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1.
Stem Cells Dev ; 24(7): 824-35, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25390472

RESUMO

Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1(+)Lin(-)CD45(-)-derived MSCs(-) (SL45-MSCs), isolated by fluorescence-activated cell sorting from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, that are, frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared with bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared with controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, more effectively than bulk BM-MSCs, an effect that may be mediated by a higher BDNF secretion.


Assuntos
Encéfalo/citologia , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/metabolismo , Fibras Nervosas/efeitos dos fármacos , Regeneração Nervosa , Animais , Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Técnicas de Cocultura , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/metabolismo , Fibras Nervosas/fisiologia , Nestina/genética , Nestina/metabolismo
2.
BMC Bioinformatics ; 14: 75, 2013 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-23452776

RESUMO

BACKGROUND: Microarrays have become a routine tool to address diverse biological questions. Therefore, different types and generations of microarrays have been produced by several manufacturers over time. Likewise, the diversity of raw data deposited in public databases such as NCBI GEO or EBI ArrayExpress has grown enormously.This has resulted in databases currently containing several hundred thousand microarray samples clustered by different species, manufacturers and chip generations. While one of the original goals of these databases was to make the data available to other researchers for independent analysis and, where appropriate, integration with their own data, current software implementations could not provide that feature.Only those data sets generated on the same chip platform can be readily combined and even here there are batch effects to be taken care of. A straightforward approach to deal with multiple chip types and batch effects has been missing.The software presented here was designed to solve both of these problems in a convenient and user friendly way. RESULTS: The virtualArray software package can combine raw data sets using almost any chip types based on current annotations from NCBI GEO or Bioconductor. After establishing congruent annotations for the raw data, virtualArray can then directly employ one of seven implemented methods to adjust for batch effects in the data resulting from differences between the chip types used. Both steps can be tuned to the preferences of the user. When the run is finished, the whole dataset is presented as a conventional Bioconductor "ExpressionSet" object, which can be used as input to other Bioconductor packages. CONCLUSIONS: Using this software package, researchers can easily integrate their own microarray data with data from public repositories or other sources that are based on different microarray chip types. Using the default approach a robust and up-to-date batch effect correction technique is applied to the data.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Bases de Dados Genéticas , Humanos
3.
Cytometry A ; 83(1): 72-5, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23165990

RESUMO

In 2006, very small embryonic-like (VSEL) stem cells were described as a pluripotent population of prospectively isolated stem cells in adult murine bone marrow (mBM) and human umbilical cord blood (hUCB). While rigorous proof of pluripotency is still lacking, murine VSEL cells have been shown to overlap with an independently identified population of neural crest derived mesenchymal stem cells (MSC). The presence of primitive mesenchymal precursors within the VSEL cell population may partially explain the findings that have led to the concept of an "embryonic-like" stem cell in mBM. However, our own studies on human VSEL cells revealed very little similarity between murine VSEL cells and their reportedly equivalent population in hUCB. On the contrary, our data strongly suggest that human VSEL cells are an aberrant and inactive population that cannot expand in vitro and has neither embryonic nor adult stem cell like properties. Here we critically re-examine the data supporting stemness and pluripotency of murine and human VSEL cells, respectively.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Embrionárias/citologia , Sangue Fetal/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Proliferação de Células , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Fenótipo
4.
PLoS One ; 7(4): e34899, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22509366

RESUMO

Very small embryonic-like (VSEL) cells have been described as putatively pluripotent stem cells present in murine bone marrow and human umbilical cord blood (hUCB) and as such are of high potential interest for regenerative medicine. However, there remain some questions concerning the precise identity and properties of VSEL cells, particularly those derived from hUCB. For this reason, we have carried out an extensive characterisation of purified populations of VSEL cells from a large number of UCB samples. Consistent with a previous report, we find that VSEL cells are CXCR4(+), have a high density, are indeed significantly smaller than HSC and have an extremely high nuclear/cytoplasmic ratio. Their nucleoplasm is unstructured and stains strongly with Hoechst 33342. A comprehensive FACS screen for surface markers characteristic of embryonic, mesenchymal, neuronal or hematopoietic stem cells revealed negligible expression on VSEL cells. These cells failed to expand in vitro under a wide range of culture conditions known to support embryonic or adult stem cell types and a microarray analysis revealed the transcriptional profile of VSEL cells to be clearly distinct both from well-defined populations of pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridisation. These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL cells are an aberrant and inactive population that is not comparable to murine VSEL cells.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Sangue Fetal/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores CXCR4/metabolismo , Aneuploidia , Diferenciação Celular , Linhagem da Célula/genética , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
5.
Exp Hematol ; 37(2): 294-301, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19101070

RESUMO

OBJECTIVE: The ABCG2 transporter has been identified as a determinant of the side population (SP) in murine bone marrow and a potential marker for primitive stem cells. To assess the potential of the SP phenotype and ABCG2 expression to identify stem cells in human umbilical cord blood (hUCB), we have examined directly the relationship between SP; expression of ABCG2, CD133, and CD34; and hematopoietic potential in UCB samples. MATERIALS AND METHODS: Multicolor fluorescence activated cell sorting analysis was combined with the Hoechst SP procedure to allow the simultaneous detection of the SP phenotype together with surface markers in cells from fresh and cryopreserved UCB. Sorted populations were analyzed for cobblestone area-forming cell (CAFC) activity and by quantitative reverse transcriptase polymerase chain reaction for expression of mRNA from the ABC transporters ABCG2, MDR1, and MRP1. ABCG2(+) cells were enriched by magnetic-activated cell sorting for stringent analysis. RESULTS: hUCB-derived SP cells were negative for ABCG2, but comprise approximately 20% CD133(+)/CD34(+) cells. Sorted SP cells from UCB were enriched 20-fold for week 13 CAFC activity, while magnetic-activated cell sorting-enriched ABCG2(+) cells retained no hematopoietic activity either in CAFC or liquid cultures. There was no significant difference in the SP frequency, immunophenotype, or CAFC potential of fresh and cryopreserved UCB. ABCG2 mRNA was not enriched in the SP and was specifically diminished ninefold in CD133 cells, which were eightfold enriched for MDR1 mRNA. CONCLUSION: We find no evidence for an association of ABCG2 with SP activity or hematopoietic progenitor function in hUCB.


Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Antígenos de Diferenciação/biossíntese , Sangue Fetal/metabolismo , Regulação da Expressão Gênica/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Neoplasias/biossíntese , Antígeno AC133 , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Antígenos CD/biossíntese , Antígenos CD34/biossíntese , Sangue Fetal/citologia , Glicoproteínas/biossíntese , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Peptídeos
6.
Cells Tissues Organs ; 188(1-2): 150-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18216460

RESUMO

Despite more than 40 years of experience with the use of haematopoietic stem cells (HSC) in the clinic and the identification of a plethora of regulatory signals of clear relevance to their function, it has proven remarkably difficult to amplify these cells efficiently in culture without a concurrent loss of potential. Based on considerations of haematopoietic environments in the embryo and the adult, on published observations of metabolic compartmentalisation between neighbouring cells in other tissues, and on considerations of the selective pressures acting on the evolution of generative and regenerative systems, we propose that the amplification of HSC may be tied obligatorily to limiting metabolic conditions provided by the niche. We suggest that this conceptually simple arrangement could combine the support of HSC with the containment of self-renewal activity within a rare and highly defined set of physiological sites, whilst avoiding the accumulation of potentially leukaemogenic mutations in the stem cell pool.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos , Hematopoese , Humanos
7.
Differentiation ; 75(2): 100-11, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17316380

RESUMO

Cord blood-derived CD133+ cells have a degree of non-hematopoietic potential and express transcripts of pluripotency markers including Oct-4, Sox-2, Rex-1, and leukemia inhibitory factor (LIF) receptor, as well as markers of progenitor cells, such as HoxB4, brachyury, and nestin. Having shown by transcriptome analysis that the mouse embryonic fibroblast (MEF) cells routinely used to maintain pluripotent embryonic stem cells express transcripts of the WNT/BMP families of signaling factors, we have assessed the effects on proliferation and differentiation of CD133+ cells of medium conditioned (CM) by MEF, by NIH3T3, and by NIH3T3 cells stably expressing WNT1, WNT3a, WNT4, WNT5a, and WNT11. Cultivation of CD133+ cells in MEF-CM led to a significant increase in cell number after 7 days of culture, while WNT-1, WNT3a-, and WNT11-CM increased the cell number significantly by 14 days of culture. During this period, WNT3a-CM increased the proportion of nestin-expressing cells and increased the ratio of blast-like cells to macrophages, suggesting that these signaling molecules contribute to the maintenance of an undifferentiated, blast-like phenotype. The number of cells expressing the endothelial-related marker CD31+ was significantly increased following culture in WNT5a- and WNT11-CM, whereas the number of cells positive for von Willebrand (vW) factor was maintained during 14 days of culture only in the presence of WNT4-CM. In addition, WNT5a-CM led to increased beta-catenin mRNA levels and the presence of beta-catenin protein in the cytoplasm and nucleus, consistent with the activation of the WNT signaling pathway. We conclude that in vitro conditioning of CD133+ cells by media containing specific WNT signaling factors influences the non-hematopoietic potential of CD133+ cells and dynamically alters the expression of the neural stem/progenitor cell marker nestin and the endothelial-related cell surface markers CD31 and vW factor.


Assuntos
Antígenos CD/metabolismo , Diferenciação Celular , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Sangue Fetal/citologia , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Proteínas Wnt/fisiologia , Antígeno AC133 , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/metabolismo , Nestina , Células-Tronco Pluripotentes/metabolismo , Gravidez , Transdução de Sinais , beta Catenina/metabolismo
8.
Blood ; 108(5): 1533-41, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16675709

RESUMO

The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline-dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fraction of the bone marrow. In addition, conditional transgene activation also was detected in a very minor population of endothelial cells and in the kidney. However, no activation of the reporter transgene was found in the brain of adult mice. These findings suggested that the expression of tetracycline-responsive reporter genes recapitulated the known endogenous expression pattern of SCL. Our data therefore demonstrate that exogenously inducible and reversible expression of selected transgenes in myeloid, megakaryocytic, erythroid, and c-kit-expressing lineage-negative bone marrow cells can be directed through SCL regulatory elements. The SCL knock-in mouse presented here represents a powerful tool for studying normal and malignant hematopoiesis in vivo.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Eritrócitos/fisiologia , Granulócitos/fisiologia , Hematopoese/fisiologia , Megacariócitos/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas/genética , Tetraciclina/farmacologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Primers do DNA , Eritrócitos/citologia , Citometria de Fluxo , Regulação da Expressão Gênica , Genes Reporter , Genótipo , Granulócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T
9.
Biotechnol Prog ; 21(1): 58-69, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15903241

RESUMO

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.


Assuntos
Amônia/metabolismo , Glutamina/metabolismo , Ácido Pirúvico/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Meios de Cultura Livres de Soro , Técnicas de Cultura/métodos , Relação Dose-Resposta a Droga , Glutamina/farmacologia , Ácido Pirúvico/farmacologia , Fatores de Tempo
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