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1.
Ugeskr Laeger ; 172(38): 2616-9, 2010 Sep 20.
Artigo em Dinamarquês | MEDLINE | ID: mdl-20920407

RESUMO

Mesenchymal stem cells (MSC) are capable of multilineage differentiation into cells like osteoblasts, chondrocytes or adipocytes. MSCs can be isolated from bone marrow and expanded ex vivo for up to 25-40 population doublings while maintaining genetic stability and differentiation potential. MSCs have great potential in the field of tissue engineering and regenerative medicine where cartilage and bone conditions which are non-treatable or show very slow improvement can be effectively handled. Several clinical trials have been performed using MSC and show very promising results.


Assuntos
Doenças Ósseas/terapia , Doenças das Cartilagens/terapia , Transplante de Células-Tronco Mesenquimais , Animais , Doenças Ósseas/cirurgia , Transplante de Medula Óssea , Doenças das Cartilagens/cirurgia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Fraturas Ósseas/cirurgia , Fraturas Ósseas/terapia , Humanos , Osteoartrite/cirurgia , Osteoartrite/terapia , Resultado do Tratamento
2.
PLoS One ; 5(5): e10900, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20531935

RESUMO

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced a 9-42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8(+) T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types.


Assuntos
Membrana Celular/imunologia , Antígenos HLA-B/genética , Células-Tronco Mesenquimais/imunologia , Células Satélites de Músculo Esquelético/imunologia , Alelos , Citotoxicidade Imunológica/imunologia , Regulação para Baixo/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Humanos , Interferon gama/imunologia , Espaço Intracelular/imunologia , Cinética , Células-Tronco Mesenquimais/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Satélites de Músculo Esquelético/citologia
3.
J Biol Chem ; 285(19): 14438-49, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20223822

RESUMO

Mechanisms controlling human multipotent mesenchymal (stromal) stem cell (hMSC) differentiation into osteoblasts or adipocytes are poorly understood. We have previously demonstrated that Wnt signaling in hMSC enhanced osteoblast differentiation and inhibited adipogenesis by comparing two hMSC cell lines overexpressing mutated forms of the Wnt co-receptor LRP5: T253I (hMSC-LRP5(T253)) and T244M (hMSC-LRP5(T244)) conducting high and low level of Wnt signaling, respectively. To explore the underlying molecular mechanisms, we compared gene expression profiles of hMSC-LRP5(T253) and hMSC-LRP5(T244) treated with Wnt3a using whole genome expression microarrays and found that TNFRSF19 is differentially up-regulated between the two cells lines. Bioinformatic analysis and dual luciferase assay of its promoter revealed that TNFRSF19 transcript 2 (TNFRSF19.2) is a target of canonical Wnt signaling. Knocking down TNFRSF19 in hMSC-LRP5(T253) cells decreased Wnt3a-induced osteoblast differentiation marker alkaline phosphate activity and its overexpression in hMSC-LRP5(T244) cells increased alkaline phosphate activity. In addition, TNFRSF19 was negatively regulated by adipogenic transcription factor CCAAT/enhancer-binding proteins (C/EBP). Knocking down TNFRSF19 in hMSC-LRP5(T253) cells or its overexpression in hMSC-LRP5(T244) cells significantly increased or decreased adipogenesis, respectively. In conclusion, we revealed a novel function of TNFRSF19 as a factor mediating differentiation signals that determine the hMSC differentiating fate into osteoblasts or adipocytes.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Proteínas Relacionadas a Receptor de LDL/metabolismo , Células-Tronco Mesenquimais/citologia , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Wnt/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Receptores do Fator de Necrose Tumoral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção
4.
J Bone Miner Res ; 22(11): 1720-31, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17680723

RESUMO

UNLABELLED: Genetic mutations in the LRP5 gene affect Wnt signaling and lead to changes in bone mass in humans. Our in vivo and in vitro results show that activated mutation T253I of LRP5 enhances osteogenesis and inhibits adipogenesis. Inactivating mutation T244M of LRP5 exerts opposite effects. INTRODUCTION: Mutations in the Wnt co-receptor, LRP5, leading to decreased or increased canonical Wnt signaling, result in osteoporosis or a high bone mass (HBM) phenotype, respectively. However, the mechanisms whereby mutated LRP5 causes changes in bone mass are not known. MATERIALS AND METHODS: We studied bone marrow composition in iliac crest bone biopsies from patients with the HBM phenotype and controls. We also used retrovirus-mediated gene transduction to establish three different human mesenchymal stem cell (hMSC) strains stably expressing wildtype LRP5 (hMSC-LRP5(WT)), LRP5(T244) (hMSC-LRP5(T244), inactivation mutation leading to osteoporosis), or LRP5(T253) (hMSC-LRP5(T253), activation mutation leading to high bone mass). We characterized Wnt signaling activation using a dual luciferase assay, cell proliferation, lineage biomarkers using real-time PCR, and in vivo bone formation. RESULTS: In bone biopsies, we found increased trabecular bone volume and decreased bone marrow fat volume in patients with the HBM phenotype (n = 9) compared with controls (n = 5). The hMSC-LRP5(WT) and hMSC-LRP5(T253) but not hMSC-LRP5(T244) transduced high level of Wnt signaling. Wnt3a inhibited cell proliferation in hMSC-LRP5(WT) and hMSC-LRP5(T253), and this effect was associated with downregulation of DKK1. Both hMSC-LRP5(WT) and hMSC-LRP5(T253) showed enhanced osteoblast differentiation and inhibited adipogenesis in vitro, and the opposite effect was observed in hMSC-LRP5(T244). Similarly, hMSC-LRP5(WT) and hMSC-LRP5(T253) but not hMSC-LRP5(T244) formed ectopic mineralized bone when implanted subcutaneously with hydroxyapatite/tricalcium phosphate in SCID/NOD mice. CONCLUSIONS: LRP5 mutations and the level of Wnt signaling determine differentiation fate of hMSCs into osteoblasts or adipocytes. Activation of Wnt signaling can thus provide a novel approach to increase bone mass by preventing the age-related reciprocal decrease in osteogenesis and increase in adipogenesis.


Assuntos
Adipogenia/genética , Diferenciação Celular/genética , Proteínas Relacionadas a Receptor de LDL/genética , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese/genética , Adulto , Animais , Densidade Óssea/genética , Linhagem Celular , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Fenótipo , Transdução Genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A
5.
J Immunother ; 30(1): 29-39, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17198081

RESUMO

A general hindrance to progress in adoptive cellular therapy is the lack of detailed knowledge of the fate of transferred cells in the body of the recipient. In this study, we present a novel technique for tracking of 124I-labeled cells in situ, which combines the high spatial resolution of magnetic resonance imaging with the high sensitivity and spatial accuracy of positron emission tomography. We have used this technique, together with determination of tissue radioactivity, flow cytometry, and microscopy, to characterize and quantitate the specific accumulation of transferred CD8+ T cells in tumor tissue in a mouse model. Transgenic CD8+ T cells, specific for the ovalbumin peptide SIINFEKL, were adoptively transferred to recipients carrying a subcutaneous tumor of the ovalbumin-expressing malignant melanoma cell line B16-OVA. The number of SIINFEKL-specific CD8+ cells in the tumor tissue was determined by flow cytometry each day for 8 consecutive days after adoptive transfer. From low levels 1 day after injection, their number gradually increased until day 5 when an average of 3.3x10(6) SIINFEKL-specific cells per gram tumor tissue was found. By applying the combined positron emission tomography/magnetic resonance imaging technique we were able to determine the position of the transferred, 124I-labeled SIINFEKL-specific T cells in 3 dimensions in recipient mice, and could demonstrate a highly significant accumulation of the 124I label in and around the subcutaneous B16-OVA tumors compared with normal tissue. Accumulation of 124I was significantly higher in B16-OVA than in B16 tumors not expressing the OVA antigen.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Receptores de Retorno de Linfócitos/imunologia , Animais , Proteínas do Ovo/imunologia , Feminino , Radioisótopos do Iodo , Imageamento por Ressonância Magnética , Masculino , Melanoma Experimental/diagnóstico por imagem , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fragmentos de Peptídeos , Tomografia por Emissão de Pósitrons
6.
Brain Res Mol Brain Res ; 105(1-2): 153-6, 2002 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-12399118

RESUMO

In one of our mouse colonies a reeler-like phenotype appeared spontaneously. The brain histology was identical to the known reeler phenotype. Northern and Western blot analysis and a complementation test showed that the defect is located to the reelin gene. Southern blot and PCR analysis together with information obtained from sequence databases revealed that this defective reelin gene had an approximately 24-kb intragenic deletion comprising exons 13-20.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Camundongos Mutantes Neurológicos/genética , Mutação/genética , Animais , Sequência de Bases/genética , Moléculas de Adesão Celular Neuronais/deficiência , Cerebelo/anormalidades , Cerebelo/fisiopatologia , DNA/análise , DNA/genética , Regulação para Baixo/genética , Éxons/genética , Proteínas da Matriz Extracelular/deficiência , Feminino , Deleção de Genes , Masculino , Camundongos , Proteínas do Tecido Nervoso , Fenótipo , RNA Mensageiro/metabolismo , Proteína Reelina , Serina Endopeptidases
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