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2.
Nat Commun ; 11(1): 5292, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087715

RESUMO

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.


Assuntos
Técnicas de Reprogramação Celular/métodos , Células Endoteliais/citologia , Hepatócitos/citologia , Células-Tronco/citologia , Animais , Ductos Biliares/citologia , Ductos Biliares/fisiologia , Agregação Celular , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Células Endoteliais/fisiologia , Feminino , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/fisiologia , Fator 6 Nuclear de Hepatócito/genética , Fator 6 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Hepatócitos/transplante , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Células-Tronco/fisiologia
3.
Nat Metab ; 2(9): 958-973, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32868922

RESUMO

Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. Here, we show that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal's appetite for sugar, the key nutrient fuelling this metabolic pathway. We provide functional evidence that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. Our findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal's preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes.


Assuntos
Apetite/fisiologia , Reprogramação Celular/fisiologia , Drosophila/metabolismo , Açúcares , Animais , Diversidade de Anticorpos , Carcinogênese , Carboidratos da Dieta/farmacologia , Corpo Adiposo/metabolismo , Feminino , Preferências Alimentares , Fome/fisiologia , Ovário/fisiologia , Via de Pentose Fosfato , Inanição
4.
Nat Commun ; 11(1): 4337, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859897

RESUMO

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 µM ouabain 100 nM blebbistatin) or chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLMWT), transgenic (PLM3SA), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23Na, 31P, 13C NMR followed by 1H-NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camito may be a new approach to ameliorate metabolic dysregulation in heart failure.


Assuntos
Reprogramação Celular/fisiologia , Citoplasma/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Sódio/metabolismo , Animais , Modelos Animais de Doenças , Metabolismo Energético , Técnicas de Introdução de Genes , Coração , Hipertrofia , Preparação de Coração Isolado , Masculino , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Wistar , Sódio/sangue , Trocador de Sódio e Cálcio/efeitos dos fármacos , Tiazepinas/farmacologia
5.
PLoS One ; 15(8): e0226235, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797046

RESUMO

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with ß-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.


Assuntos
Celobiose/metabolismo , Zymomonas/crescimento & desenvolvimento , Zymomonas/metabolismo , Adaptação Fisiológica/fisiologia , Biomassa , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Celulose/metabolismo , Expressão Gênica/genética , Glucose/metabolismo , Hidrolases/metabolismo , Proteômica , Sacarase/metabolismo , Sacarose/metabolismo , Zymomonas/genética , beta-Glucosidase/metabolismo
6.
Nat Commun ; 11(1): 4055, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792504

RESUMO

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.


Assuntos
Cromatina/metabolismo , Glucose/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunoprecipitação da Cromatina , Epigênese Genética/genética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
7.
Am J Med Sci ; 360(3): 279-286, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32829780

RESUMO

BACKGROUND: The essential role of 6-phosphogluconate dehydrogenase (6PGD), the enzyme catalyzing the oxidative pentose phosphate pathway, in tumor growth and metabolism has garnered attention in recent years. In this work, we are the first to demonstrate that aberrant activation of 6PGD is a feature in renal cell carcinoma (RCC) and is critically involved in renal carcinogenesis and chemo- and immuno-resistance. MATERIALS AND METHODS: 6PGD expression and activity were systematically analyzed in normal and malignant renal cells and tissues. The roles of 6PGD and its downstream mechanism were investigated using gain-of-function and loss-of-function approaches. RESULTS: 6PGD expression and enzyme activity were increased in RCC cells and patients' samples. Activation of 6PGD via gain-of-function approach promoted growth of normal kidney but not RCC cells, and alleviated the efficacy of chemotherapeutic (e.g., 5-FU) and immunotherapeutic (e.g., IFN-α) agents. In contrast, 6PGD inhibition using siRNA knockdown and pharmacological inhibitor physcion augmented the inhibitory effects of 5-FU and IFN-α in RCC. Mechanistic studies demonstrated that 6PGD inhibition activated AMPK signaling, leading to ACC1 enzyme inhibition and reduction of lipid synthesis. In addition, 6PGD inhibition disrupted NADPH and NADH homeostasis in RCC cells as shown by the decreased level of NADPH and NADH, and suppressed SIRT-1 activity. AMPK inhibition by siRNA knockdown reversed the inhibitory effects of physcion, demonstrating that the effect of 6PGD inhibition is AMPK activation dependent. CONCLUSIONS: Our work provides preclinical evidence that 6PGD inhibition may represent a potential therapeutic strategy to augment the efficacy of RCC standard of care drugs.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Carcinoma de Células Renais/terapia , Reprogramação Celular/fisiologia , Neoplasias Renais/terapia , Fosfogluconato Desidrogenase/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Fluoruracila/uso terapêutico , Técnicas de Silenciamento de Genes , Humanos , Imunoterapia , Interferon-alfa/uso terapêutico , Rim/patologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , NADP/fisiologia , Fosfogluconato Desidrogenase/antagonistas & inibidores , Fosfogluconato Desidrogenase/genética , RNA Interferente Pequeno , Regulação para Cima
8.
Mol Cell ; 79(4): 660-676.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32755593

RESUMO

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.


Assuntos
Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Fígado/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Reprogramação Celular/fisiologia , Cromatina/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Camundongos Endogâmicos C57BL , Domínios Proteicos , Sítio de Iniciação de Transcrição
9.
Prostate ; 80(13): 1145-1156, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659025

RESUMO

BACKGROUND: Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ induced cancer stem cells and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. METHODS: Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by quantitative real-time polymerase chain reaction, Western blots, and immunofluorescence for lineage marker expression, and fluorescence activated cell sorting analyses and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. RESULTS: Overexpression of CXCL12γ promotes the reprogramming of cells with a differentiated luminal phenotype to a nonluminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated nonluminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAMLo /CD49fHi /CD24Lo /CD44Hi cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CONCLUSIONS: CXCL12γ supports the reprogramming of epithelial cells into nonluminal cell-derived stem cells, which facilitates gland development.


Assuntos
Quimiocina CXCL12/biossíntese , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Próstata/crescimento & desenvolvimento , Animais , Reprogramação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Xenoenxertos , Humanos , Masculino , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Camundongos , Próstata/citologia , Próstata/metabolismo , Isoformas de Proteínas
10.
Cancer Treat Rev ; 88: 102060, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32619863

RESUMO

Phenotypic plasticity of malignant melanoma is a well-known phenomenon. Several translational studies and small case series have reported this clinical and biological entity, particularly in metastatic melanoma, showing frequent aberrant expression of non-melanocytic differentiation markers of different lineages, posing remarkable challenges due to several alternative differential diagnoses including undifferentiated carcinoma and sarcomas. When melanoma loses its typical morpho-phenotype by routinely used diagnostic immunohistochemical markers, it is defined as "dedifferentiated melanoma". Historically, this process was closely related to diagnostic interpretative difficulties. In recent years, however, dedifferentiation has been increasingly recognized as an important biological phenomenon that demonstrates the phenotypic and genetic plasticity of melanoma, and specifically the non-irreversibility of the multistep cancerogenesis. Furthermore, dedifferentiation emerged as a general hallmark of cancer evolution and a common denominator of cross-resistance to both targeted and immunotherapy. In this review, we summarize the histopathological features, the genetic and epigenetic bases underlying the dedifferentiated phenotype in melanomas and provide additional support that dedifferentiation is a mechanism of resistance to immunotherapy and targeted therapy.


Assuntos
Melanoma/genética , Melanoma/terapia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Animais , Desdiferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Epigênese Genética , Humanos , Melanoma/patologia , Neoplasias Cutâneas/patologia
11.
Proc Natl Acad Sci U S A ; 117(26): 15160-15171, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541026

RESUMO

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1ß, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1ß and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis.


Assuntos
Reprogramação Celular/fisiologia , Nefrite Lúpica/metabolismo , Animais , Células Cultivadas , Dinoprostona/genética , Dinoprostona/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Glicólise/fisiologia , Humanos , Imunoglobulina G/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Rim/citologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio , Receptores de IgG/genética , Receptores de IgG/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1866(10): 165841, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32439610

RESUMO

Metabolic pathways leading to the synthesis, uptake, and usage of the nonessential amino acid serine are frequently amplified in cancer. Serine encounters diverse fates in cancer cells, including being charged onto tRNAs for protein synthesis, providing head groups for sphingolipid and phospholipid synthesis, and serving as a precursor for cellular glycine and one-carbon units, which are necessary for nucleotide synthesis and methionine cycle reloading. This review will focus on the participation of serine and glycine in the mitochondrial one-carbon (SGOC) pathway during cancer progression, with an emphasis on the genetic and epigenetic determinants that drive SGOC gene expression. We will discuss recently elucidated roles for SGOC metabolism in nucleotide synthesis, redox balance, mitochondrial function, and epigenetic modifications. Finally, therapeutic considerations for targeting SGOC metabolism in the clinic will be discussed.


Assuntos
Reprogramação Celular/fisiologia , Glicina/metabolismo , Neoplasias/metabolismo , Serina/metabolismo , Animais , Carbono/metabolismo , Humanos , Redes e Vias Metabólicas , Metionina/metabolismo , Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição
13.
Nat Commun ; 11(1): 2150, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358519

RESUMO

Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT.


Assuntos
Histonas/metabolismo , MicroRNAs/genética , Placenta/metabolismo , Proteínas Repressoras/genética , Animais , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Feminino , Impressão Genômica , Camundongos , Família Multigênica/genética , Gravidez , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
14.
J Biomed Sci ; 27(1): 56, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312260

RESUMO

BACKGROUND: Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. METHODS: Induced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition. RESULTS: BN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro. CONCLUSIONS: This study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.


Assuntos
Reprogramação Celular/fisiologia , Queratinócitos/fisiologia , Células Cultivadas/fisiologia , Técnicas de Reprogramação Celular , Feminino , Humanos , Técnicas In Vitro , Masculino , Fenômenos Fisiológicos da Pele
15.
Cell Physiol Biochem ; 54(2): 271-286, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233339

RESUMO

BACKGROUND/AIMS: Pericytes (PCs) are multipotent vascular precursors that play a critical physiological role in the development and maintenance of blood vessel integrity. In this study, we aim to characterize PCs isolated from human abdominal adipose tissue and develop an integration-free induced pluripotent stem cells (iPSCs) using episomal vectors. METHODS: The ultrastructure of adipose tissue-derived PCs was determined using scanning and transmission electron microscopy. The expression of mesenchymal stem cells (MSCs) and pericyte markers were examined using flow cytometry and PCR analysis. PCs were induced to adipogenic, osteogenic and myogenic lineages, and their angiogenic potential was determined using tube formation assay. We further established pericyte reprogramming protocol using episomal vectors. RESULTS: Our data showed that human adipose tissue-derived PCs uniformly expressed MSCs, CD105 and CD73, and PCs markers, desmin, and alpha smooth muscle actin (α-SMA), while lacked the expression of HLA-DR and the hematopoietic markers CD34, CD11b and CD45. Ultrastructure analysis showed typical internal structure for the PCs with a characteristic prominent eccentric nuclei and cytoplasmic invaginations forming a caveolar system. Functional analysis showed efficient differentiation into adipocytes, osteocytes, and myocyte-like cells. Adipose tissue-derived PCs showed angiogenic potential using tube-forming assay. To determine further application of these cells for personalized therapy, we reprogrammed PCs into induced pluripotent stem cells (iPSCs) using episomal vectors. Reprogrammed cells gradually lost their fusiform shape, acquired the epithelial cell morphology and formed colonies. Furthermore, reprogrammed cells successfully expressed the pluripotency markers OCT4, Nanog, SSEA-4, and ß-catenin, an early reprogramming marker. CONCLUSION: The accessibility and abundance of human fat supports the application of adipose derived PCs as a novel and promising source of cell therapy and regenerative medicine.


Assuntos
Tecido Adiposo/citologia , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Pericitos/citologia , 5'-Nucleotidase/metabolismo , Actinas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/ultraestrutura , Linhagem da Célula , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Desmina/metabolismo , Endoglina/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Musculares/citologia , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteogênese/genética , Pericitos/metabolismo , Pericitos/ultraestrutura , Antígenos Embrionários Estágio-Específicos/metabolismo , beta Catenina/metabolismo
16.
Nat Commun ; 11(1): 1545, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210226

RESUMO

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.


Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular/fisiologia , Senescência Celular/fisiologia , RNA Mensageiro/metabolismo , Rejuvenescimento/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Animais , Células Cultivadas , Condrócitos , Metilação de DNA/fisiologia , Células Endoteliais , Epigênese Genética/fisiologia , Feminino , Fibroblastos , Perfilação da Expressão Gênica , Humanos , Microscopia Intravital , Masculino , Camundongos , Pessoa de Meia-Idade , Células Musculares , Cultura Primária de Células , Células-Tronco , Adulto Jovem
17.
Nat Commun ; 11(1): 1507, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198345

RESUMO

Tumor cells often reprogram their metabolism for rapid proliferation. The roles of long noncoding RNAs (lncRNAs) in metabolism remodeling and the underlying mechanisms remain elusive. Through screening, we found that the lncRNA Actin Gamma 1 Pseudogene (AGPG) is required for increased glycolysis activity and cell proliferation in esophageal squamous cell carcinoma (ESCC). Mechanistically, AGPG binds to and stabilizes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). By preventing APC/C-mediated ubiquitination, AGPG protects PFKFB3 from proteasomal degradation, leading to the accumulation of PFKFB3 in cancer cells, which subsequently activates glycolytic flux and promotes cell cycle progression. AGPG is also a transcriptional target of p53; loss or mutation of TP53 triggers the marked upregulation of AGPG. Notably, inhibiting AGPG dramatically impaired tumor growth in patient-derived xenograft (PDX) models. Clinically, AGPG is highly expressed in many cancers, and high AGPG expression levels are correlated with poor prognosis, suggesting that AGPG is a potential biomarker and cancer therapeutic target.


Assuntos
Reprogramação Celular/fisiologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Glicólise , Fosfofrutoquinase-2/metabolismo , Pseudogenes/fisiologia , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pseudogenes/genética , RNA Longo não Codificante/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Sci Rep ; 10(1): 3939, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127560

RESUMO

Although human induced pluripotent stem cell (hiPSC) lines are karyotypically normal, they retain the potential for mutation in the genome. Accordingly, intensive and relevant quality controls for clinical-grade hiPSCs remain imperative. As a conceptual approach, we performed RNA-seq-based broad-range genetic quality tests on GMP-compliant human leucocyte antigen (HLA)-homozygous hiPSCs and their derivatives under postdistribution conditions to investigate whether sequencing data could provide a basis for future quality control. We found differences in the degree of single-nucleotide polymorphism (SNP) occurring in cells cultured at three collaborating institutes. However, the cells cultured at each centre showed similar trends, in which more SNPs occurred in late-passage hiPSCs than in early-passage hiPSCs after differentiation. In eSNP karyotyping analysis, none of the predicted copy number variations (CNVs) were identified, which confirmed the results of SNP chip-based CNV analysis. HLA genotyping analysis revealed that each cell line was homozygous for HLA-A, HLA-B, and DRB1 and heterozygous for HLA-DPB type. Gene expression profiling showed a similar differentiation ability of early- and late-passage hiPSCs into cardiomyocyte-like, hepatic-like, and neuronal cell types. However, time-course analysis identified five clusters showing different patterns of gene expression, which were mainly related to the immune response. In conclusion, RNA-seq analysis appears to offer an informative genetic quality testing approach for such cell types and allows the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Variações do Número de Cópias de DNA/genética , Genótipo , Teste de Histocompatibilidade , Homozigoto , Humanos , Cariotipagem , RNA-Seq , Transcriptoma/genética
19.
Nat Mater ; 19(7): 797-806, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32066931

RESUMO

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)-Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK-Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell's environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies.


Assuntos
Reprogramação Celular/fisiologia , Matriz Extracelular/fisiologia , Oncogenes/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Microscopia/métodos , Oncogenes/genética , Pâncreas/citologia , Análise de Sequência de RNA
20.
Am J Physiol Lung Cell Mol Physiol ; 318(4): L671-L683, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32073882

RESUMO

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.


Assuntos
Furões/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Feminino , Fibroblastos/citologia , Humanos , Masculino
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