RESUMO
TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.
Assuntos
Antineoplásicos , Osteossarcoma , Humanos , Animais , Camundongos , Proteínas Hedgehog/metabolismo , Ligantes , Transdução de Sinais , Antineoplásicos/farmacologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Cílios/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.
Assuntos
Cisteína , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Cisteína/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Modelos Animais de Doenças , Linhagem Celular Tumoral , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.
Assuntos
Fosfatidilinositol 3-Quinases , Espermatogênese , Diferenciação Celular/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espermatogênese/genética , Espermatogônias , Células-Tronco/metabolismo , Testículo/metabolismoRESUMO
Atypical teratoid rhabdoid tumour (ATRT) is a rare but highly aggressive undifferentiated solid tumour arising in the central nervous system and predominantly affecting infants and young children. ATRT is exclusively characterized by the inactivation of SMARCB1, a member of the SWI/SNF chromatin remodelling complex that is essential for the regulation of large sets of genes required for normal development and differentiation. Histone deacetylase inhibitors (HDACi) are a promising anticancer therapy and are able to mimic the normal acetylation functions of SMARCB1 in SMARCB1-deficient cells and drive multilineage differentiation in extracranial rhabdoid tumours. However, the potential efficacy of HDACi in ATRT is unknown. Here, we show that human ATRT cells are highly responsive to the HDACi panobinostat and that sustained treatment leads to growth arrest, increased cell senescence, decreased clonogenicity and induction of a neurogenesis gene-expression profile. Furthermore, in an orthotopic ATRT xenograft model, continuous panobinostat treatment inhibits tumour growth, increases survival and drives neuronal differentiation as shown by the expression of the neuronal marker, TUJ1. Collectively, this preclinical study supports the therapeutic potential of panobinostat-mediated differentiation therapy for ATRT.
RESUMO
We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.
Assuntos
Adenocarcinoma de Pulmão/patologia , Antineoplásicos/farmacologia , Carboplatina/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases , Rad51 Recombinase , Análise de Célula Única , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.
Assuntos
Autofagia , Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Hedgehog/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas de Ligação a Retinoblastoma/genética , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Different cell types possess different copies of mtDNA to support their specific requirements for cellular metabolism. Cell-specific mtDNA copy numbers are established through cell-specific mtDNA replication during cell differentiation. However, cancer cells are trapped in a "pseudo-differentiated" state as they fail to expand mtDNA copy number. Global DNA methylation can regulate this process, as induced DNA demethylation promotes differentiation of cancer cells and expansion of mtDNA copy number. RESULTS: To determine the role that mtDNA methylation plays in regulating mtDNA replication during tumorigenesis, we have characterized the patterns of mtDNA methylation using glioblastoma and osteosarcoma tumor models that have different combinations of mtDNA genotypes and copy number against common nuclear genome backgrounds at different stages of tumor progression. To ensure the reliability of the findings, we have applied a robust experimental pipeline including three approaches, namely whole-mtDNA bisulfite-sequencing with mtDNA-genotype-specific analysis, pyrosequencing, and methylated immunoprecipitation against 5mC and 5hmC. We have determined genotype-specific methylation profiles, which were modulated through tumor progression. Moreover, a strong influence from the nuclear genome was also observed on mtDNA methylation patterns using the same mtDNA genotype under different nuclear genomes. Furthermore, the numbers of mtDNA copy in tumor-initiating cells affected mtDNA methylation levels in late-stage tumors. CONCLUSIONS: Our findings highlight the influences that the nuclear and mitochondrial genomes have in setting mtDNA methylation patterns to regulate mtDNA copy number in tumorigenesis. They have important implications for assessing global DNA methylation patterns in tumorigenesis and the availability of mtDNA template for mtDNA replication.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Encefálicas/genética , Metilação de DNA , DNA Mitocondrial/genética , Glioblastoma/genética , Mitocôndrias/genética , Osteossarcoma/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Animais , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Genótipo , Humanos , Imunoprecipitação , Camundongos , Transplante de Neoplasias , Sequenciamento Completo do GenomaRESUMO
Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.
Assuntos
Ativinas/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Platina/uso terapêutico , Células A549 , Animais , Carboplatina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Folistatina/uso terapêutico , Humanos , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.
Assuntos
Âmnio/citologia , Diferenciação Celular/genética , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Hepatócitos/citologia , Carcinogênese/genética , Sobrevivência Celular , Dosagem de Genes , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Mitocôndrias/metabolismoRESUMO
Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry-based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2-17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res; 77(16); 4279-92. ©2017 AACR.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sarcoma Sinovial/tratamento farmacológico , Sarcoma Sinovial/enzimologia , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Estudos de Coortes , Crizotinibe , Feminino , Humanos , Indazóis , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Fosforilação , Proteômica , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/biossíntese , Sarcoma Sinovial/genética , Transdução de Sinais , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.
Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Células-Tronco Pluripotentes Induzidas/citologia , Trifosfato de Adenosina/biossíntese , Células Cultivadas , Reprogramação Celular/genética , Variações do Número de Cópias de DNA , Metilação de DNA , DNA Polimerase Dirigida por DNA/fisiologia , Expressão Gênica , Biblioteca Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microesferas , Desnaturação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação Oxidativa , Consumo de Oxigênio , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Replication of mitochondrial DNA (mtDNA) is important for ensuring that cells have sufficient mtDNA copy number to meet their specific requirements for the generation of cellular energy through oxidative phosphorylation. A number of transcription and replication factors are required for this process, with a key factor being the nuclear-encoded mtDNA-specific DNA polymerase γ. DNA polymerase γ has a catalytic subunit (POLGA), whose gene has been shown to be DNA methylated at exon 2. This methylation is considered to be one of the key mechanisms that regulate mtDNA copy number. These findings have made it of great importance to establish optimal methods for investigating the effects of DNA methylation on mtDNA replication. Here, we provide methods to determine the extent of DNA methylation at exon 2 of POLGA as well as other gene targets of interest. We also show how mtDNA copy number is assessed and, from these two outputs, define the efficiency of mtDNA replication by calculating the mtDNA-replicative efficiency index.
Assuntos
Metilação de DNA/genética , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Dosagem de Genes/genética , Células Cultivadas , DNA Polimerase gama , Humanos , Imunoprecipitação/métodos , Mitocôndrias/genética , Globinas beta/genéticaRESUMO
The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.
Assuntos
Astrócitos/citologia , Complexo I de Transporte de Elétrons/metabolismo , Deleção de Genes , Doença de Leigh/genética , Neurogênese , Animais , Astrócitos/metabolismo , Linhagem Celular , Complexo I de Transporte de Elétrons/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
BACKGROUND: Intravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs. METHODS: Human AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects. RESULTS: HSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-ß1 (TGF-ß1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen-G1, a hAEC-derived factor, significantly decreased TGF-ß1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL. CONCLUSIONS: Human AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.
Assuntos
Líquido Amniótico/citologia , Colágeno/biossíntese , Meios de Cultivo Condicionados/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Líquido Amniótico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fator de Crescimento Transformador beta/biossínteseRESUMO
BACKGROUND AIMS: Multiple sclerosis (MS) is considered to be a T-cell-mediated disease. Although MS remits with corticosteroid treatment, the disease relapses on discontinuation of therapy. Human amniotic epithelial cells (hAEC) from the placenta are readily accessible in large quantities and have anti-inflammatory properties. Previously we reported that hAEC given near disease onset ameliorated clinical signs and decreased myelin oligodendrocyte glycoprotein (MOG)-specific immune responses in MOG-induced experimental autoimmune encephalomyelitis (EAE), an experimental MS model. METHODS: To examine the therapeutic effect of hAEC in a clinically relevant setting, we first treated MOG peptide-induced EAE mice with a corticosteroid, prednisolone, in drinking water to induce remission. hAEC were then infused intravenously into the remitted mice. Anti-MOG antibodies in serum were detected by enzyme-linked immunoassay. Splenocyte proliferation was assessed by (3)H-thymidine incorporation. Immune cell subpopulations in spleens and lymph nodes and secreted cytokines in splenocyte culture were quantified by flow cytometry. Central nervous system histology was examined with the use of hematoxylin and eosin, Luxol fast blue and immunostaining. RESULTS: With cessation of prednisolone treatment, hAEC delayed EAE relapse for 7 days, and, after another 7 days, largely remitted disease in six of eight responder mice. Splenocyte proliferation was suppressed, anti-MOG35-55 antibodies in serum were decreased and interleukin-2 and interleukin-5 production by splenocytes were elevated after hAEC treatment. In the central nervous system, hAEC-treated mice had decreased demyelination and fewer macrophages in the inflammatory infiltrates. hAEC treatment also increased CD4(+)CD25(+)FoxP3(+) regulatory T cells in inguinal lymph nodes. CONCLUSIONS: These data demonstrate that the therapeutic effects of hAEC after corticosteroid treatment in an MS model probably are the consequence of peripheral immunoregulation. We suggest that hAEC may have potential as a cell therapy for remitted MS.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Encefalomielite Autoimune Experimental/terapia , Células Epiteliais/transplante , Esclerose Múltipla Recidivante-Remitente/terapia , Líquido Amniótico/citologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Proliferação de Células/efeitos dos fármacos , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Células Epiteliais/citologia , Feminino , Humanos , Camundongos , Esclerose Múltipla Recidivante-Remitente/induzido quimicamente , Esclerose Múltipla Recidivante-Remitente/patologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/metabolismo , Placenta/citologia , Prednisolona/administração & dosagem , GravidezRESUMO
Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro.
Assuntos
Âmnio/citologia , Células Epiteliais/fisiologia , Hepatócitos/metabolismo , Alginatos/química , Biomarcadores/metabolismo , Cápsulas , Diferenciação Celular , Sobrevivência Celular , Meios de Cultivo Condicionados , Feminino , Expressão Gênica , Ácido Glucurônico/química , Células Hep G2 , Ácidos Hexurônicos/química , Humanos , Placenta/citologia , GravidezRESUMO
Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-ß following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury.
Assuntos
Células-Tronco Adultas/citologia , Lesão Pulmonar/terapia , Células-Tronco Adultas/transplante , Âmnio/citologia , Animais , Bleomicina/farmacologia , Células da Medula Óssea/citologia , Colágeno/metabolismo , Células Epiteliais/citologia , Células Epiteliais/transplante , Feminino , Fibrose , Humanos , Inflamação/metabolismo , Inflamação/terapia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Receptores de Interleucina-1/antagonistas & inibidoresRESUMO
Xenotransplantation of microencapsulated fetal pig islet-like cell clusters (FP ICCs) offers a potential cellular therapy for type 1 diabetes. Although microcapsules prevent direct contact of the host immune system with the xenografted tissue, poor graft survival is still an issue. This study aimed to characterise the nature of the host immune cells present on the engrafted microcapsules and effects on encapsulated FP ICCs that were transplanted into immunocompetent mice. Encapsulated FP ICCs were transplanted into the peritoneal cavity of C57BL/6 mice. Grafts retrieved at days 1, 3, 7, 14 and 21 post-transplantation were analysed for pericapsular fibrotic overgrowth (PFO), cell viability, intragraft porcine gene expression, macrophages, myofibroblasts and intraperitoneal murine cytokines. Graft function was assessed ex vivo by insulin secretion studies. Xenogeneic immune response to encapsulated FP ICCs was associated with enhanced intragraft mRNA expression of porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 seen within the first two weeks post-transplantation. This was associated with the recruitment of host macrophages, infiltration of myofibroblasts and collagen deposition leading to PFO which was evident from day 7 post-transplantation. This was accompanied by a decrease in cell viability and loss of FP ICC architecture. The only pro-inflammatory cytokine detected in the murine peritoneal flushing was TNF-α with levels peaking at day 7 post transplantation. This correlated with the onset of PFO at day 7 implying activated macrophages as its source. The anti-inflammatory cytokines detected were IL-5 and IL-4 with levels peaking at days 1 and 7, respectively. Porcine C-peptide was undetectable at all time points post-transplantation. PFO was absent and murine intraperitoneal cytokines were undetectable when empty microcapsules were transplanted. In conclusion, this study demonstrated that the macrophages are direct effectors of the xenogeneic immune response to encapsulated FP ICCs leading to PFO mediated by a combination of both pro- and anti-inflammatory cytokines.
Assuntos
Antígenos/imunologia , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/imunologia , Macrófagos/imunologia , Transplante Heterotópico , Alginatos/química , Animais , Biomarcadores/metabolismo , Movimento Celular , Citocinas/biossíntese , Citocinas/imunologia , Feto , Ácido Glucurônico/química , Rejeição de Enxerto/patologia , Ácidos Hexurônicos/química , Imunocompetência , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Macrófagos/citologia , Camundongos , Miofibroblastos/citologia , Miofibroblastos/imunologia , Cavidade Peritoneal , Suínos , Transplante HeterólogoRESUMO
Human hepatocyte transplantation is being trialled in lieu of orthotopic liver transplants for patients with acute and chronic liver diseases. Stem cells that can be differentiated into hepatocyte-like cells may replace human hepatocytes that are difficult to source, culture and in critically short supply. Hepatocyte-like cells have been derived from embryonic and adult tissue stem cells using a combination of growth factors and chemical inducers. Stem cells derived from the human placenta have gained interest due to the unlimited supply of placental tissue, minimal issues associated with stem cell retrieval from placental tissue and the large yields of stem cells that can be obtained. Placental stem cells have been characterised and differentiated into hepatocyte-like cells. This review summarises the literature relating to the differentiation of human placental stem cells into hepatocyte-like cells, the characterisation of the differentiated cells, testing the functionality of the hepatocyte-like cells in pre-clinical animal models of liver disease and biomaterials used for culturing and transplantation of these cells into extra-hepatic sites.
Assuntos
Hepatócitos/citologia , Hepatopatias/terapia , Regeneração Hepática , Placenta/citologia , Células-Tronco/citologia , Adulto , Diferenciação Celular , Feminino , Humanos , GravidezRESUMO
Hepatocyte transplantation is being trialled as an alternative to whole organ transplant for patients with acute liver failure and liver specific metabolic diseases. Due to the scarcity of human hepatocytes, hepatocyte-like cells (HLC) generated from stem cells may become a viable alternative to hepatocyte transplantation. Human amniotic epithelial cells (hAEC) from the placenta have stem cell-like properties and can be differentiated into HLC. Naïve hAEC have low immunogenicity and exert immunomodulatory effects that may facilitate allogeneic transplantation. However, whether the immunogenicity and immunomodulatory properties alter with differentiation into HLC are unknown. We further characterized HLC generated from hAEC, examined changes in human leucocyte antigens (HLA) and co-stimulatory molecules and effects exerted by the HLC on human peripheral blood mononuclear cells (PBMC). HLC derived from hAEC expressed proteins found in hepatocytes, had CYP3A4 drug metabolizing enzyme activity and secreted urea. IFN-γ treatment increased HLA Class IA, Class II and co-stimulatory molecule CD40 expression in the HLC. IFN-γ treated HLC stimulated proliferation of PBMC in one-way mixed lymphocyte reactions and were more immunogenic than undifferentiated hAEC. However, the HLC showed immunomodulatory properties and inhibited mitogen induced PBMC proliferation in vitro. PBMC proliferation may have been inhibited by IL-6, TGF-ß1, PGE2 and HLA-G secreted by the HLC. The retention of immunomodulatory properties may enable HLC grafts to survive for longer periods despite the immunogenicity of the HLC.