosis (IAP) family with bifunctional properties involved in controlling apoptosis and cell division, respectively. To elucidate the contribution of survivin in the intestine we analysed mice with a specific deletion of survivin in intestinal epithelial cells (IECs). Methods: Survivinfl mice were crossbred to Villin-Cre or Villin-CreERT2 mice, resulting in conditional knockout mice (SurvivinΔIEC) or inducible knockout mice (SurviviniΔIEC). SurviviniΔIEC and control mice were histologically investigated by immuno¬histochemistry. The gene and protein expression pattern of IECs of Survivini ΔIEC mice was analysed by quantitative PCR and Western-Blot. To analyse the timepoint of the survivin deletion and intestinal epithelial cell death we focused on the in-vitro cultivation of organoids from SurviviniΔIEC mice. Results: We initially intended to generate mice with a specific deletion of the survivin gene in IECs but mice were embryonic lethal. Therefore, we created mice with an inducible deletion of the survivin gene in IECs, triggered by a daily injection of tamoxifen. Immunohistochemical staining of control gut tissue revealed a localisation of survivin in the crypt compartment. Detailed analysis showed specific presence of survivin in transient amplifying cells and stem cells. The specific deletion of survivin in IECs leads to a fatal destruction of the intestinal epithelium. IECs of Survivini ΔIEC mice showed abnormally enlarged nuclei, suggesting a defect in cell cycle progression. Furthermore, we observed accumulations of encapsulated debri and a massive dislocation of the crypt compartment. Western-blot analysis revealed elevated protein levels of activated caspase-3 and caspase-8, indicating dysregulated cell death of survivin depleted IECs. Quantitative PCR showed reduced gene expression levels of stem cell genes like lgr5 and olfm4, as well as for the proliferation marker ki67. Conclusions: The dramatic phenotype of Survivini ΔIEC mice demonstrated the absolute necessity of survivin in the intestinal epithelium. The lack of survivin results in a complete loss of the crypt-villous structure and the prominent formation of enlarged nuclei, arrested in cell cycle. The loss of stem cells as well as transient amplifying cells demonstrates the important contribution of survivin for the intestinal stem cell niche and the preservation of epithelial cells in the gut.

Tu1732 Conditional Intestinal-Epithelial Specific HuR Deletion Modifies Murine Intestinal Tumorigenesis

Background and aims: The intestinal epithelium is a vigorously self-renewing tissue. Stem cells and transient-amplifying cells are located at the base of the crypts and give rise to terminally differentiated cells which migrate up the villous tip. A tightly regulated balance between proliferation and cell death is of utmost importance. Survivin is a unique member of the inhibitor of apoptosis (IAP) family with bifunctional properties involved in controlling apoptosis and cell division, respectively. To elucidate the contribution of survivin in the intestine we analysed mice with a specific deletion of survivin in intestinal epithelial cells (IECs). Methods: Survivinfl mice were crossbred to Villin-Cre or Villin-CreERT2 mice, resulting in conditional knockout mice (SurvivinΔIEC) or inducible knockout mice (SurviviniΔIEC). SurviviniΔIEC and control mice were histologically investigated by immuno¬histochemistry. The gene and protein expression pattern of IECs of Survivini ΔIEC mice was analysed by quantitative PCR and Western-Blot. To analyse the timepoint of the survivin deletion and intestinal epithelial cell death we focused on the in-vitro cultivation of organoids from SurviviniΔIEC mice. Results: We initially intended to generate mice with a specific deletion of the survivin gene in IECs but mice were embryonic lethal. Therefore, we created mice with an inducible deletion of the survivin gene in IECs, triggered by a daily injection of tamoxifen. Immunohistochemical staining of control gut tissue revealed a localisation of survivin in the crypt compartment. Detailed analysis showed specific presence of survivin in transient amplifying cells and stem cells. The specific deletion of survivin in IECs leads to a fatal destruction of the intestinal epithelium. IECs of Survivini ΔIEC mice showed abnormally enlarged nuclei, suggesting a defect in cell cycle progression. Furthermore, we observed accumulations of encapsulated debri and a massive dislocation of the crypt compartment. Western-blot analysis revealed elevated protein levels of activated caspase-3 and caspase-8, indicating dysregulated cell death of survivin depleted IECs. Quantitative PCR showed reduced gene expression levels of stem cell genes like lgr5 and olfm4, as well as for the proliferation marker ki67. Conclusions: The dramatic phenotype of Survivini ΔIEC mice demonstrated the absolute necessity of survivin in the intestinal epithelium. The lack of survivin results in a complete loss of the crypt-villous structure and the prominent formation of enlarged nuclei, arrested in cell cycle. The loss of stem cells as well as transient amplifying cells demonstrates the important contribution of survivin for the intestinal stem cell niche and the preservation of epithelial cells in the gut.

Linking Morphogen and Chromatin in the Hair Follicle

In this issue of Developmental Cell, Xiong etal. (2013) identify a critical role for the chromatin remodeler Brg1 in hair follicle stem cell maintenance and epidermal repair. Brg1 interacts with the Shh signaling pathway to create a positive feedback loop that fuels hair follicle growth.

Progressive Alopecia Reveals Decreasing Stem Cell Activation Probability during Aging of Mice with Epidermal Deletion of DNA Methyltransferase 1

To examine the roles of epigenetic modulation on hair follicle regeneration, we generated mice with a K14Cre-mediated loss of DNA methyltransferase (DNMT) 1. The mutant shows an uneven epidermal thickness and alterations in hair follicle size. When formed, hair follicle architecture and differentiation appear normal. Hair subtypes exist but hair fibers are shorter and thinner. Hair numbers appear normal at birth but gradually decrease to fewer than 50% of control in 1 year old mice. Sections of old mutant skin shows follicles in prolonged telogen with hyperplastic sebaceous glands. Anagen follicles in mutants exhibit decreased proliferation and increased apoptosis in matrix transient amplifying cells. Although K15 positive stem cells in the mutant bulge are comparable in number to the control, their ability to proliferate and become activated to form a hair germ is reduced. As mice age, residual DNMT activity declines further and the probability of successful anagen reentry decreases, leading to progressive alopecia. Paradoxically, there is increased proliferation in the epidermis which also shows aberrant differentiation. These results highlight the importance of DNA methylation in maintaining stem cell homeostasis during the development and regeneration of ectodermal organs.

Abstract 5219: The role of MEK and Antizyme in keratinocyte stem cell expansion and differentiation

Abstract Transgenic mice provide a valuable system to study the development of skin tumorigenesis. Previous work from our lab has shown that mice overexpressing constitutively active MEK in the basal layer of the epidermis (K14-MEK mice) exhibit epidermal hyperplasia and spontaneous skin tumor development. These tumors express high levels of ornithine decarboxylase (ODC), the critical regulatory enzyme in the polyamine biosynthetic pathway. Although polyamines are required for normal cell growth, elevated levels of polyamines and ODC activity have been associated with neoplastic transformation and tumor promotion in a variety of tissues. Antizyme (AZ) suppresses ODC activity by targeting ODC for degradation. We have observed that mice overexpressing AZ and MEK in the skin (MEK/AZ mice) showed decreased tumor development compared to mice expressing MEK alone. This has led to the hypothesis that the upregulation of ODC caused by overexpressing constitutively active MEK will result in expansion of epidermal stem cells and decreased keratinocyte differentiation, in addition to the epidermal hyperplasia and tumorigenesis already seen. Overexpression of AZ decreases ODC activity and should reverse these phenotypes. Cell division in epidermal stem cells is asymmetrical, providing one replacement stem cell and a second transient amplifying (TA) cell. TA cells reside within the interfolliclar epidermis (IFE) and after several rounds of division, give rise to terminally differentiated cells in the suprabasal layers of the epidermis expressing early differentiation markers K1/K10 and late differentiation markers loricrin and involucrin. To determine the effect of MEK and AZ expression on stem cell expansion, newborn pups and adult mice were injected with BrdU using different wash out periods to label TA cells and epidermal stem cells. Our results showed that skin from K14-MEK mice had a significant increase in the number of TA cells within the IFE and hair follicle, while mice expressing both the K14-MEK and K5-AZ transgenes showed no difference in number of TA cells compared to wild-type littermates. Additionally, K14-MEK mice had a significant increase in the number of stem cells in the bulge region of the follicle, while AZ expression repressed the MEK-mediated increase in this cell population. Examination of primary keratinocyte cultures from the four genotypes showed that K14-MEK keratinocytes had decreased levels of both early and late differentiation markers compared to wild-type and K5-AZ cells, and were resistant to Ca+2-induced differentiation. MEK/AZ keratinocytes expressed similar levels of differentiation markers as those from wild-type and K5-AZ mice, indicating that AZ was able to attenuate MEK-induced suppression of differentiation. Our results suggest that ODC and polyamines are key regulators of epidermal stem cell expansion and proliferation/differentiation of keratinocyte progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5219. doi:1538-7445.AM2012-5219