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1.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
2.
Sci Rep ; 10(1): 8061, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415167

RESUMO

CBX7 is a polycomb group protein, and despite being implicated in many diseases, its role in cell proliferation has been controversial: some groups described its pro-proliferative properties, but others illustrated its inhibitory effects on cell growth. To date, the reason for the divergent observations remains unknown. While several isoforms for CBX7 were reported, no studies investigated whether the divergent roles of CBX7 could be due to distinct functions of CBX7 isoforms. In this study, we newly identified mouse CBX7 transcript variant 1 (mCbx7v1), which is homologous to the human CBX7 gene (hCBX7v1) and is expressed in various mouse organs. We revealed that mCbx7v1 and hCBX7v1 encode a 36 kDa protein (p36CBX7) whereas mCbx7 and hCBX7v3 encode a 22 kDa protein (p22CBX7). This study further demonstrated that p36CBX7 was localized to the nucleus and endogenously expressed in proliferating cells whereas p22CBX7 was localized to the cytoplasm, induced by serum starvation in both human and mouse cells, and inhibited cell proliferation. Together, these data indicate that CBX7 isoforms are localized in different locations in a cell and play differing roles in cell proliferation. This varying function of CBX7 isoforms may help us understand the distinct function of CBX7 in various studies.


Assuntos
Regulação da Expressão Gênica , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores , Proliferação de Células , Imunofluorescência , Humanos , Espaço Intracelular/metabolismo , Camundongos , Complexo Repressor Polycomb 1/química , Isoformas de Proteínas , Transporte Proteico
3.
Circ Res ; 120(5): 848-861, 2017 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-28003219

RESUMO

RATIONALE: Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. OBJECTIVE: We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. METHODS AND RESULTS: We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR+ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. CONCLUSIONS: This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.


Assuntos
Técnicas de Reprogramação Celular/métodos , Células Endoteliais/fisiologia , Fibroblastos/fisiologia , Fatores de Transcrição/biossíntese , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Membro Posterior/irrigação sanguínea , Membro Posterior/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Nus , Neovascularização Fisiológica/fisiologia , Fatores de Transcrição/genética
4.
Exp Neurobiol ; 26(6): 321-328, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29302199

RESUMO

Huntington disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive dysfunction caused by expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin (HTT). In patients, the number of glutamine residues in polyQ tracts are over 35, and it is correlated with age of onset, severity, and disease progression. Expansion of polyQ increases the propensity for HTT protein aggregation, process known to be implicated in neurodegeneration. These pathological aggregates can be transmitted from neuron to another neuron, and this process may explain the pathological spreading of polyQ aggregates. Here, we developed an in vivo model for studying transmission of polyQ aggregates in a highly quantitative manner in real time. HTT exon 1 with expanded polyQ was fused with either N-terminal or C-terminal fragments of Venus fluorescence protein and expressed in pharyngeal muscles and associated neurons, respectively, of C. elegans. Transmission of polyQ proteins was detected using bimolecular fluorescence complementation (BiFC). Mutant polyQ (Q97) was transmitted much more efficiently than wild type polyQ (Q25) and forms numerous inclusion bodies as well. The transmission of Q97 was gradually increased with aging of animal. The animals with polyQ transmission exhibited degenerative phenotypes, such as nerve degeneration, impaired pharyngeal pumping behavior, and reduced life span. The C. elegans model presented here would be a useful in vivo model system for the study of polyQ aggregate propagation and might be applied to the screening of genetic and chemical modifiers of the propagation.

5.
Stem Cell Reports ; 5(6): 1239-1249, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26651608

RESUMO

Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.


Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Ventrículos do Coração/citologia , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/citologia , Potenciais de Ação , Animais , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Camundongos , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/genética
6.
Bioorg Med Chem Lett ; 25(3): 621-5, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25537268

RESUMO

Four new glabretal-type triterpenoids, dictabretols A-D (1-4), were isolated by activity-guided fractionation from the root bark of Dictamnus dasycarpus T. (Rutaceae) using an in vitro antiproliferative assay on T cells using splenocytes. The structures of these compounds were determined by spectroscopic methods, including 2D NMR experiments. Compounds were evaluated for their immunosuppressive activity on T cells and demonstrated inhibition of proliferation of activated T cells, up to IC50 of 1.5µM.


Assuntos
Dictamnus/química , Imunossupressores/química , Triterpenos/química , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Dictamnus/metabolismo , Imunossupressores/isolamento & purificação , Imunossupressores/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Conformação Molecular , Casca de Planta/química , Casca de Planta/metabolismo , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Triterpenos/isolamento & purificação , Triterpenos/farmacologia
7.
ACS Nano ; 8(10): 10815-25, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25210842

RESUMO

A significant barrier to the therapeutic use of stem cells is poor cell retention in vivo. Here, we evaluate the therapeutic potential and long-term engraftment of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated in an injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand Arg-Gly-Asp-Ser (PA-RGDS) in experimental myocardial infarction (MI). We cultured rat neonatal CMs in PA-RGDS for 7 days and found that more than 90% of the CMs survived. Next, we intramyocardially injected mouse CM cell line HL-1 CMs with or without PA-RGDS into uninjured hearts. Histologic examination and flow cytometry analysis of digested heart tissues showed approximately 3-fold higher engraftment in the mice that received CMs with PA-RGDS compared to those without PA-RGDS. We further investigated the therapeutic effects and long-term engraftment of mESC-CMs with PA-RGDS on MI in comparison with PBS control, CM-only, and PA-RGDS only. Echocardiography demonstrated that the CM-only and CM+PA-RGDS groups showed higher cardiac function at week 2 compared to other groups. However, from 3 weeks, higher cardiac function was maintained only in the CM+PA-RGDS group; this was sustained for 12 weeks. Confocal microscopic examination of the cardiac tissues harvested at 14 weeks demonstrated sustained engraftment and integration of mESC-CMs into host myocardium in the CM+PA-RGDS group only. This study for the first time demonstrated that PA-RGDS encapsulation can enhance survival of mESC-derived CMs and improve cardiac function post-MI. This nanomatrix gel-mediated stem cell therapy can be a promising option for treating MI.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Embrionárias/citologia , Coração/fisiopatologia , Miócitos Cardíacos/citologia , Nanoestruturas , Animais , Ratos
8.
Circulation ; 128(17): 1897-909, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-23995537

RESUMO

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.


Assuntos
Transplante de Células/métodos , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/isolamento & purificação , Potenciais de Ação/fisiologia , Animais , Biomarcadores , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citometria de Fluxo/métodos , Humanos , Camundongos , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Nanotecnologia , Conformação de Ácido Nucleico , Células-Tronco Pluripotentes/fisiologia , Sondas RNA/química , Sondas RNA/isolamento & purificação , RNA Mensageiro/química , Troponina I/genética , Troponina T/genética
9.
In Vitro Cell Dev Biol Anim ; 49(5): 360-70, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23605804

RESUMO

Polycomb group (PcG) proteins, which are conserved from invertebrates to mammals, are associated with epigenetic regulation of many cell fates. The activities of PcG proteins are largely associated with modulation of specific immune reactions. However, no study has attempted to explore the role of Phc2, a subunit of polycomb repressive complex 1, on helper T (Th) cell activation. Presently, Phc2 expression was down-regulated in activated Th cells. The ectopic expression of Phc2 in Th cells inhibited Th cell proliferation and secretion of interleukin-2 from Th cells upon antigen-specific activation. Phc2 may act as a negative regulator that inhibits the activity of Th cells.


Assuntos
Ativação Linfocitária/imunologia , Complexo Repressor Polycomb 2/imunologia , Complexo Repressor Polycomb 2/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Primers do DNA/genética , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Auxiliares-Indutores/metabolismo
10.
Anim Biotechnol ; 19(4): 237-42, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18855249

RESUMO

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.


Assuntos
Proteína Kangai-1/genética , Suínos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Feminino , Proteína Kangai-1/biossíntese , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Suínos/imunologia
11.
Vet Immunol Immunopathol ; 120(3-4): 254-9, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17692929

RESUMO

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.


Assuntos
Antígenos CD/genética , Regulação da Expressão Gênica , Suínos/genética , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Clonagem Molecular , DNA Complementar/genética , Imuno-Histoquímica , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/metabolismo , Tetraspanina 28
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