Search

Many reptiles are able to continuously replace their teeth through life, an ability attributed to the existence of epithelial stem cells. Tooth replacement occurs in a spatially and temporally regulated manner, suggesting the involvement of diffusible factors, potentially over long distances. Here, we locally disrupted tooth replacement in the leopard gecko (Eublepharis macularius) and followed the recovery of the dentition. We looked at the effects on local patterning and functionally tested whether putative epithelial stem cells can give rise to multiple cell types in the enamel organs of new teeth. Second generation teeth with enamel and dentine were removed from adult geckos. The dental lamina was either left intact or disrupted in order to interfere with local patterning cues. The dentition began to reform by 1 month and was nearly recovered by 2–3 months as shown in μCT scans and eruption of teeth labeled with fluorescent markers. Microscopic analysis showed that the dental lamina was fully healed by 1 month. The deepest parts of the dental lamina retained odontogenic identity as shown by PITX2 staining. A pulse-chase was carried out to label cells that were stimulated to enter the cell cycle and then would carry BrdU forward into subsequent tooth generations. Initially we labeled 70–78% of PCNA cells with BrdU. After a 1-month chase, the percentage of BrdU + PCNA labeled cells in the dental lamina had dropped to 10%, consistent with the dilution of the label. There was also a population of single, BrdU-labeled cells present up to 2 months post surgery. These BrdU-labeled cells were almost entirely located in the dental lamina and were the likely progenitor/stem cells because they had not entered the cell cycle. In contrast fragmented BrdU was seen in the PCNA-positive, proliferating enamel organs. Homeostasis and recovery of the gecko dentition was therefore mediated by a stable population of epithelial stem cells in the dental lamina.

Label-retaining cells, which are characterized by dormancy or slow cycling, may be identified in a number of human normal and cancer tissues, and these cells demonstrate stem cell potential. In glioblastoma, label-retaining assays to enrich glioma stem cells remain to be fully investigated. In the present study, glioblastoma sphere cells cultured in serum-free medium were initially stained with the cell membrane fluorescent marker DiI. The fluorescence intensity during cell proliferation and sphere reformation was observed. At 2 weeks, the DiI-retaining cells were screened by fluorescence-activated cell sorting and compared phenotypically with the DiI-negative cells in terms of in vitro proliferation, clonogenicity and multipotency and for in vivo tumorigenicity, as well as sensitivity to irradiation and temozolomide treatment. It was observed that DiI-retaining cells accounted for a small proportion, <10%, within the glioblastoma spheres and that DiI-retaining cells proliferated significantly more slowly compared with DiI-negative cells (P=0.011, P=0.035 and P=0.023 in the of NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines). Significantly increased clonogenicity (P=0.002, P=0.034 and P=0.016 in the NCH441, NCH644 and NCH421k glioblastoma sphere cell lines) and three-lineage multipotency were observed in DiI-retaining cells in vitro compared with DiI-negative cells. As few as 100 DiI-retaining cells were able to effectively generate tumors in the immunocompromised mouse brain, whereas the same number of DiI-negative cells possessed no such ability, indicating the increased tumorigenicity of DiI-retaining cells compared with DiI-negative cells. Furthermore, DiI-retaining cells demonstrated significant resistance following irradiation (P=0.012, P=0.024 and P=0.036) and temozolomide (P=0.003, P=0.005 and P=0.029) compared with DiI-negative cells in the NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines, respectively. It was concluded that label-retaining cells in glioblastoma spheres manifest clear stem cell features and that the label-retaining assay may be utilized to further enrich glioma stem cells cultured under serum-free conditions for additional study. [ABSTRACT FROM AUTHOR]

Background It is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse. Results Using an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal. Conclusions We conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slowcycling CSCs and a post-inflammatory onset of an aggressive tumor. [ABSTRACT FROM AUTHOR]

Objective: This study has investigated the existence of label-retaining cell and its distribution in gastric cancer, in the hope that this information will assist investigations on gastric cancer stem cells. Methods: The gastric carcinoma cell line BGC-823 was labeled with BrdU in vitro and then engrafted into the right axilla of nude mice, which developed tumors. Label-retaining cells were quantified by immunohistochemical methods. Results: BrdU positive cells constituted about 96% of the cells in xenograft tumors after 10 days. Subsequently, BrdU positive cells gradually decreased, at the 80th day, label-retaining cells steadily occupied about 0.5%. This set of population cell localized in the margin of cancer nests, which had no difference in cellular morpha. Conclusion: The study demonstrates the presence of label-retaining cells in human gastric cancer xenografts in nude mice and the label-retaining cells may be related with cancer stem cells, which are most likely the cause for spread, metastasis and recurrence. [ABSTRACT FROM AUTHOR]

Adult taste buds are maintained by the lifelong proliferation of epithelial stem and progenitor cells, the identities of which have remained elusive. It has been proposed that these cells reside either within the taste bud (intragemmal) or in the surrounding epithelium (perigemmal). Here, we apply three different in vivo approaches enabling single-cell resolution of proliferative history to identify putative stem and progenitor cells associated with adult mouse taste buds. Experiments were performed across the circadian peak in oral epithelial proliferation (04:00 h), a time period in which mitotic activity in taste buds has not yet been detailed. Using double label pulse-chase experiments, we show that defined intragemmal cells (taste and basal) and perigemmal cells undergo rapid, sequential cell divisions and thus represent potential progenitor cells. Strikingly, mitotic activity was observed in taste cells previously thought to be postmitotic (labelled cells occur in 30% of palatal taste buds after 1 h of BrdU exposure). Basal cells showed expression of the transcription factor p63, required for maintaining the self-renewal potential of various epithelial stem cell types. Candidate taste stem cells were identified almost exclusively as basal cells using the label-retaining cell approach to localize slow-cycling cells (0.06 ± 0.01 cells per taste bud; n = 436 taste buds). Together, these results indicate that both stem- and progenitor-like cells reside within the mammalian taste bud. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSC) have been defined by their ability to establish long-term hematopoiesis in myelo-ablated hosts. Prospective isolation using combinations of cell-surface markers and/or dye exclusion can yield highly purified and nearly homogeneous phenotypically defined cells that repopulate irradiated hosts. Although highly informative, these types of analyses may not necessarily reflect ongoing homeostatic hematopoiesis. HSCs are also described as being quiescent. This has been demonstrated by cell cycle analysis of phenotypically defined HSCs. Some studies have challenged the existence of truly quiescent HSCs, suggesting that they continuously cycle, albeit with very slow kinetics. Here we present a pulse–chase system based on the controllable incorporation of H2B-GFP into nucleosomes, which allows the identification, purification, and functional analysis of viable label-retaining cells. Our data complement and extend recent studies using similar strategies. These, together with our present studies, find a rare, quiescent or dormant subset within the population of stringently defined HSC phenotypes. To date, three types of niches, endosteal, vascular, and reticular, have been described; herein we review the cellular and spatial nature of these microenvironments. We propose that HSC label-retention combined with genetically manipulated stem cell niches will allow us to determine their anatomical architecture, to address HSC cell fate proliferation kinetics, and to begin to dissect the molecular cross talk among stem cells and niche cells in vivo during both normal and perturbed homeostasis. [ABSTRACT FROM AUTHOR]

Somatic stem cells (SSCs) are defined as a rare population of undifferentiated cells residing in differentiated tissues and organs. Different studies and diverse approaches suggest the presence of SSCs in human and murine endometria. Owing to the lack of specific phenotypic markers to identify endometrial SSCs, they are defined by their functional properties. The embryological origin of the uterus and the putative implication of bone marrow in endometrial regeneration suggest their presumed mesenchymal origin. Nevertheless, more information is needed regarding the physiology of endometrial stem cells to understand the gynecological disorders associated with abnormal endometrial proliferation, such as endometriosis and endometrial cancer. [ABSTRACT FROM AUTHOR]

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 μm coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphereforming cells detected in individual 400 μm sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 ± 276) or colonies (4275 ± 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 ± 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 ± 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques. [ABSTRACT FROM AUTHOR]

Abstract: In response to changing signals, quiescent hematopoietic stem cells (HSCs) can be induced to an activated cycling state and provide multi-lineage hematopoietic cells to the whole body via blood vessels. However, the precise localization of quiescent HSCs in bone marrow microenvironment is not fully characterized. Here, we performed whole-mount immunostaining of bone marrow and found that BrdU label-retaining cells (LRCs) definitively reside in the sinusoidal hypoxic zone distant from the “vascular niche”. Although LRCs expressed very low level of a well-known HSC marker, c-kit in normal circumstances, myeloablation by 5-FU treatment caused LRCs to abundantly express c-kit and proliferate actively. These results demonstrate that bone marrow LRCs reside in the sinusoidal hypoxic niche, and function as a regenerative cell pool of HSCs. [Copyright &y& Elsevier]

The kidney is capable of regeneration. In response to a variety of insults, renal epithelial tubular cells dedifferentiate into an immature phenotype, proliferate, migrate to the injured area, and redifferentiate into mature polarized epithelial cells. In animal models of acute kidney injury induced by renal ischemia or renal toxins, various growth factors, transcription factors, chemokines, and extracellular matrix components have been demonstrated to be involved in the regeneration process. Recent research has suggested the existence of renal stem/progenitor cells in the kidney and their involvement in renal regeneration. In this review, we will focus on the mechanisms of tubular regeneration after kidney injury, particularly on label-retaining cells actively engaged in this process, and discuss their potential as targets of regenerative therapy for various kidney diseases. [ABSTRACT FROM AUTHOR]

Hair follicles repeatedly regress and reconstitute themselves, suggesting the presence of intrinsic tissue stem cells. Using label-retaining cell technique to detect slow-cycling stem cells, hair follicle stem cells were detected in the bulge region of the outer root sheath, which provides the insertion point for the arrector pili muscle and marks the bottom of the permanent portion of hair follicles. Later studies elucidated important stem cell characteristics of the bulge cells, including high proliferative capacity and multipotency to regenerate the pilosebaceous unit as well as epidermis. Isolation of living bulge cells is now feasible. In addition, microarray analyses revealed the global gene expression profile of the bulge cells. However, most of those studies were performed in mouse hair follicles and our understanding of human bulge cells has been limited. Recently, remarkable progress was made in human bulge cell biology. The morphologically ill-defined human bulge boundary was precisely determined by the distribution of label-retaining cells. Laser capture microdissection enabled accurate isolation of human bulge cells and control cell populations. Microarray comparison analyses between isolated bulge and nonbulge cells elucidated the molecular signature of human bulge cells and identified cell surface markers for living bulge cell isolation. Importantly, isolated living human bulge cells demonstrated stem cell characteristics in vitro. In this review, recent advances in hair follicle bulge cell research are summarized, especially focusing on the characterization and isolation of human bulge cells. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have identified some unusually persistent label-retaining cells in the hair follicles of mice, and have investigated their role in hair growth. Three-dimensional reconstruction of dorsal underfur follicles from serial sections made 14 mo after complete labeling of epidermis and hair follicles in neonatal mice disclosed the presence of highly persistent label-retaining cells associated with the first-generation follicle involved in the production of the first wave of hairs, commonly called the bulge. The label-retaining cells were most often found on the ventral surface of the first-generation follicle, five cell positions from the base, near the attachment site of the arrector pilorum muscle. No label-retaining cells were found in the hair canal, sebaceous gland, or hair germ. These label-retaining cells remained in the follicle following induction of anagen by plucking of the hairs. Surprisingly, they were not part of the first wave of mitotic activity following plucking, but instead underwent mitosis beginning 42 h after plucking. Label-retaining cells or their labeled daughters were not found in the hair germs through 48 h following induction of anagen by plucking, but instead remained in their subsebaceous follicular location even upon completion of the hair growth cycle 21 d later. These label-retaining cells are, therefore, unlikely to contribute to the formation of a new anagen follicle. [ABSTRACT FROM AUTHOR]