Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface.

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at approximately 15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic "cell doubling clock. " These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.     

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.     

Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells

Conceptual and technical advances in neural stem cell biology are being applied to the study of human brain tumours. These studies suggest that human brain tumours are organized as a hierarchy and are maintained by a small number of tumour cells that have stem cell properties. Most of the bulk population of human brain tumours comprise cells that have lost the ability to initiate and maintain tumour growth. Although the cell of origin for human brain tumours is uncertain, recent evidence points towards the brain's known proliferative zones. The identification of brain tumour stem cells has important implications for understanding brain tumour biology and these cells may be critical cellular targets for curative therapy.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Expansion of retinal stem cells and their progeny using cell microcarriers in a bioreactor

Blindness as a consequence of degenerative eye diseases (e.g., age-related macular degeneration and retinitis pigmentosa) is a major health problem and numbers are expected to increase by up to 50% by 2020. Unfortunately, adult mouse and human retinal stem cells (RSCs), unlike fish and amphibians , are quiescent in vivo and do not regenerate following disease or injury. To replace lost cells, we used microcarriers (MCs) in a suspension stirring bioreactor to help achieve numbers suitable for differentiation and transplantation. We achieved a significant 10-fold enrichment of RSC yield compared to conventional static culture techniques using a combination of FACTIII MCs and relative hypoxia (5%) inside the bioreactor. We found that hypoxia (5% O₂) was associated with better RSC expansion across all platforms; and this can be attributed to hypoxia-induced increases in survival and/or symmetric division of stem cells. In the future, we will target the differentiation of RSCs and their progeny toward rod and cone photoreceptor phenotypes using FACTIII MCs inside bioreactors to expand their populations in order to produce the large numbers of cells needed for transplantation.     

The scaling method of spatial distribution of keratinocytes in the basal layer of epidermis in norm and during the development of psoriatic lesion

On the basis of the modern conceptions postulating that the growing layer of normal epidermis contains two populations of keratinocytes differing greatly in the rate of mitotic division (stem and transient cells), a hypothesis was proposed that explains the reason for the establishment of the portion of the proliferating fraction close to 60%. A relationship between this value and the distribution of keratinocytes of the basal layer in space was demonstrated, and formulae for its calculation were derived. Possible pathways of the transition from the normal spatial configuration to the distribution typical for the focal lesion in psoriasis were analyzed when all stem cells begin to divide vigorously, and the size of the population of transient cells is 100%.     

Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis

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 alpha6beta4 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 beta-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.     

Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum

In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions—the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100β. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish.     

Dynamics between stem cells, niche, and progeny in the hair follicle

Here, we exploit the hair follicle to define the point at which stem cells (SCs) become irreversibly committed along a differentiation lineage. Employing histone and nucleotide double-pulse-chase and lineage tracing, we show that the early SC descendents en route to becoming transit-amplifying cells retain stemness and slow-cycling properties and home back to the bulge niche when hair growth stops. These become the primary SCs for the next hair cycle, whereas initial bulge SCs become reserves for injury. Proliferating descendents further en route irreversibly lose their stemness, although they retain many SC markers and survive, unlike their transit-amplifying progeny. Remarkably, these progeny also home back to the bulge. Combining purification and gene expression analysis with differential ablation and functional experiments, we define critical functions for these non-SC niche residents and unveil the intriguing concept that an irreversibly committed cell in an SC lineage can become an essential contributor to the niche microenvironment.     

The relationship between the doubling time of human lung tumor volume and the labelling index]

The value of the labelling index (incubation with H3-thymidine) was compared with the doubling time of tumour volume (by repeated roentgenograms) for 3 tumours of human lungs: two squamous cell carcinomas taken at various differentiation levels and a hamartoma. The minimum doubling time (178 days) was shown for the squamous cell carcinoma with a lower differentiation level and the greatest labelling index value (19.0%). Hamartoma (a non-malignant tumour of lung) displayed the longest doubling time (1250 days) and the least labelling index value (3.8%).     

Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin

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.     

E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor

Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor.     

Periostin: a bridge between cancer stem cells and their metastatic niche

Only a minority of cancer cells have the potential to initiate metastatic growth, but the factors that limit metastatic colonization remain mostly unknown. Malanchi et?al. (2012) recently demonstrated that stromal periostin is crucial for metastatic colonization by regulating the interactions between breast cancer stem cells and their metastatic niche.     

The relationship between phytoplankton biovolume and chlorophyll in a deep oligotrophic lake: decoupling in their spatial and temporal maxima

The seasonal distributions of phytoplankton biovolume and chlorophylla content were monitored for 14 months in a deep oligotrophic,high mountain lake (Redó, Pyrenees). An allometric relationshipof chlorophyll with biovolume was found throughout the periodstudied, with a correlation coefficient of 0.66. However, therelationship changed with season and the taxonomic compositionof the phytoplankton. Both parameters showed a similar seasonalpattern, but differences in space and time were observed. Thechlorophyll maximum was recorded deeper and later than thatof phytoplankton biovolume. While the biovolume maximum wasrelated to an improvement in conditions for growth (nutrientinput during column mixing periods), and reflected an increasein biomass, the chlorophyll maximum was related to changes incell pigment content, and to spatial or successional trendsin species dominance. Flagellated chrysophytes predominatedat the chlorophyll maxima. Chlorophyll content per unit of phytoplanktonbiovolume fluctuated greatly throughout the year, dependingon light intensity, temperature and phytoplankton composition.Of the main groups of phytoplankton in the lake, the dinoflagellates,which dominated the summer epilimnion phytoplankton community,recorded the lowest pigment content per biovolume (which isconsistent with their size). Higher chlorophyll contents perbiovolume were found in the deep hypolimnion and during thewinter cover period associated with small cells such as somespecies of chlorococcales chlorophytes. When flagellated chrysophyteswere predominant, a broad range of chlorophyll values per biovolumewas found and there was no significant correlation between thetwo biomass indices. These findings reaffirm the need to treatphytoplankton biomass estimates with caution, in particularwhen conducting primary production studies. While our resultsshow that changes in chlorophyll content per cell occur as aphotoacclimation response along a vertical profile, they alsopoint out a component of the successional trends which appearin a phytoplankton growth phase in a lake.     

中国陆域地表人类活动与自然环境的空间关系研究

在人类活动主导人地矛盾的当代背景下,从空间关联角度测度人类活动与自然环境相互作用是推动区域可持续发展的基本前提。以2015年数据为基础,通过选取特征变量构建综合测度指标体系,利用小波多尺度分析、双变量局域空间自相关、人地系统耦合协调度模型等方法多层次刻画中国陆域地表人类活动与自然环境的空间相关关系、空间匹配关系、空间耦合关系。研究结果表明:①人类活动与自然地理基础的空间相关关系随着地域尺度增大而趋于增强,说明自然环境条件对人类活动格局的基础性作用;②人类活动与自然资源支撑的空间匹配关系呈现非均衡性特征,华北地区人类活动规模与自然资源支撑空间错位显著、区域可持续发展面临严峻挑战;③人类活动与自然环境承载的空间耦合协调度从东南向西北递减,调整人类活动的结构与方式是化解区域人地矛盾的理性选择。     

The relationship between diet quality and basal metabolic rate in endotherms: insights from intraspecific analysis

In this article, we review intraspecific studies of basal metabolic rate (BMR) that address the correlation between diet quality and BMR. The "food-habit hypothesis" stands as one of the most striking and often-mentioned interspecific patterns to emerge from studies of endothermic energetics. Our main emphasis is the explicit empirical comparison of predictions derived from interspecific studies with data gathered from within-species studies in order to explore the mechanisms and functional significance of the putative adaptive responses encapsulated by the food-habit hypothesis. We suggest that, in addition to concentrating on the relationship among diet quality, internal morphology, and BMR, new studies should also attempt to unravel alternative mechanisms that shape the interaction between diet and BMR, such as enzymatic plasticity, and the use of energy-saving mechanisms, such as torpor. Another avenue for future study is the measurement of the effects of diet quality on other components of the energy budget, such as maximum thermogenic and sustainable metabolic rates. It is possible that the effects of diet quality operate on such components rather than directly on BMR, which might then push or pull along changes in these traits. Results from intraspecific studies suggest that the factors responsible for the association between diet and BMR at an ecological timescale might not be the same as those that promoted the evolution of this correlation. Further analyses should consider how much of a role the proximate and ultimate processes have played in the evolution of BMR.     

Selective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture

We report on a three dimensional (3D)-organotypic culture in vitro for selective growth and expansion of human corneal epithelial stem cells. Limbal corneal explants were cultured on porous collagen sponges submerged in Epilife medium containing 10% fetal bovine serum. The fragments were analyzed by immunohistochemistry for the expression and distribution of a spectrum of corneal epithelium markers: p63, CK-19, CK-3, Ki-67, pan-cytokeratins and vimentin. Early in culture the epithelium began to exfoliate losing its differentiated high-zone layers into the medium, maintaining only basal and few parabasal cells (mostly both p63 and CK-19 positive), which had remained attached to the specimen. After 14 days a new epithelium was formed displaying an increasing prominence of basal and suprabasal cells that, sliding onto the whole explant, showed the tendency to underlay stromal tissue and infiltrate into the underlaying sponge. After 21 days, sponge and fragments were incubated with trypsin-EDTA and dispersed epithelial cells were pipetted on a feeder monolayer of mitomycin-c-treated murine NIH.3T3 fibroblasts. Colonies of undifferentiated epithelial cells (p63, CK-19 and Ki-67 positive, CK-3 negative) were obtained: their cells, if seeded onto a collagen matrix containing embedded primary human corneal fibroblasts as feeder, provided the basic building blocks for reconstructing in vitro a 3D-multilayered corneal epithelium.