RESUMO
Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce , Desenvolvimento Muscular , Músculo Esquelético , Regeneração , Animais , Camundongos , Músculo Esquelético/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Desenvolvimento Muscular/genética , Regeneração/genética , Regulação para Cima , Células Satélites de Músculo Esquelético/metabolismo , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Sistema de Sinalização das MAP Quinases , Camundongos Knockout , Diferenciação CelularRESUMO
Chloride intracellular channel protein 4 (CLIC4) has been implicated in different types of cancers, but the role of CLIC4 in the development of gastric cancer (GC) remains unknown. We analyzed the expression of CLIC4 in 102 pairs of gastric adenocarcinomas by western blot and real-time PCR. Our data revealed that the expression of CLIC4 is reduced in GC tumor tissues compared with adjacent normal tissues. The expression levels of CLIC4 correlate inversely with the clinical stage of GC. CLIC4 expression is lowest in MKN45 cells, which have the highest tumorigenic potential and express the highest levels of cancer stem cell markers CD44 and OCT4, compared with N87 and AGS cells. Exogenous overexpression of CLIC4 downregulated the expression of CD44 and OCT4, and inhibited migration, invasion and epithelial-mesenchymal transition (EMT). Moreover, anchorage-independent growth of GC cells was decreased and the cells became more sensitive to 5-fluorouracil and etoposide treatment when CLIC4 was overexpressed. The ability of N87 cells to form tumors in nude mice was enhanced when CLIC4 was silenced. We, for the first time, demonstrate that CLIC4 suppresses tumor growth by inhibiting cancer cell stemness and EMT.
Assuntos
Biomarcadores Tumorais/metabolismo , Canais de Cloreto/antagonistas & inibidores , Transição Epitelial-Mesenquimal , Células-Tronco Neoplásicas/patologia , Neoplasias Gástricas/patologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: DAXX is a transcription repressor that has been implicated in several types of cancers, but its role in the development of gastric cancer remains unknown. METHODS: We analysed the expression of DAXX in 83 pairs of gastric cancer samples, including neoplastic and adjacent tissues, and correlated the expression levels with clinical stages. We also investigated the molecular mechanisms by which DAXX downregulation promotes cancer growth using both in vitro and in vivo models. RESULTS: DAXX was downregulated in advanced gastric cancer samples. The expression of DAXX inversely correlates with that of cancer stem cell markers CD44 and Oct4 in gastric cancer lines. DAXX overexpression in gastric cancer cells inhibited migration, invasion and epithelial- mesenchymal transition (EMT). The inhibition of EMT was achieved through the repression of SNAI3, a key inducer of EMT, by recruiting HDAC-1 into the nucleus. Using a xenograft mouse model, we demonstrated that the MKN45 cells formed smaller tumours when DAXX was overexpressed. Wild-type AGS cells were not able to form tumours in nude mice, but in contrast, formed visible tumours when DAXX was silenced in the cells. CONCLUSION: We for the first time demonstrated that DAXX functions as a tumour suppressor in gastric cancer by inhibiting stem cell growth and EMT.
Assuntos
Proteínas Correpressoras/genética , Transição Epitelial-Mesenquimal/genética , Chaperonas Moleculares/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/genética , Adulto , Idoso , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Neoplasias Gástricas/patologiaRESUMO
FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in hematological malignancies. FLT3 internal tandem duplication (FLT3-ITD) mutations located in juxtamembrane domain (JMD) and tyrosine kinase domain 1 (TKD1) regions account for two-thirds of all FLT3 mutations. The outcome of patients remains unsatisfactory, with low survival rates. It is not yet known whether the different mutations within the FLT3 gene are all associated with patient outcome. In addition, the cause of FLT3-ITD in-frame duplication events remains unknown. Although there are some published studies investigating the FLT3-ITD mutation and its clinical implications in Chinese acute myeloid leukemia (AML) patients, sample sizes tend to be small and detailed molecular profiles of FLT3 mutations are lacking in these studies. In our study, 227 FLT3-ITD sequences were analyzed from 227 Chinese de novo AML patients. ITD were next classified into 3 types based on molecular profiles of insertion DNA sequences: DNA complete duplication (type I), DNA partial duplication (type II) and complete random sequence (type III). From the 154 patients, we confirmed that high ITD allelic ratio (≥.5) and allogeneic stem cell transplant treatment under CR1 are independent prognostic factors. We also presented evidence that ITD integration sites in the hinge region or beta1-sheet region are an unfavorable prognostic factor in adult AML patients with FLT3-ITD mutations. These findings may help to decipher the mechanisms of FLT3-ITD in-frame duplication events and stratify patients when considering different therapeutic combinations.
Assuntos
Leucemia Mieloide Aguda/terapia , Transplante de Células-Tronco/métodos , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , China , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Prognóstico , Domínios Proteicos , Indução de Remissão , Tamanho da Amostra , Análise de Sobrevida , Transplante Homólogo , Adulto JovemRESUMO
RUNX1 is a transcription factor that is not expressed in uninjured muscles, but can be detected in denervated muscles, suggesting a role of RUNX1 in muscle's response to injury. However, the role of RUNX1 in muscle's response to ischemia has not been reported. Our study showed that Runx1 is up regulated in skeletal muscle during ischemia reperfusion induced injury. Over-expression of Runx1 in C2C12â¯cells inhibits myogenic differentiation, but promotes proliferation of myoblasts. Consistent with these findings, we found that Runx1 expression was decreased in differentiated satellite cells. Our results indicate that Runx1 regulates muscle regeneration by promoting proliferation of satellite cells.
Assuntos
Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Isquemia , Músculo Esquelético/fisiologia , Regeneração , Células Satélites de Músculo Esquelético/citologia , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Desenvolvimento Muscular , MioblastosRESUMO
It is known that moderate exercise can prevent the development of cardiovascular diseases, but the exact molecular mechanisms mediating cardioprotective effect of exercise remain unknown. Emerging evidence suggests that exercise has great impact on the biogenesis of exosomes, which have been found in both interstitial fluid and circulation, and play important roles in cellular communication. Exosomes carry functional molecules such as mRNAs, microRNA, and specific proteins, which can be used in the early diagnosis and targeted therapy of a variety of diseases. Our review focus on the current knowledge on exosome production, secretion, uptake and how exercise influence exosome content. We also highlight recent research development in exosome based approach for cardiac repair.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Exercício Físico/fisiologia , Exossomos/genética , MicroRNAs/genética , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Animais , Biomarcadores/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Exossomos/metabolismo , Regulação da Expressão Gênica , HumanosRESUMO
Cardiovascular diseases resulting from ischemic heart diseases remain to be the main causes of heart failure and death despite significant advances in medical treatment. The development of new therapies for heart failure is thus required to improve the outcome in these patients, and this has led to the development of cell-based therapies. Animal studies showed interesting results using various cell types. Some stem cell based therapies have been tested in clinical trials. Although the results were encouraging, challenges remain. Tumorigenic potential, immune rejection, and low engraftment and survival rate of transplant cells have hindered the widespread application of stem cells in the clinic. Fortunately, exosome based therapy could avoid these problems associated with cell therapy. Future research should focus on how various molecules are sorted into exosomes and this information will help to design better exosomes for treatment of cardiovascular diseases. Recent studies suggest that exosome content can vary depending on how cells are challenged. It would be important to find out exactly what types of cellular stress is needed for producing most useful exosomes. Alternatively, specific molecules can be introduced into exosomes by genetic engineering in order to treat specific conditions and to improve efficacy.
Assuntos
Doenças Cardiovasculares/cirurgia , Células-Tronco Embrionárias/transplante , Exossomos/transplante , Miocárdio/patologia , Miócitos Cardíacos/transplante , Regeneração , Transplante de Células-Tronco , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Células-Tronco Embrionárias/metabolismo , Exossomos/genética , Exossomos/metabolismo , Exossomos/patologia , Regulação da Expressão Gênica , Humanos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Recuperação de Função Fisiológica , Transdução de Sinais , Transplante de Células-Tronco/efeitos adversosRESUMO
E-cadherin, CD44v6, and IMP3 expression in partial, complete, and invasive hydatidiform moles (HMs) was evaluated. High E-cadherin expression with low CD44v6 expression was observed in partial, complete, and invasive HMs, as well as in normal placental tissues; and there was no significant difference in E-cadherin and CD44v6 expression among the four groups. However, IMP3 expression was gradually decreased in the order of normal placental tissues, partial HMs, complete HMs, and invasive HMs; wherein, invasive HMs had the lowest level. Low IMP3 expression may serve as a prognostic biomarker for HMs, and IMP3 may play a certain role in HMs progression.
Assuntos
Caderinas/genética , Receptores de Hialuronatos/genética , Mola Hidatiforme/diagnóstico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Hemorragia Uterina/diagnóstico , Adulto , Biomarcadores/metabolismo , Caderinas/metabolismo , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Idade Gestacional , Humanos , Receptores de Hialuronatos/metabolismo , Mola Hidatiforme/classificação , Mola Hidatiforme/genética , Mola Hidatiforme/metabolismo , Pessoa de Meia-Idade , Gravidez , Primeiro Trimestre da Gravidez , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologia , Hemorragia Uterina/genética , Hemorragia Uterina/metabolismo , Hemorragia Uterina/patologiaRESUMO
BACKGROUND: The type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase promotes the survival of an aggressive subtype of T-cell lymphoma by interacting with nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein. NPM-ALK(+) T-cell lymphoma exhibits much higher levels of IGF-IR than normal human T lymphocytes. The mechanisms underlying increased expression of IGF-IR in this lymphoma are not known. We hypothesized that upregulation of IGF-IR could be attributed to previously unrecognized defects that inherently exist in the transcriptional machinery in NPM-ALK(+) T-cell lymphoma. METHODS AND RESULTS: Screening studies showed substantially lower levels of the transcription factors Ikaros isoform 1 (Ik-1) and myeloid zinc finger 1 (MZF1) in NPM-ALK(+) T-cell lymphoma cell lines and primary tumor tissues from patients than in human T lymphocytes. A luciferase assay supported that Ik-1 and MZF1 suppress IGF-IR gene promoter. Furthermore, ChIP assay showed that these transcription factors bind specific sites located within the IGF-IR gene promoter. Forced expression of Ik-1 or MZF1 in the lymphoma cells decreased IGF-IR mRNA and protein. This decrease was associated with downregulation of pIGF-IR, and the phosphorylation of its interacting proteins IRS-1, AKT, and NPM-ALK. In addition, overexpression of Ik-1 and MZF1 decreased the viability, proliferation, migration, and anchorage-independent colony formation of the lymphoma cells. CONCLUSIONS: Our results provide novel evidence that the aberrant decreases in Ik-1 and MZF1 contribute significantly to the pathogenesis of NPM-ALK(+) T-cell lymphoma through the upregulation of IGF-IR expression. These findings could be exploited to devise new strategies to eradicate this lymphoma.
Assuntos
Citocinas/genética , Regulação para Baixo/genética , Fatores de Transcrição Kruppel-Like/genética , Linfoma de Células T/genética , Proteínas Tirosina Quinases/genética , Receptores de Somatomedina/genética , Fatores de Transcrição/genética , Células 3T3 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Fosforilação/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Receptor IGF Tipo 1 , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
The ribosome is a multi-unit complex that translates mRNA into protein. Ribosome biogenesis is the process that generates ribosomes and plays an essential role in cell proliferation, differentiation, apoptosis, development, and transformation. The mTORC1, Myc, and noncoding RNA signaling pathways are the primary mediators that work jointly with RNA polymerases and ribosome proteins to control ribosome biogenesis and protein synthesis. Activation of mTORC1 is required for normal fetal growth and development and tissue regeneration after birth. Myc is implicated in cancer development by enhancing RNA Pol II activity, leading to uncontrolled cancer cell growth. The deregulation of noncoding RNAs such as microRNAs, long noncoding RNAs, and circular RNAs is involved in developing blood, neurodegenerative diseases, and atherosclerosis. We review the similarities and differences between eukaryotic and bacterial ribosomes and the molecular mechanism of ribosome-targeting antibiotics and bacterial resistance. We also review the most recent findings of ribosome dysfunction in COVID-19 and other conditions and discuss the consequences of ribosome frameshifting, ribosome-stalling, and ribosome-collision. We summarize the role of ribosome biogenesis in the development of various diseases. Furthermore, we review the current clinical trials, prospective vaccines for COVID-19, and therapies targeting ribosome biogenesis in cancer, cardiovascular disease, aging, and neurodegenerative disease.
Assuntos
COVID-19 , Neoplasias , Doenças Neurodegenerativas , Humanos , Gravidez , Feminino , Vacinas contra COVID-19/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , COVID-19/metabolismo , Ribossomos/genética , Proteínas Ribossômicas/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , RNA não Traduzido , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
Microplastics (MPs) in sewage pose significant threats to aquatic system. Surface flow wetland (SFW) is a common natural wetland type, and is also used as a cheap and easy-to-build sewage treatment system for small and scattered settlements. However, seasonal variation patterns of MPs in sewage removed by SFW are still limited. Therefore, a field investigation was conducted in an SFW that has been operated for 17 years. The concentration of microplastics in the influent of the SFW (CMPs, in) ranged from 56 ± 6 to 250 ± 14 items L-1. The dominant plastic types were fibers and polyethylene terephthalate (PET). CMPs, in were high in summer and winter, significantly related to the seasonal dressing habits. The removal efficiencies of MPs in SFW were 48.03-92.32 % in different seasons, and the mechanisms of MP removal were different with traditional pollutants. Before flowing out occasionally or by heavy precipitation, MPs were primarily trapped in the SFW and underwent certain oxidation. Simulation experiments demonstrated that 47.5-92.9 % of MPs would be trapped in the SFW, and plants would significantly enhance the trapping capacities. This study sheds light on the seasonal variation characteristics and patterns of MPs in actual sewage, and clarifies the fate of MPs in a long-term operation SFW.
RESUMO
The regenerative capacity of skeletal muscle is dependent on satellite cells. The circadian clock regulates the maintenance and function of satellite cells. Cryptochrome 2 (CRY2) is a critical component of the circadian clock, and its role in skeletal muscle regeneration remains controversial. Using the skeletal muscle lineage and satellite cell-specific CRY2 knockout mice (CRY2scko), we show that the deletion of CRY2 enhances muscle regeneration. Single myofiber analysis revealed that deletion of CRY2 stimulates the proliferation of myoblasts. The differentiation potential of myoblasts was enhanced by the loss of CRY2 evidenced by increased expression of myosin heavy chain (MyHC) and myotube formation in CRY2-/- cells versus CRY2+/+ cells. Immunostaining revealed that the number of mononucleated paired box protein 7 (PAX7+) cells associated with myotubes formed by CRY2-/- cells was increased compared with CRY2+/+ cells, suggesting that more reserve cells were produced in the absence of CRY2. Loss of CRY2 leads to the activation of the ERK1/2 signaling pathway and ETS1, which binds to the promoter of PAX7 to induce its transcription. CRY2 deficient myoblasts survived better in ischemic muscle. Therefore, CRY2 is essential in regulating skeletal muscle repair.
RESUMO
Tumor development relies on the stemness of cancer stem cells, which is regulated by environmental cues. Previous studies have shown that zyxin can inhibit the expression of genes for embryonic stem cell status. In the present study, the expression levels of zyxin protein in cancer tissues and adjacent noncancerous tissues from 73 gastric cancer patients with different clinical stages were analyzed by Western blot. We showed that the relative expression levels of zyxin in gastric cancer tissues (cancer tissues/adjacent tissues) were significantly downregulated in advanced clinical stages. Overexpression of zyxin inhibited the stemness and epithelial-mesenchymal transition (EMT) processes in gastric cancer cells. Zyxin also inhibited the proliferation, migration, and invasion but increased the sensitivity of cancer cells to drugs. Overexpression of zyxin in MKN45 cells inhibited tumor growth in nude mice. We show that the interactions between zyxin and SIRT1 led to the upregulation of SIRT1, reduced acetylation levels of histone H3 K9 and K23, decreased transcription levels of SNAI 1/2, and inhibition of the EMT process. This study demonstrated that zyxin negatively regulates the progression of gastric cancer by inhibiting the stemness of cancer stem cells and EMT. Our findings shed new light on the pathogenesis of gastric cancer.
RESUMO
Epithelial-mesenchymal transition (EMT) generates tumor cells with stem cell properties. The aim of our study was to investigate the effects of adipose tissue-derived stem cells (ASCs) on EMT of cancer cells and to further investigate the mechanisms involved. We demonstrate that conditioned medium from ASCs induces breast cancer cells (4T1) to express mesenchymal markers such as fibronectin, alpha smooth muscle actin and vimentin. Flow cytometry analyses show that ASC-conditioned medium promotes the expansion of CD44high/CD24low cancer stem cells. Soft agar assays using T47D, BT474 and MCF-7 breast cancer cells reveals that ASC conditioned medium promotes the anchorage-independent growth of cancer cells. These effects were inhibited by a neutralizing antibody against platelet-derived growth factor-D (PDGF-D). Furthermore, PDGF-D treated breast cancer cells grow faster in a mouse model, and this effect could be neutralized by a PDGF antibody. In conclusion, our data show that tissue-resident stem cells interact with the cancer microenvironment via PDGF-D, induce EMT in the cancer cells in a paracrine fashion, thereby increasing the number of cancer stem cells and increase tumor growth in a PDGF dependent manner. Our findings shed new light on mechanisms where local tissue-resident stem cells are able to promote the growth of breast cancer cells. Possibly this could open up a novel selective therapeutic strategy targeting EMT pathways and the specific communication between tissue-resident normal stem cell and cancer stem cells, assuming that the blockage of PDGF-D pathways is critical for tumor growth but would not affect normal tissue homeostasis.
Assuntos
Tecido Adiposo/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Linfocinas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Tecido Adiposo/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Linfocinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of this study is to evaluate the role of adipose tissue resident stromal cells on tumor cell invasion. Our data show that a subpopulation of adipose tissue derived stromal cells expressing Nestin, NG2, α-smooth muscle actin and PDGFR-α migrate toward the cancer cells. Microarray analysis revealed the upregulation of IL-8 in the migrated cells. We demonstrated that stromal cell derived IL-8 promote the invasion and the anchorage-independent growth of cancer cells. We conclude that human breast cancer cells attract a subpopulation of stromal cells that secrete IL-8 to promote tumor cell invasion in a paracrine fashion.
Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Neoplasias da Mama/imunologia , Interleucina-8/imunologia , Invasividade Neoplásica/imunologia , Células Estromais/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Interleucina-8/genética , Invasividade Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente Tumoral , Regulação para CimaRESUMO
Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell-based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue-derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant DiI and then treated with fusion-inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte-like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole-cell voltage-clamp analysis demonstrated action potentials in beating fused cells. RT-PCR analysis using rat- or human-specific myosin heavy chain primers revealed that the myosin heavy-chain expression in fused cells was derived from rat cardiomyocytes. Real-time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Fusão Celular , Células Cultivadas , Humanos , Mitomicina/farmacologia , Inibidores da Síntese de Ácido Nucleico , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND INFORMATION: Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony-forming ability and differentiation potential of four human cell types in vitro: commercially available skin-derived fibroblasts [hSDFs (human skin-derived fibroblasts)], adipose tissue-derived stem cells [hASCs (human adipose tissue-derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. RESULTS: hSDFs, hASCs and WI38 exhibited a similar spindle-like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell-associated gene expressions by performing real-time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5-fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell-derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. CONCLUSIONS: These findings suggest that (i) so-called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony-forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Adulto , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Fibroblastos/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , FenótipoRESUMO
Skeletal muscle regeneration relies on satellite cells that can proliferate, differentiate, and form new myofibers upon injury. Emerging evidence suggests that misregulation of satellite cell fate and function influences the severity of Duchenne muscular dystrophy (DMD). The transcription factor Pax7 determines the myogenic identity and maintenance of the pool of satellite cells. The circadian clock regulates satellite cell proliferation and self-renewal. Here, we show that the CLOCK-interacting protein Circadian (CIPC) a negative-feedback regulator of the circadian clock, is up-regulated during myoblast differentiation. Specific deletion of Cipc in satellite cells alleviates myopathy, improves muscle function, and reduces fibrosis in mdx mice. Cipc deficiency leads to activation of the ERK1/2 and JNK1/2 signaling pathways, which activates the transcription factor SP1 to trigger the transcription of Pax7 and MyoD. Therefore, CIPC is a negative regulator of satellite cell function, and loss of Cipc in satellite cells promotes muscle regeneration.