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1.
Cell Prolif ; 41(1): 12-27, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18211283

RESUMEN

OBJECTIVES: To generate non-haematopoietic tissues from mobilized haematopoietic CD133(+) stem cells. MATERIALS AND METHODS: Mobilized peripheral blood CD133(+) cells from adult healthy donors were used. In vitro ability of highly enriched CD133(+) cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. RESULTS: We found that a recently identified population of CD45(+) adherent cells generated in vitro after culture of highly purified CD133(+) cells for 3-5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. CONCLUSION: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.


Asunto(s)
Antígenos CD/inmunología , Glicoproteínas/inmunología , Células Madre Multipotentes/citología , Péptidos/inmunología , Antígeno AC133 , Adulto , Secuencia de Bases , Diferenciación Celular , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Dihidroxifenilalanina/biosíntesis , Dopamina/biosíntesis , Humanos , Técnicas In Vitro , Células Madre Multipotentes/inmunología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
2.
Blood Cells Mol Dis ; 27(3): 667-76, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11482882

RESUMEN

Through differential screening of mouse hematopoietic stem cell (HSC) and progenitor-subtracted cDNA libraries we have identified a progenitor cell-specific transcript that represents a novel gene, named Hepp (hematopoietic progenitor protein). The mouse Hepp gene encodes a protein of 237 amino acids with no detectable known functional domains or motifs. Lack of invertebrate orthologs and a high degree of evolutionary conservation of the peptide sequence in vertebrate species (zebrafish, mouse, human) suggest that the Hepp gene could have conserved although as yet unknown function in vertebrates. Mouse Hepp shows a restricted expression pattern in adult tissues and is transcribed at a very low level in heart, lung, spleen, and thymus and at a higher level in muscle. During embryonic hematopoiesis Hepp is not expressed in mouse fetal liver HSC (Sca-1(+)c-kit(+)AA4.1(+)Lin(-) cells), but is abundantly transcribed in the population of hematopoietic progenitors (AA4.1(-) cells). Similarly, during adult hematopoiesis Hepp is not transcribed in the highly enriched population of bone marrow HSC (Rh-123(low)Sca-1(+)c-kit(+)Lin(-) cells), but its expression is upregulated as a greater heterogeneous population of bone marrow HSC (Lin(-)Sca-1(+) cells) differentiates into progenitors (Lin(-)Sca-1(-) cells) and more mature lymphoid and myeloid cell types. A restricted pattern of expression in adult tissues and preferential expression in both fetal and adult hematopoietic progenitors and mature blood cells suggest that Hepp could be involved in molecular regulation of HSC and progenitor cell lineage commitment and differentiation.


Asunto(s)
Células Sanguíneas/metabolismo , Proteínas de Ciclo Celular/genética , Clonación Molecular , Genes/genética , Células Madre Hematopoyéticas/metabolismo , Proteínas Musculares/genética , Animales , Secuencia de Bases , Médula Ósea/metabolismo , ADN Complementario/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Distribución Tisular
3.
Blood Cells Mol Dis ; 27(1): 320-33, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11358394

RESUMEN

Through differential screening of mouse hematopoietic stem cell (HSC) and progenitor subtracted cDNA libraries we have identified a HSC-specific transcript that represents a novel RING finger gene, named FLRF (fetal liver ring finger). FLRF represent a novel evolutionarily highly conserved RING finger gene, present in Drosophila, zebrafish, Xenopus, mouse, and humans. Full-length cDNA clones for mouse and human gene encode an identical protein of 317 amino acids with a C3HC4 RING finger domain at the amino terminus. During embryonic hematopoiesis FLRF is abundantly transcribed in mouse fetal liver HSC (Sca-1+c-kit+AA4.1+Lin- cells), but is not expressed in progenitors (AA4.1-). In adult mice FLRF is not transcribed in a highly enriched population of bone marrow HSC (Rh-123lowSca-1+c-kit+Lin- cells). Its expression is upregulated in a more heterogeneous population of bone marrow HSC (Lin-Sca-1+ cells), downregulated as they differentiate into progenitors (Lin-Sca-1- cells), and upregulated as progenitors differentiate into mature lymphoid and myeloid cell types. The human FLRF gene that spans a region of at least 12 kb and consists of eight exons was localized to chromosome 12q13, a region with frequent chromosome aberrations associated with multiple cases of acute myeloid leukemia and non-Hodgkin's lymphoma. The analysis of the genomic sequence upstream of the first exon in the mouse and human FLRF gene has revealed that both putative promoters contain multiple putative binding sites for several hematopoietic (GATA-1, GATA-2, GATA-3, Ikaros, SCL/Tal-1, AML1, MZF-1, and Lmo2) and other transcription factors, suggesting that mouse and human FLRF expression could be regulated in a developmental and cell-specific manner during hematopoiesis. Evolutionary conservation and differential expression in fetal and adult HSC and progenitors suggest that the FLRF gene could play an important role in HSC/progenitor cell lineage commitment and differentiation and could be involved in the etiology of hematological malignancies.


Asunto(s)
Proteínas Portadoras/genética , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Ratones/genética , Oligopéptidos/genética , Factores de Edad , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Feto , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Alineación de Secuencia , Ubiquitina-Proteína Ligasas , Dedos de Zinc/genética
4.
Curr Opin Microbiol ; 3(3): 316-21, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10851166

RESUMEN

Analysis of differential gene expression is a classic tool in experimental biology. Broadly applicable new methods to identify and quantitative differential mRNA profiles, such as long distance differential display PCR and cDNA microarrays, promise to greatly accelerate understanding of mechanisms of development, differentiation, and disease.


Asunto(s)
ADN Complementario/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Regulación Bacteriana de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , ARN Mensajero/genética
5.
Anal Biochem ; 259(2): 235-44, 1998 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-9618202

RESUMEN

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long-distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, approximately 60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.


Asunto(s)
Clonación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Northern Blotting , Línea Celular , ADN Complementario , Expresión Génica , Técnicas Genéticas , Ratones , ARN Mensajero/genética
6.
Methods Mol Med ; 7: 223-40, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-24493430

RESUMEN

A variety of genetic and acquired diseases could conceivably be treated by gene therapy targeted to hematopoietic stem cells (HSC). Inevitably, the effort to develop reliable methods of gene transfer into stem cells has raised many questions about their biology and role in the development and maintenance of hematopoiesis. As a result, we currently have a convergence of research goals in the areas of stem cell biology and gene therapy. Murine models for stem cell transduction have played a useful role in establishing two basic principles: retroviral vectors can transduce pluripotent self-renewing hematopoietic stem cells and retroviral vectors can express foreign gene products in the differentiated progeny of stem cells. Murine models also have allowed the identification of several key factors that allow efficient transduction of stem cells and each of these is dealt with here. However, methods for stem cell transduction that are effective with mouse cells have only been partially successful in dog, nonhuman primate, and human models. Whereas scale-up of stem cell transduction procedures for human applications will present unique technical problems, mouse models may yet provide further insight into the mechanisms of efficient stem cell gene transfer that can then be used to design enhanced and reproducible protocols.

8.
Proc Natl Acad Sci U S A ; 92(9): 3673-7, 1995 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-7731963

RESUMEN

We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice.


Asunto(s)
Adenosina Desaminasa/genética , Envejecimiento/fisiología , Genes Letales , Hígado/patología , Adenosina Desaminasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Nucleótidos de Desoxiadenina/metabolismo , Femenino , Genotipo , Edad Gestacional , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/patología , Homeostasis , Leucocitos/citología , Leucocitos/enzimología , Leucocitos/patología , Hígado/embriología , Hígado/enzimología , Ratones , Ratones Mutantes , Mutagénesis , Embarazo , Purinas/metabolismo , Mapeo Restrictivo
9.
J Immunol ; 154(3): 1157-66, 1995 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-7822791

RESUMEN

We attempted to isolate novel receptor tyrosine kinase, which may play a role in hematopoietic development by screening for expressed sequences with conserved tyrosine kinase catalytic domains. Among the known tyrosine kinases identified in this screen, we found a gene with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids and placing it as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, we have found that ryk expression is not ubiquitous. Expression increased in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk Ab demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells whereas Ryk+ cells represent only 0.3% of the Lin- population. We conclude that ryk expression is regulated during hematopoietic development by lineage commitment and stage of maturation.


Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Superficie Celular/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células de la Médula Ósea , Línea Celular , Mapeo Cromosómico , Femenino , Citometría de Flujo , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa
10.
Blood ; 82(9): 2673-83, 1993 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-8219220

RESUMEN

Approximately 4% to 5% of all bone marrow (BM) cells and 8% to 9% of low density BM cells from FVB/N and BALB/c mice (Ly-6a haplotype) show high to intermediate expression of Ly-6E.1 antigen, recognized by the Sca-1 antibody. Functional properties of enriched cells expressing Ly-6E.1-allelic form of Sca-1 antigen were analyzed and correlated with the properties of cells expressing the carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). Using equilibrium density centrifugation and fluorescence-activated cell sorting, Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ cells were isolated and their splenic colony-forming unit (CFU-S) cell content, radioprotection ability, and long-term reconstitution capacity determined. Enriched Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+ and Lin-Sca-1+WGA+ cells gave rise to 1 CFU-S12 cell out of 26, 20, 21, and 15 sorted cells, respectively. When transplanted into lethally irradiated recipients (100 to 500 cells/mouse) all populations rescued 70% to 100% of recipients in a 30-day radioprotection assay and mediated survival of 40% to 80% of recipients 6 months after transplantation. Using transgenic mice as cell donors we have shown that 12 to 16 weeks after transplantation of 100 Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ cells, 40% to 80% of recipients had donor cells in BM, spleen, thymus, and lymph nodes. These results indicate that the population of cells expressing Ly-6E.1 form of Sca-1 antigen in two analyzed mouse strains with Ly-6a haplotype contains CFU-S and long-term repopulating cells. Furthermore, the data suggest that, at least in FVB/N mice, day-12 CFU-S cells and cells with long-term repopulating capacity simultaneously express Ly-6E.1 form of Sca-1 antigen and WGA-binding molecules.


Asunto(s)
Antígenos Ly/análisis , Antígenos Ly/genética , Células de la Médula Ósea , Células Madre Hematopoyéticas/fisiología , Proteínas de la Membrana/análisis , Aglutininas del Germen de Trigo/metabolismo , Animales , Antígenos Ly/fisiología , Secuencia de Bases , Trasplante de Médula Ósea , Supervivencia Celular , Haplotipos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Protección Radiológica
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