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1.
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.
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
4.
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
5.
Nucleic Acids Res ; 48(16): 9037-9052, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32735658

RESUMO

Epigenetic regulation is important for establishing lineage-specific gene expression during early development. Although signaling pathways have been well-studied for regulation of trophectoderm reprogramming, epigenetic regulation of trophectodermal genes with histone modification dynamics have been poorly understood. Here, we identify that plant homeodomain finger protein 6 (PHF6) is a key epigenetic regulator for activation of trophectodermal genes using RNA-sequencing and ChIP assays. PHF6 acts as an E3 ubiquitin ligase for ubiquitination of H2BK120 (H2BK120ub) via its extended plant homeodomain 1 (PHD1), while the extended PHD2 of PHF6 recognizes acetylation of H2BK12 (H2BK12Ac). Intriguingly, the recognition of H2BK12Ac by PHF6 is important for exerting its E3 ubiquitin ligase activity for H2BK120ub. Together, our data provide evidence that PHF6 is crucial for epigenetic regulation of trophectodermal gene expression by linking H2BK12Ac to H2BK120ub modification.


Assuntos
Cromatina/genética , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/genética , Acetilação , Animais , Reprogramação Celular/genética , Histonas/genética , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Ubiquitinação/genética
6.
Nat Commun ; 11(1): 2717, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483112

RESUMO

Somatic inactivating mutations of ARID1A, a SWI/SNF chromatin remodeling gene, are prevalent in human endometrium-related malignancies. To elucidate the mechanisms underlying how ARID1A deleterious mutation contributes to tumorigenesis, we establish genetically engineered murine models with Arid1a and/or Pten conditional deletion in the endometrium. Transcriptomic analyses on endometrial cancers and precursors derived from these mouse models show a close resemblance to human uterine endometrioid carcinomas. We identify transcriptional networks that are controlled by Arid1a and have an impact on endometrial tumor development. To verify findings from the murine models, we analyze ARID1AWT and ARID1AKO human endometrial epithelial cells. Using a system biology approach and functional studies, we demonstrate that ARID1A-deficiency lead to loss of TGF-ß tumor suppressive function and that inactivation of ARID1A/TGF-ß axis promotes migration and invasion of PTEN-deleted endometrial tumor cells. These findings provide molecular insights into how ARID1A inactivation accelerates endometrial tumor progression and dissemination, the major causes of cancer mortality.


Assuntos
Carcinogênese/genética , Carcinoma Endometrioide/genética , Reprogramação Celular/genética , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Fatores de Transcrição/genética , Animais , Carcinogênese/metabolismo , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Endométrio/citologia , Endométrio/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fatores de Transcrição/metabolismo
7.
Jpn J Clin Oncol ; 50(8): 839-846, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32577751

RESUMO

NCYM is an antisense transcript of MYCN oncogene and promotes tumor progression. NCYM encodes a de novo protein whose open reading frame evolved from noncoding genomic regions in the ancestor of Homininae. Because of its topology, NCYM is always co-amplified with MYCN oncogene, and the mutual regulations between NCYM and MYCN maintain their expressions at high levels in MYCN-amplified tumors. NCYM stabilizes MYCN by inhibiting GSK3ß, whereas MYCN stimulates transcription of both NCYM and MYCN. NCYM mRNA and its noncoding transcript variants MYCNOS have been shown to stimulate MYCN expression via direct binding to MYCN promoter, indicating that both coding and noncoding transcripts of NCYM induce MYCN expression. In contrast to the noncoding functions of NCYM, NCYM protein also promotes calpain-mediated cleavage of c-MYC. The cleaved product called Myc-nick inhibits cell death and promotes cancer cell migration. Furthermore, NCYM-mediated inhibition of GSK3ß results in the stabilization of ß-catenin, which promotes aggressiveness of bladder cancers. These MYCN-independent functions of NCYM showed their clinical significance in MYCN-non-amplified tumors, including adult tumors. This year is the 30th anniversary of the identification of NCYM/MYCNOS gene. On this special occasion, we summarize the current understanding of molecular functions and the clinical significance of NCYM and discuss future directions to achieve therapeutic strategies targeting NCYM.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Reprogramação Celular/genética , Retroalimentação Fisiológica , Humanos , Neuroblastoma/patologia , Resultado do Tratamento
8.
PLoS One ; 15(5): e0232759, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453737

RESUMO

SUMMARY: Reprogramming autologous adult cells to pluripotent cells allows for relatively safe cell replacement therapy. This can be achieved by nuclear transfer, cell fusion, or induced pluripotent stem cell technology However, the epigenetic memory of the cell is considered as a great challenge facing the complete reprograming of cells by these methods. Introducing oocyte-specific factors into differentiated cells may present a promising approach by mimicking cellular reprogramming during fertilization. METHODS: Human bone marrow mesenchymal stromal cells (hBM-MSCs) were cultured with different concentrations of human metaphase II (M II) oocyte extract (0.1, 1, 5, 10, 30 ng/µl). Reprogramming was assessed at various exposure times (1, 4, 7 days). Cells were tested for their proliferation rate, morphological changes, expression of pluripotency markers, expression of mesenchymal to epithelial transition markers, and mitochondrial rejuvenation. (mitochondrial localization, morphological changes, bioenergetics, transmembrane potential, and levels of reactive oxygen species, ROS). RESULTS: Treatment of human BM-MSCs with 10 ng/µl oocyte extract resulted in increased cell proliferation, which was associated with the upregulation of the pluripotency genes OCT-4, NANOG, and SOX-2 and a concomitant downregulation of mesenchymal-specific genes. MSCs exhibited small, immature round mitochondria with few swollen cristae localized proximal to the cell nucleus. This was accompanied by morphological cell changes, a metabolic shift towards oxidative phosphorylation, a high mitochondrial membrane potential, and increased ROS production. CONCLUSION: These data show that treatment with 10 ng/µl human MII-phase oocyte extract induced genetic and mitochondrial reprogramming of human BM-MSCs to a more embryonic phenotype.


Assuntos
Extratos Celulares/farmacologia , Reprogramação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oócitos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fatores de Tempo
9.
Nat Commun ; 11(1): 2285, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385277

RESUMO

Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell transcriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions.


Assuntos
Adenocarcinoma de Pulmão/genética , Reprogramação Celular/genética , Neoplasias Pulmonares/genética , Análise de Sequência de RNA , Análise de Célula Única , Imunidade Adaptativa , Adenocarcinoma de Pulmão/irrigação sanguínea , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Linhagem da Célula , Progressão da Doença , Células Endoteliais/patologia , Humanos , Ligantes , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Células Mieloides/patologia , Miofibroblastos/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Neovascularização Patológica/patologia , Fenótipo , Receptores de Superfície Celular/metabolismo , Células Estromais/metabolismo , Análise de Sobrevida
10.
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
11.
PLoS One ; 15(5): e0232934, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428045

RESUMO

AIMS: Much work has been done to find markers of cancer stem cells (CSCs) that distinguish them from the tumor bulk cells and normal cells. Recent CSC research has applied the induced pluripotent stem cell (iPSC) concept. In this study, we investigated the expression of a panel of iPSC markers in primary colon adenocarcinoma (CA)-derived cell lines. MATERIALS AND METHODS: Expression of iPSC markers by CA-derived primary cell lines was interrogated using immunocytochemistry, western blotting and RT-qPCR. The stem cell function of these cells was then assessed in vitro using differentiation and tumorsphere assays. RESULTS: Expression of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC was more widespread in high-grade CA (HGCA) cell lines than low-grade CA (LGCA) cell lines, as demonstrated by western blotting and RT-qPCR. These cells could be induced to differentiate down the three embryonic lineages. Cells derived from HGCA were more capable of forming tumorspheres than those derived from LGCA. EpCAM sorting revealed that a population enriched for EpCAMHigh cells formed larger tumorspheres than EpCAMLow cells. Pluripotency markers, SSEA4 and TRA-1-60, were co-expressed by a small subpopulation of cells that also co-expressed SOX2 in 75% and OCT4 in 50% of the cell lines. CONCLUSIONS: CA-derived primary cell lines contain tumorsphere-forming cells which express key pluripotency genes and can differentiate down 3 embryonic lineages, suggesting a pluripotent CSC-like phenotype. There appear to be two iPSC-like subpopulations, one with high EpCAM expression which forms larger tumorspheres than another with low EpCAM expression. Furthermore, these cells can be characterized based on iPSC marker expression, as we have previously demonstrated in the original CA tumor tissues.


Assuntos
Adenocarcinoma/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Reprogramação Celular/genética , Colo/citologia , Colo/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Ligação a DNA/análise , Genes Homeobox , Genes myc , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/análise , Proteína Homeobox Nanog/análise , Fator 3 de Transcrição de Octâmero/análise , Cultura Primária de Células , Fatores de Transcrição SOXB1/análise , Fatores de Transcrição/análise
12.
Nat Commun ; 11(1): 2364, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398665

RESUMO

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.


Assuntos
Autorrenovação Celular/genética , Regulação da Expressão Gênica/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Linhagem Celular , Reprogramação Celular/genética , Células-Tronco Embrionárias , Elementos Facilitadores Genéticos/genética , Mutação com Ganho de Função , Técnicas de Silenciamento de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Dedos de Zinco
13.
Genomics Proteomics Bioinformatics ; 18(1): 16-25, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32445708

RESUMO

The generation of induced pluripotent stem cells through somatic cell reprogramming requires a global reorganization of cellular functions. This reorganization occurs in a multi-phased manner and involves a gradual revision of both the epigenome and transcriptome. Recent studies have shown that the large-scale transcriptional changes observed during reprogramming also apply to long non-coding RNAs (lncRNAs), a type of traditionally neglected RNA species that are increasingly viewed as critical regulators of cellular function. Deeper understanding of lncRNAs in reprogramming may not only help to improve this process but also have implications for studying cell plasticity in other contexts, such as development, aging, and cancer. In this review, we summarize the current progress made in profiling and analyzing the role of lncRNAs in various phases of somatic cell reprogramming, with emphasis on the re-establishment of the pluripotency gene network and X chromosome reactivation.


Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , RNA Longo não Codificante/fisiologia , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Longo não Codificante/genética , Transcriptoma , Cromossomo X
14.
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
15.
Gene ; 741: 144552, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32165297

RESUMO

Hypoxia, as a form of stress, plays a critical role in oncogenesis, including metabolic reprogramming. Mitochondrial, the centers of energy production, re-balance mitochondria dynamic to maintain cell survival during high levels of environmental stresses. NDRG1 is a hypoxia-inducible protein that is involved in various human cancers, including HCC. However, little is known about whether NDRG1 participants in the quality control of mitochondrial in times of stress. Here, we firstly showed that how NDRG1 exerted its role through mediating mitochondrial dynamic in HCC cells under hypoxia. Initially, we identified that NDRG1 expression varies with oxygen content. NDRG1 silencing notably induced cell apoptosis under hypoxia, while no obviously change of wildtype cells in hypoxia compared with that in normoxia. Further analysis revealed that NDRG1 silencing in HCC cells led to increase of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx, which meant mitochondrial damage were induced. In the analysis of mitochondria, we found that more released cytochrome c located in cytosolic with NDRG1 knockdown in hypoxia, which may be due to mitochondria division. And the following experiment proved that more fragmented mitochondria were presented in NDRG1 silencing cells, as well as destroyed mitochondrial membrane potential with evidence by JC-1 was verified. Moreover, these trends could be reversed by Mdivi1. Further research showed that NDRG1 silencing disrupt hypoxia-enhanced aerobic glycolysis through effectively decreased glucose uptake, lactate output and ECAR value. In sum, we provide the first direct evidence that NDRG1-driven change in mitochondrial dynamics and aerobic glycolysis maintain cells survival in HCC during hypoxia.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Reprogramação Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Apoptose/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Citocromos c/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Humanos , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/genética , Dinâmica Mitocondrial/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Hipóxia Tumoral/genética , Proteína bcl-X/genética
16.
Chem Biol Interact ; 323: 109055, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32171851

RESUMO

Alcoholism synergizes the development of the hepatocellular carcinoma (HCC) in patients infected with hepatitis B or C virus (HBV or HCV). Tumor-initiating stem-like cells (TICs) are refractory to therapy and have expression of stemness transcription factors. Leaky-gut-derived endotoxin stimulates TLR4-NANOG pathway that skews asymmetric cell division and that metabolically reprograms hepatocytes/liver progenitor cells, leading to self-renewal. TICs isolated from mouse HCC models or human HCCs are tumorigenic and have p53 degradation via phosphorylation of the protective protein NUMB and its dissociation from p53 by the oncofetal protein TBC1D15. Furthermore, dysregulation of lncRNA promotes genesis of TICs, leading to HCC development. This review describes roles of cell fate decision, metabolic reprogramming and lncRNA for TIC genesis and liver oncogenesis. This project was supported by NIH grants 1R01AA018857-01, 5R21AA025470, P50AA11999 (Animal Core, Morphology Core, and Pilot Project Program), R24AA012885 (Non-Parenchymal Liver Cell Core) and pilot project funding (5P30DK048522-13).


Assuntos
Linhagem da Célula , Reprogramação Celular/genética , Etanol/efeitos adversos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/genética , Animais , Linhagem da Célula/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Biochem Biophys Res Commun ; 525(3): 563-569, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115145

RESUMO

Supercentenarians (≥110-year-old, SC) are a uniquely informative population not only because they surpass centenarians in age, but because they appear to age more slowly with fewer incidences of chronic age-related disease than centenarians. We reprogramed donor B-lymphoblastoid cell lines (LCL) derived from a 114-year-old (SC), a 43-year-old healthy disease-free control (HDC) and an 8-year-old with a rapid aging disease (Hutchinson-Gilford progeria syndrome (HGPS)) and compared SC-iPSC to HDC-iPSC and HGPS-iPSCs. Reprogramming to pluripotency was confirmed by pluripotency marker expression and differentiation to 3 germ-layers. Each iPSC clone differentiated efficiently to mesenchymal progenitor cells (MPC) as determined by surface marker expression and RNAseq analysis. We identified supercentenarian and HGPS associated gene expression patterns in the differentiated MPC lines that were not evident in the parental iPSC lines. Importantly, telomere length resetting occurred in iPSC from all donors albeit at a lower incidence in supercentenarian iPSCs. These data indicate the potential to use reprogramming to reset both developmental state and cellular age in the "oldest of the old." We anticipate that supercentenarian iPSC and their differentiated derivatives will be valuable tools for studying the underlying mechanisms of extreme longevity and disease resistance.


Assuntos
Senescência Celular , Células-Tronco Pluripotentes Induzidas/citologia , Doadores de Tecidos , Adulto , Idoso de 80 Anos ou mais , Diferenciação Celular/genética , Linhagem Celular , Reprogramação Celular/genética , Criança , Células Clonais , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Homeostase do Telômero , Transcriptoma/genética
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.
Nucleic Acids Res ; 48(7): 3869-3887, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32016422

RESUMO

Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.


Assuntos
Reprogramação Celular/genética , DNA/metabolismo , RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Ligação Proteica , Domínios Proteicos , Processamento de RNA , Fatores de Transcrição SOXB1/química
20.
Nat Commun ; 11(1): 608, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001704

RESUMO

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in ß-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFß and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.


Assuntos
Reprogramação Celular/genética , Genoma , Transdução de Sinais/genética , Proteína 9 Associada à CRISPR/metabolismo , Sinalização do Cálcio , Engenharia Genética , Células HEK293 , Humanos , Imunomodulação , Inflamação/genética , Inflamação/patologia , Insulina/metabolismo , Interleucina-12/metabolismo , Células Jurkat , Ativação Linfocitária/imunologia , Potenciais da Membrana , Linfócitos T/imunologia , Transgenes
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