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1.
Talanta ; 229: 122259, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33838770

RESUMO

Investigation of stem cell-like property in cancer cells is important for the development of new therapeutic drugs targeting at malignant tumors. Currently, the standard approach for identifying cancer stem cell-like cells relies on the recognition of stem cell surface markers. However, the reliability remains controversial among biologists. In the current work, a dielectrophoretic and impedimetric hybrid microfluidic platform was developed for capturing single cells and characterizing their stem cell-like property. Single cells were captured in 20 µm trapping wells by dielectrophoretic force and their impedance spectra were measured by an impedance analyzer. The result showed that different cancer cell lines could be differentiated by impedance magnitude ranging between 2 and 20 kHz. Moreover, cancer cells and cancer stem cell-like cells could be categorized by a 2-dimensional graph of the impedance magnitudes at 2 and 20 kHz. The stem cell-like property in cancer cells was verified by stem cell surface markers and single-cell derived colony assay. Comparing with bio-chemical approach, i.e., surface markers, bio-physical approach, i.e., cell impedance, is a label-free technique to identify cancer stem cell-like cells.


Assuntos
Técnicas Analíticas Microfluídicas , Neoplasias , Células-Tronco , Linhagem Celular , Impedância Elétrica , Microfluídica , Reprodutibilidade dos Testes , Células-Tronco/fisiologia
2.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799809

RESUMO

Nails are highly keratinized skin appendages that exhibit continuous growth under physiological conditions and full regeneration upon removal. These mini-organs are maintained by two autonomous populations of skin stem cells. The fast-cycling, highly proliferative stem cells of the nail matrix (nail stem cells (NSCs)) predominantly replenish the nail plate. Furthermore, the slow-cycling population of the nail proximal fold (nail proximal fold stem cells (NPFSCs)) displays bifunctional properties by contributing to the peri-nail epidermis under the normal homeostasis and the nail structure upon injury. Here, we discuss nail mini-organ stem cells' location and their role in skin and nail homeostasis and regeneration, emphasizing their importance to orchestrate the whole digit tip regeneration. Such endogenous regeneration capabilities are observed in rodents and primates. However, they are limited to the region adjacent to the nail's proximal area, indicating the crucial role of nail mini-organ stem cells in digit restoration. Further, we explore the molecular characteristics of nail mini-organ stem cells and the critical role of the bone morphogenetic protein (BMP) and Wnt signaling pathways in homeostatic nail growth and digit restoration. Finally, we investigate the latest accomplishments in stimulating regenerative responses in regeneration-incompetent injuries. These pioneer results might open up new opportunities to overcome amputated mammalian digits and limbs' regenerative failures in the future.


Assuntos
Células Epidérmicas/citologia , Unhas/citologia , Regeneração , Pele/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Células Epidérmicas/fisiologia , Extremidades/fisiologia , Homeostase/fisiologia , Humanos , Células-Tronco/fisiologia
3.
Mol Med Rep ; 23(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33655325

RESUMO

Stem cell therapy is considered a novel treatment modality for critical diseases. Adipose tissue is a rich and easily accessible source of stem cells. Adipose­derived stem cells (ADSCs) can be expanded ex vivo and possess characteristics similar to those derived from the bone marrow. However, the quality of ADSCs can be affected by age, underlying disease or the lifestyle of individuals. The aim of the present study was to explore the association between age and ADSC activity, including paracrine and differentiation potential. Adipose tissues from young (age <30 years) and elderly (age >70 years) groups were obtained, and ADSCs from each group were cultured in vitro. The effect of age on ADSC activity was investigated in vitro by evaluating the proliferation rate, adipo/osteogenic differentiation potential and cytokine profile using ELISA. The results revealed that increased age reduced cell activity and increased the doubling time of ADSCs, without causing profound morphological changes. The paracrine action of ADSCs was markedly altered by increased age, as demonstrated by reduced expression levels of vascular endothelial growth factor, stromal cell­derived factor­1α and hepatocyte growth factor. Differentiation of ADSCs into osteoblasts or adipocytes rarely occurred in the elderly group compared with the young group. Overall, these results indicate that age may affect the cellular function of ADSCs and should be considered prior to ADSC transplantation.


Assuntos
Tecido Adiposo , Diferenciação Celular , Citocinas , Regulação da Expressão Gênica , Células-Tronco/fisiologia , Adulto , Fatores Etários , Idoso , Células Cultivadas , Quimiocina CXCL12/genética , Feminino , Fator de Crescimento de Hepatócito/genética , Humanos , Masculino , Osteogênese , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética
4.
Nat Commun ; 12(1): 1502, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33686070

RESUMO

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.


Assuntos
Proteína BRCA1/genética , Transformação Celular Neoplásica/genética , Neoplasias Mamárias Experimentais/genética , Fenobarbital/metabolismo , Análise de Célula Única/métodos , Células-Tronco/patologia , Animais , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Mutação , Células-Tronco/fisiologia , Microambiente Tumoral/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Neuron ; 109(7): 1150-1167.e6, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33600763

RESUMO

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.


Assuntos
Hipotálamo/citologia , Hipotálamo/crescimento & desenvolvimento , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/embriologia , Linhagem da Célula , Ácido Glutâmico/fisiologia , Proteínas de Homeodomínio/metabolismo , Hipotálamo/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/fisiologia , Células-Tronco/fisiologia , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Hipotalâmico Ventromedial/citologia , Núcleo Hipotalâmico Ventromedial/embriologia , Núcleo Hipotalâmico Ventromedial/metabolismo , Ácido gama-Aminobutírico/fisiologia
6.
Biochem Biophys Res Commun ; 545: 14-19, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33529805

RESUMO

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.


Assuntos
Interferon gama/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/patologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sistemas Computacionais , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Interferon gama/fisiologia , Mucosa Intestinal/fisiologia , Camundongos , Camundongos Transgênicos , Celulas de Paneth/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Interferon/metabolismo , Nicho de Células-Tronco/efeitos dos fármacos , Nicho de Células-Tronco/fisiologia , Células-Tronco/fisiologia
7.
Methods Mol Biol ; 2224: 87-98, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606208

RESUMO

Type 1 diabetes (T1D) is an autoimmune disease, where insulin-producing ß-cells in the pancreas are inappropriately recognized and destroyed by immune cells. Islet transplantation is the most successful cell-based therapy for T1D individuals who experience frequent and severe life-threatening hypoglycemia. However, this therapy is extremely restricted owing to the limited availability of donor pancreas. In recent years, significant progress has been made in generating ß-cells from stem/progenitor cells using different approaches of in vitro differentiation. The insulin production from such in vitro generated ß-cells is still far less than that observed in islet ß-cells. We employed a novel strategy to improve the efficiency of progenitor cell differentiation by performing partial mouse pancreas resection after transplanting in vitro generated insulin-producing cells under the kidney capsule of these mice. Pancreas resection (pancreatectomy) has been shown to induce regenerative pathways, leading to regeneration of almost the entire resected pancreas over 3-5 weeks in mice. We found that in our method, regenerating mouse pancreas promotes better graft differentiation/maturation and insulin production from transplanted cells. In this chapter, we detail the protocols used for transplantation of in vitro differentiated cells in immunocompromised mice, partial pancreatectomy in host (NOD scid) mice, and assessment of graft function. We believe that our protocols provide a solid platform for further studies aimed at understanding growth/differentiation molecules secreted from regenerating pancreas that promote graft maturation.


Assuntos
Diferenciação Celular/fisiologia , Pâncreas/fisiologia , Animais , Diabetes Mellitus Tipo 1/fisiopatologia , Células Secretoras de Insulina/fisiologia , Transplante das Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pancreatectomia/métodos , Células-Tronco/fisiologia
8.
Methods Mol Biol ; 2224: 153-182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606214

RESUMO

Hematopoiesis in the mouse and other mammals occurs in several waves and arises from distinct anatomic sites. Transgenic mice expressing fluorescent reporter proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to more restricted progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene, survey available fluorescent probes, and describe methods for confirming and analyzing transgene expression in the hematopoietic tissues of the embryo, fetus, and postnatal/adult animal.


Assuntos
Genes Reporter/genética , Hematopoese/genética , Proteínas Luminescentes/genética , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/fisiologia , Feminino , Feto/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco/fisiologia , Transgenes/genética
9.
Life Sci ; 273: 119270, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33640402

RESUMO

Stem cells (SCs) are clonogenic cells that develop into the specialized cells which later responsible for making up various types of tissue in the human body. SCs are not only the appropriate source of information for cell division, molecular and cellular processes, and tissue homeostasis but also one of the major putative biological aids to diagnose and cure various degenerative diseases. This study emphasises on various research outputs that occurred in the past two decades. This will give brief information on classification, differentiation, detection, and various isolation techniques of SCs. Here, the various signalling pathways which includes WNT, Sonic hedgehog, Notch, BMI1 and C-met pathways and how does it effect on the regeneration of various classes of SCs and factors that regulates the potency of the SCs are also been discussed. We also focused on the application of SCs in the area of regenerative medicine along with the cellular markers that are useful as salient diagnostic or curative tools or in both, by the process of reprogramming, which includes diabetes, cancer, cardiovascular disorders and neurological disorders. The biomarkers that are mentioned in various literatures and experiments include PDX1, FOXA2, HNF6, and NKX6-1 (for diabetes); CD33, CD24, CD133 (for cancer); c-Kit, SCA-1, Wilm's tumor 1 (for cardiovascular disorders); and OCT4, SOX2, c-MYC, EN1, DAT and VMAT2 (for neurological disorders). In this review, we come to know the advancements and scopes of potential SC-based therapies, its diverse applications in clinical fields that can be helpful in the near future.


Assuntos
Diferenciação Celular , Medicina Regenerativa , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Humanos , Transdução de Sinais
10.
Nat Commun ; 12(1): 1318, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637744

RESUMO

Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/fisiologia , Desenvolvimento Muscular/fisiologia , Músculos/metabolismo , Células-Tronco/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Mutação , Proteína MyoD/genética , Proteína MyoD/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição HES-1/metabolismo , Transcriptoma
11.
Science ; 371(6526)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446527

RESUMO

Multicellular organisms are composed of cells connected by ancestry and descent from progenitor cells. The dynamics of cell birth, death, and inheritance within an organism give rise to the fundamental processes of development, differentiation, and cancer. Technical advances in molecular biology now allow us to study cellular composition, ancestry, and evolution at the resolution of individual cells within an organism or tissue. Here, we take a phylogenetic and phylodynamic approach to single-cell biology. We explain how "tree thinking" is important to the interpretation of the growing body of cell-level data and how ecological null models can benefit statistical hypothesis testing. Experimental progress in cell biology should be accompanied by theoretical developments if we are to exploit fully the dynamical information in single-cell data.


Assuntos
Linhagem da Célula , Filogenia , Análise de Célula Única , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Biologia Celular/tendências , Humanos , Células-Tronco/citologia , Células-Tronco/fisiologia
12.
Nutrients ; 13(1)2021 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-33477388

RESUMO

There is evidence demonstrating that heart failure (HF) occurs in 1-2% of the global population and is often accompanied by comorbidities which contribute to increasing the prevalence of the disease, the rate of hospitalization and the mortality. Although recent advances in both pharmacological and non-pharmacological approaches have led to a significant improvement in clinical outcomes in patients affected by HF, residual unmet needs remain, mostly related to the occurrence of poorly defined strategies in the early stages of myocardial dysfunction. Nutritional support in patients developing HF and nutraceutical supplementation have recently been shown to possibly contribute to protection of the failing myocardium, although their place in the treatment of HF requires further assessment, in order to find better therapeutic solutions. In this context, the Optimal Nutraceutical Supplementation in Heart Failure (ONUS-HF) working group aimed to assess the optimal nutraceutical approach to HF in the early phases of the disease, in order to counteract selected pathways that are imbalanced in the failing myocardium. In particular, we reviewed several of the most relevant pathophysiological and molecular changes occurring during the early stages of myocardial dysfunction. These include mitochondrial and sarcoplasmic reticulum stress, insufficient nitric oxide (NO) release, impaired cardiac stem cell mobilization and an imbalanced regulation of metalloproteinases. Moreover, we reviewed the potential of the nutraceutical supplementation of several natural products, such as coenzyme Q10 (CoQ10), a grape seed extract, Olea Europea L.-related antioxidants, a sodium-glucose cotransporter (SGLT2) inhibitor-rich apple extract and a bergamot polyphenolic fraction, in addition to their support in cardiomyocyte protection, in HF. Such an approach should contribute to optimising the use of nutraceuticals in HF, and the effect needs to be confirmed by means of more targeted clinical trials exploring the efficacy and safety of these compounds.


Assuntos
Suplementos Nutricionais , Insuficiência Cardíaca/terapia , Animais , Antioxidantes/administração & dosagem , Citrus/química , Suplementos Nutricionais/estatística & dados numéricos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Extrato de Sementes de Uva/administração & dosagem , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Malus/química , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/fisiologia , Miocárdio/citologia , Óxido Nítrico/metabolismo , Apoio Nutricional , Olea/química , Extratos Vegetais/administração & dosagem , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Ubiquinona/administração & dosagem , Ubiquinona/análogos & derivados
13.
Cell Mol Life Sci ; 78(7): 3299-3315, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33449148

RESUMO

The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review focuses on research conducted on mammalian salivary gland development, particularly on the differentiation of acinar, ductal, and myoepithelial cells. We discuss recent studies that provide conceptual advances in the understanding of the molecular mechanisms of salivary gland development. In addition, we describe the organogenesis of submandibular glands (SMGs), model systems used for the study of SMG development, and the key signaling pathways as well as cellular processes involved in salivary gland development. The findings from the recent studies elucidating the identity of stem/progenitor cells in the SMGs, and the process by which they are directed along a series of cell fate decisions to form functional glands, are also discussed. Advances in genetic tools and tissue engineering strategies will significantly increase our knowledge about the mechanisms by which signaling pathways and cells establish tissue architecture and function during salivary gland development, which may also be conserved in the growth and development of other organ systems. An increased knowledge of organ development mechanisms will have profound implications in the design of therapies for the regrowth or repair of injured tissues. In addition, understanding how the processes of cell survival, expansion, specification, movement, and communication with neighboring cells are regulated under physiological and pathological conditions is critical to the development of future treatments.


Assuntos
Diferenciação Celular , Organogênese , Glândulas Salivares/citologia , Transdução de Sinais , Células-Tronco/citologia , Animais , Humanos , Glândulas Salivares/fisiologia , Células-Tronco/fisiologia
14.
Science ; 371(6524): 52-57, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33384370

RESUMO

Neuroendocrine (NE) cells are epithelial cells that possess many of the characteristics of neurons, including the presence of secretory vesicles and the ability to sense environmental stimuli. The normal physiologic functions of solitary airway NE cells remain a mystery. We show that mouse and human airway basal stem cells sense hypoxia. Hypoxia triggers the direct differentiation of these stem cells into solitary NE cells. Ablation of these solitary NE cells during hypoxia results in increased epithelial injury, whereas the administration of the NE cell peptide CGRP rescues this excess damage. Thus, we identify stem cells that directly sense hypoxia and respond by differentiating into solitary NE cells that secrete a protective peptide that mitigates hypoxic injury.


Assuntos
Diferenciação Celular , Hipóxia/patologia , Células Neuroendócrinas/fisiologia , Oxigênio/fisiologia , Células-Tronco/fisiologia , Traqueia/citologia , Anaerobiose , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Contagem de Células , Deleção de Genes , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Mutantes , Células Neuroendócrinas/citologia , Prolil Hidroxilases/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Transativadores/genética
15.
Nat Commun ; 12(1): 448, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469008

RESUMO

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Queratinócitos/fisiologia , Reepitelização/genética , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Autorrenovação Celular/genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Íntrons/genética , Poliadenilação/genética , Cultura Primária de Células , Fatores de Transcrição/genética
16.
Nat Commun ; 12(1): 692, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514709

RESUMO

Skeletal muscle has remarkable regeneration capabilities, mainly due to its resident muscle stem cells (MuSCs). In this review, we introduce recently developed technologies and the mechanistic insights they provide to the understanding of MuSC biology, including the re-definition of quiescence and Galert states. Additionally, we present recent studies that link MuSC function with cellular heterogeneity, highlighting the complex regulation of self-renewal in regeneration, muscle disorders and aging. Finally, we discuss MuSC metabolism and its role, as well as the multifaceted regulation of MuSCs by their niche. The presented conceptual advances in the MuSC field impact on our general understanding of stem cells and their therapeutic use in regenerative medicine.


Assuntos
Músculo Esquelético/citologia , Doenças Musculares/terapia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Músculo Esquelético/fisiologia , Doenças Musculares/fisiopatologia , Regeneração/fisiologia
17.
Methods Mol Biol ; 2147: 63-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840811

RESUMO

Biofabrication is revolutionizing substitute tissue manufacturing. Skeletal stem cells (SSCs) can be blended with hydrogel biomaterials and printed to form three-dimensional structures that can closely mimic tissues of interest. Our bioink formulation takes into account the potential for cell printing including a bioink nanocomposite that contains low fraction polymeric content to facilitate cell encapsulation and survival, while preserving hydrogel integrity and mechanical properties following extrusion. Clay inclusion to the nanocomposite strengthens the alginate-methylcellulose network providing a biopaste with unique shear-thinning properties that can be easily prepared under sterile conditions. SSCs can be mixed with the clay-based paste, and the resulting bioink can be printed in 3D structures ready for implantation. In this chapter, we provide the methodology for preparation, encapsulation, and printing of SSCs in a unique clay-based bioink.


Assuntos
Bioimpressão/métodos , Regeneração Óssea/fisiologia , Argila/química , Nanocompostos/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Substitutos Ósseos/síntese química , Substitutos Ósseos/química , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Tinta , Microtecnologia/métodos , Impressão Tridimensional , Silicatos/química , Células-Tronco/citologia , Células-Tronco/fisiologia , Engenharia Tecidual/instrumentação
18.
Methods Mol Biol ; 2202: 51-61, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857345

RESUMO

Reactive oxygen species (ROS) may severely affect the biochemical viability of most cells. However, ROS may act also as key second messengers regulating important physiological functions in eukaryotic organisms. Of special interest is the potential role of ROS in the regulation of stem cell function and tissue homeostasis and regeneration in adult mammalian tissues. In this context, the hair follicle constitutes an excellent experimental model to study this aspect of ROS biology.Here we present a robust protocol to promote a sustained growth of ex vivo cultured human hair follicles based on the induction of a transient/modulable production of nonlethal endogenous ROS levels in the tissue through a protoporphyrin IX-dependent photodynamic procedure. The light-switchable ROS production activates hair follicle stem cell niches, induces cell proliferation, and maintains the growth/anagen phase for long time. This approach constitutes a complementary experimental tool to study the physiological roles of ROS in human tissues.


Assuntos
Técnicas de Cultura de Células/métodos , Folículo Piloso/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Adulto , Proliferação de Células/fisiologia , Células Cultivadas , Cabelo/fisiologia , Folículo Piloso/metabolismo , Humanos , Nicho de Células-Tronco/fisiologia , Células-Tronco/fisiologia
19.
Eur J Endocrinol ; 184(1): 155-168, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33112291

RESUMO

Objective: The term Multiple Symmetric Lipomatosis (MSL) describes a heterogeneous group of rare monogenic disorders and multifactorial conditions, characterized by upper-body adipose masses. Biallelic variants in LIPE encoding hormone-sensitive lipase (HSL), a key lipolytic enzyme, were implicated in three families worldwide. We aimed to further delineate LIPE-related clinical features and pathophysiological determinants. Methods: A gene panel was used to identify pathogenic variants. The disease features were reviewed at the French lipodystrophy reference center. The immunohistological, ultrastructural, and protein expression characteristics of lipomatous tissue were determined in surgical samples from one patient. The functional impact of variants was investigated by developing a model of adipose stem cells (ASCs) isolated from lipomatous tissue. Results: We identified new biallelic LIPE null variants in three unrelated patients referred for MSL and/or partial lipodystrophy. The hallmarks of the disease, appearing in adulthood, included lower-limb lipoatrophy, upper-body and abdominal pseudo-lipomatous masses, diabetes and/or insulin resistance, hypertriglyceridemia, liver steatosis, high blood pressure, and neuromuscular manifestations. Ophthalmological investigations revealed numerous auto-fluorescent drusen-like retinal deposits in all patients. Lipomatous tissue and patient ASCs showed loss of HSL and decreased expression of adipogenic and mature adipocyte markers. LIPE-mutated ASCs displayed impaired adipocyte differentiation, decreased insulin response, defective lipolysis, and mitochondrial dysfunction. Conslusions: Biallelic LIPE null variants result in a multisystemic disease requiring multidisciplinary care. Loss of HSL expression impairs adipocyte differentiation, consistent with the lipodystrophy/MSL phenotype and associated metabolic complications. Detailed ophthalmological examination could reveal retinal damage, further pointing to the nervous tissue as an important disease target.


Assuntos
Diferenciação Celular/genética , Lipodistrofia/genética , Lipomatose Simétrica Múltipla/genética , Modelos Genéticos , Esterol Esterase/genética , Adipócitos/fisiologia , Tecido Adiposo/citologia , Idoso , Alelos , Feminino , Variação Genética , Humanos , Pessoa de Meia-Idade , Fenótipo , Células-Tronco/fisiologia , Síndrome
20.
Methods Mol Biol ; 2258: 29-40, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340352

RESUMO

Lineage-tracing experiments aim to identify and track the progeny and/or fate of cells. The use of inducible recombinases and fluorescent reporters has been instrumental in defining cellular hierarchies and allowing for the identification of stem cells in an unperturbed in vivo setting. The refinement of these approaches, labeling single cells, and the subsequent quantitative analysis of the clonal dynamics have allowed the comparison of different stem cell populations as well as establishing different mechanisms of cellular replenishment during steady-state homeostasis as well as during morphogenesis and disease. Utilizing this approach, it is now possible to establish the cellular hierarchy in a given tissue and the frequency of cell fate decisions on a population basis, thus providing a comprehensive analysis of cellular behavior in vivo. Although in this chapter we describe a protocol for lineage tracing of cells from fetal intestinal epithelium to the adult intestine, this approach can be widely applied to quantitatively assess the cell fate of any fetal cell during morphogenesis.


Assuntos
Linhagem da Célula , Rastreamento de Células , Intestinos/fisiologia , Microscopia de Fluorescência , Morfogênese , Células-Tronco/fisiologia , Animais , Linhagem da Célula/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Intestinos/embriologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Masculino , Camundongos Transgênicos , Transdução de Sinais
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