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1.
APMIS ; 129 Suppl 142: 1-30, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34399444

RESUMO

Over the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient-derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient-specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high-throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole-image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular-like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11-day period. The parental tumours were analysed by next-generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS-mutated parental tumours showed a significantly higher growth rate compared to their wild-type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell-derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine.


Assuntos
Adenocarcinoma/patologia , Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/patologia , Animais , Diferenciação Celular/fisiologia , Corpos Embrioides/patologia , Células Endoteliais/patologia , Humanos , Macrófagos/patologia , Camundongos , Células-Tronco/patologia , Linfócitos T/patologia , Microambiente Tumoral/fisiologia
2.
J Immunol ; 207(4): 1078-1086, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34341172

RESUMO

Emergency granulopoiesis, also known as demand-adapted granulopoiesis, is defined as the response of an organism to systemic bacterial infections, and it results in neutrophil mobilization from reservoir pools and increased myelopoiesis in the bone marrow. Indirect and direct initiating mechanisms of emergency granulopoiesis have been hypothesized. However, the detailed mechanism of hyperactive myelopoiesis in the bone marrow, which leads to granulocyte left shift, remains unknown. In this study, we report that TLR4 is expressed on granulo-monocytic progenitors, as well as mobilized human peripheral blood CD34+ cells, which account for 0.2% of monocytes in peripheral blood, and ∼ 10% in bone marrow. LPS, a component of Gram-negative bacteria that results in a systemic bacterial infection, induces the differentiation of peripheral blood CD34+ cells into myelocytes and monocytes in vitro via the TLR4 signaling pathway. Moreover, CD34+ cells directly responded to LPS stimulation by activating the MAPK and NF-κB signaling pathways, and they produced IL-6 that promotes emergency granulopoiesis by phosphorylating C/EBPα and C/EBPß, and this effect was suppressed by the action of an IL-6 receptor inhibitor. This work supports the finding that TLR is expressed on human hematopoietic stem and progenitor cells, and it provides evidence that human hematopoietic stem and progenitor cells can directly sense pathogens and produce cytokines exerting autocrine and/or paracrine effects, thereby promoting differentiation.


Assuntos
Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Receptor 4 Toll-Like/metabolismo , Adaptação Fisiológica/fisiologia , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Células Precursoras de Granulócitos/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Monócitos/metabolismo , Mielopoese/fisiologia
3.
FASEB J ; 35(9): e21837, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383985

RESUMO

Overwhelming evidence indicates that excessive stimulation of innate immune receptors of the NOD-like receptor (NLR) family causes significant damage to multiple tissues, yet the role of these proteins in bone metabolism is not well known. Here, we studied the interaction between the NLRP3 and NLRC4 inflammasomes in bone homeostasis and disease. We found that loss of NLRP3 or NLRC4 inflammasome attenuated osteoclast differentiation in vitro. At the tissue level, lack of NLRP3, or NLRC4 to a lesser extent, resulted in higher baseline bone mass compared to wild-type (WT) mice, and conferred protection against LPS-induced inflammatory osteolysis. Bone mass accrual in mutant mice correlated with lower serum IL-1ß levels in vivo. Unexpectedly, the phenotype of Nlrp3-deficient mice was reversed upon loss of NLRC4 as bone mass was comparable between WT mice and Nlrp3;Nlrc4 knockout mice. Thus, although bone homeostasis is perturbed to various degrees by the lack of NLRP3 or NLRC4, this tissue appears to function normally upon compound loss of the inflammasomes assembled by these receptors.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Diferenciação Celular/fisiologia , Homeostase/fisiologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/metabolismo , Osteólise/metabolismo
4.
FASEB J ; 35(9): e21799, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339055

RESUMO

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.


Assuntos
Linhagem da Célula/fisiologia , Miofibroblastos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Matriz Extracelular/fisiologia , Humanos , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Fenótipo , Transcrição Genética/fisiologia
5.
FASEB J ; 35(9): e21824, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34370353

RESUMO

Crosstalk between multiple components underlies the formation of mature vessels. Although the players involved in angiogenesis have been identified, mechanisms underlying the crosstalk between them are still unclear. Using the ex vivo aortic ring assay, we set out to dissect the interactions between two key angiogenic signaling pathways, vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGFß), with members of the lysyl oxidase (LOX) family of matrix modifying enzymes. We find an interplay between VEGF, TGFß, and the LOXs is essential for the formation of mature vascular smooth muscle cells (vSMC)-coated vessels. RNA sequencing analysis further identified an interaction with the endothelin-1 pathway. Our work implicates endothelin-1 downstream of TGFß in vascular maturation and demonstrate the complexity of processes involved in generating vSMC-coated vessels.


Assuntos
Endotelina-1/metabolismo , Neovascularização Patológica/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
FASEB J ; 35(9): e21816, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34396583

RESUMO

Proper physiological function of mammalian airways requires the differentiation of basal stem cells into secretory or multiciliated cells, among others. In addition, the self-renewal ability of these basal stem cells is crucial for developing a quick response to toxic agents in order to re-establish the epithelial barrier function of the airways. Although these epithelial missions are vital, little is known about those mechanism controlling airway epithelial regeneration in health and disease. p53 has been recently proposed as the guardian of homeostasis, promoting differentiation programs, and antagonizing a de-differentiation program. Here, we exploit mouse and human tracheal epithelial cell culture models to study the role of MDM2-p53 signaling in self-renewal and differentiation in the airway epithelium. We show that p53 protein regulation by MDM2 is crucial for basal stem cell differentiation and to keep proper cell proliferation. Therefore, we suggest that MDM2/p53 interaction modulation is a potential target to control regeneration of the mammalian airway epithelia without massively affecting the epithelium integrity and differentiation potential.


Assuntos
Diferenciação Celular/fisiologia , Epitélio/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Mucosa Respiratória/metabolismo , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Feminino , Homeostase/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Traqueia/metabolismo
7.
Immunity ; 54(7): 1511-1526.e8, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260887

RESUMO

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.


Assuntos
Homeostase/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Apoptose/fisiologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Chlorocebus aethiops , Feminino , Humanos , Pulmão/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Metástase Neoplásica/patologia , Proteômica/métodos , Transdução de Sinais/fisiologia
8.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34279592

RESUMO

Despite striking parallels between the fields of developmental biology and adult tissue homeostasis, these are disconnected in contemporary research. Although development describes tissue generation and homeostasis describes tissue maintenance, it is the balance between stem cell proliferation and differentiation that coordinates both processes. Upstream signalling regulates this balance to achieve the required outcome at the population level. Both development and homeostasis require tight regulation of stem cells at the single-cell level and establishment of patterns at the tissue-wide level. Here, we emphasize that the general principles of embryonic development and tissue homeostasis are similar, and argue that interactions between these disciplines will be beneficial for both research fields.


Assuntos
Desenvolvimento Embrionário , Homeostase , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Drosophila , Humanos , Modelos Biológicos , Transdução de Sinais , Células-Tronco
9.
Aging (Albany NY) ; 13(13): 17190-17201, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34229300

RESUMO

Emerging evidence proves that exosomes contain specific microRNAs(miRNAs) contribute to osteogenic differentiation of bone marrow stem cells (BMSCs). However, the role and mechanism of bone marrow stem cells (BMSCs)-derived exosomes overexpressing miR-424-5p in osteoblasts remains unclear. Firstly, the BMSCs-derived exosomes were isolated, and identified by Western blot with the exosome surface markers CD9, CD81 and CD63. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the level of miR-424-5p in exosomes, and western blot was implemented to verify the WIF1/Wnt/ß-catenin expression. The binding association between miR-424-5p and WIF1 was determined by the dual-luciferase reporter gene assay. Functional enhancement experiments were adopted to determine the role of exosome-carried miR-424-5p and WIF1/Wnt/ß-catenin in osteogenic differentiation. ALP staining was adopted, and levels of RUNX2, OCN, and OPN were monitored using qRT-PCR to determine osteogenic differentiation. As a result, In vivo experiments showed that RUNX2, OCN and OPN levels decreased and the ALP activity was dampened after miR-424-5p overexpression in exosomes. Besides, exosomes overexpressing miR-424-5p attenuated osteogenic development via WIF1/Wnt/ß-catenin. Our findings may bring evidence for miR-424-5p as a new biomarker for the treatment of osteoporosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Exossomos/metabolismo , MicroRNAs/genética , Osteoblastos/metabolismo , Osteogênese/genética , Células-Tronco/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Osteocalcina/genética
10.
Aging (Albany NY) ; 13(13): 16957-16973, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253690

RESUMO

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.


Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Miofibroblastos/fisiologia , Telomerase/fisiologia , Actinas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Telomerase/biossíntese , Telomerase/genética
11.
Anticancer Res ; 41(7): 3567-3572, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230152

RESUMO

BACKGROUND/AIM: Medullary carcinoma (MC) of the colon is a rare subtype of colorectal adenocarcinoma (CRC) with unique histomorphology and frequent mismatch repair (MMR) deficiency. MC with exclusive squamous differentiation has not been reported. We report an unusual case of MC with squamous differentiation and tested this differentiation potential in other MMR-deficient CRC cases. CASE REPORT: A 68-year-old woman presented with a large ascending colon mass and biopsy showed squamoid tumor morphology with immunoprofile concerning for squamous cell carcinoma (SCC). She underwent right hemicolectomy. Immunohistochemistry and next-generation sequencing (NGS) were performed for tumor classification. Macroscopically, the tumor was large and locally advanced. It metastasized to the lung without lymph node metastasis. Microscopically, the tumor cells were monotonous with cytological features of both MC and SCC. Immunostains were diffusely positive for p40 and CK5/6, but negative for other lineage markers including CDX2, CK20, and SATB2. The tumor was MMR deficient with loss of MLH1 and PMS2. NGS confirmed BRAF V600E mutation. In comparison, a tissue microarray comprising 64 previously diagnosed MMR deficient CRC was tested for squamous differentiation, and only 1 case showed focal CK5/6 expression, but none was positive for p40. CONCLUSION: MC with exclusive squamous differentiation not only posed significant diagnostic challenges, but also unveiled unrecognized differentiation plasticity in this tumor type.


Assuntos
Carcinoma Medular/patologia , Carcinoma de Células Escamosas/patologia , Diferenciação Celular/fisiologia , Neoplasias do Colo/patologia , Idoso , Carcinoma Medular/genética , Carcinoma de Células Escamosas/genética , Diferenciação Celular/genética , Colo/patologia , Neoplasias do Colo/genética , Feminino , Humanos , Mutação/genética
12.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299331

RESUMO

BACKGROUND: Due to its prominence in the regulation of metabolism and inflammation, adipose tissue is a major target to investigate alterations in insulin action. This hormone activates PI3K/AKT pathway which is essential for glucose homeostasis, cell differentiation, and proliferation in insulin-sensitive tissues, like adipose tissue. The aim of this work was to evaluate the impact of chronic and intermittent high glucose on the expression of biomolecules of insulin signaling pathway during the differentiation and maturation of human visceral preadipocytes. METHODS: Human visceral preadipocytes (HPA-V) cells were treated with high glucose (30 mM)during the proliferation and/or differentiation and/or maturation stage. The level of mRNA (by Real-Time PCR) and protein (by Elisa tests) expression of IRS1, PI3K, PTEN, AKT2, and GLUT4 was examined after each culture stage. Furthermore, we investigated whether miR-29a-3p, miR-143-3p, miR-152-3p, miR-186-5p, miR-370-3p, and miR-374b-5p may affect the expression of biomolecules of the insulin signaling pathway. RESULTS: Both chronic and intermittent hyperglycemia affects insulin signaling in visceral pre/adipocytes by upregulation of analyzed PI3K/AKT pathway molecules. Both mRNA and protein expression level is more dependent on stage-specific events than the length of the period of high glucose exposure. What is more, miRs expression changes seem to be involved in PI3K/AKT expression regulation in response to hyperglycemic stimulation.


Assuntos
Hiperglicemia/metabolismo , Gordura Intra-Abdominal/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Humanos , Hiperglicemia/patologia , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Gordura Intra-Abdominal/citologia , Gordura Intra-Abdominal/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais
13.
Methods Mol Biol ; 2320: 3-7, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302642

RESUMO

The ability to differentiate pluripotent stem cells to cardiomyocyte lineages (PSC-CMs) has opened the door to new disease models and innovative drug and cell therapies for the heart. Nevertheless, further advances in the differentiation protocols are needed to fulfill the promise of PSC-CMs. Obstacles that remain include deriving PSC-CMs with proper electromechanical properties, coalescing them into functional tissue structures, and manipulating the genome to test the impact mutations have on arrhythmias and other heart disorders. This chapter gives a brief consideration of these challenges and outlines current methodologies that offer partial solutions.


Assuntos
Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Cardiopatias/terapia , Humanos , Mutação/genética
14.
Methods Mol Biol ; 2320: 23-27, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302644

RESUMO

Regenerative medicine using human-induced pluripotent stem cells (hiPSCs) is a promising approach to treat heart failure. However, a large number of cells are required to achieve the desired therapeutic effect. The stirring-type suspension culture method allows a large-scale production of hiPSC-derived cardiomyocytes (more than 1 × 108 cells/100 mL), leading to a stable cell supply. Here, we describe a method to scale-up hiPSC-derived cardiomyocyte production with a high differentiation efficiency.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Camundongos
15.
Methods Mol Biol ; 2320: 29-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302645

RESUMO

The human adult heart consists of approximately four billion cardiomyocytes, which do not possess self-renewal abilities. Severe myocardial infarction and dilated cardiomyopathy result in the loss of more than a billion cardiomyocytes. Induced pluripotent stem cells (iPSCs) can differentiate into various types of cells. Due to this ability, these cells could potentially serve as a new resource for cell therapy. Many studies have utilized cardiomyocytes derived from iPSCs for myocardial regeneration therapy. To obtain large number of cardiomyocytes for transplantation, we need to develop effective methods that would allow us to dissociate multiple cardiomyocyte aggregates simultaneously. Here, we describe a method to efficiently dissociate large number of iPSC-derived cardiomyocyte aggregates.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Infarto do Miocárdio/terapia
16.
Methods Mol Biol ; 2320: 35-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302646

RESUMO

The most common method for isolating cells of interest is an antibody method that recognizes cell surface antigens. However, specific surface antigens for many cell types have not been identified. We have developed the microRNA (miRNA)-responsive synthetic mRNA systems (miRNA switches), which isolate target cells based on endogenous miRNA activity. In this chapter, we describe protocols for isolating human pluripotent stem cell (hPSC)-derived cardiomyocytes using miRNA switches with or without cell sorting.


Assuntos
MicroRNAs/genética , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Separação Celular/métodos , Citometria de Fluxo/métodos , Humanos , RNA Mensageiro/genética
17.
Methods Mol Biol ; 2320: 81-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302650

RESUMO

The present protocol describes a method to generate cylindrical engineered cardiac tissues (ECTs) composed of cardiovascular cell lineages induced from human induced pluripotent stem cells (hiPSCs). Cardiomyocytes, endothelial cells, and vascular mural cells induced from hiPSCs are mixed with gel matrix and poured into a tissue mold with posts. By culture day 14, the mixed culture matures into a cylindrical ECT which beats spontaneously and synchronously. Cardiomyocytes align to the long axis of the ECT. The ECTs generated by the present method may be regarded as a surrogate of human myocardium and be served as researches in cardiac regenerative medicine, disease modeling, drug discovery, and cardiac toxicity tests.


Assuntos
Linhagem da Célula/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Engenharia Tecidual/métodos
18.
Methods Mol Biol ; 2320: 91-100, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302651

RESUMO

Induced pluripotent stem cells (iPSCs) have been utilized to study physiological development and also the pathogenesis of heart diseases. iPS-derived cardiomyocytes and engineered cardiac tissues provide a promising capacity for investigating cardiac development and disease modeling. In addition to protocols for cardiac differentiation and 3D cardiac tissue construction, the establishment of protocols for the comprehensive evaluation of the physiological and/or pathophysiological properties for the iPS-derived cells/tissues are indispensable.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Cardiopatias/terapia , Humanos , Fenótipo , Engenharia Tecidual/métodos
19.
Methods Mol Biol ; 2320: 101-110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302652

RESUMO

FluoVolt, a membrane potential dye, has been used to depict the action potentials of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) in order to classify the cardiac cell subtype, evaluate long QT syndrome, and conduct cardiotoxic drug-responsive tests. To apply FluoVolt, users must prepare the hiPSC-CMs, assess the dye loadings, and apply the excitation light. Here we describe the steps to measure action potentials from single hiPSC-CMs and hiPSC-CM monolayers using this dye.


Assuntos
Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Corantes/química , Corpos Embrioides/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Células Cultivadas , Humanos , Síndrome do QT Longo/terapia
20.
Methods Mol Biol ; 2320: 135-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302655

RESUMO

Human iPSC-derived cardiomyocytes (hiPSC-CMs) are expected to be used in regenerative therapies and drug discovery for heart failure. hiPSC-CMs are a mixture of mainly ventricular CMs (VCMs) and also of atrial CMs (ACMs) and pacemaker cells. Here we describe a method to enrich VCM and ACM differentiation and to characterize these subtypes by gene expression analysis using qRT-PCR and by electrophysiological properties using the patch-clamp method. The differentiated VCMs and ACMs highly express VCM and ACM marker genes, respectively. Furthermore, both subtypes show specific properties of action potentials.


Assuntos
Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Átrios do Coração/citologia , Humanos , Técnicas de Patch-Clamp/métodos
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