Kailin R. Mesa

Spatial organization within a niche as a determinant of stem-cell fate.

Stem-cell niches in mammalian tissues are often heterogeneous and compartmentalized; however, whether distinct niche locations determine different stem-cell fates remains unclear. To test this hypothesis, here we use the mouse hair follicle niche and combine intravital microscopy with genetic lineage tracing to re-visit the same stem-cell lineages, from their exact place of origin, throughout regeneration in live mice. Using this method, we show directly that the position of a stem cell within the hair follicle niche can predict whether it is likely to remain uncommitted, generate precursors or commit to a differentiated fate. Furthermore, using laser ablation we demonstrate that hair follicle stem cells are dispensable for regeneration, and that epithelial cells, which do not normally participate in hair growth, re-populate the lost stem-cell compartment and sustain hair regeneration. This study provides a general model for niche-induced fate determination in adult tissues.

Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.

Tracking stem cell fate in time and space After injury and during homeostasis, tissues rely on the balance of cell loss and renewal. Rompolas et al. visualized individual stem cells over their lifetime in the epidermis of live mice. Tracking stem cells over multiple generations revealed that tissue homeostasis in the mouse epidermis is not maintained by asymmetric cell division as previously thought, but through the coordination of sibling cell fate and lifetimes. Furthermore, differentiating stem cells reused the existing spatial organization of the epidermis. Science, this issue p. 1471 In mice a single stem cell population maintains the adult epidermis by coordinating cell fate and using existing tissue organization. Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cell self-renewal, commitment, and functional integration into the tissue remain unsolved. Using imaging techniques in live mice, we captured the lifetime of individual cells in the ear and paw epidermis. Our data suggest that epidermal stem cells have equal potential to either divide or directly differentiate. Tracking stem cells over multiple generations reveals that cell behavior is not coordinated between generations. However, sibling cell fate and lifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, we demonstrated that differentiating stem cells integrate into preexisting ordered spatial units of the epidermis. This study elucidates how a tissue is maintained by both temporal and spatial coordination of stem cell behaviors.

Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool

Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-β activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis.

Alveolar Macrophages and Toll-like Receptor 4 Mediate Ventilated Lung Ischemia Reperfusion Injury in Mice

Background:Ischemia-reperfusion (I-R) injury is a sterile inflammatory process that is commonly associated with diverse clinical situations such as hemorrhage followed by resuscitation, transient embolic events, and organ transplantation. I-R injury can induce lung dysfunction whether the I-R occurs in the lung or in a remote organ. Recently, evidence has emerged that receptors and pathways of the innate immune system are involved in recognizing sterile inflammation and overlap considerably with those involved in the recognition of and response to pathogens. Methods:The authors used a mouse surgical model of transient unilateral left pulmonary artery occlusion without bronchial involvement to create ventilated lung I-R injury. In addition, they mimicked nutritional I-R injury in vitro by transiently depriving cells of all nutrients. Results:Compared with sham-operated mice, mice subjected to ventilated lung I-R injury had up-regulated lung expression of inflammatory mediator messenger RNA for interleukin-1&bgr;, interleukin-6, and chemokine (C-X-C motif) ligand-1 and -2, paralleled by histologic evidence of lung neutrophil recruitment and increased plasma concentrations of interleukin-1&bgr;, interleukin-6, and high-mobility group protein B1 proteins. This inflammatory response to I-R required toll-like receptor-4 (TLR4). In addition, the authors demonstrated in vitro cooperativity and cross-talk between human macrophages and endothelial cells, resulting in augmented inflammatory responses to I-R. Remarkably, the authors found that selective depletion of alveolar macrophages rendered mice resistant to ventilated lung I-R injury. Conclusions:The data reveal that alveolar macrophages and the pattern recognition receptor toll-like receptor-4 are involved in the generation of the early inflammatory response to lung I-R injury.

Activation of endothelial TLR2 by bacterial lipoprotein upregulates proteins specific for the neutrophil response

The vascular endothelium is integrally involved in the host response to infection and in organ failure during acute inflammatory disorders such as sepsis. Gram-negative and Gram-positive bacterial lipoproteins circulate in sepsis and can directly activate the endothelium by binding to endothelial cell (EC) TLR2. In this report, we perform the most comprehensive analysis to date of the immune-related genes regulated after activation of endothelial TLR2 by bacterial di- and triacylated lipopeptides. We found that TLR2 activation specifically induces the expression of the genes IL-6, IL-8, CSF2, CSF3, ICAM1 and SELE by human umbilical vein ECs and human lung microvascular ECs. These proteins participate in neutrophil recruitment, adherence and activation at sites of inflammation. Significantly, our studies demonstrate that TLR2-mediated EC responses are specifically geared towards recruitment, activation, and survival of neutrophils and not mononuclear leukocytes, that ECs do not require priming by other inflammatory stimuli to respond to bacterial lipopeptides and, unlike mononuclear leukocytes, TLR2 agonists do not induce ECs to secrete TNF-α. This study suggests that endothelial TLR2 may be an important regulator of neutrophil trafficking to sites of infection in general, and that direct activation of lung endothelial TLR2 may contribute to acute lung injury during sepsis.

The Dynamic Duo: Niche/Stem Cell Interdependency.

Summary Most tissues in our bodies undergo constant cellular turnover. This process requires a dynamic balance between cell production and elimination. Stem cells have been shown in many of these tissues to be the major source of new cells. However, despite the tremendous advances made, it still remains unclear how stem cell behavior and activity are regulated in vivo. Furthermore, we lack basic understanding for the mechanisms that coordinate niche/stem cell interactions to maintain normal tissue homeostasis. Our lab has established a novel imaging approach in live mice using the skin as a model system to investigate these fundamental processes in both physiological and pathological settings such as cancer, with the goal of understanding how tissues successfully orchestrate tissue regeneration throughout the lifetime of an organism.

Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice

Tissue repair is fundamental to our survival as tissues are challenged by recurrent damage. During mammalian skin repair, cells respond by migrating and proliferating to close the wound. However, the coordination of cellular repair behaviours and their effects on homeostatic functions in a live mammal remains unclear. Here we capture the spatiotemporal dynamics of individual epithelial behaviours by imaging wound re-epithelialization in live mice. Differentiated cells migrate while the rate of differentiation changes depending on local rate of migration and tissue architecture. Cells depart from a highly proliferative zone by directionally dividing towards the wound while collectively migrating. This regional coexistence of proliferation and migration leads to local expansion and elongation of the repairing epithelium. Finally, proliferation functions to pattern and restrict the recruitment of undamaged cells. This study elucidates the interplay of cellular repair behaviours and consequent changes in homeostatic behaviours that support tissue-scale organization of wound re-epithelialization.

Intravital imaging of hair follicle regeneration in the mouse

Hair follicles are mammalian skin organs that periodically and stereotypically regenerate from a small pool of stem cells. Hence, hair follicles are a widely studied model for stem cell biology and regeneration. This protocol describes the use of two-photon laser-scanning microscopy (TPLSM) to study hair regeneration within a living, uninjured mouse. TPLSM provides advantages over conventional approaches, including enabling time-resolved imaging of single hair follicle stem cells. Thus, it is possible to capture behaviors including apoptosis, proliferation and migration, and to revisit the same cells for in vivo lineage tracing. In addition, a wide range of fluorescent reporter mouse lines facilitates TPLSM in the skin. This protocol also describes TPLSM laser ablation, which can spatiotemporally manipulate specific cellular populations of the hair follicle or microenvironment to test their regenerative contributions. The preparation time is variable depending on the goals of the experiment, but it generally takes 30–60 min. Imaging time is dependent on the goals of the experiment. Together, these components of TPLSM can be used to develop a comprehensive understanding of hair regeneration during homeostasis and injury.

ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

Background: Endothelial cell (EC) Toll-like receptor 2 (TLR2) signaling induces inflammatory events. Results: NF-κB, p38-MAPK, JNK, and ERK5 promote, whereas MEK1 suppresses, EC TLR2 signaling. Conclusion: ERK5 is a newly identified mediator of TLR2 signaling, and TLR2 signaling pathways differ in ECs and monocytes. Significance: TLR2 signaling differences can be exploited therapeutically for endothelial-specific or leukocyte-specific inflammatory responses. Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.

Epidermal stem cells self-renew upon neighboring differentiation

Many adult tissues are dynamically sustained by the rapid turnover of stem cells. Yet, how cell fates such as self-renewal and differentiation are orchestrated to achieve long-term homeostasis remains elusive. Studies utilizing clonal tracing experiments in multiple tissues have argued that while stem cell fate is balanced at the population level, individual cell fate - to divide or differentiate – is determined intrinsically by each cell seemingly at random ( 1 2 3 4 5). These studies leave open the question of how cell fates are regulated to achieve fate balance across the tissue. Stem cell fate choices could be made autonomously by each cell throughout the tissue or be the result of cell coordination ( 6 7). Here we developed a novel live tracking strategy that allowed recording of every division and differentiation event within a region of epidermis for a week. These measurements reveal that stem cell fates are not autonomous. Rather, direct neighbors undergo coupled opposite fate decisions. We further found a clear ordering of events, with self-renewal triggered by neighbor differentiation, but not vice-versa. Typically, around 1-2 days after cell delamination, a neighboring cell entered S/G2 phase and divided. Functional blocking of this local feedback showed that differentiation continues to occur in the absence of cell division, resulting in a rapid depletion of the epidermal stem cell pool. We thus demonstrate that the epidermis is maintained by nearest neighbor coordination of cell fates, rather than by asymmetric divisions or fine-tuned cell-autonomous stochastic fate choices. These findings establish differentiation-dependent division as a core feature of homeostatic control, and define the relevant time and length scales over which homeostasis is enforced in epithelial tissues.