RESUMEN
Wound healing is a complex physiological process involving various cell types and signaling pathways. The capability to observe the dynamics of wound repair offers valuable insights into the effects of genetic modifications, pharmaceutical interventions or other experimental manipulations on the skin-repair process. Here, we provide a comprehensive protocol for a full-thickness, excisional skin-wound-healing assay in mice, which can easily be performed by any scientist who has received an animal welfare course certificate and can be completed within ~3 h, depending on the number of animals. Crucially, we highlight the importance of considering key aspects of the assay that can dramatically contribute to the reliability and reproducibility of these experiments. We thoroughly discuss the experimental design, necessary preparations, wounding technique and analysis. In addition, we discuss the use of lineage-tracing techniques to monitor cell migration, differentiation and the contribution of different cell populations to the repair process. Overall, we explore key aspects of the skin-wound-healing assay, supplying a detailed procedure and guidelines essential for decreasing variability and obtaining reliable and reproducible results.
Asunto(s)
Piel , Cicatrización de Heridas , Ratones , Animales , Piel/metabolismo , Reproducibilidad de los Resultados , Diferenciación Celular , Movimiento CelularRESUMEN
Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in "loser" cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis.
Asunto(s)
Competencia Celular , Factor de Necrosis Tumoral alfa , Animales , Proteína X Asociada a bcl-2 , Cicatrización de Heridas/fisiología , Folículo Piloso , Células Madre , MamíferosRESUMEN
Alzheimer's disease (AD) is characterized by toxic protein accumulation in the brain. Ubiquitination is essential for protein clearance in cells, making altered ubiquitin signaling crucial in AD development. A defective variant, ubiquitin B + 1 (UBB+1), created by a non-hereditary RNA frameshift mutation, is found in all AD patient brains post-mortem. We now detect UBB+1 in human brains during early AD stages. Our study employs a 3D neural culture platform derived from human neural progenitors, demonstrating that UBB+1 alone induces extracellular amyloid-ß (Aß) deposits and insoluble hyperphosphorylated tau aggregates. UBB+1 competes with ubiquitin for binding to the deubiquitinating enzyme UCHL1, leading to elevated levels of amyloid precursor protein (APP), secreted Aß peptides, and Aß build-up. Crucially, silencing UBB+1 expression impedes the emergence of AD hallmarks in this model system. Our findings highlight the significance of ubiquitin signalling as a variable contributing to AD pathology and present a nonclinical platform for testing potential therapeutics.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , Transducción de Señal , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Técnicas de Cultivo Tridimensional de CélulasRESUMEN
Cell competition describes the process in which cells of greater fitness are capable of sensing and instructing elimination of lesser fit mutant cells. Since its discovery in Drosophila, cell competition has been established as a critical regulator of organismal development, homeostasis, and disease progression. It is therefore unsurprising that stem cells (SCs), which are central to these processes, harness cell competition to remove aberrant cells and preserve tissue integrity. Here, we describe pioneering studies of cell competition across a variety of cellular contexts and organisms, with the ultimate goal of better understanding competition in mammalian SCs. Furthermore, we explore the modes through which SC competition takes place and how this facilitates normal cellular function or contributes to pathological states. Finally, we discuss how understanding of this critical phenomenon will enable targeting of SC-driven processes, including regeneration and tumor progression.
Asunto(s)
Competencia Celular , Fenómenos Fisiológicos Celulares , Animales , Competencia Celular/genética , Células Madre , Drosophila , MamíferosRESUMEN
Mitotic spindle orientation (SO) is a conserved mechanism that governs cell fate and tissue morphogenesis. In the developing epidermis, a balance between self-renewing symmetric divisions and differentiative asymmetric divisions is necessary for normal development. While the cellular machinery that executes SO is well characterized, the extrinsic cues that guide it are poorly understood. Here, we identified the basal cell adhesion molecule (BCAM), a ß1 integrin coreceptor, as a novel regulator of epidermal morphogenesis. In utero RNAi-mediated depletion of Bcam in the mouse embryo did not hinder ß1 integrin distribution or cell adhesion and polarity. However, Bcam depletion promoted apoptosis, thinning of the epidermis, and symmetric cell division, and the defects were reversed by concomitant overexpression of the apoptosis inhibitor Xiap. Moreover, in mosaic epidermis, depletion of Bcam or Xiap induced symmetric divisions in neighboring wild-type cells. These results identify apoptosis and epidermal architecture as extrinsic cues that guide SO in the developing epidermis.
Asunto(s)
Integrina beta1 , Huso Acromático , Animales , Apoptosis , División Celular , Polaridad Celular , Epidermis , Integrina beta1/metabolismo , Ratones , Huso Acromático/metabolismoRESUMEN
The presence of distinct stem cells that maintain the interfollicular epidermis is highly debated. Here, we report a population of keratinocytes, marked by Thy1, in the basal layer of the interfollicular epidermis. We find that epidermal cells expressing differential levels of Thy1 display distinct transcriptional signatures. Thy1+ keratinocytes do not express T cell markers, express a unique transcriptional profile, cycle significantly slower than basal epidermal progenitors and display significant expansion potential in vitro. Multicolor lineage tracing analyses and mathematical modeling reveal that Thy1+ basal keratinocytes do not compete neutrally alike interfollicular progenitors and contribute long-term to both epidermal replenishment and wound repair. Importantly, ablation of Thy1+ cells strongly impairs these processes, thus indicating the non-redundant function of Thy1+ stem cells in the epidermis. Collectively, these results reveal a distinct stem cell population that plays a critical role in epidermal homeostasis and repair.
Asunto(s)
Células Epidérmicas , Células Madre , Animales , Diferenciación Celular/fisiología , Epidermis/metabolismo , Queratinocitos/metabolismo , Ratones , Células Madre/metabolismoRESUMEN
Anchored cells of the basal epidermis constantly undergo proliferation in an overcrowded environment. An important regulator of epidermal proliferation is YAP, which can be controlled by both cell-matrix and cell-cell interactions. Here, we report that THY1, a GPI-anchored protein, inhibits epidermal YAP activity through converging molecular mechanisms. THY1 deficiency leads to increased adhesion by activating the integrin-ß1-SRC module. Notably, regardless of high cellular densities, the absence of THY1 leads to the dissociation of an adherens junction complex that enables the release and translocation of YAP. Due to increased YAP-dependent proliferation, Thy1-/- mice display enhanced wound repair and hair follicle regeneration. Taken together, our work reveals THY1 as a crucial regulator of cell-matrix and cell-cell interactions that controls YAP activity in skin homeostasis and regeneration.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Epidermis/metabolismo , Homeostasis , Ratones , Piel/metabolismoRESUMEN
In various placental mammals, the bidirectional exchange of cells during pregnancy can lead to the acquisition of genetically unique cells that can persist in both mother and child for decades. Over the years, it has become increasingly clear that this phenomenon, termed fetomaternal microchimerism may play key roles in a number of biological processes. In this perspective, we explore the concept of fetomaternal microchimerism and outline how fetal microchimeric cells are detected and immunologically tolerated within the maternal setting. Moreover, we discuss undertakings in the field that hint at the significant plasticity of fetal microchimeric cells and their potential roles in promoting maternal wound healing. Finally, we explore the multifaceted roles of fetal microchimeric cells in cancer development and progression. A deeper understanding of fetomaternal chimerism in healthy and diseased states will be key toward developing more efficient anti-cancer treatments and regenerative therapies.
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Quimerismo , Neoplasias , Animales , Niño , Femenino , Feto , Humanos , Mamíferos , Intercambio Materno-Fetal , Neoplasias/genética , Placenta , Embarazo , Cicatrización de HeridasRESUMEN
The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase-8 as a pro-survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ-specific manner. In particular, we find that deletion of Caspase-8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase-8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut-vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function.
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Caspasa 8 , Células Endoteliales , Mucosa Intestinal , Animales , Caspasa 8/metabolismo , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Enteritis/enzimología , Enteritis/patología , Homeostasis , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Intestino Delgado/enzimología , Intestino Delgado/patología , RatonesRESUMEN
The hair follicle is a unique mini organ that undergoes continuous cycles of replenishment. While hair follicle formation was long thought to occur strictly during embryogenesis, it is now becoming increasingly clear that hair follicles can regenerate from the wound bed. Here, we provide an overview of the recent advancements in the field of Wound Induced Hair Neogenesis (WIHN) in mice. We briefly outline the hair follicle morphogenic process and discuss the major features of adult hair follicle regeneration. We examine the role of distinct cell types and review the contribution of specific signaling pathways to the WIHN phenotype. The phenomenon of neogenic hair regeneration provides an important platform, which may offer new insights into mammalian regeneration in the adult setting.
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Piel , Cicatrización de Heridas , Animales , Cabello , Folículo Piloso , Mamíferos , Ratones , Transducción de Señal/genética , Cicatrización de Heridas/genéticaRESUMEN
Damaged or superfluous cells are typically eliminated by apoptosis. Although apoptosis is a cell-autonomous process, apoptotic cells communicate with their environment in different ways. Here we describe a mechanism whereby cells under apoptotic stress can promote survival of neighbouring cells. We find that upon apoptotic stress, cells release the growth factor FGF2, leading to MEK-ERK-dependent transcriptional upregulation of pro-survival BCL-2 proteins in a non-cell autonomous manner. This transient upregulation of pro-survival BCL-2 proteins protects neighbouring cells from apoptosis. Accordingly, we find in certain cancer types a correlation between FGF-signalling, BCL-2 expression and worse prognosis. In vivo, upregulation of MCL-1 occurs in an FGF-dependent manner during skin repair, which regulates healing dynamics. Importantly, either co-treatment with FGF-receptor inhibitors or removal of apoptotic stress restores apoptotic sensitivity to cytotoxic therapy and delays wound healing. These data reveal a pathway by which cells under apoptotic stress can increase resistance to cell death in surrounding cells. Beyond mediating cytotoxic drug resistance, this process also provides a potential link between tissue damage and repair.
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Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Cicatrización de HeridasRESUMEN
Stem cells (SCs) play a key role in homeostasis and repair. While many studies have focused on SC self-renewal and differentiation, little is known regarding the molecular mechanism regulating SC elimination and compensation upon loss. Here, we report that Caspase-9 deletion in hair follicle SCs (HFSCs) attenuates the apoptotic cascade, resulting in significant temporal delays. Surprisingly, Casp9-deficient HFSCs accumulate high levels of cleaved caspase-3 and are improperly cleared due to an essential caspase-3/caspase-9 feedforward loop. These SCs are retained in an apoptotic-engaged state, serving as mitogenic signaling centers by continuously releasing Wnt3 and instructing proliferation. Investigating the underlying mechanism, we reveal a caspase-3/Dusp8/p38 module responsible for Wnt3 induction, which operates in both normal and Casp9-deleted HFSCs. Notably, Casp9-deleted mice display accelerated wound repair and de novo hair follicle regeneration. Taken together, we demonstrate that apoptotic cells represent a dynamic SC niche, from which emanating signals drive SC proliferation and tissue regeneration.
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Caspasa 3/genética , Caspasa 9/genética , Fosfatasas de Especificidad Dual/genética , Regeneración/genética , Proteína Wnt3/genética , Animales , Apoptosis/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Autorrenovación de las Células/genética , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Ratones , Nicho de Células Madre/genética , Células Madre/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in ß-catenin target genes, suggesting that YAP is required for nuclear ß-catenin activity. Mechanistically, nuclear YAP interacted with ß-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, ß-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/ß-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. SIGNIFICANCE: These findings show that YAP cooperates with ß-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2116/F1.large.jpg.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinogénesis , Línea Celular Tumoral , Transdiferenciación Celular , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Ratones , Proteínas Musculares/metabolismo , Células Madre Neoplásicas/patología , Proteómica , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/prevención & control , Neoplasias de la Mama Triple Negativas/terapia , Regulación hacia Arriba , Proteínas Wnt/metabolismo , Proteínas Señalizadoras YAP , beta Catenina/genéticaRESUMEN
Cell suicide pathways, termed regulated cell death (RCD), play a critical role in organismal development, homeostasis, and pathogenesis. Here, we provide an overview of key RCD modalities, namely apoptosis, entosis, necroptosis, pyroptosis, and ferroptosis. We explore how various RCD modules serve as a defense mechanism against the emergence of cancer as well as the manner in which they can be exploited to drive oncogenesis. Furthermore, we outline current therapeutic agents that activate RCD and consider novel RCD-based strategies for tumor elimination. SIGNIFICANCE: A variety of antitumor therapeutics eliminate cancer cells by harnessing the devastating potential of cellular suicide pathways, emphasizing the critical importance of RCD in battling cancer. This review supplies a mechanistic perspective of distinct RCD modalities and explores the important role they play in tumorigenesis. We discuss how RCD modules serve as a double-edged sword as well as novel approaches aimed at selectively manipulating RCD for tumor eradication.
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Neoplasias/patología , Muerte Celular Regulada , HumanosRESUMEN
BACKGROUND: Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks. METHODS: We administered renal tubule-forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of "renovascular units" comprising both vessels and tubules with potential interaction. RESULTS: Directly injecting vessel-forming cells and renal tubule-forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule-forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal-epithelial transition in renal tubule-forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule-forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo. CONCLUSIONS: Combined cell therapy of vessel-forming cells and renal tubule-forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.
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Células Endoteliales/citología , Glomérulos Renales/citología , Túbulos Renales/citología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Técnicas de Cocultivo , Humanos , Ratones , Neovascularización FisiológicaRESUMEN
End-stage renal disease is a worldwide epidemic requiring renal replacement therapy. Harvesting tissue from failing kidneys and autotransplantation of tissue progenitors could theoretically delay the need for dialysis. Here we use healthy and end-stage human adult kidneys to robustly expand proliferative kidney epithelial cells and establish 3D kidney epithelial cultures termed "nephrospheres." Formation of nephrospheres reestablishes renal identity and function in primary cultures. Transplantation into NOD/SCID mice shows that nephrospheres restore self-organogenetic properties lost in monolayer cultures, allowing long-term engraftment as tubular structures, potentially adding nephron segments and demonstrating self-organization as critical to survival. Furthermore, long-term tubular engraftment of nephrospheres is functionally beneficial in murine models of chronic kidney disease. Remarkably, nephrospheres inhibit pro-fibrotic collagen production in cultured fibroblasts via paracrine modulation, while transplanted nephrospheres induce transcriptional signatures of proliferation and release from quiescence, suggesting re-activation of endogenous repair. These data support the use of human nephrospheres for renal cell therapy.
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Riñón/lesiones , Riñón/patología , Esferoides Celulares/patología , Cicatrización de Heridas , Animales , Diferenciación Celular , Proliferación Celular , Enfermedad Crónica , Modelos Animales de Enfermedad , Células Epiteliales/patología , Fibrosis , Humanos , Riñón/fisiopatología , Ratones Endogámicos NOD , Ratones SCID , Insuficiencia Renal Crónica/patología , Esferoides Celulares/trasplanteRESUMEN
Most studies on the skin focus primarily on the hair follicle and interfollicular epidermis, whereas little is known regarding the homeostasis of the sebaceous gland (SG). The SG has been proposed to be replenished by different pools of hair follicle stem cells and cells that resides in the SG base, marked by Blimp1. Here, we demonstrate that single Blimp1+ cells isolated from mice have the potential to generate SG organoids in vitro. Mimicking SG homeostasis, the outer layer of these organoids is composed of proliferating cells that migrate inward, undergo terminal differentiation and generating lipid-filled sebocytes. Performing confocal microscopy and mass-spectrometry, we report that these organoids exhibit known markers and a lipidomic profile similar to SGs in vivo. Furthermore, we identify a role for c-Myc in sebocyte proliferation and differentiation, and determine that SG organoids can serve as a platform for studying initial stages of acne vulgaris, making this a useful platform to identify potential therapeutic targets.
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Diferenciación Celular , Proliferación Celular , Organoides/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Glándulas Sebáceas/metabolismo , Animales , Epidermis/metabolismo , Epidermis/ultraestructura , Técnicas In Vitro , Metabolismo de los Lípidos , Espectrometría de Masas , Ratones , Microscopía Confocal , Organoides/ultraestructura , Glándulas Sebáceas/ultraestructura , Células Madre/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Although much is known regarding intestinal stem cell (ISC) self-renewal and differentiation, the specific mechanisms used for their elimination is unclear. We recently discovered that the pro-apoptotic protein ARTS, a Septin4 isoform, interacts with X-linked inhibitor of apoptosis (XIAP) in the ISC niche to regulate stem cell survival during intestinal homeostasis and regeneration. These findings point to an intriguing avenue of translational research, examining how manipulation of stem cell apoptosis through the ARTS/XIAP module can affect stem-cell-dependent processes.