Fiona M. Watt

Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression

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.

Extracellular-matrix tethering regulates stem-cell fate

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

Stem cell patterning and fate in human epidermis.

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.

Autophagy mediates the mitotic senescence transition

As a stress response, senescence is a dynamic process involving multiple effector mechanisms whose combination determines the phenotypic quality. Here we identify autophagy as a new effector mechanism of senescence. Autophagy is activated during senescence and its activation is correlated with negative feedback in the PI3K-mammalian target of rapamycin (mTOR) pathway. A subset of autophagy-related genes are up-regulated during senescence: Overexpression of one of those genes, ULK3, induces autophagy and senescence. Furthermore, inhibition of autophagy delays the senescence phenotype, including senescence-associated secretion. Our data suggest that autophagy, and its consequent protein turnover, mediate the acquisition of the senescence phenotype.

Role of integrins in regulating epidermal adhesion, growth and differentiation

Mammalian epidermis is renewed throughout life by proliferation of a multipotential stem cell population and terminal differentiation of stem cell progeny. In recent years, extracellular matrix receptors of the integrin family have been identified as important regulators of epidermal homeostasis, influencing the balance between stem cell renewal and differentiation. Integrin expression is altered when the epidermis is damaged or diseased, and there is good evidence that specific integrins can contribute positively or negatively to pathogenesis. In this review I summarize what is known about the expression and function of epidermal integrins, and highlight the challenges for future research.

Changes in keratinocyte adhesion during terminal differentiation: Reduction in fibronectin binding precedes α5β1 integrin loss from the cell surface

Abstract During terminal differentiation keratinocytes move out of the basal layer of the epidermis and thereby lose contact with the basement membrane. We show that terminal differentiation in culture involves loss of adhesiveness to fibronectin, laminin, and collagen types I and IV. The adheslve changes precede, by several hours, loss of the α 2 β 1 , α 3 β 1 , and α 5 β 1 integrins from the cell surface. Keratinocyte adhesion to fibronectin is mediated by the α 5 β 1 integrin, and the decrease in adhesion of intact cells to fibronectin is correlated with a decrease in the ability of α 5 β 1 receptors to bind fibronectin. Thus modulation of integrin function early in terminal differentiation may be an early event determining cell migration out of the basal layer.

Role of the extracellular matrix in regulating stem cell fate

The field of stem cells and regenerative medicine offers considerable promise as a means of delivering new treatments for a wide range of diseases. In order to maximize the effectiveness of cell-based therapies — whether stimulating expansion of endogenous cells or transplanting cells into patients — it is essential to understand the environmental (niche) signals that regulate stem cell behaviour. One of those signals is from the extracellular matrix (ECM). New technologies have offered insights into how stem cells sense signals from the ECM and how they respond to these signals at the molecular level, which ultimately regulate their fate.

Stimulation of human epidermal differentiation by Delta–Notch signalling at the boundaries of stem-cell clusters

BACKGROUND Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.

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 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.

Lrig1 Expression Defines a Distinct Multipotent Stem Cell Population in Mammalian Epidermis

Summary Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence. We show that, in mouse epidermis, Lrig1 defines the hair follicle junctional zone adjacent to the sebaceous glands and infundibulum. Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo. Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays. However, during homeostasis and on retinoic acid stimulation, they are bipotent, contributing to the sebaceous gland and interfollicular epidermis. β-catenin activation increases the size of the junctional zone compartment, and loss of Lrig1 causes a selective increase in β-catenin-induced ectopic hair follicle formation in the interfollicular epidermis. Our results suggest that Lrig1-positive cells constitute a previously unidentified reservoir of adult mouse interfollicular epidermal stem cells.