c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.     

Chromatin remodeling and stem cell theory of relativity

The field of stem cell biology is currently being redefined. Stem cell (hematopoietic and non-hematopoietic) differentiation has been considered hierarchical in nature, but recent data suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. The stem cell (hematopoietic and non-hematopoietic) phenotype, the total differentiation capacity (hematopoietic and non-hematopoietic), gene expression as well as other stem cell functional characteristics (homing, receptor and adhesion molecule expression) vary throughout a cell-cycle transit widely. This seems to be dependent on shifting chromatin and gene expression with cell-cycle transit. The published data on DNA methylation, histone acetylation, and also RNAi, the major regulators of gene expression, conjoins very well and provides an explanation for the major issues of stem cell biology. Those features of stem cells mentioned above can be rather difficult to apprehend when a classical hierarchy biology view is applied, but they become clear and easier to understand once they are correlated with the underlining epigenetic changes. We are entering a new era of stem cell biology the era of "chromatinomics." We are one step closer to the practical use of cellular therapy for degenerative diseases.     

The rate of apoptosis in post mitotic neutrophil precursors of normal and neutropenic humans

Abstract. Using data on the fraction of post-mitotic neutrophil precursors (CD15⁺ cells) displaying positive markers for apoptosis in 12 normal humans, and a simple mathematical model, we have estimated the apoptotic rate to be about 0.28/day in this compartment. This implies that the influx of myelocytes into the post-mitotic compartment exceeds twice the granulocyte turnover rate (GTR), and that about 55% of the cells entering this compartment die before being released into the blood. The normal half life of apoptotic post-mitotic neutrophil precursors is calculated to be 10.4 h. Comparable calculations for patients indicate apoptosis rates in the post-mitotic compartment of about 17 times normal for one myelokathexis patient and rates of about 13 times normal for the one cyclical neutropenic patient and two severe congenital neutropenic patients. The estimated half life for apoptotic post-mitotic neutrophil precursors in the myelokathexis patient was about 0.4 h, 1.4 h in the cyclical neutropenia patient, and about 0.6 h in the severe congenital neutropenic patients.     

Flow cytometric BrdUrd-pulse-chase study of heat-induced cell-cycle progression delays

The flow cytometric, bromodeoxyuridine (BrdUrd)-pulse-chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse the cell-cycle perturbations induced by heat shock. Exponentially growing, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43°C for 5,10 or 15 min. The cells were then incubated in a BrdUrd-free medium and, at various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd-labelled divided and undivided, and unlabelled G₁, G₁S, and G₂) were defined in the resulting dual-parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell-cycle progression were assessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct modes of alteration in cell progression: 1 a delay in cell transit (either out of or into a given compartment), and 2 a decrease in the rate of cell transit. Hyperthermia produced a delay in the exit of cells from the G₁ compartment of ≈ 16 min per minute of heat at 43°C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 min at 43°C. Above this threshold the delay in exit of cells from the BrdUrd-labelled, undivided compartment was 25 min per minute of heat at 43°C. The more complex dose-response function of this latter compartment may reflect the fact that it includes two cell-cycle phases, S and G₂+ M. The decrease in the rate of transit out of G₂ for cells heated in G₂ was significantly larger than that for any other compartment, consistent with previous studies, which showed a G₂ accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell-cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.     

Epidermal cell proliferation. I. Changes with time in the proportion of isolated, paired and clustered labelled cells in sheets of murine epidermis

A new technical approach to analysing labelled cells in sheets of epidermis is presented. The changes in the proportion of isolated single labelled cells, paired or clusters of 3, 4, or more than 4, labelled cells in sheets of epidermis from the back of the mouse have been analysed at various times up to 500 h after 3HTdR administration at either 03.00 h or 15.00 h. The technique is not dependent on the relative number of labelled cells (i.e. the labelling index) but on the spatial distribution of labelled cells. The data cannot be adequately explained on the basis of a simple homogeneous stem cell population in the basal layer but can be better understood on the basis of an hierarchical stem cell-dividing transit proliferative model. The data are consistent with an average cell cycle time of about 100 h but there are suggestions of considerable cell kinetic heterogeneity. The data also suggest that the amount of lateral cell movement within the basal layer is small. The results may suggest that some stem cells either loose label in a manner similar to that suggested by Cairns (1975) i.e. through a process of selective segregation of their DNA strands, or that they have an extremely short S phase duration as postulated earlier (Potten et al. 1982). The present data have been extensively mathematically modelled in an accompanying paper. The model which best fits all the data is an hierarchical scheme with three cell divisions in the transit population but some branches of the lineage may be prematurely terminated by the early production of post-mitotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Psoriasis: hyperproliferation cannot induce characteristic epidermal morphology

Abstract A mathematical model of cell renewal in epidermis is proposed for describing how psoriatic lesions might develop, based on available cell kinetic data for normal and psoriatic epidermis. Our simulations clearly demonstrate that an increase in the turnover rate in the germinative cell population cannot alone induce the typical psoriatic tissue architecture (i.e. increased number of germinative cells). Two perturbations are needed to account for the morphology of clinically-stable psoriatic lesions. The first corresponds to a temporary disturbance of the steady state of the germinative layer, resulting in limited growth of this compartment; the second perturbation corresponds to a reduction in transit time in the differentiated compartment. Moreover, our simulation, based on a widely-accepted hypothesis of homeostatic control of tissue kinetics, demonstrates that the primary cause of typical psoriatic morphology is probably an alteration in epidermal maturation. In this view, depletion of differentiated cells at the surface is the stimulus for the increased cell production rate in the germinative population.     

Psoriasis: hyperproliferation cannot induce characteristic epidermal morphology

A mathematical model of cell renewal in epidermis is proposed for describing how psoriatic lesions might develop, based on available cell kinetic data for normal and psoriatic epidermis. Our simulations clearly demonstrate that an increase in the turnover rate in the germinative cell population cannot alone induce the typical psoriatic tissue architecture (i.e. increased number of germinative cells). Two perturbations are needed to account for the morphology of clinically-stable psoriatic lesions. The first corresponds to a temporary disturbance of the steady state of the germinative layer, resulting in limited growth of this compartment; the second perturbation corresponds to a reduction in transit time in the differentiated compartment. Moreover, our simulation, based on a widely-accepted hypothesis of homeostatic control of tissue kinetics, demonstrates that the primary cause of typical psoriatic morphology is probably an alteration in epidermal maturation. In this view, depletion of differentiated cells at the surface is the stimulus for the increased cell production rate in the germinative population.     

The involvement of Gab1 and PI 3-kinase in beta1 integrin signaling in keratinocytes

The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. Beta1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these beta1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of beta1 integrins, we established HaCaT cells with high beta1 integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing beta1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high beta1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the beta1 integrin-mediated regulation of the epidermal stem cell compartment.     

Epidermal Stem Cells

Epidermis contains a compartment of stem cells but currently there is no common criterion to recognize individual stem cells with any confidence. Epidermis appears to contain stem cells of different levels of maturity and it is very likely that the main repository of epidermal stem cells is located in the hair follicle from which cells can emigrate into epidermis and also give rise to follicular and sebaceous keratinocytes. Epidermis consists of proliferative units containing stem and transit-amplifying cells, but the exact size of a proliferative unit cannot be measured accurately. The available data suggest that populations of stem and transit-amplifying cells are not discrete but represent a continuum from cells with a high self-renewal capacity and a low probability of differentiation to those with low self-renewal capacities and high commitments to differentiation. Stem cells occupy a special niche that provides a microenvironment, including an adhesion of stem cells to the basal membrane and their paracrine interactions with neighbor epidermal and mesenchymal cells. The fate of an epidermal stem cell depends on its prehistory and microenvironment.     

Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis

To clarify the mechanisms that support the continuity of actively cycling tissues of long-lived organisms, we investigated the composition of a mouse spermatogenic stem cell system by pulse-chase of the undifferentiated spermatogonia, the population responsible for stem cell functions, in combination with transplantation and regeneration assays after pulse-labeling. We demonstrate that in addition to "actual stem cells," which are indeed self-renewing, a second population ("potential stem cells") also exists, which is capable of self-renewing but do not self-renew in the normal situation. Potential stem cells rapidly turn over in normal testes, suggesting that they belong to the transit-amplifying, rather than the dormant, population. During the long natural course, actual stem cells are occasionally lost and compensated for by progeny of their neighbors. In this process, potential stem cells are postulated to shift their modes from transit amplification to self-renewal, thus playing an essential role to ensure spermatogenesis integrity.     

Simulating psoriasis by altering transit amplifying cells

Computational models of tissue homeostasis will facilitate a deeper understanding of many diseases. They link molecular networks, cellular differentiation and the spatial and temporal organization of tissues. Here we show an approach which is able to computationally turn a healthy in silico epidermis into one with four central properties of psoriatic epidermis. We achieve this by altering a single simulation parameter in the cellular differentiation program of the simulated epidermal keratinocytes: the fractional time period during which transit amplifying cells proliferate (tau). Prolonging tau results in the four main pathological characteristics of psoriatic skin: (1) an absolute increase of the germinative compartment, (2) an absolute increase of the differentiated compartment, (3) a higher proportion of germinative cells and (4) a marked reduction in turnover time. The prolongation of tau is able to increase the proliferation capacity of the epidermal tissue without altering the cell cycle frequency.     

Persistence to anti-cancer treatments in the stationary to proliferating transition

The heterogeneous responses of clonal cancer cells to treatment is understood to be caused by several factors, including stochasticity, cell-cycle dynamics, and different micro-environments. In a tumor, cancer cells may encounter fluctuating conditions and transit from a stationary culture to a proliferating state, for example this may occur following treatment. Here, we undertake a quantitative evaluation of the response of single cancerous lymphoblasts (L1210 cells) to various treatments administered during this transition. Additionally, we developed an experimental system, a “Mammalian Mother Machine,” that tracks the fate of thousands of mammalian cells over several generations under transient exposure to chemotherapeutic drugs. Using our developed system, we were able to follow the same cell under repeated treatments and continuously track many generations. We found that the dynamics of the transition between stationary and proliferative states are highly variable and affect the response to drug treatment. Using cell-cycle markers, we were able to isolate a subpopulation of persister cells with distinctly higher than average survival probability. The higher survival rate encountered with cell-cycle phase specific drugs was associated with a significantly longer time-till-division, and was reduced by a non cell-cycle specific drug. Our results suggest that the variability of transition times from the stationary to the proliferating state may be an obstacle hampering the effectiveness of drugs and should be taken into account when designing treatment regimens.     

Epidermal kinetic alterations required to generate the psoriatic phenotype: a reappraisal

Objectives: Although there have been major advances in understanding immunopathogenesis of psoriasis, the basic processes causing psoriatic morphology remain to be identified. Materials and methods: Our group has designed a systematic review of studies (1962–2009) on keratinocyte kinetics in psoriasis. We obtained data from MEDLINE, PubMed, Current Contents, reference lists and specialist textbooks. A general equation for evolution of the differentiated epidermis has been analysed. Necessary conditions for observed qualitative change in homeostasis between normal skin and established psoriatic lesions were determined. Results and discussion: Increase in the number of cell divisions (or imbalance in symmetric division rates of committed progenitor cells) and/or decrease in physiological apoptosis in the germinative compartment, together with feedback loops that limit thickening of the skin, are required to generate psoriatic morphology, that is, to increase the absolute size but decrease relative size of the differentiated cell compartment with respect to the germinative compartment.     

An analysis of the growth of the retinal cell population in embryonic chicks yielding proliferative ratios, numbers of proliferative and non-proliferative cells and cell-cycle times for successive generations of cell cycles

Growth curves of the retinal cell population of embryonic chicks were fitted by a branching-process model of cell population growth, thereby estimating the proliferative ratios and mean cell-cycle times of the generations of cell cycles that underlie retinal growth. The proliferative ratio determines the proportion of cells that divides in the next generation, so the numbers of proliferative and non-proliferative cells in each generation of cell cycles were obtained. The mean cell-cycle times determine the times over which the generations are extant. Assuming growth starts from one cell in generation 0, the proliferative cells reach 3.6 × 10⁶ and the non-proliferative cells reach 1.1 × 10⁶ by generation 23. The next four generations increase the proliferative cell numbers to 13.9 × 10⁶ and produce 20.1 × 10⁶ non-proliferative cells. In the next five generations in the end phase of growth, non-proliferative cells are produced in large numbers at an average of 13.9 × 10⁶ cells per generation as the retinal lineages are completed. The retinal cell population reaches a maximum estimated here at 98.2 × 10⁶ cells. The mean cell-cycle time estimates range between 6.8 and 10.1 h in generations before the end phase of growth and between 10.6 and 17.2 h in generations in the end phase. The retinal cell population growth is limited by the depletion of the proliferative cell population that the production of non-proliferative cells entails. The proliferative ratios and the cell-cycle-time distribution parameters are the likely determinants of retinal growth rates. The results are discussed in relation to other results of spatial and temporal patterns of the cessation of cell cycling in the embryonic chick retina.     

Patterns of cell division and the risk of cancer

Epidermal and intestinal tissues divide throughout life to replace lost surface cells. These renewing tissues have long-lived basal stem cell lineages that divide many times, each division producing one stem cell and one transit cell. The transit cell divides a limited number of times, producing cells that move up from the basal layer and eventually slough off from the surface. If mutation rates are the same in stem and transit divisions, we show that minimal cancer risk is obtained by using the fewest possible stem divisions subject to the constraints imposed by the need to renew the tissue. In this case, stem cells are a necessary risk imposed by the constraints of tissue architecture. Cairns suggested that stem cells may have lower mutation rates than transit cells do. We develop a mathematical model to study the consequences of different stem and transit mutation rates. Our model shows that stem cell mutation rates two or three orders of magnitude less than transit mutation rates may favor relatively more stem divisions and fewer transit divisions, perhaps explaining how renewing tissues allocate cell divisions between long stem and short transit lineages.     

Multifaceted control of adult SVZ neurogenesis by the vascular niche

The subventricular zone is one of the 2 germinal niches of the adult brain where neural stem cells (NSC) generate new neurons and glia throughout life. NSC behavior is controlled by the integration of intrinsic signals and extrinsic cues provided by the surrounding microenvironment, or niche. Within the niche, the vasculature has emerged as a critical compartment, to which both neural stem cells and transit-amplifying progenitors are closely associated. A key function of the vasculature is to deliver blood-borne and secreted factors that promote proliferation and lineage progression of committed neural progenitors. We recently found that, in contrast to the established role of soluble cues, juxtacrine signals on vascular endothelial cells maintain neural stem cells in a quiescent and undifferentiated state through direct cell-cell interactions. In this perspective, we discuss how, through these apparently opposing signals, the vascular niche might coordinate stem cell decisions between maintenance and proliferation.     

A comprehensive model of the crypts of the small intestine of the mouse provides insight into the mechanisms of cell migration and the proliferation hierarchy

A comprehensive model has been formulated for the proliferative behaviour of the crypts of the small intestine based on individual cell to cell relationships rather than on the average effects of all cells. The model accommodates a wide range of cell kinetic data and provides an insight into the mechanisms involved in cell movement within the columnar sheet of cells and into the relationship between the stem cells and their progeny. The model permits the number of stem cells and transit generations to be estimated. The number of stem cells is predicted to be not less than 4 and not more than 16 per crypt with cell cycle times of between 12 and 32 h respectively. Certain conclusions can be drawn concerning the mechanisms involved in the initial cell displacements after cell division. The model also allows an estimation of parameters which cannot be measured directly such as the degree of cell generation disorder and the amount of dispersion of cells within a cell lineage.