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Epidermal Label-Retaining Cells: Background and Recent Applications
Abstract
Epidermal label-retaining cells (LRC) can be identified by giving neonatal mice repeated injections of 3H-thymidine or 5-bromo-2'-deoxyuridine and then finding the cells that are still labelled in adulthood. Although label retention is simply a marker of the proliferative history of a cell, there is, nevertheless, evidence that it is a characteristic of epidermal stem cells. Here we review the early literature on LRC and then highlight two recent applications. We describe how LRC can be visualized by whole-mount labelling of the epidermis and how purified LRC can be used to screen for markers of the epidermal stem cell compartment.
... Mice were entrained to restricted 12-hr light/dark cycles. Where indicated, LRC was performed by injecting mice with bromodeoxyuridine (50 mg/g body weight) as described previously (Bickenbach and Chism, 1998;Braun and Watt, 2004). For further details see Supplemental Experimental Procedures. ...
... weeks (Squarize et al., 2013). LRC was performed by injecting mice with BrDU 50 µg/g of body weight) as described (Bickenbach and Chism, 1998) (Braun and Watt, 2004). ...
... Braun, K.M., and Watt, F.M. (2004). Epidermal label-retaining cells: background and recent applications. ...
... Traditionally, epithelial tissues have been thought to be maintained and repaired solely by a pool of stem/progenitor cells (SCs) that balance proliferation and differentiation to preserve tissue integrity [1][2][3][4][5][6][7] . However, it has now become widely accepted that epithelial cell fate is more dynamic than originally thought 8 . ...
... For many decades, squamous epithelial cells were thought to be structured in a rigid hierarchy where stem cells were responsible for tissue maintenance and regeneration, while committed and differentiated cells only contributed to the formation of the epithelial barrier [5][6][7] . Mounting evidence over the last two decades has now established that epithelial cell behavior present a remarkable plasticity 8 . ...
The ability of epithelial cells to rewire their cell fate program beyond their physiological repertoire has become a new paradigm in stem cell biology. This plasticity leaves behind the concept of strict stem cell hierarchies, opening up new exciting questions about its limits and underlying regulation. Here we developed a heterotypic 3D culture system to study the mechanisms modulating changes in the identity of adult esophageal epithelial cells. We demonstrate that, when exposed to the foreign stroma of adult skin, esophageal cells transition towards hair follicle identity and architecture. Heterotypic transplantation experiments recapitulated this cell fate conversion process in vivo. Single-cell RNA sequencing and histological analysis, capturing the temporality of this process, reveal that most esophageal cells switching towards skin identity remain in an intermediate state marked by a transient regenerative profile and a particularly strong hypoxic signature. Inhibition of HIF1a establishes the central role of this pathway in regulating epithelial cell plasticity, driving cells away from their transition state in favor of cell fate conversion.
... These studies have been conducted in mouse uteri marked with bromodeoxyuridine (BrdU). 52,[54][55][56][57] BrdU is a molecular analogue of thiamine and is used by the cells in place of the original nitrogen base during cellular multiplication periods. As a result of this procedure, cellular populations with a slow division cycle are identified as stem/progenitor cells or label-retaining cells (LRC), 55 whereas cells that divide rapidly (which do not correspond to stem cells) dilute the label. ...
... 52,[54][55][56][57] BrdU is a molecular analogue of thiamine and is used by the cells in place of the original nitrogen base during cellular multiplication periods. As a result of this procedure, cellular populations with a slow division cycle are identified as stem/progenitor cells or label-retaining cells (LRC), 55 whereas cells that divide rapidly (which do not correspond to stem cells) dilute the label. In these studies, immunohistochemistry was also used to locate epithelial, stromal and myometrial cells positive for BrdU, which revealed their locations and some components of the cellular niche. ...
Mesenchymal stem cells (MSCs) have been isolated from the endometrium of humans, mice, cows, pigs and ewes. Typically, these cells are detected in the deep regions of the endometrium, closer to the union with the myometrium. MSCs possess characteristics such as clonogenicity and multipotentiality since they can differentiate in vitro into adipogenic, chondrogenic and osteogenic lineages. These cells can be induced to differentiate in vitro not only into the mesodermal lineage but also into the endodermal and ectodermal lineages. Therefore, MSCs show a great regenerative capacity for various organs and tissues, including the endometrium. Some advantages of endometrial MSCs compared with other MSC sources are their immune modulating activity, their ease of obtainment, and the amount of sample that may be collected. The study of endometrial MSCs in domestic animals is a new and promising field because increasing our understanding of the physiology and biology of these cells may lead to a better understanding of the physiopathology of reproductive diseases, and the development of treatment methods for infertility problems. In other veterinary medicine fields, MSCs can be used for the treatment of autoimmune diseases, cardiac affections, musculoskeletal and articular lesions, muscle degeneration, type 1 diabetes, urinary tract diseases, neurodegenerative processes and tumours. Finally, MSCs are also an important clinical tool for tissue engineering and regenerative medicine. The aim of this review is to present an updated outlook of the knowledge regarding endometrial MSCs and their possible applications in veterinary medicine.
... TPA is also known to initiate the proliferation of slow cycling putative stem cells from within the bulge region of the hair follicle (18), and it is the expansion of these cells that gives rise to papillomas in the chemically induced mouse model (12,19). As stem cells divide very infrequently in the adult mouse epidermis, DNA LRCs have been used to mark stem cell populations (20,21). Injection of BrdU into neonatal mice, which are undergoing rapid tissue expansion, results in the labelling of all dividing cells. ...
... LRCs are those cells that have retained the label in adult life and represent cells that are not dividing or have undergone limited cell division but retain regenerative capacity (22). The hair follicles within the tail epidermis are larger than in the dorsal skin and therefore have a larger number of LRCs, thus facilitating quantitative analysis (17,21). These LRCs within the hair follicle are mainly located within the non-cycling region below the sebaceous gland in the tail epidermis (17) (Figure 2A, visualized in red). ...
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates signals downstream of integrin and growth factor
activation. Previously, we have shown that skin-specific loss of fak prevents chemically induced skin carcinogenesis in mice following phorbol ester treatment. In this study, we show that skin-specific
deletion of fak prevents mobilization of stem cells within the bulge region of the hair follicle, which are the precursors of papillomas
following phorbol ester treatment. We also show that phorbol ester treatment results in activation of-catenin within the skin
and that FAK is required for β-catenin-induced stem cell mobilization. In addition, inhibition of Src kinase activity, a major
binding partner of FAK also prevents stem cell mobilization. We show that FAK is required for the nuclear localization of
β-catenin in the skin following phorbol ester treatment and the transcriptional activation of the β-catenin target gene c-Myc. This provides the first evidence of cross-talk between integrin and Wnt signalling pathways in the control of epidermal
stem cells and the early events associated with skin carcinogenesis.
... The holoclones are thought to correspond to the label-retaining stem cell population and the paraclones to the transient amplifying cells. These cells are distinguished based on differences in label-retention [28], cell surface marker expression, proliferation frequency, and ability to grow as clones in culture [11,11,28,64,96,98,117]. In murine epidermis cell populations have been distinguished as epidermal stem cells which are label-retaining and occasionally give rise to an identical daughter stem cell (symmetrical division) and a transient amplifying cell (asymmetrical division). ...
... The holoclones are thought to correspond to the label-retaining stem cell population and the paraclones to the transient amplifying cells. These cells are distinguished based on differences in label-retention [28], cell surface marker expression, proliferation frequency, and ability to grow as clones in culture [11,11,28,64,96,98,117]. In murine epidermis cell populations have been distinguished as epidermal stem cells which are label-retaining and occasionally give rise to an identical daughter stem cell (symmetrical division) and a transient amplifying cell (asymmetrical division). ...
Background:
The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue.
Scope of review:
A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer.
Major conclusions:
An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis.
General significance:
Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.
... They are then followed during a "chase" period when rapidly cycling cells dilute out their label, and only quiescent cells, so called "label-retaining" cells (LRCs), retain label. Prior work using this method identified LRCs, presumed to be stem cells, in the IFE basal layer (Potten 1974;Braun and Watt 2004). LRCs in the IFE were previously thought to represent the sole stem cell population in this region, and non-LRCs were thought to be progenitor cells. ...
Adult mammals retain the remarkable ability to regenerate hair follicles after wounding. Wound-induced hair neogenesis (WIHN) in many ways recapitulates embryogenesis. The origin of the stem cells that give rise to a nascent hair follicle after wounding and the role of mesenchymal cells and signaling pathways responsible for this regenerative phenomenon are slowly being elucidated. WIHN provides a potential therapeutic window for manipulating cell fate by the introduction of factors during the wound healing process to enhance hair follicle formation.
... For label-retaining cell experiments, 10-day-old mice were injected with 5-bromodeoxy-uridine (BrdU, 50 μg/g) intraperitoneally (i.p) every 12 h for 48 h [26]. After a 60-day chase period, the dorsal skin was isolated. ...
Background
Hair follicle stem cells (HFSC) play an essential role in the maintenance of hair homeostasis; during the hair cycle, HFSC remain quiescent for most of its duration. The hairpoor mouse (+ /Hr Hp ), an animal model of Marie-Unna hypotrichosis (MUHH), overexpresses hairless in the bulge, inner root sheath, and outer root sheath of HF and shows the same phenotype as in MUHH patients manifesting sparse hair with progression to alopecia with age. The aim of this study was to gain an understanding of the hair cycle and the status of HFSC during the hair cycle of the hairpoor mouse in order to delineate the pathogenesis of MUHH.
Methods
H&E staining was performed in order to define the state of the hair follicle. FACS analysis and immunostaining were performed at the 1st and 2nd telogen stages for observation of the HFSC. A label retaining assay was performed to determine the quiescent state of hair follicles. qRT-PCR was performed to determine expression of factors involved in niche signaling and Wnt signaling.
Results
We observed a drastic decrease in the number of hair follicles after the 1st telogen, followed by an intensified disturbance in the hair cycle with shorter anagen as well as 2nd telogen in the hairpoor mouse. A dramatic reduction in the number of CD34 expressing bulges as well as cells was observed at the telogen of the HFs, with prominent high proliferation of bulge cells, suggesting the loss of HFSC quiescence in the hairpoor mouse. The increased cell proliferation in HF was reiterated following the synchronization of the hair cycle, leading to acceleration of HF cycling. Reduced expression of Fgf18 and Bmp6, the factors involved in HFSC quiescence, was observed in the HFSC niche of the hairpoor mouse. In addition, disturbed expression of Wnt signaling molecules including Wnt7b , Wnt10b , and Sfrp1 was observed, which induced the telogen-to-anagen transition of HFs in the hairpoor mouse.
Conclusions
These results indicate that the quiescent state of HFSC is not properly maintained in the hairpoor mouse, consequently leading HFs to the completely disarrayed hair cycle. These findings may provide an understanding of an underlying mechanism for development of alopecia with age in MUHH patients.
... To label slowly cycling stem cells, neonatal mice can be subjected to long-term DNA labeling with tritiated thymidine or BrdU, the rationale being that as the skin is expanding in the first days after birth, both stem and transit-amplifying cells are dividing and will be labeled (Bickenbach, 1981;Braun and Watt, 2004). In adult mice, the transit-amplifying cells will continue to divide and will therefore lose the DNA label, whereas the stem cells will divide infrequently and therefore retain the label (label-retaining cells) (Bickenbach, 1981). ...
The human epidermis is one of the first tissues in which the existence of stem cells was recognized and is one of the few in which ex vivo expansion for tissue repair is established clinically. Nevertheless, the nature of stem cells has been elusive. Using clonal growth assays of cultured keratinocytes as a quantitative measure of their abundance, several candidate stem cell markers have been described. Recently, the volume and quality of single-cell RNA-sequencing datasets have increased exponentially, providing new opportunities to explore the nature of epidermal stem cells and test the validity of in vitro experimental models.
... Side population (SP) cells, identified as a small population of cells capable of effluxing the vital DNA-binding dye, Hoechst 33342 by dual wavelength flow cytometry, may be used as an assay of potential stem/progenitor cells in a cell population (Challen and Little, 2006). Another approach uses label retention of DNA synthesis labels, such as bromodeoxyuridine (BrdU), in studies which may indirectly predict potential stem/progenitor cell populations retaining the label following a chase period while their proliferating progeny rapidly dilute the label to histologically non-detectable levels (Braun and Watt, 2004). Further evidence is required to functionally verify the stem or progenitor identity of cells labeled by both approaches. ...
The human endometrium undergoes approximately 450 cycles of proliferation, differentiation, shedding and regeneration over a woman’s reproductive lifetime. The regenerative capacity of the endometrium is attributed to stem/progenitor cells residing in the basalis layer of the tissue. Mesenchymal stem cells have been extensively studied in the endometrium, whereas endometrial epithelial stem/progenitor cells have remained more elusive. This review details the discovery of human and mouse endometrial epithelial stem/progenitor cells. It highlights recent significant developments identifying putative markers of these epithelial stem/progenitor cells that reveal their in vivo identity, location in both human and mouse endometrium, raising common but also different viewpoints. The review also outlines the techniques used to identify epithelial stem/progenitor cells, specifically in vitro functional assays and in vivo lineage tracing. We will also discuss their known interactions and hierarchy and known roles in endometrial dynamics across the menstrual or estrous cycle including re-epithelialization at menses and regeneration of the tissue during the proliferative phase. We also detail their potential role in endometrial proliferative disorders such as endometriosis.
... While retention of nuclear labels essentially defines the proliferative history of cells, studies based on long-term pulse-chase experiments and on the repair of radiation-induced damage in epidermis and intestine [13][14][15] showed that LRCs were spatially segregated in these tissues. This led to the proposal that cell cycle characteristics and spatial organization of these cells could describe their identities: slow cycling cells are stem cells (located in the basal layer, in a protected position) and fast cycling cells represent the transient amplifying cells that terminally differentiate after a finite number of divisions following a unidirectional stream starting from the basal layer 14,16,17 . Although the search for novel stem cells based on quiescence is complicated, also considering the fact that the majority of adult cells are not dividing, studies on LRCs (and therefore defining the quiescent state as a "stem cell trait") were successfully applied in the quest to uncover adult stem cells in several tissues, such as the hair follicle 18 , skin (reviewed in 19 ), sweat glands 20,21 , teeth 22 , pancreas 23 , and intestine 24 . ...
Dysfunction of the salivary gland and irreversible hyposalivation are the main side effects of radiotherapy treatment for head and neck cancer leading to a drastic decrease of the quality of life of the patients. Approaches aimed at regenerating damaged salivary glands have been proposed as means to provide long-term restoration of tissue function in the affected patients. In studies to elucidate salivary gland regenerative mechanisms, more and more evidence suggests that salivary gland stem/progenitor cell behavior, like many other adult tissues, does not follow that of the hard-wired professional stem cells of the hematopoietic system. In this review, we provide evidence showing that several cell types within the salivary gland epithelium can serve as stem/progenitor-like cells. While these cell populations seem to function mostly as lineage-restricted progenitors during homeostasis, we indicate that upon damage specific plasticity mechanisms might be activated to take part in regeneration of the tissue. In light of these insights, we provide an overview of how recent developments in the adult stem cell research field are changing our thinking of the definition of salivary gland stem cells and their potential plasticity upon damage. These new perspectives may have important implications on the development of new therapeutic approaches to rescue radiation-induced hyposalivation.
... La quiescence n'est plus considérée comme une caractéristique spécifique des cellules souches. En effet, la majorité des cellules d'un organisme adulte sont des cellules différenciées non prolifératives(Cotsarelis, Sun, and Lavker 1990;Braun and Watt 2004;Post and Clevers 2019). Mais l'utilisation de souris transgéniques inductibles exprimant une protéine histone-GFP a permis par le même principe d'identifier et de caractériser les cellules souches se divisant lentement de la niche du follicule pileux(Tumbar et al. 2004) L'utilisation de souris transgéniques permettant de suivre le devenir des cellules in vivo (lineage tracing). ...
La peau est en permanence exposée à des stress extrinsèques notamment aux rayonnements ultraviolets, principale cause de cancer cutané. Les cancers cutanés non mélanocytaires (NMSC) sont divisés en deux types : le carcinome basocellulaire (BCC) et le carcinome spinocellulaire (SCC). Les BCC surviennent après une exposition aigüe et précoce aux UVs au cours de l'enfance et présentent un faible potentiel métastatique. Les BCC dérivent des cellules souches du follicule pileux. Alors que les SCC se développent après une exposition chronique à de faibles doses d’UV et présentent un potentiel métastatique plus élevé. Les SCC dérivent de cellules souches de l’épiderme inter folliculaire. Le ratio BCC/SCC est de 4 pour 1 dans la population générale. Au laboratoire, nous nous intéressons à la pathologie Xeroderma Pigmentosum (XP). XP est une maladie génétique récessive autosomique rare. En Europe, la majorité des patients XP sont porteurs de mutations du gène XPC entrainant l'absence d’une protéine fonctionnelle. Le rôle canonique de XPC est la reconnaissance des dommages à l’ADN induits par les UVs et permet la réparation de l’ADN par le mécanisme de réparation par excision de nucléotide (NER). Les patients XP-C présentent une hypersensibilité aux rayons UV et développent très précocement des cancers cutanés et particulièrement des SCC avec une incidence plus élevée. L'objectif de ma thèse est d’étudier les mécanismes moléculaires sous-jacents à la susceptibilité des kératinocytes XP-C au SCC. En utilisant des kératinocytes primaires humains de patients XP-C ou d’individus sains, nous avons développé un protocole pour mimer une exposition solaire chronique en les exposant de manière récurrente à de faibles doses d’UV. Nous avons choisi d’effectuer une approche non biaisée en effectuant un séquençage génomique entier de kératinocytes XP-C et WT exposés ou non aux UV. Comme attendu, les kératinocytes XP-C chroniquement irradiés présentent de nombreuses mutations particulièrement dans les séquences codantes. L’analyse bio-informatique de ces données révèle un défaut dans des gènes impliqués dans l'organisation de la chromatine. De façon intéressante, chez les kératinocytes non exposés aux UV, nous observons une augmentation de mutation dans les séquences codantes, l’analyse des gènes portant des mutations stop a mis en évidence un défaut dans les réseaux de gènes impliqués dans l'organisation de chromatine. Ces résultats suggèrent un état plus ouvert de chromatine dans les kératinocytes XP-C amenant l’hypothèse que les cellules XP-C présenteraient un phénotype cellule souche de l’épiderme. Pour valider ce modèle, nous avons utilisé des kératinocytes primaires humains issus de patients. Nous avons montré que des kératinocytes sains dont l’organisation de la chromatine a été altérée, présentent un défaut de différenciation terminale comparable à celui observé dans nos cellules XP-C. De plus, ce phénotype est complémenté par la réexpression de la protéine XPC fonctionnelle. Nous avons montré que les kératinocytes XP-C forment majoritairement des clones issus de cellules souches dans un test de clonogénicité. Ils présentent une expression plus élevée de marqueurs de cellules souches spécifiques de l’épiderme inter folliculaire. Les résultats obtenus au cours de ma thèse, grâce à notre modèle in silico et à nos résultats in vitro, suggèrent une nouvelle piste dans la compréhension de la susceptibilité des patients XP-C à développer des SCC. Le changement d’identité cellulaire lié à XP-C entraine la présence d’une chromatine plus ouverte et une identité plus proche des cellules souches, cellules à l’origine des SCC. Ce phénotype étant directement lié à l’absence de la protéine XPC fonctionnelle, nous mettons en évidence un nouveau rôle de XP-C dans l’organisation de la chromatine et la régulation de l’expression du génome. Ces travaux ouvrent donc de nouvelles perspectives dans l’approche des pathologies liées à une déficience en XPC.
... If CSC behaves similarly, quiescent CSCs would be intrinsically resistant to common cancer drugs or radiation therapy, as these treatments usually only target actively dividing cells [61]. As mentioned above, the property of quiescence has been used to isolate CSCs after ACD: daughter cells retaining labeled nucleotides or fluorescent lipid markers were regarded as quiescent and hence enriched CSCs [60,[80][81][82]. Some reports have provided experimental support that cancer dormancy might be explained by the label-retaining cancer cell population [61,83]. ...
Stem cells including cancer stem cells (CSC) divide symmetrically or asymmetrically. Usually symmetric cell division makes two daughter cells of the same fate, either as stem cells or more differentiated progenies; while asymmetric cell division (ACD) produces daughter cells of different fates. In this review, we first provide an overview of ACD, and then discuss more molecular details of ACD using the well- characterized Drosophila neuroblast system as an example. Aiming to explore the connections between cell heterogeneity in cancers and the critical need of ACD for self-renewal and generating cell diversity, we then examine how cell division symmetry control impacts common features associated with CSCs, including niche competition, cancer dormancy, drug resistance, epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET), and cancer stem cell plasticity. As CSC may underlie resistance to therapy and cancer metastasis, understanding how cell division mode is selected and executed in these cells will provide possible strategies to target CSC.
... Slow-cycling cells were not positioned randomly in the basal layer of the epidermis: each of these columns contained a single slowly cycling cell (Potten, 1974). Together with studies of cell- cycle kinetics following radiation-induced damage repair, this led Potten and Morris (1988) to propose that the cell-cycle characteristics of basal layer keratinocytes indicate their identity: slow cycling cells are stem cells and rapidly cycling cells represent the transit-amplifying cells, the latter leaving the basal layer after a few rounds of division to terminally differentiate (Braun and Watt, 2004). ...
Classic studies on hematopoiesis indicate that blood cell numbers are maintained by rare, hard-wired, transplantable stem cells (SCs). Subsequent studies in other organs have implicitly assumed that all SC hierarchies follow the design of the hematopoietic system. Lineage tracing techniques have revolutionized the study of solid tissue SCs. It thus appears that key characteristics of the hematopoietic SC hierarchy (rarity of SCs, specific marker expression, quiescence, asymmetric division, and unidirectional differentiation) are not generalizable to other tissues. In light of these insights, we offer a revised, generalizable definition of SC function: the ability to replace lost tissue through cell division.
... weeks (Squarize et al., 2013). LRC was performed by injecting mice with BrDU 50 µg/g of body weight) as described (Bickenbach and Chism, 1998) (Braun and Watt, 2004). ...
... Mice were entrained to restricted 12-hr light/dark cycles. Where indicated, LRC was performed by injecting mice with bromodeoxyuridine (50 mg/g body weight) as described previously (Bickenbach and Chism, 1998;Braun and Watt, 2004). For further details see Supplemental Experimental Procedures. ...
Tissue integrity requires constant maintenance of a quiescent, yet responsive, population of stem cells. In the skin, hair follicle stem cells (HFSCs) that reside within the bulge maintain tissue homeostasis in response to activating cues that occur with each new hair cycle or upon injury. We found that PTEN, a major regulator of the PI3K-AKT pathway, controlled HFSC number and size in the bulge and maintained genomically stable pluripotent cells. This regulatory function is central for HFSC quiescence, where PTEN-deficiency phenotype is in part regulated by BMAL1. Furthermore, PTEN ablation led to downregulation of BMI-1, a critical regulator of adult stem cell self-renewal, and elevated senescence, suggesting the presence of a protective system that prevents transformation. We found that short- and long-term PTEN depletion followed by activated BMAL1, a core clock protein, contributed to accumulation of HFSC.
... The slow-cycling population assay can be used to distinguish CSCs from progenitor cells, which determines the incorporation of labeled precursor of nucleotides for cellular DNA synthesis [81]. The slow-cycling cells, such as CSCs, maintaining sufficient labels allow their detection by the anti-BrdU antibody staining or radioactive label [82]. ...
The CSC concept is still an evolving model moving forward. Recent lineage tracing strategy has proven the existence and functions of CSCs in progression of solid tumors. CSCs with intrinsic activities of chemoresistance are the culprit in tumor recurrence and the root of treatment failures. Challenges remain on how to efficiently identify and target CSCs in vivo and monitor CSCs posttreatment. Cancer biology has proofed that traditional clonal evolution, and CSC models are not exclusive but co-exist with each other in tumorigenesis. Therefore, future elucidation of molecular mechanisms underlying CSC biology should open a new window of efficacious novel therapy strategies that eliminate both CSCs and non-CSCs.
... Adult stem cells, that maintain proliferative epithelia, have a high proliferative capacity but divide infrequently (Braun and Watt, 2004). H-Tdr or the thymidine analogue BrdU are incorporated into the DNA of dividing cells during S-phase of the cell cycle, and because of their slowly cycling nature, the label is retained in putative stem cells but is diluted from their more proliferative daughter cells. ...
Limbal stem cells maintain the corneal epithelium through a process of clonal growth and
ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of
their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6
weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to
represent chords of clonally related, inwardly migrating cells. By measuring the number and
width of stripes and correcting for the effects of different proportions of LacZ-positive cells,
an estimate of the number of coherent stem cell clones maintaining the tissue can be derived.
Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is
reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks.
An automated method was developed using image analysis software to analyse these striping
patterns. This method produced results that did not differ significantly from the above. The
dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6
heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at
15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77
transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics,
estimated clone number was similarly reduced to ~50 with no further decline. Mice
heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an
increase in coherent clones. When the corneal epithelium is injured the surrounding
epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of
healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted
during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged
using time-lapse confocal microscopy. This technique demonstrated that clones remain
contiguous throughout their migration. Healing of peripheral wounds was observed to form
de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away
from and towards the limbal region.
... Whole mounts of mouse tail epidermis were prepared as previously described (Braun and Watt 2004). Briefly, whole skin was removed from the tail of mice and incubated for 3 hours at 37 o C in 5mM EDTA/PBS. ...
... However, reliable and specific stem cell markers for epidermal stem cells and their transient amplifying cell progeny are still lacking. Therefore, rather than relying upon a single stem cell marker, a panel of markers have been identified to isolate the epidermal stem cells [9,[15][16][17]. ...
Stem cells have been found to reside in the different organs of the human body localized in specific niches. One such mini-organ is hair follicle wherein stem cells of epidermal origin have been reported. In the present review article we have reviewed in brief about the melanocyte stem cells of the hair follicle tissue, their identification, surface marker characteristics and role in the cell based therapy for vitiligo.
... 20,21 Additionally, the validity of the long-term label-retaining assay, employed for the identification of stem cells in various organs, would be problematic. [32][33][34][35][36][37] This protocol is based on the notion that stem cells divide rarely and/or have a very long cell cycle time. Therefore, the long-term label-retaining property of a cell would document its stemness while the progressive dilution of the label would identify the generated progeny. ...
Following the recognition that hematopoietic stem cells improve the outcome of myocardial infarction in animal models, bone marrow mononuclear cells, CD34-positive cells and mesenchymal stromal cells have been introduced clinically. The intracoronary or intramyocardial injection of these cell classes has been shown to be safe and to produce a modest but significant enhancement in systolic function. However, the identification of resident cardiac stem cells in the human heart (hCSCs) has created great expectation concerning the potential implementation of this category of autologous cells for the management of the human disease. Although phase 1 clinical trials have been conducted with encouraging results, the search for the most powerful hCSC for myocardial regeneration is in its infancy. This manuscript discusses the efforts performed in our laboratory to characterize the critical biological variables that define the growth reserve of hCSCs. Based on the theory of the immortal DNA template, we propose that stem cells retaining the old DNA represent one of the most powerful cells for myocardial regeneration. Similarly, the expression of insulin-like growth factor-1 receptors in hCSCs recognizes a cell phenotype with superior replicating reserve. However, the impressive recovery in ventricular hemodynamics and anatomy mediated by clonal hCSCs carrying the "mother" DNA underscores the clinical relevance of this hCSC class for the treatment of human heart failure.
... Slow turnover rate has been demonstrated by label retaining studies in animal models. However, there are several pitfalls related to the use of label retention as a marker of stem cells [106] . The duration of the DNA labelling period was typically less than one week in most label retaining studies [8][9][10][11] . ...
A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface.
... Rapidly dividing transitamplifying cells (TACs) dilute their DNA label with cell divisions. LRCs are the cells that can retain a DNA label after a prolonged period due to infrequent cycling, and have been identified as somatic SCs in some organs [17,18]. 5-Ethynyl-2deoxyuridine (EdU) is a thymidine analogue that can be incorporated into the newly synthesized DNA of replicating cells, and has been used to identify endogenous SCs [19]. ...
Self-renewal and differentiation of endogenous stem cells (SCs) are essential for adult tissue homoeostasis and intrinsic healing capacity. In this study, we hypothesize that penis contains a small population of endogenous SCs, which might help rejuvenation of damaged erectile function. In this study, 60 newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous SCs. Twelve weeks later, 48 rats underwent bilateral cavernous nerves injury and were randomized into gavage feeding of solvent (vehicle group) or icariside II (0.5, 1.5, and 4.5 mg/kg/day, respectively). Twelve sham-operated rats received vehicle treatment and served as control. The treatments were continued for 4 weeks followed by a washout period of 72 h. Results showed that ICA II treatment significantly restored erectile function and effectively prevented distortion of normal neural anatomy, smooth muscle atrophy, and collagen deposition compared with the vehicle group. The numbers of label-retaining cells (LRCs) coexpressing EdU and differentiated phenotypes (smooth muscle marker α-SMA or Schwann cell marker S100) were significantly higher in the three ICA II-treated groups than those in vehicle group in a dose-dependent manner. In addition, the changing trend of p38 mitogen-activated protein kinase (MAPK) activity in the penis between groups was same as that of the number of differentiated LRCs. Together, these results suggest that the underlying mechanisms of ICA II in ameliorating erectile function and pathological changes appear to involve enhanced endogenous SCs differentiation, which might be regulated by p38 MAPK signaling pathway.
... This ensured that the majority of cells were labeled. Labeling was followed by a chase period in which the label was diluted out through numerous cell divisions in the proliferating cells, but was retained in a small population of infrequently cycling stem/progenitor cells, or label retaining cells (Braun and Watt 2004). Immunohistochemistry was used to localize BrdU+ epithelial, stromal, and myometrial LRC, revealing their locations and proportions in the stem cell niche. ...
The human uterus is unique in that it exhibits a tremendous regenerative capacity that enables cyclical regeneration and remodeling throughout a woman's reproductive life. This plasticity of the reproductive system has recently been highlighted. Regeneration and remodeling in the female reproductive tract alludes to the existence of endometrial and myometrial stem cell systems, which has been supported by increasing experimental evidence. Characterization of these stem cells, along with the study of the mechanisms controlling their regeneration, will improve the understanding of the physiology and pathophysiology of the female reproductive tract.
... These results indicate rapid turnover of double positive HSCs cells in TG mice. Evidence indicates that at least two types of cell population exist: the slow cycling designated as stem cells and rapidly cycling cells as transit amplifying cells [30][31][32]. Rapid turnover of double positive HSCs cells in TG mice may be the effect of overexpression of PKC in TG mice and its associated cytokines such as TNF and G-CSF [33]. Additionally, the level or percentage of BrdU retention in the stem cell populations is not consistently uniform. ...
To find clues about the mechanism by which kinase C epsilon (PKC ε ) may impart susceptibility to ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinomas (SCC), we compared PKC ε transgenic (TG) mice and their wild-type (WT) littermates for (1) the effects of UVR exposures on percent of putative hair follicle stem cells (HSCs) and (2) HSCs proliferation. The percent of double HSCs (CD34+ and α 6-integrin or CD34+/CD49f+) in the isolated keratinocytes were determined by flow cytometric analysis. Both single and chronic UVR treatments (1.8 kJ/m(2)) resulted in an increase in the frequency of double positive HSCs in PKC ε TG mice as compared to their WT littermates. To determine the rate of proliferation of bulge region stem cells, a 5-bromo-2'-deoxyuridine labeling (BrdU) experiment was performed. In the WT mice, the percent of double positive HSCs retaining BrdU label was 28.4 ± 0.6% compared to 4.0 ± 0.06% for the TG mice, an approximately 7-fold decrease. A comparison of gene expression profiles of FACS sorted double positive HSCs showed increased expression of Pes1, Rad21, Tfdp1 and Cks1b genes in TG mice compared to WT mice. Also, PKC ε over expression in mice increased the clonogenicity of isolated keratinocytes, a property commonly ascribed to stem cells.
... Label retaining cells (LRCs) were identified in the laCL of incisors by giving neonatal mice repeated injections of BrdU (Harada et al., 1999;Seidel et al., 2010). Although the label retention is simply a marker of the proliferative history of a cell, LRCs represent a subset of tissue stem cells because of retaining the label over a long period of time (Braun and Watt, 2004). To address the LRC number and their distribution in laCL of Bcl11b S826G/KO incisors, we injected BrdU repeatedly into the abdominal cavity of P3 mice and harvested incisor tissues in P21 mice (Fig. 5A). ...
Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of Serine at position 826 for Glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5 days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells.
... Although, overall, net asymmetric division is required to maintain the SC pool and produce TACs, in some systems SCs need not always divide asymmetrically but may also divide symmetrically to produce two TACs or two SCs, providing a stochastic mechanism whereby some SC lineages expand and others are lost (Nakagawa et al. 2007;Snippert et al. 2010;Lopez-Garcia et al. 2010;Klein and Simons 2011). The adult stem cells that maintain these epithelia divide relatively infrequently; however, they have the potential to divide indefinitely (Braun and Watt 2004). Identification of the stem cells has, therefore, exploited these features using incorporation of tritiated thymidine ( 3 H-TdR) or the thymidine analogue bromodeoxyuridine (BrdU) into the DNA of dividing cells during S-phase of the cell cycle (Potten and Loeffler 1990). ...
Maintenance of the corneal epithelium is essential for vision and is a dynamic process incorporating constant cell production, movement and loss. Although cell-based therapies involving the transplantation of putative stem cells are well advanced for the treatment of human corneal defects, the scientific understanding of these interventions is poor. No definitive marker that discriminates stem cells that maintain the corneal epithelium from the surrounding tissue has been discovered and the identity of these elusive cells is, therefore, hotly debated. The key elements of corneal epithelial maintenance have long been recognised but it is still not known how this dynamic balance is co-ordinated during normal homeostasis to ensure the corneal epithelium is maintained at a uniform thickness. Most indirect experimental evidence supports the limbal epithelial stem cell (LESC) hypothesis, which proposes that the adult corneal epithelium is maintained by stem cells located in the limbus at the corneal periphery. However, this has been challenged recently by the corneal epithelial stem cell (CESC) hypothesis, which proposes that during normal homeostasis the mouse corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium with LESCs only contributing during wound healing. In this chapter we review experimental studies, mostly based on animal work, that provide insights into how stem cells maintain the normal corneal epithelium and consider the merits of the alternative LESC and CESC hypotheses. Finally, we highlight some recent research on other stem cell systems and consider how this could influence future research directions for identifying the stem cells that maintain the corneal epithelium.
... Cells with characteristics similar to those described above for germ layer lineage mesodermal stem cells have been isolated from newborn, adolescent, and adult mice, rats and humans [1,4], geriatric mice and geriatric humans [1,4], and adult rabbits, dogs, goats, sheep, and pigs [1,4]. Cells with characteristics similar to those described above for germ layer lineage mesodermal stem cells have also been isolated from the skeletal muscle of mice, rabbits, dogs, goats, sheep, and pigs [1,4], from the dermis of mice and rats [1,4], from the connective tissue layers surrounding the myocardium of mice and rats [1,4], from granulation tissue derived from rabbits and humans [1,4], and from other tissue sites, including brain, bone marrow, breast, blood, and the connective tissues associated with bone, cartilage, fat, nerves, and organs of the respiratory system, gastrointestinal system, and urinary system [1][2][3][4][5][29][30][31][32][33][34][35][36]. Germ layer lineage ectodermal stem cells have been isolated from meninges, brain tissues, skeletal muscle, dermis, fat, and skin [2,[24][25][26][27][28]. ...
... Kurzrock et al. localized label retaining cells to the basal layer of rat bladder urothelium [3]. Pulse labelling with bromo-deoxyuridine (BrdU) followed by long term observation allows to distinguish mitotically active TA cells, which gradually dilute the label at each round of DNA replication, from stem cells, which because of slow cycling and, possibly, retention of the same DNA strand (the hypothetical immortal DNA strand) throughout many replications retain the label [4,5]. One year after the BrdU pulse, ~ 10% of bladder urothelium basal cells were still BrdU-positive, strongly implicating this minor fraction as urothelial stem cells [3]. ...
Urothelium is a special type of stratified epithelium that lines the distal portion of the urinary tract. For a long time, basal urothelial cells have been suspected to include a population of urothelial stem cells. Recent experiments identifying label-retaining cells as well as lineage tracing analyses corroborate this notion. There are striking morphological and antigenic similarities between basal or differentiated urothelial cells and the corresponding cells in some urothelial carcinomas. In this respect, basal cell-specific markers provide good candidates to identify urothelial carcinoma stem cells, e.g. specific cytokeratins (CK5, CK14, CK17) or adhesion molecules (specific integrin subspecies, CD44). Common properties of the stem cells of normal urothelium and urothelial cancer have thus emerged. Both are characterized by a remarkable plasticity and both rely on reciprocal interactions with stromal fibroblasts. However, the stem cells of individual urothelial carcinomas appear to differ considerably and may contribute to the heterogeneity of this disease. The presence, quantity, and particular biological nature of urothelial carcinoma stem cells in each case may thus carry important clinical information that might allow a rationale stratification of urothelial cancer patients for treatment in the near future. Keywords: urothelium, urothelial cancer, stem cells, epithelial-stromal interactions, cell culture models, clinical risk assessment.
... Early TA cells are slow-cycling and undergo a few cell divisions, some of which may be asymmetric; late TA cells undergo a state of rapid, yet restricted cell proliferation that effectively builds up the critical mass necessary for differentiation. This arrangement of tissue regenerative processes as a hierarchy thus ensures that tissue-specific stem cells retain a long-term regenerative capacity, [29] whereas progenitors characterized by a finite division capacity eventually stop dividing and differentiate along tissue-specific lineages into mature cell types, and on completing their functionality, ultimately undergo apoptosis. [30] The process also ensures a long-term genetic stablility, as a large majority of the de novo mutations arising during the processes of cell proliferation , commitment, and differentiation in the hierarchy will undergo a process of natural elimination, as all the differentiated cells will ultimately undergo apoptosis . ...
Introduction Stem Cells in the Life of an Organism Cancer Stem Cells Self-Renewal and Differentiation in CSCs CSC Plasticity as Regulated by Intrinsic and Extrinsic Stem Cell Factors Conclusions and Future Perspectives References
... Cells with characteristics similar to those described above for germ layer lineage mesodermal stem cells have been isolated from newborn, adolescent, and adult mice, rats and humans [1,4], geriatric mice and geriatric humans [1,4], and adult rabbits, dogs, goats, sheep, and pigs [1,4]. Cells with characteristics similar to those described above for germ layer lineage mesodermal stem cells have also been isolated from the skeletal muscle of mice, rabbits, dogs, goats, sheep, and pigs [1,4], from the dermis of mice and rats [1,4], from the connective tissue layers surrounding the myocardium of mice and rats [1,4], from granulation tissue derived from rabbits and humans [1,4], and from other tissue sites, including brain, bone marrow, breast, blood, and the connective tissues associated with bone, cartilage, fat, nerves, and organs of the respiratory system, gastrointestinal system, and urinary system [1][2][3][4][5][29][30][31][32][33][34][35][36]. Germ layer lineage ectodermal stem cells have been isolated from meninges, brain tissues, skeletal muscle, dermis, fat, and skin [2,[24][25][26][27][28]. ...
This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.
... Owing to this infrequently dividing nature, the stem cells in animals that incorporate DNA synthesis labels such as tritiated thymidine or bromodeoxyuridine (BrdU) tend to remain " labelled " for longer than TACs, whose more rapid cycling soon dilutes the label below detection levels. Thus, the identification of label-retaining cells (LRCs) is often used as a stem cell marker (Braun and Watt, 2004; Alison et al., 2006). Some stem cells appear to have devised a strategy for maintaining genome integrity; another cause of " label retention " . ...
The origin and propagation of neoplastic growth appear to be from stem cells, initially affecting normal stem cells and then involving neoplastic stem cells whose identity will be paramount for successful chemotherapeutic targeting. Many, if not all cancers contain a minority population of self-renewing stem cells, the cancer stem cells (CSCs) that are entirely responsible for sustaining the tumour as well as giving rise to proliferating but progressively differentiating cells that contribute to the cellular heterogeneity typical of many solid tumours. Thus, the bulk of the tumour is often not the clinical problem; the identification of CSCs and the factors that regulate their behaviour are likely to have an enormous bearing on the way we treat neoplastic disease in the future. This chapter summarizes our knowledge of the origins of some cancers from normal stem cells, the evidence for the existence of cancer stem cells and illustrates some of the stem cell renewal pathways that are frequently aberrant in cancer.
Background
Hair follicle stem cells (HFSC) play an essential role in the maintenance of hair homeostasis during the hair cycle in which HFSC remain quiescent most of its duration. The hairpoor mouse (+/HrHp), an animal model of Marie-Unna Hypotrichosis (MUHH), exhibits over-expression of Hairless in the bulge, inner root sheath and outer root sheath of HF and exhibits the same phenotype as in MUHH patients manifesting sparse hair with progression to alopecia with age. In this study, we aimed to understand the hair cycle and the status of HFSC during the hair cycle of the hairpoor mouse to delineate the pathogenesis of MUHH.
Methods
To define the state of the hair follicle, H&E staining was performed. FACS analysis and immunostaining was utilized at the 1st and 2nd telogen stages to observe the HFSCs. Label retaining assay was carried out to determine the quiescent state of hair follicle. Expression of factors in involved in the niche signaling and the Wnt signaling was determined using qRT-PCR.
Results
We found that the number of hair follicle was drastically decreased after 1st telogen, then followed by the intensified disturbance in the hair cycle with shorter anagen as well as 2nd telogen in the hairpoor mouse. The number of CD34 expressing bulges as well as cells were dramatically reduced at the telogen of the HFs and the high proliferation of bulge cells was prominent, suggesting the loss of HFSC quiescence in the hairpoor mouse. The increased cell proliferation in HF was reiterated following the synchronization of hair cycle, leading to accelerating HF cycling. The expression of Fgf18 and Bmp6, the factors involved in the HFSC quiescence, was reduced in the HFSC niche of the hairpoor mouse. Furthermore, expression of Wnt signaling molecules including Wnt7b, Wnt10b and Sfrp1 were disturbed inducing the telogen to anagen transition of HFs in the hairpoor mouse.
Conclusions
These results indicate that the quiescent state of HFSC is not properly maintained in the hairpoor mouse and consequently leading HFs to the completely disarrayed hair cycle. These findings may provide an understanding of an underlying mechanism by which alopecia develops with age in MUHH patients.
Intestinal tissue, and specifically its mucosal layer, is a complex and gradient-rich environment. Gradients of soluble factor (BMP, Noggin, Notch, Hedgehog, and Wnt), insoluble extracellular matrix proteins (laminins, collagens, fibronectin, and their cognate receptors), stromal stiffness, oxygenation, and sheer stress induced by luminal fluid flow at the crypt-villus axis controls and supports healthy intestinal tissue homeostasis. However, due to current technological challenges, very few of these features have so far been included in in vitro intestinal tissue mimetic platforms. In this review, the tightly defined and dynamic microenvironment of the intestinal tissue is presented in detail. Additionally, the authors introduce the current state-of-the-art intestinal tissue mimetic platforms, as well as the design drawbacks and challenges they face while attempting to capture the complexity of the intestinal tissue’s physiology. Finally, the compositions of an “idealized” mimetic system is presented to guide future developmental efforts.
The prostate develops by epithelial budding and branching processes that occur during fetal and postnatal stages. The adult prostate demonstrates remarkable regenerative capacity, with the ability to regrow to its original size over multiple cycles of castration and androgen administration. This capacity for controlled regeneration prompted the search for an androgen-independent epithelial progenitor in benign prostatic hyperplasia (BPH) and prostate cancer (PCa). BPH is hypothesized to be a reawakening of ductal branching, resulting in the formation of new proximal glands, all while androgen levels are decreasing in the aging male. Advanced prostate cancer can be slowed with androgen deprivation, but resistance eventually occurs, suggesting the existence of an androgen-independent progenitor. Recent studies indicate that there are multiple castration-insensitive epithelial cell types in the proximal area of the prostate, but not all act as progenitors during prostate development or regeneration. This review highlights how recent cellular and anatomical studies are changing our perspective on the identity of the prostate progenitor.
Little is known about the origin of the inductive signal that translates the amputation stress into a cooperative cellular response. By studying the process underlying the reformation of lost body parts in the anthozoan cnidarian Nematostella vectensis, we identified a regeneration-inducing structure that, via a tissue crosstalk, is responsible for the initiation of the repair program. We further revealed for the first time in anthozoan cnidarians, that fast and slow-cycling/quiescent stem cells respond to the amputation stress and actively participate in the reformation of lost body parts. Importantly, a synergic interaction of both stem cell populations is required to complete the regeneration process. Our findings suggest that the emergence/loss of structure complexity/compartmentalization influences the proprieties of tissue plasticity, changes the competence of a tissue to reprogram and, in the context of regeneration, the capacity of the tissue to emit or respond to a regeneration-inducing signal.
The molecular circuitry directing tissue development and homeostasis is hardwired by genetic programs but may also be subject to fine-tuning or major modification by environmental conditions. It remains unclear whether such malleability is at work—particularly in tissues directly in contact with the environment—and contributes to their optimal maintenance and resilience. The protein kinase p38α is activated by physiological cues that signal tissue damage and neoplastic transformation. Here, we found that p38α phosphorylated and thereby destabilized p63, a transcription factor essential for epidermal development. Through this regulatory mechanism, p38α limited the frequency of keratinocytes with stem cell properties and tumorigenic potential. Correspondingly, epidermal loss of p38α expression or activity promoted or correlated with carcinogenesis in mouse and human skin, respectively. Genetic mouse models revealed a tumorigenic mechanism from p38α loss through p63-mediated suppression of the matrix metalloprotease MMP13. These findings illustrate a previously uncharacterized epidermal tumor–suppressive mechanism in which stress-activated signaling induces the contraction of stem cell–like keratinocyte pools.
The skin is an organ defined by its ability to act as a barrier to both mechanical and chemical stresses. Skin's barrier function is mostly achieved by epidermal keratinocyte (KC) stem cell (SC) regeneration and then stratification, terminal differentiation, and shedding of their progeny cells. As the barrier with the environment, an individual's KCs may be exposed to environmentally or systemically delivered toxins. Among these are naturally occurring inorganic arsenic, human‐made aryl hydrocarbons, e.g. dioxin, and bacterially derived toxins. The balance of SC division and progeny KC differentiation is crucial to maintaining a healthy epidermis; loss of regulation can result in KC hyperplasia and epidermis overgrowth or hypoplasia and skin ulceration. In this chapter, we examine KC responses to such toxins and the possible mechanisms responsible for their dysregulation of the regeneration/differentiation balance crucial to skin barrier maintenance.
This review discusses the changes in stem and progenitor populations that occur with aging and, more specifically, changes of the epidermis that occur with aging. The consensus of opinion is that changes responsible for aging of tissues occur not only in the stem cell pool itself but also in the transit-amplifying cell compartment and in the stem cell environment. In order to study aging of epidermal stem cells, it is essential to isolate epidermal stem cells at the single cell level to better define them at a molecular level. It will also be important to study the intrinsic and extrinsic changes that occur in the environment/niche of the epidermal stem cell with aging.
Introduction Human Prostate Biology, Gland Architecture and Pathological Alterations Prostate Epithelial Stem Cells Prostate Cancer Stem Cells Stem Cell Tracking in the Prostate Conclusions and Future Perspectives References
Human epidermis is a continuously renewing tissue where equilibrium between new born and lost cells is maintained by asymmetric division of stem cells producing two unequal daughter cells; one with the ability to perpetuate itself and another committed to differentiate into several cell lineages reconstituting epidermis and its appendages. Recent findings suggest that altered stem cells, called tumor initiating or cancer stem cells, are at the origin of tumor development. Accordingly, it is believed that targeted eradication of these cells, holds the promise to cure cancer. Our studies were design to identify and characterize cancer stem cells in skin tumors and to evaluate involvement of Epidermal Growth Factor Receptor (EGFR) signalling pathway in controlling their self renewal and differentiation function. We found rare mitotic keratinocytes with asymmetrically distributed EGFR and determined that its prevalence on the surface was linked to normal and cancer cell fate. Although essential for epithelial cell proliferation, differentiation and survival, EGFR was not present on the surface of cells satisfying criteria for stem cells such as quiescence, competence to produce functionally distinct daughters, high proliferative and clonogenic potential, sphere formation ability and expression of stem cell markers. In contrast, keratinocytes displaying EGFR acquired a more differentiated phenotype, demonstrating that EGFR controls a switch from the stem to transient amplifying cell compartment. This switch was associated with changes in the expression profile of cell cycle, survival and mitochondria controlling proteins that varied between normal and cancer cells. Based on our findings, we propose a model in which EGFR functions as a determinant of keratinocytes fate that balances quiescence and proliferation/differentiation of epidermal stem cells during mitosis. This balance is clearly malfunctioning in cancer. Since, as we previously showed keratinocytes differentiation shares some aspects of apoptotic pathway and is stimulated by reactive oxygen species (ROS), we further propose that acquisition of EGFR on cell surface activates cell proliferation and consequently, ROS production. Importantly, our study strongly suggests that EGFR negative cells may constitute the tumor reproductive compartment responsible for tumor maintenance and development, thus providing mechanistic insights into the relative inefficacy of current anti-EGFR therapies.
Advances in aging biology indicate that stem cells have a crucial role in organ maturation and aging. Studies have demonstrated molecular and biochemical changes in tissue-resident progenitor cells and their microenvironments during chronological aging of tissues such as the heart [1], brain [2], and hematopoietic system [3]. In this chapter knowledge in the field of aging and stem cells derived from tissues other than the epidermis is reviewed, and the challenges of studying aging stem cells discussed. Subsequently, epidermal stem cells are reviewed and changes in progenitor populations of the epidermis that occur with age discussed. Finally, the body of knowledge specifically related to the aging of epidermal stem cells and the implications of stem cell aging for carcinogenesis are examined.
Lineage tracing allows the destiny of a stem cell (SC) and its progeny to be followed through time. In order to track their long-term fate, SC must be permanently marked to discern their distribution, division, displacement and differentiation. This information is essential for unravelling the mysteries that govern their replenishing activity while they remain anchored within their niche microenvironment. Modern-day lineage tracing uses inducible genetic recombination to illuminate cells within embryonic, newborn and adult tissues, and the advent of powerful high-resolution microscopy has enabled the behaviour of labelled cells to be monitored in real-time in a living organism. The simple structural organization of the mammalian cornea, including its accessibility and transparency, renders it the ideal tissue to study SC fate using lineage tracing assisted by non-invasive intravital microscopy. Despite more than a century of research devoted to understanding how this tissue is maintained and repaired, many limitations and controversies continue to plague the field, including uncertainties about the specificity of current SC markers, the number of SC within the cornea, their mode of division, their location, and importantly the signals that govern cell migration. This communication will highlight historical discoveries as well as recent developments in the corneal SC field; more specifically how the progeny of these cells are mobilised to replenish this dynamic tissue during steady-state, disease and transplantation. Also discussed is how insights gleaned from animal studies can be used to advance our knowledge of the fundamental mechanisms that govern modelling and remodelling of the human cornea in health and disease.
Copyright © 2015. Published by Elsevier Ltd.
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.
The epidermis and its appendages, the hair follicle and sebaceous gland, have the capacity to constantly regenerate throughout adult life. Postnatal hair follicles undergo a cyclic mode of tissue homeostasis, defined by periods of growth, degeneration, and rest. A multipotent population of stem cells residing within the hair follicle bulge not only generates the hair lineages during each hair cycle, but also transiently contributes to the repair of epidermis following wounding. In this chapter, we provide methods for identifying epidermal stem cells and investigating their proliferative and apoptotic characteristics. We introduce whole-mount and flow cytometry techniques, which complement each other by permitting visualization of the epidermal stem cell compartment in situ and assessment of the phenotype of purified cells. These techniques can easily be adapted to characterize novel putative epidermal stem or progenitor cell populations. By applying whole-mount and flow cytometry techniques to characterize normal and genetically modified mice with skin defects, we expect to learn more about the factors that regulate stem cell self-renewal and differentiation.
Four categories of cells have been previously identified in postnatal mammals, including humans. These categories are differentiated cells, cell- and tissue-committed progenitor cells, germ layer lineage-committed stem cells and lineage-uncommitted pluripotent stem cells. Differentiated cells are variable in size. They form the physiological functional components of the tissue. Progenitor cells are also variable in size and are the immediate precursors of the differentiated cells. … Recently, a 5th category of cell was discovered in postnatal tissues. These putative stem cells are less than 1 micron in size. They demonstrate a normal karyotype. These cells are the immediate precursors to the pluripotent stem cells. They can form all somatic cells of the body and spermatogonia. These cells have shown extensive capabilities for self-renewal. They will form multiple layers of cells post confluence. Shared and unique characteristics will be discussed for the five categories of adult-derived cells.
The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells.
The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells.
The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.
Pluristratified epithelia of adult vertebrate skin continuously regenerate from stem cells, and the question still arises as to whether those cells are committed to the production of only one cell lineage, or in contrast they conserve their embryonic pluripotentiality. In order to investigate the abilities of adult cultured as well as wound healing epidermis, heterospecific fibroblast-keratinocyte recombinations were performed, which allow unquestionable identification of the cells implicated in the structures that differentiate. Adult human cultured breast epidermal cells and full-thickness wound healing from human facial skin and foreskin were associated with either rabbit embryonic trichogenic dermis or cultured dermal papilla cells of adult rat, before grafting onto nude mice for two weeks to one month. In situ hybridization with a human specific sequence Alu probe labeled the human cells, whereas implanted rabbit or rat and host mouse cells were distinguished by the Hoechst staining of their nuclei. The results show that human adult cultured breast epidermal cells are able to form hair buds and to participate in hair follicle formation, while adult healing epidermis from a sparsely hairy skin as the human face or the dorsal skin of nude mouse, or even from a glabrous epidermis as the human foreskin, are able to differentiate pilosebaceous units. Although a follicular origin of the involved keratinocytes cannot be excluded in the three first cases, the formation of hair and sebaceous glands by foreskin keratinocytes of children 2 to 10 years-old establishes the cutaneous appendage ability of the interfollicular epidermal stem cells. The formation of interspecies mosaic follicles also highlights the fact that there must be a significant level of commonality in the interactive signaling molecules used by epithelial cells from different species.
Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of beta1 integrin expression, and expression of cytokeratin 19.
Stem cells are currently in the news for two reasons: the successful cultivation of human embryonic stem cell lines and reports
that adult stem cells can differentiate into developmentally unrelated cell types, such as nerve cells into blood cells. Both
intrinsic and extrinsic signals regulate stem cell fate and some of these signals have now been identified. Certain aspects
of the stem cell microenvironment, or niche, are conserved between tissues, and this can be exploited in the application of
stem cells to tissue replacement therapy.
The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [(3)H]thymidine ([(3)H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [(3)H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of alpha(6) integrin and low to undetectable expression of the transferrin receptor (CD71) termed alpha(6)(bri)CD71(dim) cells, are enriched for epithelial stem cells because they represent a minor ( approximately 8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing approximately 70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed alpha(6)(bri)CD71(bri), representing the majority ( approximately 60%) of basal keratinocytes that are actively cycling, and importantly contain approximately 70% of [(3)H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71(dim) cells in the hair follicle bulge region, a well documented location for KSCs.
The beta-catenin/TCF signaling pathway is essential for the maintenance of epithelial stem cells in the small intestine. c-Myc a downstream target of beta-catenin/TCF (ref. 2), can induce differentiation of epidermal stem cells in vitro. To determine the role of c-Myc in epidermal stem cells in vivo, we have targeted expression of human MYC2 to the hair follicles and the basal layer of mouse epidermis using a keratin 14 vector (K14.MYC2). Adult K14.MYC2 mice gradually lose their hair and develop spontaneous ulcerated lesions due to a severe impairment in wound healing; their keratinocytes show impaired migration in response to wounding. The expression of beta1 integrin, which is preferentially expressed in epidermal stem cells is unusually low in the epidermis of K14.MYC2 mice. Label-retaining analysis to identify epidermal stem cells reveals a 75% reduction in the number of stem cells in 3-month-old K14.MYC2 mice, compared with wildtype mice. We conclude that deregulated expression of c-Myc in stem cells reduces beta1 integrin expression, which is essential to both keratinocyte migration and stem cell maintenance.
Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of beta1 integrin expression, and expression of cytokeratin 19.
Activation of Myc (c-Myc) causes epidermal cells to exit the stem cell compartment and differentiate into sebocytes and interfollicular epidermis at the expense of the hair lineages. To investigate how Myc exerts these effects we analysed the transcription of more than 10,000 genes following Myc activation in the basal layer of mouse epidermis for 1 or 4 days. The major classes of induced genes were involved in synthesis and processing of RNA and proteins, in cell proliferation and in differentiation. More than 40% of the downregulated genes encoded cell adhesion and cytoskeleton proteins. Repression of these genes resulted in profound changes in the adhesive and motile behaviour of keratinocytes. Myc activation inhibited cell motility and wound healing, correlating with decreased expression of a large number of extracellular matrix proteins. Cell adhesion and spreading were also impaired, and this correlated with decreased expression of the α6β4 integrin, decreased formation of hemidesmosomes and decreased assembly of the actomyosin cytoskeleton. We propose that Myc stimulates exit from the stem cell compartment by reducing adhesive interactions with the local microenvironment or niche, and that the failure of hair differentiation reflects an inability of keratinocytes to migrate along the outer root sheath to receive hair inductive stimuli.
Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that differentiate along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. As stem cells divide infrequently in adult mouse epidermis, they can be visualised as DNA label-retaining cells (LRC). With whole-mount labelling, we can examine large areas of interfollicular epidermis and many hair follicles simultaneously, enabling us to evaluate stem cell markers and examine the effects of different stimuli on the LRC population. LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. LRC reside throughout the permanent region of the hair follicle,where they express keratin 15 and lie in a region of high α6β4 integrin expression. LRC are not significantly depleted by successive hair growth cycles. They can, nevertheless, be stimulated to divide by treatment with phorbol ester, resulting in near complete loss of LRC within 12 days. Activation of Myc stimulates epidermal proliferation without depleting LRC and induces differentiation of sebocytes within the interfollicular epidermis. Expression of N-terminally truncated Lef1 to block β-catenin signalling induces transdifferentiation of hair follicles into interfollicular epidermis and sebocytes and causes loss of LRC primarily through proliferation. We conclude that LRC are more sensitive to some proliferative stimuli than others and that changes in lineage can occur with or without recruitment of LRC into cycle.
The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
A model is suggested for the organization of the epidermis based on the ordered structure of the differentiating layers, as demonstrated by published work. This ordered structure enables one to look through the epidermis at the basal cell nuclei and to see their arrangement beneath the differentiating column of cells. In dorsal skin from male DBA-2 mice there are 10.6 basal nuclei beneath a column of cells. The central nucleus of the group responds slightly earlier and more effectively than the rest to stimulation, is cycling more slowly than the majority of the basal nuclei and may spend significant periods of time out of cycle.
The skin appears to contain a series of fairly independent proliferative units, each of which contain ten or eleven basal nuclei and eight to ten superficial cells of which only the youngest one or two retain their nuclei. At the centre of each group of basal nuclei is a cell that behaves differently from the rest and which is present in the skin in numbers compatible with the number of clonogenic cells. It is suggested that this represents the basic stem cell of the unit.
The structure of mouse tail epidermis has been investigated from day 15 of embryonic development and a qualitative and quantitative analysis of the localization of Langerhans cells has been carried out using the ATPase method.
Langerhans cells occur exclusively in regions where a granular layer is present in the developing epidermis. The appearance of both a granular layer and evenly distributed Langerhans cells is noted for the first time at embryonic days 16 to 17. At postnatal day 3 the number of Langerhans cells as well as the thickness of the granular layer are increased although the characteristic scale-interscale pattern of adult mouse tail skin is not yet outlined. This occurs first around postnatal day 9 and is characterized by a gradual reduction of the granular layer in the regions surrounding the forming scales. Exactly the same areas are occupied by Langerhans cells.
This pattern of distribution is maintained in the adult animal. Repeated treatment of adult mouse tail skin with vitamin A results in a conversion of the para-keratotic epidermis into an ortho-keratinizing one, so that a granular layer is present throughout the whole epidermis. Concomitantly, Langerhans cells can clearly be demonstrated in the scale regions.
The formation of new hair follicles was quantitatively demonstrated in the tail skin of adult mice in the course of a two-stage carcinogenesis experiment with 7,12-dimethylbenz(a)anthracene as an initiator and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate as a promoter, as well as in experiments with 12-O-tetradecanoylphorbol-13-acetate alone. Two kinds of follicular neogenesis could be distinguished. The most frequently encountered type was characterized by the organization of new follicles from the upper neck and orifice regions of already existing follicles. During their development, these new follicles remained in close apposition to the original follicles but, after having reached a critical size, split off to form fully independent follicles. In the second, type of follicular neogenesis, which occurred very rarely, the new follicles seemed to arise directly from the epidermis between two sets of hair triads; however, these follicles never reached their final stage and did not produce hairs. The formation of new hair follicles may be explained by a "dedifferentiation" of epidermal cells caused by the tumor promoter. Because of the paucity and advanced stage of the papillomas formed in tail skin after long-term treatment with 12-O-tetradecanoylphorbol-13-acetate, no reliable comment as to whether the papillomas derive from the hair follicles can be made.
A study was made on the stem cell concepts. When we are talking about stem cells, we do not necessarily, and certainly not always talk about hemopoietic stem cells. There are many kinds of stem cells in the adult organism and there is one definition only which can apply to all these stem cells, and that is: a self-maintaining capacity. This means that stem cells can maintain their numbers for at least one life span of the organism. This means that 'stemness' implies an extensive self-maintaining capacity, and we mean self-maintaining capacity for many cell cycles, which in the mouse in most cases of 200 cell cycles, and in man - especially with a hemopoietic or intestinal stem cell system - well in excess of a thousand cell cycles. Every cell in the body, after the zygote and the first few cleavage divisions, is already a differentiated cell. That is: stem cells in the late embryonic crypt stem cells, skin epithelial stem cells or liver stem cell, are already differentiated cells. It is true that there are some stem cells which can give rise to further differentiated products, but not all stem cells do so. The author has given in his paper some of his ideas, about G(o) and genetic housekeeping, proliferation control and cell interaction, which like all concepts are pure fiction until such time that appropriate experimental evidence corroborates them. But at least, with the hemopoietic stem cells assay methods are available and with a little bit of clear thinking, clear definition - according to the author - and proper use of the available assays, the ideas can be tested. (E. Szirmai - Stuttgart) - Stuttgart, GFR)
Adult rat pelage follicle dermal papilla cells induced follicle neogenesis and external hair growth when associated with adult footpad skin epidermis. They thus demonstrated a capacity to completely change the structural arrangement and gene expression of adult epidermis--an ability previously undocumented for cultured adult cells. Isolation chambers ensured that de novo follicle formation must have occurred by eliminating the possibility of cellular contributions, and/or inductive influences, from local skin follicles. These findings argue against previous suggestions of vibrissa follicle specificity, and imply that the potential for hair follicle induction may be common to all adult papilla cells.
The mammalian hair follicle is a treasure waiting to be discovered by more molecular geneticists. How can a tiny cluster of apparently uniform epithelial cells, adjacent to a tiny cluster of uniform mesenchymal cells, give rise to five or six concentric cylinders, each of which is composed of cells of a distinctive type that synthesize their own distinctive set of proteins? There is now evidence that several growth factors, cell adhesion molecules and other molecules play important roles in the regulation of this minute organ.
The aims of the present study were to characterize the phenotype, growth kinetics, and proliferative activation in culture of a population of poorly differentiated homogeneously small (HS) keratinocytes. These slow-cycling cells were separated by unit gravity sedimentation from a population of actively proliferating basal keratinocytes in newborn rat skin. This population (approximately 1% of the total basal keratinocytes) consisted of extremely small cells with little cytoplasm or RNA. Their positive KL4 staining demonstrates that they were keratinocytes. HS keratinocytes did not, however, contain epidermal calcium binding protein. Acridine orange, bivariate Hoechst, and ethidium bromide flow cytometry of in vitro bromodeoxyuridine-labeled cells as well as Ki67 staining showed that HS keratinocytes were in the G0 stage of the cell cycle and did not actively proliferate in vivo. [3H]thymidine label-retaining cells were found only in the HS cell population, showing that HS cells may originate from a central position in the epidermal proliferative unit. Growth of HS cells in vitro was characterized by a delayed but progressive increase in RNA before entry into the cell cycle. The clonogenic efficiency of HS cells in primary culture was much less than that of larger cells. Subclones of HS cell colonies exceeded primary colonies in their cloning efficiency and proliferative potential, suggesting that HS cells, although normally prevented from dividing, retain a high self-renewal capacity. They also maintain the ability to differentiate. The results are consistent with the concept that HS cell population may represent the epidermal-specific progenitor cells which act as stem cells in this tissue.
Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.
Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.
Cellular topography within the highly polarized surface epithelia can be used to identify the location of the stem cells. In some instances, this can be quite precise and allows the characteristics of stem cells to be studied. Our current knowledge of the stem cell population in murine epidermis and small intestinal crypts is reviewed. In the epidermis, the stem cells would appear to make up about 10% of the basal layer and are distributed towards the centre of the basal layer component of the epidermal proliferative unit. These cells have a long cell cycle and are probably the same cells that retain both tritiated thymidine and radioactively labelled carcinogens for long periods of time. This label retention permits the labelling of the putative stem cell compartment. Over recent years, there has been an accumulation of information indicating various types of heterogeneity within the basal layer, much of which can be interpreted in relation to cellular hierarchies. In the small intestine, cell positions can be fairly precisely identified and the stem cell zone identified. Complex modelling of a wide range of cell kinetic experiments suggests that each crypt contains between 4 and 16 steady state functional stem cells. Radiobiological experiments suggest that up to 32 cells may be capable of clonal regeneration. The repopulation of the clonogenic cell compartment has been determined and the doubling time measured to be 19.7 h. Such studies should throw further light on the behaviour of stem cells and identify the timing of periods of increased and decreased cell proliferation (activation and suppression of controls).
The distribution and persistence of radioactively labeled benzo(a)pyrene [B(a)P] in the skin of adult female SENCAR mice were investigated by autoradiography of epidermal whole mounts and cross-sections at intervals following a single initiating application of 200 nmol of either [3H]B(a)P (2 mCi) or [14C]B(a)P (23 muCi). One day after treatment, the entire thickness of the skin was labeled; the grain density was greatest over hair follicles, sebaceous glands, and interfollicular epidermis. At 1 and 2 weeks, decreases in the nuclear grain density were consistent with the overall pattern of epidermal renewal. One month after treatment, carcinogen label-retaining cells made up approximately 2% of the interfollicular basal cells. They were also present in the hair follicles, approximately 4 and 5% of basal cells in the infundibulum and external root sheath, respectively. They were rare in the germ region and dermal papilla. Carcinogen label-retaining cells were compared with slowly cycling [3H]thymidine label-retaining cells and "maturing" basal cells, two distinct proliferative subsets of adult murine epidermis. Carcinogen label-retaining cells were found to have characteristics of the slowly cycling cells: (a) most of the carcinogen labeled nuclei were found in the central regions of the epidermal proliferative units; (b) treatment of the carcinogen label-retaining cells with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate elicited labeled mitoses within 1 day, and a general decrease in grain density over basal nuclei. In contrast, maturing basal cells 4 days after a single injection of [3H]thymidine were found at the periphery of the epidermal proliferative units. Within 1 day after treatment with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate, maturing basal cells were displaced to the suprabasal layers. Double isotope-double emulsion autoradiographs demonstrated doubly labeled cells 1 month after continuous labeling with [3H]thymidine and [14C]B(a)P and provide evidence that the radioactive carcinogen is retained by the slowly cycling [3H]thymidine label-retaining cells. These observations suggest that a slowly cycling population of epidermal cells may be relevant to the initiation phase of two-stage carcinogenesis.
A subpopulation of epithelial cells which retains a tritiated thymidine label (termed label-retaining cells, LRCs) has been previously demonstrated in skin and oral mucosae of mice and hamsters. To examine the rate of decrease in the number of LRCs and the changes in degree of labelling, young mice were labelled with tritiated thymidine and the rate at which label was diluted from basal keratinocytes assessed for up to 90 days. The number of LRCs in each tissue examined decreased from 15 to 90 days after labelling with the epidermal tissues maintaining a higher percentage of LRCs than the oral mucosae. Grain counts for LRCs in each tissue at each time period indicated that the number of silver grains overlying LRCs also decreased with time. The observed decrease in numbers of LRCs and the change in their degree of labelling with time suggest that such cells divide slowly, a property associated with stem cells.
The purpose of this investigation was to characterize the [3H]thymidine label-retaining and the "maturing" classes of basal cells from the dorsal epidermis of adult SENCAR mice and to compare their early cellular kinetic responses to topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Autoradiography of epidermal whole mounts and cross sections demonstrated that injection of [3H]thymidine every 6 h for 1 week labeled 95% of the basal nuclei, including those in the central region of the epidermal proliferative units. One month later, the labeling index was reduced to 2%; 90% of the label-retaining cells were within a nuclear diameter of the central suprabasal column of the proliferative units. When mice were treated with 2 micrograms of TPA 1 month after labeling, mitotic label-retaining cells were found within 22 h after treatment. Seventy-five percent of the label-retaining cells remained on the basal layer through the 28-h experimental period. In contrast, the basal labeling index following a 1-h pulse of [3H]thymidine was 5%. Eighty-five percent of the labeled cells were found in the periphery of the proliferative units. By 4 days after pulse labeling, most of the originally labeled cells had divided, although vertical cross sections indicated that 92% remained on the basal layer. When mice were treated with TPA on day 4, labeled cells were rarely found in mitosis. Instead, about 60% of the labeled cells were displaced to the suprabasal layers. These observations suggest that 2 classes of epidermal basal cells have different early responses to TPA treatment: the label-retaining cells proliferate, and most of the "maturing" cells continue to differentiate.
Within 24 hr after injection of mice on day 10 of pregnancy, embryos exposed to a teratogenic dose of 3H-bromodeoxyuridine (3H-BrdU) have smaller wet weights than control embryos exposed to a trace dose of 3H-thymidine (3H-TdR) or a trace dose of 3H-BrdU. This difference increased by 48 hr after injection. Measurements of total embryo DNA content indicate that these lighter embryos contain fewer cells than controls. If cell death plays a role in the observed growth retardation following administration of 3H-BrdU, one would expect label to be lost from the embryo DNA fraction. Following injection of a trace dose of 3H-TdR or 3H-BrdU, total embryo DNA label rapidly reached peak levels and remained at those levels for at least 48 hr after injection. However, within 48 hr of administering a teratogenic dose of 3H-BrdU, total embryo DNA label had decreased to 25% of peak levels. Histological evidence of cell death was found in the embryos, although it is not yet clear whether cell death was sufficiently extensive to account for the amount of label lost from the DNA fraction. Since none of these BrdU-induced cytotoxic effects were found in the maternal liver, spleen, kidney, or placenta, it is concluded that BrdU probably exerts its teratogenic effects directly on the embryo.
THE stratum corneum of mammalian skin usually appears in histological preparations as a loosely arranged layer of squames with little organization. The absence of organization is incompatible with the important barrier function of this layer and it has been suggested that histological processing severely distorts the true structure1. The boundaries of the cells of the stratum corneum can be clearly seen after treating unfixed sections with sodium hydroxide solution, because this causes the stratum corneum to swell but leaves the cell membranes of this layer intact2,3.
Statistical analysis of clustering of mitotic figures in adult human epidermis shows that this clustering is highly significant, in excess of what can be explained by chance. To explain this, some cause for the clustering can be looked for.
The results are consistent with the existence of a mitosis stimulating factor that is balanced by a diurnally varying anti-mitotic factor or factors—perhaps the adrenal steroid chalone complex of present theory. Such a mitosis stimulating factor would have the characteristics of an epidermal antichalone.
THE cells of the stratum corneum of mammalian skin, although usually
regarded as being randomly arranged, have been shown to be aligned in
regular columns1. This arrangement was suggested to result
from a regular pattern of cell migration from the basal layer. The
available evidence, however, links cell migration to cell division and
suggests that cell division is a random process2.
Continuous (repeated) labelling studies in mouse epidermis indicate that nearly all cells are labelled after about 100 hr. Percentage labelled mitoses studies ([3H]TdR at 15.00 and 03.00 hours) have a first peak that does not reach 100% and has a half-width of about 10 hr. Small second and third peaks can be detected at about 90 and 180 hr, respectively. The changes with time in the number of labelled cells show a difference dependent on the time of day of [3H]TdR administration. Both curves show an early doubling in labelled cells which then decline, forming a peak of labelled cells. A second peak occurs at about 120 hr. This is followed by a progressive decline with no further peaks until values of about 1% labelling are obtained at 340 hr.
These experiments have been investigated mathematically. A computer programme has been devized that permits all three types of experiments to be analysed simultaneously. More importantly, it can analyse situations with a heterogeneity in cell cycle parameters in all proliferative subpopulations.
Various models for epidermal cell replacement have been considered. The data as a whole can best be explained if the basal layer contains at least two distinct subpopulations of cells and an exponentially decaying post-mitotic population with a half-life of about 30 hr. The proliferative sub-populations must be characterized by near integer differences in the length of cycle, the precursor (stem) compartment having the longer cycle. An inverse relationship is required for the length of S, i.e. the shortest time for the stem cells.
A full range of cell kinetic parameters can be calculated and are tabulated for the most appropriate model system which is one involving three transit proliferating subpopulations.
Within human epidermis there are two types of proliferating keratinocyte: stem cells, which have high proliferative potential, and transit-amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. We show that, in vivo, stem cells express higher levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins than transit-amplifying cells and that this can be used both to determine the location of stem cells within the epidermis and to isolate them directly from the tissue. The distribution of stem cells and transit-amplifying cells is not random: patches of integrin-bright and integrin-dull cells have a specific location with respect to the epidermal-dermal junction that varies between body sites and that correlates with the distribution of S phase cells. Stem cell patterning can be recreated in culture, in the absence of dermis, and appears to be subject to autoregulation.
We have examined the growth capacity of keratinocytes isolated from human scalp hair follicles. Like the keratinocytes of glabrous epidermis, most of the colony-forming cells are classified as holoclones or meroclones when analyzed in a clonal assay. Some of them have extensive growth potential, as they are able to undergo at least 130 doublings. Therefore, the hair follicle, like the epidermis, contains keratinocytes with the expected property of stem cells: an extensive proliferative capacity permitting the generation of a large amount of epithelium. We have also examined the distribution of clonogenic keratinocytes within the hair follicle. Several hundred colony-forming cells are concentrated at a region below the midpoint of the follicle and outside the hair bulb. This region lies deeper than the site of insertion of the arrector pili muscle, which corresponds with the position of the bulge when the latter can be identified. In contrast, few colony-forming cells are present in the hair bulb, where most of the mitotic activity is observed during the active growth phase of the follicle. Paraclones, which are present both in the midregion and in the bulb of hair follicles, are unlikely to be the transient amplifying cells expected from kinetic studies.
The epidermis and its related appendages such as the hair follicle constitute the epithelial compartment of the skin. The exact location and distribution of the keratinocyte colony-forming cells within the epidermis or its appendages are unknown. We report that in the rat vibrissa, keratinocyte colony-forming cells are highly clustered in the bulge-containing region. Approximately 95% of the total colonies formed in culture from fractionated vibrissae were in this location and fewer than 4% were located in the matrix area of the follicle. Finer dissection of the bulge-containing region located the colony-forming cells in the small part containing the bulge itself. The segregation of keratinocyte colony-forming cells in the bulge confirms the hypothesis that the bulge is the reservoir of the stem cells responsible for the long-term growth of the hair follicle and perhaps of the epidermis as well.
The epidermis is believed to contain two types of proliferating cells: stem cells and cells with a lower capacity for self-renewal and higher probability of undergoing terminal differentiation (transit amplifying cells). We report that keratinocytes with characteristics of stem cells can be isolated from cultured human epidermis on the basis of high surface expression of beta 1 integrins and rapid adhesion to extracellular matrix (ECM) proteins. Among keratinocytes there was a log linear relationship between the relative level of beta 1 integrins on the cell surface and proliferative capacity; furthermore, the cells with the highest colony-forming efficiency adhered most rapidly to type IV collagen, fibronectin, or keratinocyte ECM. Proliferating keratinocytes that adhered more slowly had characteristics of transit amplifying cells: after one to five rounds of division, all of their daughters underwent terminal differentiation. Since stem cells can be isolated to greater than 90% purity on the basis of their adhesive properties, it will now be possible to investigate the mechanisms that regulate the fate of their progeny.
Continuously renewing epithelia contain small undifferentiated stem cells capable of self-renewal and maintenance of the differentiating cell population. In murine epidermis stem cells have been identified as label-retaining cells (LRCs) by long-term retention of tritiated thymidine or BrdU. It has been suggested that epidermal stem cells adhere to basement membranes through differential expression of specific integrins. To determine whether we could use a specific integrin to enrich for murine epidermal stem cells, we tested adherence of LRCs to several substrates. Regardless of the substrate used, approximately 10% of total basal cells and 100% of LRCs adhered in 10 min. In our medium specifically formulated for murine keratinocytes, rapidly adherent stem cells formed large colonies and could be used to form a structurally complete epidermis in organotypic culture. They showed a fivefold greater transient transfection efficiency than total basal cells, and when individual adherent cells were transduced with a retroviral vector, they formed large clones. Although these stem cells grew more slowly than the total basal cell population, they could be subcultured more times. Our results indicate that murine epidermal stem cells can be selected by rapid attachment to a substrate, but not by one specific integrin, and that they can be expanded in culture if the appropriate conditions are maintained.
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.
In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.
Slowly cycling label-retaining epidermal cells were identified by light microscopic autoradiography in the dorsal epidermis and hair follicles of adult mice 8-10 weeks after twice daily injection of [3H]dT on days three through five after birth. Pulse-labelled epidermal cells were identified in the epidermis and hair follicles of 7-8 week old mice 1 h after a single injection of [3H]dT at 8.00 a.m. For mice of both groups, epidermal cells including those from the hair follicles were harvested by trypsinization and were cultured from low density on feeder layers of irradiated Swiss mouse 3T3. On days 2, 4, 5, 7, 10 and 12, the cultures were fixed and processed for light microscopic autoradiography, and the distribution of labelled nuclei was quantified. On day 2 of culture, both label-retaining cells (LRC) and pulse labelled cells (PLC) were found primarily as single cells. After five days, LRC were found as pairs and clusters having silver grain counts consistent with their division. In contrast, PLC remained primarily as single cells. These results suggest that LRC may divide to form colonies (are clonogenic) whereas PLC are rarely clonogenic. The significance of this experiment is that it suggests that the LRC may not only be persistent in the epidermis, but that they may also be cells with relatively greater proliferative potential than the PLC and are thus likely to be stem cells.
In rodents, the hair follicle stem cells lie in a well-defined bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of beta1 integrin and low levels of E-cadherin and beta- and gamma-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of beta1 integrin subunits, E-cadherin, and beta- and gamma-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65-84 d EGA) and later to the outermost cells of hair peg (85-104 d EGA). In the bulbous hair peg (105-135 d EGA) and in the differentiated lanugo hair follicle (>135 d EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium.
A skin cancer seen in the clinic is in reality the culmination of a long history, only the later stages of which are easily observed. Progression from normal growth to the neoplastic, and later to the metastatic, stage of disease may involve multiple changes in cellular phenotypes and patterns of gene expression. Nonetheless, the early events in the pathway that leads from exposure to mutagenic agents to the formation of skin papillomas or carcinomas can be viewed as a two-step process of tumor initiation and tumor promotion. Fundamental questions remain about this process. In particular, the identity and the biological properties have yet to be defined of those cells, existing within a larger population of basal keratinocytes, that are specifically subject to tumor initiation. In addition, the behavior of these cells before, during, and after tumor promotion remains uncertain. The rare cells in this tissue that are capable of becoming pre-neoplastic when treated with a carcinogen exhibit many of the properties expected of keratinocyte stem cells. Here, I consider the evidence that they are indeed stem cells, and I explore the implications of this hypothesis for carcinogenesis in the skin.
Based on the hierarchical stem cell model of cellular replacement — originally described by Gilbert and Lajtha (1, 2) for the hematopoietic bone marrow, and later discussed by Buick (3) and by Hume (4) — a stem cell represents a self-renewing cell that may also produce proliferative cells, which undergo a series of amplification divisions prior to terminal maturation. Stem cells are thought to be quiescent or to cycle slowly under normal circumstances and to be protected by the architecture of the tissue. They should also be capable, when appropriately stimulated, of extensive proliferation to produce terminally differentiated progeny, and they should be capable of self-renewing in vivo and in vitro in order to maintain their own population as well as that of the terminally differentiated cells. Furthermore, some but not all stem cells are multipotential, that is, capable of producing more than one terminally differentiated lineage. Given this definition, do the targets of carcinogen action meet this definition of a stem cell (5)?
Keratinocyte stem cells have not yet been isolated, but some of their features are now becoming evident. From work in my laboratory and others, it is now possible to identify subpopulations with varying proliferative potential within the basal layer of the epidermis. This, along with other characteristics, suggests that some of these cells correspond to true stem cells. Keratinocyte subpopulations can be defined by various characteristics — location within the tissue, responses to chemical treatments, mitotic behavior in vivo or in vitro, morphology, and expression of marker proteins. Each of these parameters helps define a subpopulation that may contain stem cells, but none of them, taken individually, provides an accepted operational definition of the keratinocyte stem cell. Together, however, these parameters show great promise for the identification and isolation of keratinocyte stem cells. I will focus on the properties of these candidate stem cell populations and the possible identities among them, as well as on the evidence that these cells are the targets of two-stage carcinogenesis.
The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis.
To produce conditional expression of genes in the mouse epidermis we have generated transgenic mouse lines in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter. The transactivator lines were crossed with the tetOlacZ indicator line to test for transactivation in vivo. In the absence of doxycycline, the K5/tTA line induced beta-galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical doxycycline caused a dose- and time-dependent suppression of beta-galactosidase mRNA levels and enzyme activity. In the K5/rTA lines, doxycycline induced beta-galactosidase activity between 3- and 50-fold higher depending on the founder line, and this occurred within 24-48 h after dosing. Histochemical analysis of all lines localized beta-galactosidase expression to the basal layer of the epidermis and the outer root sheath of the hair follicle, as well as other keratin 5 positive tissues. In several K5/rTA lines, skin-specific transactivation was restricted to the hair follicle. Treatment of these double transgenic mice with 12-O-tetradecanoyl-phorbol-13-acetate caused rapid migration of beta-galactosidase marked cells from the hair follicle through the interfollicular epidermis, demonstrating the usefulness of this specific double transgenic for fate mapping cells in the epidermis. These results show that the tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and suggest that it should be amenable to suppression and activation of foreign genes during development and specific pathologic conditions relevant to the epidermis.
In spite of its clinical importance, little is known about the stem-cell compartment of the human oesophageal epithelium [1,2]. The epithelial basal layer consists of two distinct zones, one overlying the papillae of the supporting connective tissue (PBL) and the other covering the interpapillary zone (IBL) [3]. In examining the oesophageal basal layer, we found that proliferating cells were rare in the IBL and a high proportion of mitoses were asymmetrical, giving rise to one basal daughter and one suprabasal, differentiating daughter. In the PBL, mitoses were more frequent and predominantly symmetrical. The IBL was characterised by low expression of ?1 integrins and high expression of the beta2 laminin chain. By combining fluorescence-activated cell sorting (FACS) with in vitro clonal analysis, we obtained evidence that the IBL is enriched for stem cells. A normal oesophageal epithelium with asymmetric divisions was reconstituted on denuded oesophageal connective tissue. In contrast, asymmetric divisions were not sustained on skin connective tissue, and the epithelium formed resembled epidermis. We propose that stem cells located in the IBL give rise to differentiating daughters through asymmetric divisions in response to cues from the underlying basement membrane. Until now, stem-cell fate in stratified squamous epithelia was believed to be achieved largely through populational asymmetry [4-6].
The upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands, and epidermis, i.e., all the lineages of the hairy skin. At the time when hair production ceases and when the lower region of the follicle undergoes major structural changes, the lower region contains a significant number of clonogenic keratinocytes, and can then respond to morphogenetic signals. This demonstrates that multipotent stem cells migrate to the root of the follicle to produce whisker growth. Moreover, our results indicate that the clonogenic keratinocytes are closely related, if not identical, to the multipotent stem cells, and that the regulation of whisker growth necessitates a precise control of stem cell trafficking.
Continuous renewal of the epidermis and its appendages throughout life depends on the proliferation of a distinct population of cells called stem cells. We have used in situ retrovirus-mediated gene transfer to genetically mark cutaneous epithelial stem cells of adolescent mice, and have followed the fate of the marked progeny after at least 37 epidermal turnovers and five cycles of depilation-induced hair growth. Histological examination of serial sections of labeled pilosebaceous units demonstrated a complex cell lineage. In most instances, labeled cells were confined to one or more follicular compartments or solely to sebaceous glands. Labeled keratinocytes in interfollicular epidermis were confined to distinct columnar units representing epidermal proliferative units. The contribution of hair follicles to the epidermis was limited to a small rim of epidermis at the margin of the follicle, indicating that long term maintenance of interfollicular epidermis was independent of follicle-derived cells. Our results indicate the presence of multiple stem cells in cutaneous epithelium, some with restricted lineages in the absence of major injury.
The epidermis is maintained throughout adult life by pluripotential stem cells that give rise, via daughter cells of restricted self-renewal capacity and high differentiation probability (transit-amplifying cells), to interfollicular epidermis, hair follicles, and sebaceous glands. In vivo, transit-amplifying cells are actively cycling, whereas stem cells divide infrequently. Experiments with cultured human keratinocytes suggest that c-Myc promotes epidermal-stem cell differentiation. However, Myc is a potent oncogene that suppresses differentiation and causes reversible neoplasia when expressed in the differentiating epidermal layers of transgenic mice. To investigate the effects of c-Myc on the stem cell compartment in vivo, we targetted c-MycER to the basal layer of transgenic mouse epidermis.
The activation of c-Myc by the application of 4-hydroxy-tamoxifen caused progressive and irreversible changes in adult epidermis. Proliferation was stimulated, but interfollicular keratinocytes still underwent normal terminal differentiation. Hair follicles were abnormal, and sebaceous differentiation was stimulated at the expense of hair differentiation. The activation of c-Myc by a single application of 4-hydroxy-tamoxifen was as effective as continuous treatment in stimulating proliferation and sebocyte differentiation, and the c-Myc-induced phenotype continued to develop even after the grafting of treated skin to an untreated recipient.
We propose that transient activation of c-Myc drives keratinocytes from the stem to the transit-amplifying compartment and thereby stimulates proliferation and differentiation along the epidermal and sebaceous lineages. The ability, demonstrated here for the first time, to manipulate exit from the stem cell compartment in vivo will facilitate further investigations of the relationship between stem cells and cancer.