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1.
Nat Commun ; 11(1): 706, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024836

RESUMO

The basal cell compartment in many epithelial tissues is generally believed to serve as an important pool of stem cells. However, basal cells are heterogenous and the stem cell subpopulation within basal cells is not well elucidated. Here we uncover that the core epithelial-to-mesenchymal transition (EMT) inducer Zeb1 is expressed in a prostate basal cell subpopulation. The Zeb1+ prostate epithelial cells are multipotent prostate basal stem cells (PBSCs) that can self-renew and generate functional prostatic glandular structures at the single-cell level. Genetic ablation studies reveal an indispensable role for Zeb1 in prostate basal cell development. Utilizing unbiased single-cell transcriptomic analysis of over 9000 mouse prostate basal cells, we confirm the existence of the Zeb1+ basal cell subset. Moreover, Zeb1+ epithelial cells can be detected in mouse and human prostate tumors. Identification of the PBSC and its transcriptome profile is crucial to advance our understanding of prostate development and tumorigenesis.


Assuntos
Próstata/citologia , Células-Tronco/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Gravidez , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Ratos Sprague-Dawley , Análise de Célula Única , Via de Sinalização Wnt , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
2.
Nat Protoc ; 15(3): 1066-1081, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005982

RESUMO

Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157+ vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.


Assuntos
Técnicas Citológicas/métodos , Células Endoteliais/fisiologia , Fígado/citologia , Células-Tronco/fisiologia , Animais , Camundongos
3.
Nat Protoc ; 15(3): 1082-1097, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31965111

RESUMO

Limited methods exist to assay the direct effects of therapeutic intervention on muscle stem cell fate, proliferation or differentiation in an in vivo context. Here we provide an optimized protocol for muscle stem cell isolation and transplantation into mice to deconvolute heterogeneity within isolated stem cell populations. Viable and pure cell populations are isolated within 2 h and can then be used for therapeutic intervention or transplantation to uncover the repopulating and differentiation potential in mice, a physiologically relevant in vivo context. Effects can be assessed 9 d after transplantation. This methodology analyzes cell and sort purity prior to transplantation to improve reproducibility and outlines novel blocking steps to improve tissue staining and analysis. Experience with surgical procedures in mice is recommended before attempting this protocol. Our system is widely applicable for exploring stem cell dynamics within muscle and has already been used to study heterogeneity within muscle stem cell populations and efficacy of therapeutic intervention on isolated stem cell populations.


Assuntos
Linhagem da Célula/fisiologia , Separação Celular/métodos , Células Satélites de Músculo Esquelético/classificação , Células Satélites de Músculo Esquelético/fisiologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Citometria de Fluxo/métodos , Camundongos
4.
PLoS One ; 15(1): e0227279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999703

RESUMO

Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (GsαR201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the GsαR201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of CEBPs (CCAAT-Enhancer Binding Proteins) in both data sets, factors related to browning of white fat. This is the first analysis of the reaction of human skeletal stem/progenitor cells to the introduction of the FD mutation and we believe it provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets.


Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Cromograninas/genética , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco/fisiologia , Proteínas ADAM/metabolismo , Adipogenia/genética , Tecido Adiposo Branco/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Cromograninas/metabolismo , Simulação por Computador , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Conjuntos de Dados como Assunto , Displasia Fibrosa Óssea/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Mutação com Ganho de Função , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteogênese/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Estromais/fisiologia , Regulação para Cima
5.
Nat Commun ; 11(1): 37, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896747

RESUMO

Nutrients are absorbed solely by the intestinal villi. Aging of this organ causes malabsorption and associated illnesses, yet its aging mechanisms remain unclear. Here, we show that aging-caused intestinal villus structural and functional decline is regulated by mTORC1, a sensor of nutrients and growth factors, which is highly activated in intestinal stem and progenitor cells in geriatric mice. These aging phenotypes are recapitulated in intestinal stem cell-specific Tsc1 knockout mice. Mechanistically, mTORC1 activation increases protein synthesis of MKK6 and augments activation of the p38 MAPK-p53 pathway, leading to decreases in the number and activity of intestinal stem cells as well as villus size and density. Targeting p38 MAPK or p53 prevents or rescues ISC and villus aging and nutrient absorption defects. These findings reveal that mTORC1 drives aging by augmenting a prominent stress response pathway in gut stem cells and identify p38 MAPK as an anti-aging target downstream of mTORC1.


Assuntos
Intestinos/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células-Tronco/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Envelhecimento , Animais , Proliferação de Células , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , MAP Quinase Quinase 6/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais , Sirolimo/farmacologia , Células-Tronco/citologia , Tamoxifeno/farmacologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo
6.
Hum Genet ; 139(3): 309-331, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31324975

RESUMO

DNA damage is one of the most consistent cellular process proposed to contribute to aging. The maintenance of genomic and epigenomic integrity is critical for proper function of cells and tissues throughout life, and this homeostasis is under constant strain from both extrinsic and intrinsic insults. Considering the relationship between lifespan and genotoxic burden, it is plausible that the longest-lived cellular populations would face an accumulation of DNA damage over time. Tissue-specific stem cells are multipotent populations residing in localized niches and are responsible for maintaining all lineages of their resident tissue/system throughout life. However, many of these stem cells are impacted by genotoxic stress. Several factors may dictate the specific stem cell population response to DNA damage, including the niche location, life history, and fate decisions after damage accrual. This leads to differential handling of DNA damage in different stem cell compartments. Given the importance of adult stem cells in preserving normal tissue function during an individual's lifetime, DNA damage sensitivity and accumulation in these compartments could have crucial implications for aging. Despite this, more support for direct functional effects driven by accumulated DNA damage in adult stem cell compartments is needed. This review will present current evidence for the accumulation and potential influence of DNA damage in adult tissue-specific stem cells and propose inquiry directions that could benefit individual healthspan.


Assuntos
Envelhecimento/fisiologia , Dano ao DNA/fisiologia , Células-Tronco/fisiologia , Animais , Homeostase/fisiologia , Humanos
7.
Life Sci ; 241: 117121, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31794772

RESUMO

AIMS: This study addressed whether endogenous hepatic stem/progenitor (HSP) cells survival were related to the injured signals during liver cirrhosis. MATERIAL AND METHODS: Liver cirrhosis was induced in C57BL/6 mice by administering diethylnitrosamine (DEN) in drinking water. Hematoxylin-eosin staining and Masson's trichrome staining were used to identify infiltrative cells and connective tissues, respectively. The inflammatory activity grade and fibrotic stage, represented as G and S respectively, and evaluated by the International Simplified Grading and Staging System (ISGSS). Endogenous HSP cells (Ng2+HSP) were identified by immunofluorescence staining with an anti-neuro/glial antigen 2 (Ng2) antibody. All data were analyzed using SPSS 22.0 and GraphPad Prism 6 and Student's t-test was performed to determine statistical significance. p-values < 0.05 were considered statistically significant. KEY FINDINGS: All mice administered oral DEN developed liver fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). During the fibrosis period, observed a positive correlation of survival of endogenous HSP (Ng2+HSP) cells with inflammatory activity. As the disease developed into the cirrhotic stage, when lost correlation of endogenous HSP cells with inflammatory activity, revealed a dramatically reduced survival rate of endogenous HSP (Ng2+HSP) cells. SIGNIFICANCE: The DEN-induced liver cirrhosis could develop into three time zone of fibrosis, cirrhosis and cancer, and the histological patterns in the model are similar to those described in humans. Dramatic survival and less apoptosis of endogenous HSP (Ng2+HSP) cells was within fibrotic state, where inflammation signals is strong, indicating such time zone (1-6 weeks) during liver cirrhosis is important for mobilizing endogenous HSP-based regeneration.


Assuntos
Carcinoma Hepatocelular/terapia , Inflamação/terapia , Cirrose Hepática/terapia , Neoplasias Hepáticas Experimentais/terapia , Fígado/citologia , Células-Tronco/citologia , Animais , Carcinoma Hepatocelular/etiologia , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Inflamação/etiologia , Fígado/imunologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Neoplasias Hepáticas Experimentais/etiologia , Regeneração Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/fisiologia
8.
Am J Pathol ; 190(3): 520-534, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31866347

RESUMO

The adventitia, the outer layer of the blood vessel wall, may be the most complex layer of the wall and may be the master regulator of wall physiology and pathobiology. This review proposes a major shift in thinking to apply a functional lens to the adventitia rather than only a structural lens. Human and experimental in vivo and in vitro studies show that the adventitia is a dynamic microenvironment in which adventitial and perivascular adipose tissue cells initiate and regulate important vascular functions in disease, especially intimal hyperplasia and atherosclerosis. Although well away from the blood-wall interface, where much pathology has been identified, the adventitia has a profound influence on the population of intimal and medial endothelial, macrophage, and smooth muscle cell function. Vascular injury and dysfunction of the perivascular adipose tissue promote expansion of the vasa vasorum, activation of fibroblasts, and differentiation of myofibroblasts. This regulates further biologic processes, including fibroblast and myofibroblast migration and proliferation, inflammation, immunity, stem cell activation and regulation, extracellular matrix remodeling, and angiogenesis. A debate exists as to whether the adventitia initiates disease or is just an important participant. We describe a mechanistic model of adventitial function that brings together current knowledge and guides the design of future investigations to test specific hypotheses on adventitial pathobiology.


Assuntos
Aterosclerose/patologia , Hiperplasia/patologia , Doenças Vasculares/patologia , Tecido Adiposo/patologia , Túnica Adventícia/patologia , Matriz Extracelular/patologia , Fibroblastos/patologia , Humanos , Inflamação/patologia , Macrófagos/patologia , Modelos Biológicos , Miócitos de Músculo Liso/patologia , Miofibroblastos/patologia , Células-Tronco/fisiologia , Vasa Vasorum/patologia
9.
Int. j. morphol ; 37(4): 1564-1571, Dec. 2019. tab
Artigo em Espanhol | LILACS | ID: biblio-1040170

RESUMO

Las glándulas salivales humanas pueden ser gravemente lesionadas por la radioterapia utilizada contra neoplasias de cabeza y cuello, produciendo hiposialia y xerostomía, las cuales afectan la salud oral y sistémica, mermando la calidad de vida de la persona. Los tratamientos convencionales actuales están diseñados para disminuir los síntomas, sin actuar sobre los cambios fisiopatológicos que se dan a nivel glandular. Esta revisión intenta analizar aquellas terapias preventivas y/o curativas que están desarrollándose en el campo biomolecular y que tienen un futuro prometedor por sus características innovadoras: terapia génica, terapia con células madre y terapia con factores de crecimiento. Se evidencia un aporte adicional de la nanotecnología, la cual está mejorando las vías de aplicación de los tratamientos.


Human salivary glands can be seriously injured by the radiotherapy used against head and neck neoplasms, producing hyposialia and xerostomy, which affect oral and systemic health, diminishing the person's quality of life. Current conventional treatments are designed to reduce symptoms, without acting on the pathophysiological changes that occur at the glandular level. This review attempts to analyze those preventive and /or curative therapies that are developing in the biomolecular field and that have a promising future due to their innovative features: Gene therapy, stem cell therapy and growth factor therapy. An additional contribution of nanotechnology is evident, which is improving the routes of treatment application.


Assuntos
Humanos , Radioterapia/efeitos adversos , Doenças das Glândulas Salivares/prevenção & controle , Células-Tronco/fisiologia , Terapia Genética/métodos , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Doenças das Glândulas Salivares/terapia , Glândulas Salivares/efeitos da radiação , Xerostomia/prevenção & controle , Nanotecnologia
10.
PLoS Negl Trop Dis ; 13(12): e0007932, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31881019

RESUMO

Echinococcosis represents a major public health problem worldwide and is considered a neglected disease by the World Health Organization. The etiological agents are Echinococcus tapeworms, which display elaborate developmental traits that imply a complex control of gene expression. MicroRNAs (miRNAs), a class of small regulatory RNAs, are involved in the regulation of many biological processes such as development and metabolism. They act through the repression of messenger RNAs (mRNAs) usually by binding to the 3' untranslated region (3'UTR). Previously, we described the miRNome of several Echinococcus species and found that miRNAs are highly expressed in all life cycle stages, suggesting an important role in gene expression regulation. However, studying the role of miRNAs in helminth biology remains a challenge. To develop methodology for functional analysis of miRNAs in tapeworms, we performed miRNA knockdown experiments in primary cell cultures of Echinococcus multilocularis, which mimic the development of metacestode vesicles from parasite stem cells in vitro. First, we analysed the miRNA repertoire of E. multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterian miRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs. Using genomic information and bioinformatic algorithms for miRNA binding prediction, we found a high number of potential miR-71 targets in E. multilocularis. Inhibition of miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that block miRNA function. To this end, we evaluated a variety of chemically modified anti-miRs for miR-71 knockdown. Electroporation of primary cells with 2'-O-methyl modified anti-miR-71 led to significantly reduced miR-71 levels. Transcriptomic analyses showed that several predicted miR-71 targets were up-regulated in anti-miR-treated primary cells, including genes potentially involved in parasite development, host parasite interaction, and several genes of as yet unknown function. Notably, miR-71-silenced primary cell cultures showed a strikingly different phenotype from control cells and did not develop into fully mature metacestodes. These findings indicate an important function of miR-71 in Echinococcus development and provide, for the first time, methodology to functionally study miRNAs in a tapeworm.


Assuntos
Echinococcus multilocularis/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Animais , Células Cultivadas , Biologia Computacional , Células-Tronco/fisiologia
11.
Zhonghua Wei Chang Wai Ke Za Zhi ; 22(11): 1095-1100, 2019 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-31770844

RESUMO

Colonic organoids are three-dimensional organotypic cultures of the colonic stem cells or pluripotent stem cells. Its essence is the culture of colonic stem cells or pluripotent stem cells, and their derived intestinal epithelial cells, intestinal endocrine cells and goblet cells in basement membrane extract with specific growth factors. Colonic organoids are comprised of all major types of colonic epithelial cells and represent the architecture and function remarkably similar to those of the colonic epithelium, faithfully recapitulating the functional colonic epithelium ex vivo. As a superior basic experimental model, colonic organoids are representing advantages over conventional cell models and animal models in many aspects, such as high successful rate, short productive cycle, and high consistency with source tissue. Since first reported in 2011, colonic organoids have soon become an important topic in the field of colonic diseases. It has now been applied in the field of physiology of colonic epithelium, infectious diarrhea, ulcerative colitis, regeneration of intestinal injury, and colon tumors. In this review, we summarize the research advances of establishment and application of colonic organoids.


Assuntos
Colo/fisiologia , Mucosa Intestinal/fisiologia , Organoides/fisiologia , Células-Tronco/fisiologia , Animais , Colo/citologia , Humanos , Intestinos
12.
Nat Cell Biol ; 21(12): 1490-1503, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768046

RESUMO

Tendon injuries cause prolonged disability and never recover completely. Current mechanistic understanding of tendon regeneration is limited. Here, we use single-cell transcriptomics to identify a tubulin polymerization-promoting protein family member 3-expressing (Tppp3+) cell population as potential tendon stem cells. Through inducible lineage tracing, we demonstrate that these cells can generate new tenocytes and self-renew upon injury. A fraction of Tppp3+ cells expresses platelet-derived growth factor receptor alpha (Pdfgra). Ectopic platelet-derived growth factor-AA (PDGF-AA) protein induces new tenocyte production while inactivation of Pdgfra in Tppp3+ cells blocks tendon regeneration. These results support Tppp3+Pdgfra+ cells as tendon stem cells. Unexpectedly, Tppp3-Pdgfra+ fibro-adipogenic progenitors coexist in the tendon stem cell niche and give rise to fibrotic cells, revealing a clandestine origin of fibrotic scars in healing tendons. Our results explain why fibrosis occurs in injured tendons and present clinical challenges to enhance tendon regeneration without a concurrent increase in fibrosis by PDGF application.


Assuntos
Moléculas de Adesão Celular/metabolismo , Fibrose/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Regeneração/fisiologia , Células-Tronco/metabolismo , Tendões/metabolismo , Adipogenia/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Fibrose/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/fisiopatologia , Tendões/fisiopatologia , Tenócitos/metabolismo , Tenócitos/fisiologia
14.
Res Vet Sci ; 126: 207-212, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31610471

RESUMO

To explore the effect of epigenetic modification on the differentiation of goat adipose-derived stem cells in vitro, we used two common epigenetic modification inhibitors, trichostatin A and vorinostat, to treat cashmere goat adipose-derived stem cells and induce adipocyte differentiation. The results showed that trichostatin A and vorinostat changed the relative amounts of H3K9 acetylation and dimethylation in the upstream sequence of PPARG, increased peroxisome proliferator-activated receptor gamma (PPARG) transcription before differentiation and then promoted adipocyte differentiation, and regulated the expression of adipocyte-specific genes. We conclude that adipocyte differentiation is regulated dynamically by different histone modifications. The areas of acetylation and demethylation changed by trichostatin A and vorinostat are the basis for further research on the mechanism of PPARG promoter to regulate adipocytes differentiation and provide research theroies for using adipose-derived stem cells as donor to produce transgenic animals to improve meat quality improvement.


Assuntos
Adipogenia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Cabras/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Vorinostat/farmacologia , Acetilação/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/fisiologia , Animais , Metilação/efeitos dos fármacos , PPAR gama/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
15.
Int J Sports Med ; 40(14): 921-930, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31614382

RESUMO

Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1+cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1+cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1+cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1+cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.


Assuntos
Coração/fisiologia , Proteínas com Homeodomínio LIM/genética , Miocárdio/citologia , Miócitos Cardíacos/citologia , Condicionamento Físico Animal , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Proliferação de Células/fisiologia , Mapeamento Cromossômico , Coração/embriologia , Coração/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos C57BL
16.
J Mater Sci Mater Med ; 30(9): 101, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31473826

RESUMO

Diabetes mellitus is the most common metabolic disorder with a high mortality and morbidity rate. A new promising strategy to treat DM is pancreatic tissue engineering. We described a 3D culture system accompanied by signaling factors to differentiate hEnSCs into IPCs in the presence of nZnO. We isolated EnSCs and cultured it in DMEM/F12 medium. Nanofibrous PLA/Cs scaffold was prepared through the electrospinning method. The morphological properties of the scaffolds and cells were evaluated by SEM. MTT assay was used to investigate the metabolic activity of the hEnSCs cultured on the scaffolds and a four-stage protocol was applied to differentiate hEnSCs. The differentiated cells were tested for pancreatic markers by immunocytochemistry, qRT-PCR and DTZ staining. The results of this study revealed that hEnSCs cultured on PLA/Cs scaffold and treated with nZnO can efficiently differentiate into IPCs. The examination of differentiated cell morphology showed their near similarity with pancreatic islet cells, and DTZ staining emphasized the presence of insulin granules inside their cytoplasm. Moreover, qRT-PCR and immunofluorescent staining results showed the efficient expression of specific gene markers of IPCs in resultant differentiated cells. Moreover, PLA/CS and nZnO were able to provide a good nanoenvironment for the differentiation of hEnSCs into IPCS the in presence of other molecules.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Endométrio/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Nanofibras/química , Células-Tronco/efeitos dos fármacos , Tecidos Suporte/química , Óxido de Zinco/farmacologia , Células Cultivadas , Feminino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Teste de Materiais , Nanopartículas Metálicas/química , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Óxido de Zinco/química
17.
Elife ; 82019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502540

RESUMO

How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. We test the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, we manipulate expression of the classic temporal transcription factor Hunchback (Hb) specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then we monitor dendrite morphology and neuromuscular synaptic partnerships. We find that prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, our data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acts early in neurogenesis as a switch to control motor circuit wiring, and provide novel insight into the relationship between stem cell and circuit.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila/biossíntese , Expressão Gênica , Neurônios Motores/fisiologia , Vias Neurais/embriologia , Junção Neuromuscular/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/biossíntese , Animais , Drosophila , Neurônios Motores/citologia , Junção Neuromuscular/citologia , Células-Tronco/citologia
18.
Elife ; 82019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393263

RESUMO

Hepatocytes are regarded as the only effective cell source for cell transplantation to treat liver diseases; however, their availability is limited due to a donor shortage. Thus, a novel cell source must be developed. We recently reported that mature rodent hepatocytes can be reprogrammed into progenitor-like cells with a repopulative capacity using small molecule inhibitors. Here, we demonstrate that hepatic progenitor cells can be obtained from human infant hepatocytes using the same strategy. These cells, named human chemically induced liver progenitors (hCLiPs), had a significant repopulative capacity in injured mouse livers following transplantation. hCLiPs redifferentiated into mature hepatocytes in vitro upon treatment with hepatic maturation-inducing factors. These redifferentiated cells exhibited cytochrome P450 (CYP) enzymatic activities in response to CYP-inducing molecules and these activities were comparable with those in primary human hepatocytes. These findings will facilitate liver cell transplantation therapy and drug discovery studies.


Assuntos
Técnicas de Cultura de Células/métodos , Hepatócitos/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Lactente , Fígado/lesões , Regeneração , Transplante de Células-Tronco , Resultado do Tratamento
19.
Nutrients ; 11(8)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426533

RESUMO

Growth hormone (GH) and glutamine (Gln) stimulate the growth of the intestinal mucosa. GH activates the proliferation of intestinal stem cells (ISCs), enhances the formation of crypt organoids, increases ISC stemness markers in the intestinal organoids, and drives the differentiation of ISCs into Paneth cells and enterocytes. Gln enhances the proliferation of ISCs and increases crypt organoid formation; however, it mainly acts on the post-proliferation activity of ISCs to maintain the stability of crypt organoids and the intestinal mucosa, as well as to stimulate the differentiation of ISCs into goblet cells and possibly Paneth cells and enteroendocrine cells. Since GH and Gln have differential effects on ISCs. Their use in combination may have synergistic effects on ISCs. In this review, we summarize the evidence of the actions of GH and/or Gln on crypt cells and ISCs in the literature. Overall, most studies demonstrated that GH and Gln in combination exerted synergistic effects to activate the proliferation of crypt cells and ISCs and enhance crypt organoid formation and mucosal growth. This treatment influenced the proliferation of ISCs to a similar degree as GH treatment alone and the differentiation of ISCs to a similar degree as Gln treatment alone.


Assuntos
Glutamina/farmacologia , Hormônio do Crescimento/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células , Sinergismo Farmacológico , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco/fisiologia
20.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438545

RESUMO

Pancreatic progenitor cells (PPCs) are the primary source for all pancreatic cells, including beta-cells, and thus the proliferation and differentiation of PPCs into islet-like cell clusters (ICCs) opens an avenue to providing transplantable islets for diabetic patients. Meanwhile, mesenchymal stem cells (MSCs) can enhance the development and function of different cell types of interest, but their role on PPCs remains unknown. We aimed to explore the mechanism-of-action whereby MSCs induce the in vitro and in vivo PPC/ICC development by means of our established co-culture system of human PPCs with human fetal bone marrow-derived MSCs. We examined the effect of MSC-conditioned medium on PPC proliferation and survival. Meanwhile, we studied the effect of MSC co-culture enhanced PPC/ICC function in vitro and in vivo co-/transplantation. Furthermore, we identified IGF1 as a critical factor responsible for the MSC effects on PPC differentiation and proliferation via IGF1-PI3K/Akt and IGF1-MEK/ERK1/2, respectively. In conclusion, our data indicate that MSCs stimulated the differentiation and proliferation of human PPCs via IGF1 signaling, and more importantly, promoted the in vivo engraftment function of ICCs. Taken together, our protocol may provide a mechanism-driven basis for the proliferation and differentiation of PPCs into clinically transplantable islets.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Pâncreas/citologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Apoptose/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Células-Tronco Mesenquimais/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco/metabolismo
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