Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
Sci Rep ; 8(1): 8072, 2018 05 23.
Article in English | MEDLINE | ID: mdl-29795287

ABSTRACT

MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.


Subject(s)
Biomarkers/metabolism , Cell Lineage/genetics , Gene Expression Regulation , Mesoderm/metabolism , MicroRNAs/genetics , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/metabolism , Cell Differentiation , Cells, Cultured , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Mesoderm/cytology , Myocytes, Cardiac/cytology , Pluripotent Stem Cells/cytology
2.
J Cardiovasc Transl Res ; 11(1): 1-13, 2018 02.
Article in English | MEDLINE | ID: mdl-29019149

ABSTRACT

Leukemia inhibitory factor (LIF) is a growth factor with pleiotropic biological functions. It has been reported that LIF acts at different stages during mesoderm development. Also, it has been shown that LIF has a cytoprotective effect on neonatal murine cardiomyocytes (CMs) in culture, but little is known about the role of LIF during human cardiogenesis. Thus, we analyzed the effects of LIF on human pluripotent stem cells (PSC) undergoing cardiac differentiation. We first showed that LIF is expressed in the human heart during early development. We found that the addition of LIF within a precise time window during the in vitro differentiation process significantly increased CMs viability. This finding was associated to a decrease in the expression of pro-apoptotic protein Bax, which coincides with a reduction of the apoptotic rate. Therefore, the addition of LIF may represent a promising strategy for increasing CMs survival derived from PSCs.


Subject(s)
Cell Differentiation , Human Embryonic Stem Cells/drug effects , Leukemia Inhibitory Factor/pharmacology , Myocytes, Cardiac/drug effects , Pluripotent Stem Cells/drug effects , Apoptosis/drug effects , Cell Line , Cell Survival/drug effects , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/pathology , Humans , Leukemia Inhibitory Factor/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Phenotype , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/pathology , Time Factors , bcl-2-Associated X Protein/metabolism
3.
J Cardiovasc Transl Res ; 11(1): 14, 2018 02.
Article in English | MEDLINE | ID: mdl-29139097

ABSTRACT

Please note that Carolina Blüguermann's surname was misspelled (as Blugüermann) in this article as originally published.

4.
Stem Cell Res ; 16(2): 300-3, 2016 Mar.
Article in English | MEDLINE | ID: mdl-27345989

ABSTRACT

Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.


Subject(s)
Fibroblasts/cytology , Foreskin/cytology , Induced Pluripotent Stem Cells/cytology , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Comparative Genomic Hybridization , DNA Methylation , Humans , Induced Pluripotent Stem Cells/metabolism , Karyotype , Kruppel-Like Factor 4 , Male , Microscopy, Fluorescence , Octamer Transcription Factor-3/genetics , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism
5.
Stem Cell Reports ; 1(6): 575-89, 2013.
Article in English | MEDLINE | ID: mdl-24371811

ABSTRACT

Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166(low/neg)CD146(low/neg)CD73(+)CD44(low)BMPR1B(+)) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5-6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166(low/neg)BMPR1B(+) putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering.


Subject(s)
Cell Culture Techniques , Cell Differentiation/genetics , Chondrocytes/cytology , Chondrogenesis/genetics , Pluripotent Stem Cells/cytology , Biomarkers/metabolism , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cell Lineage , Cells, Cultured , Chondrocytes/metabolism , Flow Cytometry , Gene Expression Regulation, Developmental , Humans , Laser Capture Microdissection , Oligonucleotide Array Sequence Analysis , Pluripotent Stem Cells/metabolism , Tissue Engineering
6.
PLoS One ; 8(7): e70267, 2013.
Article in English | MEDLINE | ID: mdl-23936178

ABSTRACT

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin(TM) and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin(TM) 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin(TM) 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin(TM) and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.


Subject(s)
Anti-Bacterial Agents/pharmacology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Anti-Bacterial Agents/toxicity , Apoptosis/drug effects , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Ciprofloxacin/pharmacology , Ciprofloxacin/toxicity , Embryonic Stem Cells/cytology , Embryonic Stem Cells/microbiology , Humans , Karyotype , Macrolides/pharmacology , Macrolides/toxicity , Mycoplasma/drug effects
7.
Cell Biochem Funct ; 31(4): 271-80, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23315627

ABSTRACT

Mesenchymal stem or stromal cells (MSCs) were initially isolated from the bone marrow and received their name on the basis of their ability to differentiate into multiple lineages such as bone, cartilage, fat and muscle. However, more recent studies suggest that MSCs residing in perivascular compartments of the small and large blood vessels play a regulatory function supporting physiologic and pathologic responses of parenchymal cells, which define the functional representation of an organ or tissue. MSCs secrete or express factors that reach neighbouring parenchymal cells via either a paracrine effect or a direct cell-to-cell interaction promoting functional activity, survival and proliferation of the parenchymal cells. Previous concept of 'epithelial-stromal' interactions can now be widened. Given that MSC can also support hematopoietic, neuronal and other non-epithelial parenchymal lineages, terms 'parenchymal-stromal' or 'parenchymal-mesenchymal' interactions may better describe the supportive or 'trophic' functions of MSC. Importantly, in many cases, MSCs specifically provide supportive microenvironment for the most primitive stem or progenitor populations and therefore can play a role as 'stem/progenitor niche' forming cells. So far, regulatory roles of MSCs have been reported in many tissues. In this review article, we summarize the latest studies that focused on the supportive function of MSC. This thread of research leads to a new perspective on the interactions between parenchymal and mesenchymal cells and justifies a principally novel approach for regenerative medicine based on co-application of MSC and parenchymal cell for the most efficient tissue repair.


Subject(s)
Cell Communication , Mesenchymal Stem Cells/cytology , Animals , Humans , Neoplasms/physiopathology , Regeneration
8.
J Androl ; 33(6): 1360-70, 2012.
Article in English | MEDLINE | ID: mdl-22653965

ABSTRACT

Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved in fertilization. With the aim of examining the relevance of CRISP2 for fertility and its potential use as a target for contraception, in the present work, male and female rats were immunized with recombinant CRISP2 coupled to maltose-binding protein (MBP) and evaluated for their subsequent fertility. As controls, animals were injected with either MBP or recombinant CRISP1. Enzyme-linked immunosorbent assay of sera collected at different intervals after immunization indicated that CRISP2 immunization raised specific antibodies in both sexes, with levels that increased as a function of time. Western blot studies revealed that anti-CRISP2 sera were capable of recognizing CRISP2 in testicular, epididymal, and sperm extracts, whereas histological studies showed no evidence of autoimmune orchitis or epididymitis. Indirect immunofluorescence experiments revealed the ability of anti-CRISP2 sera to recognize the native sperm protein in fresh, capacitated, and ionophore-induced acrosome-reacted cells. Moreover, anti-CRISP2 sera significantly inhibited the sperm ability to penetrate zona-free eggs, confirming the role of CRISP2 in rat gamete fusion. In spite of the presence of circulating anti-CRISP2 antibodies capable of inhibiting the sperm fertilizing ability, mating studies revealed no effects of CRISP2 immunization on male or female fertility, in contrast to the significant inhibition observed in both sexes in animals injected with CRISP1. Together, these observations indicated the immunogenic properties of testicular CRISP2 but do not support CRISP2 as a target for immunocontraception or as a molecule responsible for generating autoimmune orchitis or immunoinfertility.


Subject(s)
Fertilization/physiology , Glycoproteins/immunology , Animals , Cell Adhesion Molecules , Contraception, Immunologic , Female , Male , Rats , Rats, Sprague-Dawley , Rats, Wistar , Recombinant Proteins/immunology , Sperm Capacitation
9.
Apoptosis ; 17(2): 132-42, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22012335

ABSTRACT

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X(L) and Bax protein levels were observed upon CVB3 infection during 5-24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.


Subject(s)
Apoptosis , Coxsackievirus Infections/metabolism , Embryonic Stem Cells/metabolism , Enterovirus/metabolism , Caspases/genetics , Caspases/metabolism , Cell Line , Cell Survival , Chromatin/metabolism , Coxsackievirus Infections/virology , DNA Fragmentation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/virology , Enterovirus/pathogenicity , Gene Expression , HeLa Cells , Humans , Signal Transduction , bcl-Associated Death Protein/genetics , bcl-Associated Death Protein/metabolism
10.
Biochem Biophys Res Commun ; 410(2): 252-7, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21651896

ABSTRACT

Induced pluripotent stem cells (iPSCs) are a promising type of stem cells, comparable to embryonic stem cells (ESCs) in terms of self-renew and pluripotency, generated by reprogramming somatic cells. These cells are an attractive approach to supply patient-specific pluripotent cells, for producing in vitro models of disease, drug discovery, toxicology and potentially treating degenerative disease circumventing immune rejection. In spite of the great advance since iPSCs' establishment, their obtention and propagation is an increasing area of great interest. In a recent work, we have shown that the conditioned medium from a bovine granulosa cell line (BGC-CM) is able to preserve the basic properties of mESCs. Therefore, based on our previous results and the reported resemblance between iPSCs and ESCs, we hypothesized that BGC-CM could provide a favorable context to culturing iPSCs. In this work, we have reprogrammed mouse embryonic fibroblasts obtaining iPSC lines, and showed that they can be propagated in BGC-CM while maintaining self-renewal and pluripotency, evidenced by expression of specific gene markers and capability of in vitro and in vivo differentiation to cell types from the three germ layers. We believe that these findings may provide a novel context to propagate iPSCs to study the molecular mechanisms involved in self-renewal and pluripotency.


Subject(s)
Culture Media, Conditioned/pharmacology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/physiology , Regeneration , Animals , Cattle , Cell Culture Techniques , Cell Line , Culture Media, Conditioned/metabolism , Female , Granulosa Cells/metabolism , Granulosa Cells/physiology , Induced Pluripotent Stem Cells/metabolism , Mice , Octamer Transcription Factor-3/metabolism
11.
Biochem Biophys Res Commun ; 410(4): 816-22, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21703227

ABSTRACT

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are a promising source of cells for regenerative medicine because of their potential of self renew and differentiation. Multiple evidences highlight the relationship of chromatin remodeling with stem cell properties, differentiation programs and reprogramming for iPSC obtention. With the purpose of finding chromatin modifying factors relevant to these processes, and based on ChIP on chip studies, we selected several genes that could be modulated by Oct4, Sox2 and Nanog, critical transcription factors in stem cells, and studied their expression profile along the differentiation in mouse and human ESCs, and in mouse iPSCs. In this work, we analyzed the expression of Gcn5l2, GTF3C3, TAF15, ATF7IP, Myst2, HDAC2, HDAC3, HDAC5, HDAC10, SUV39H2, Jarid2, and Bmi-1. We found some genes from different functional groups that were highly modulated, suggesting that they could be relevant both in the undifferentiated state and during differentiation. These findings could contribute to the comprehension of molecular mechanisms involved in pluripotency, early differentiation and reprogramming. We believe that a deeper knowledge of the epigenetic regulation of ESC will allow improving somatic cell reprogramming for iPSC obtention and differentiation protocols optimization.


Subject(s)
Chromatin/genetics , Embryonic Stem Cells/metabolism , Gene Expression Regulation , Induced Pluripotent Stem Cells/metabolism , Animals , Cell Line , Chromatin Immunoprecipitation , Humans , Mice , Transcription Factors/genetics , Transcription Factors/metabolism
12.
Stem Cell Res ; 6(1): 13-22, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20951660

ABSTRACT

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 105-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iß significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.


Subject(s)
Coxsackievirus Infections/virology , Embryoid Bodies/virology , Embryonic Stem Cells/virology , Enterovirus B, Human/physiology , Interferon-beta/pharmacology , Cell Line , Coxsackievirus Infections/genetics , Coxsackievirus Infections/metabolism , Embryoid Bodies/drug effects , Embryonic Stem Cells/drug effects , Enterovirus B, Human/drug effects , Humans , Receptors, Virus/genetics , Receptors, Virus/metabolism , Virus Replication/drug effects
SELECTION OF CITATIONS
SEARCH DETAIL
...