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1.
Cells ; 9(9)2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32825270

RESUMO

Growth factors are important regulators during organ development. For many vertebrates (but not humans) it is known how they contribute to the formation and expansion of PDX1-positive cells during pancreas organogenesis. Here, the effects of the fibroblast growth factors FGF2, FGF7, FGF10, and epidermal growth factor (EGF) on pancreas development in humans were assessed by using human pluripotent stem cells (hPSCs). During this, FGF2 was identified as a potent anti-pancreatic factor whereas FGF7, FGF10, and EGF increased the cell mass while retaining PDX1-positivity. FGF2 increased the expression of the anti-pancreatic factor sonic hedgehog (SHH) while suppressing PDX1 in a dose-dependent manner. Differentiating cells secreted SHH to the medium and we interrogated the cells' secretome during differentiation to globally examine the composition of secreted signaling factors. Members of the TGF-beta-, Wnt-, and FGF-pathways were detected. FGF17 showed a suppressive anti-pancreatic effect comparable to FGF2. By inhibition of specific branches of FGF-receptor signaling, we allocated the SHH-induction by FGF2 to MEK/ERK-signaling and the anti-pancreatic effect of FGF2 to the receptor variant FGFR1c or 3c. Altogether, we report findings on the paracrine activity of differentiating hPSCs during generation of pancreatic progenitors. These observations suggest a different role for FGF2 in humans compared to animal models of pancreas organogenesis.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Pâncreas/fisiopatologia , Diferenciação Celular , Linhagem da Célula , Humanos
2.
Sci Rep ; 9(1): 996, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700818

RESUMO

In vitro differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) represents a key step towards somatic cells of lung, liver and pancreas. For future clinical applications, mass production of differentiated cells at chemically defined conditions and free of xenogeneic substances is envisioned. In this study we adapted our previously published two-dimensional (2D) DE induction protocol to three-dimensional (3D) static suspension culture in the absence of the xenogeneic extracellular matrix Matrigel. Next, fetal calf serum and bovine serum albumin present in the standard medium were replaced by a custom-made and xeno-free B-27. This yielded in a chemically defined and xenogeneic-free 3D culture protocol for differentiation of hPSCs into DE at efficiencies similar to standard 2D conditions. This novel protocol successfully worked with different hPSC lines including hESCs and hiPSCs maintained in two different stem cell media prior to differentiation. DE cells obtained by our novel BSA-free 3D protocol could be further differentiated into PDX1- or NKX6.1-expressing pancreatic progenitor cells. Notably, upon DE differentiation, we also identified a CXCR4+/NCAM+/EpCAMlow cell population with reduced DE marker gene expression. These CXCR4+/NCAM+/EpCAMlow cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Endoderma/citologia , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Células Cultivadas , Meios de Cultura , Humanos , Pâncreas/citologia , Soroalbumina Bovina/metabolismo
3.
Stem Cell Reports ; 9(5): 1588-1603, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141233

RESUMO

Pluripotent stem cells hold great promise for regenerative medicine since they can differentiate into all somatic cells. MicroRNAs (miRNAs) could be important for the regulation of these cell-fate decisions. Profiling of miRNAs revealed 19 differentially expressed miRNAs in the endoderm and 29 in the mesoderm when analyzing FACS-purified cells derived from human embryonic stem cells. The mesodermal-enriched miR-483-3p was identified as an important regulator for the generation of mesodermal PDGFRA+ paraxial cells. Repression of its target PGAM1 significantly increased the number of PDGFRA+ cells. Furthermore, miR-483-3p, miR-199a-3p, and miR-214-3p might also have functions for the mesodermal progenitors. The endoderm-specific miR-489-3p and miR-1263 accelerated and increased endoderm differentiation upon overexpression. KLF4 was identified as a target of miR-1263. RNAi-mediated downregulation of KLF4 partially mimicked miR-1263 overexpression. Thus, the effects of this miRNA were mediated by facilitating differentiation through destabilization of pluripotency along with other not yet defined targets.


Assuntos
Diferenciação Celular , Endoderma/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Mesoderma/citologia , MicroRNAs/genética , Células Cultivadas , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mesoderma/metabolismo , MicroRNAs/metabolismo , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo
4.
Curr Protoc Stem Cell Biol ; 40: 1D.9.1-1D.9.17, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28152181

RESUMO

Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm-committed cells in high numbers in a 4-day treatment protocol. Second, a method for the purification of CXCR4-positive endoderm cells by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm-derived somatic cells. © 2017 by John Wiley & Sons, Inc.


Assuntos
Separação Celular/métodos , Endoderma/citologia , Citometria de Fluxo/métodos , Magnetismo/métodos , Células-Tronco Pluripotentes/citologia , Contagem de Células , Diferenciação Celular , Células Cultivadas , Células Alimentadoras/citologia , Imunofluorescência , Humanos , Receptores CXCR4/metabolismo
5.
Mol Cell Biol ; 36(24): 3113-3127, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27697866

RESUMO

The clinical potential of transplantation is often reduced by T cell-mediated alloresponses that cause graft rejection or graft-versus-host disease. Integrin-mediated adhesion between alloreactive T cells and antigen-presenting cells is essential for allorejection. The identity of the signaling events needed for the activation of integrins such as LFA-1 is poorly understood. Here, we identified a novel role of the protein tyrosine phosphatase SHP-1 in the regulation of murine LFA-1-mediated adhesion in an allograft setting. Upon alloactivation, SHP-1 activity is reduced, resulting in an increase in LFA-1 adhesion compared to that for syngeneically activated T cells. The importance of these differential activation properties was further indicated by small interfering RNA (siRNA) knockdown of SHP-1 in syngeneically and allogeneically stimulated T cells. Mechanistically, SHP-1 modulated the binding of SLP-76 to ADAP by dephosphorylation of the YDGI tyrosine motif of ADAP, a known docking site for the Src family kinase Fyn. This novel key role of SHP-1 in the regulation of LFA-1-mediated adhesion may provide a new insight into T cell-mediated alloresponses and may pave the way to the development of new immunosuppressive pharmaceutical agents.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aloenxertos/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fosfoproteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/química , Aloenxertos/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Células Cultivadas , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Camundongos , Fosforilação , Ligação Proteica , Linfócitos T/metabolismo
6.
Stem Cells ; 34(11): 2635-2647, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27299363

RESUMO

As known from model organisms, such as frog, fish, mouse, and chicken, the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid, TGF-ß-ligands, Wnt-, and BMP-inhibitors in the anterior half of the DE sheet. However, it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut, we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-ß-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut toward a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2, BMP-4, and RA induces the formation of CDX2-positive posterior endoderm. Thus, these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. Stem Cells 2016;34:2635-2647.


Assuntos
Proteína Morfogenética Óssea 4/genética , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Proteína Wnt3/genética , Ativinas/genética , Ativinas/metabolismo , Ativinas/farmacologia , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Linhagem Celular , Endoderma/citologia , Endoderma/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Separação Imunomagnética , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Proteína Wnt3/farmacologia , beta Catenina/genética , beta Catenina/metabolismo
7.
J Vis Exp ; (109)2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26966833

RESUMO

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.


Assuntos
Endoderma/citologia , Endoderma/fisiologia , Citometria de Fluxo/métodos , Células-Tronco Embrionárias Humanas/fisiologia , Diferenciação Celular/fisiologia , Humanos , Células Secretoras de Insulina/fisiologia , Fígado/citologia , Fígado/fisiologia , Pâncreas/citologia , Pâncreas/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais/fisiologia
8.
Mol Cell Proteomics ; 15(4): 1435-52, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26785728

RESUMO

PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors andin vitrodifferentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCsin vitro Knock-down ofPMM2by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.


Assuntos
Defeitos Congênitos da Glicosilação/patologia , Glicômica/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Fosfotransferases (Fosfomutases)/deficiência , Células Cultivadas , Defeitos Congênitos da Glicosilação/metabolismo , Perfilação da Expressão Gênica/métodos , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Fosfotransferases (Fosfomutases)/metabolismo , Polissacarídeos/metabolismo
9.
Methods Mol Biol ; 1341: 157-72, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25762297

RESUMO

Differentiation of pluripotent stem cells into cells of the definitive endoderm requires an in vitro gastrulation event. Differentiated somatic cells derived from this germ layer may then be used for cell replacement therapies of degenerative diseases of the liver, lung, and pancreas. Here we describe an endoderm differentiation protocol, which initiates the differentiation from a defined cell number of dispersed single cells and reliably yields in >70-80 % endoderm-committed cells in a short 5-day treatment regimen.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Endoderma/citologia , Células-Tronco Pluripotentes/citologia , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Via de Sinalização Wnt
10.
Methods Mol Biol ; 1341: 67-85, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25762298

RESUMO

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes, shRNAs, miRNAs, or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Lentivirus/genética , Células-Tronco Pluripotentes/metabolismo , Transdução Genética/métodos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Vetores Genéticos/isolamento & purificação , Células HEK293 , Humanos , Lentivirus/isolamento & purificação , Células-Tronco Pluripotentes/citologia , Ultracentrifugação/métodos , Ultrafiltração/métodos
11.
Stem Cells Dev ; 24(2): 190-204, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25137387

RESUMO

Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h, followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally, inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required, nonetheless yielding high cell numbers efficiently committed toward the DE. Finally, the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus, this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias , Endoderma , Células-Tronco Pluripotentes Induzidas , Ativinas/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Nodal/metabolismo , Transdução de Sinais
12.
J Tissue Eng Regen Med ; 9(4): 473-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23418163

RESUMO

Pluripotent stem cells hold great promise for regenerative medicine, due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed, get relatively old (≤ 13 years), are small in size, are relatively cost-effective and have a high genetic proximity to the human. In particular, diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable, chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However, the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds, whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus, differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds.


Assuntos
Ativinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Animais , Callithrix , Células Cultivadas , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Humanos
13.
Stem Cell Rev Rep ; 10(4): 480-93, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24913278

RESUMO

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.


Assuntos
Ativinas/metabolismo , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Mesoderma/citologia , Pâncreas/citologia , Proteínas Wnt/metabolismo , Ativinas/genética , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Citometria de Fluxo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Técnicas Imunoenzimáticas , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/genética
14.
BMC Res Notes ; 7: 273, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24779365

RESUMO

BACKGROUND: Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized. METHODS: Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated. RESULTS: CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T. CONCLUSIONS: Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.


Assuntos
Células-Tronco Embrionárias/citologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/toxicidade , Via de Sinalização Wnt/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/enzimologia , Proteínas Fetais/metabolismo , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Proteínas com Domínio T/metabolismo , Via de Sinalização Wnt/genética
15.
Stem Cell Rev Rep ; 9(5): 555-68, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23111459

RESUMO

Pluripotent cells hold great promise for cell replacement therapies in regenerative medicine. All known protocols for directed in vitro differentiation of pluripotent cells did not yield pure populations complicating the characterization of the derived cells. In addition, the risk of tumor formation due to residual undifferentiated cells is a serious unresolved problem. In the present study the tissue-specific mouse Pdx1 promoter was used to control the expression of the reporter gene GFP2 in mouse ES cells in order to purify them via FACS during in vitro differentiation. The background fluorescence of transduced ES cells hampered the purification of Pdx1-positive cells due to a contaminating population of partially undifferentiated cells. MicroRNAs (mir) are important regulators of gene expression and were used to enhance promoter specificity during differentiation towards pancreatic progenitor cells. The mouse mmu-mir-294 was found to be mainly expressed during pluripotency, whereas the expression of the mir-302 cluster was increased during early differentiation. Integration of a microRNA target site for the mmu-mir-294 into the lentiviral vector reduced the background fluorescence specifically during pluripotency and permitted re-occurrence of GFP2 expression upon differentiation. A combination of the microRNA target site with the Pdx1 promoter fragment allowed the purification of pancreatic progenitors from differentiated ES cells. This population reflected an early pancreatic progenitor population without other contaminating cell lineages. In conclusion, microRNA target sites are efficient regulatory elements to control transgene expression and to enhance tissue specificity as presented in this study facilitating the sorting and purification of Pdx1-positive pancreatic progenitor cells.


Assuntos
Células-Tronco Embrionárias/metabolismo , MicroRNAs/genética , Regiões Promotoras Genéticas/genética , Células-Tronco/metabolismo , Células 3T3 , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Células Cultivadas , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Lentivirus/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , MicroRNAs/metabolismo , Microscopia de Fluorescência , Pâncreas/citologia , Pâncreas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Transativadores/genética , Proteína Vermelha Fluorescente
16.
Islets ; 4(2): 123-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22627676

RESUMO

The common marmoset New World monkey (Callithrix jacchus), is a primate model with great potential for scientific research, including research on diabetes. However, in opposite to Rhesus and Java monkeys (Macaca mulatta and Macaca fascicularis) little is known about the marmosets islet microarchitecture, glucose transporter and pancreatic marker gene expression. In this work we analyze differences and similarities in size, shape, cellular composition and intra-islet topography between the common marmoset and the human endocrine pancreas. Different sized, circular and a-circular shaped islets of the common marmoset and human display α-cells in the whole islet organ leading to a ribbon-like islet type. The number of islets was significantly higher in the common marmoset compared with humans. However, the area of insulin-producing cells was significantly higher in the human pancreas. Intra-islet distribution pattern of δ- and ß-cells was similar in both species. The morphology of the exocrine pancreas regarding acinar and ductal cells was quite similar as confirmed by ultrastructural analysis. Additionally the ultrastructure of secretory granules from α-, δ- and ß-cells of human and non-human primate pancreas showed the same characteristics. Molecular analysis showed the presence of endocrine pancreatic marker genes like PMCA2, NCX1, SUR1, KIR6.2, MAFA, NGN3 and PDX1 also expressed in the human. For the first time we could show presence of Glut 5 and 9 transporters in addition to the low abundance transporter Glut2 and the highly expressed Glut1 glucose transporter. We propose that Callithrix jacchus displays a new animal model for diabetes research and regenerative medicine.


Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Sódio-Glucose/biossíntese , Animais , Callithrix , Modelos Animais de Doenças , Histocitoquímica , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/ultraestrutura , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Proteínas de Transporte de Sódio-Glucose/genética
17.
Cryobiology ; 63(2): 104-10, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21810414

RESUMO

Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed "induced pluripotent" cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are commonly used for ESC long term culture procedures and colony regeneration. These MEFs originate from different mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consistency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells. According to our findings, MEFs should be inactivated by irradiation (30Gy) and cryopreserved with optimal 10% DMSO at 1K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryopreserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture without detaching from the culture dish or disruption of the cell layer.


Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Células-Tronco Embrionárias/citologia , Células Alimentadoras , Animais , Técnicas de Cocultura , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células Alimentadoras/efeitos da radiação , Raios gama , Células-Tronco Pluripotentes Induzidas , Camundongos , Mycoplasma/isolamento & purificação , Controle de Qualidade
18.
J Mol Med (Berl) ; 87(10): 965-70, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19575173

RESUMO

Pseudoxanthoma elasticum (PXE) is a rare disorder predominantly affecting the skin, the eyes, and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling. In the present study, we investigated matrix metalloproteinases MMP-2 and MMP-9 in PXE patients compared to healthy controls. We analyzed the serum concentrations of MMP-2 and MMP-9 in a cohort of 69 German PXE patients and in 69 healthy, age-, and sex-matched control subjects using commercially available ELISA assays. We found elevated concentrations of both MMPs in the sera of PXE patients. MMP-2 levels were significantly higher in patients than controls (231 +/- 5.89 vs 202 +/- 5.17 ng/ml, p = 0.0002), as were MMP-9 levels (841 +/- 65.9 vs 350 +/- 30.8 ng/ml, p < 0.0001). Our findings point to an involvement of matrix metalloproteinases in PXE pathology. ECM remodeling in PXE is reflected by elevated levels of circulating MMP-2 and MMP-9. Those MMPs might, therefore, be applicable as serum markers for the matrix-degradative process in PXE.


Assuntos
Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Pseudoxantoma Elástico/sangue , Pseudoxantoma Elástico/enzimologia , Adulto , Animais , Biomarcadores/sangue , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Pseudoxantoma Elástico/genética , Pseudoxantoma Elástico/patologia
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