RESUMEN
Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.
Asunto(s)
Cromatina/metabolismo , Metilación de ADN , Genes Supresores de Tumor , Células Madre/metabolismo , Adulto , Proliferación Celular , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Histonas/metabolismo , Humanos , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Células Tumorales CultivadasRESUMEN
Relapse of acute myeloid leukemia (AML) is thought to reflect the failure of current therapies to adequately target leukemia stem cells (LSCs), the rare, resistant cells presumed responsible for maintenance of the leukemia and typically enriched in the CD34(+)CD38(-) cell population. Despite the considerable research on LSCs over the past 2 decades, the clinical significance of these cells remains uncertain. However, if clinically relevant, it is expected that LSCs would be enriched in minimal residual disease and predictive of relapse. CD34(+) subpopulations from AML patients were analyzed by flow cytometry throughout treatment. Sorted cell populations were analyzed by fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities (when present) and by transplantation into immunodeficient mice to determine self-renewal capacity. Intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity reliably distinguished leukemic CD34(+)CD38(-) cells capable of engrafting immunodeficient mice from residual normal hematopoietic stem cells that exhibited relatively higher ALDH activity. Minimal residual disease detected during complete remission was enriched for the CD34(+)CD38(-)ALDH(int) leukemic cells, and the presence of these cells after therapy highly correlated with subsequent clinical relapse. ALDH activity appears to distinguish normal from leukemic CD34(+)CD38(-) cells and identifies those AML cells associated with relapse.
Asunto(s)
Antígenos CD34/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , ADP-Ribosil Ciclasa 1/metabolismo , Adulto , Anciano , Animales , Separación Celular/métodos , Células Cultivadas , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Pronóstico , Recurrencia , Adulto JovenRESUMEN
Both plasticity and cell fusion have been suggested to have a role in germ-layer switching. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs) in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2-7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.
Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula/genética , Estratos Germinativos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Animales , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Señales (Psicología) , Medios de Cultivo Condicionados/farmacología , Líquido Extracelular/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Estratos Germinativos/citología , Supervivencia de Injerto/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Hepatocitos/citología , Regeneración Hepática/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ploidias , Cromosomas Sexuales/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34(+) stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34(+)CD38(-) CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34(+)CD38(-)ALDH(high) CML cells, similar to the normal CD34(+)CD38(-)ALDH(high) cells and less than in the total CML CD34(+) cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34(+) cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34(+) subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34(+) population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , ADP-Ribosil Ciclasa 1/biosíntesis , Adulto , Anciano , Aldehído Deshidrogenasa/biosíntesis , Animales , Antígenos CD34/biosíntesis , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Femenino , Células Madre Hematopoyéticas/inmunología , Humanos , Proteínas Inhibidoras de la Apoptosis , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Mieloblastina/biosíntesis , Mieloblastina/genética , Trasplante de Neoplasias , ARN Mensajero/biosíntesis , Survivin , Telomerasa/biosíntesis , Telomerasa/genética , Proteínas WT1/biosíntesis , Proteínas WT1/genéticaRESUMEN
A major challenge facing investigators working in the field of hematopoietic stem cell (HSC) biology has been to develop a strategy to purify rare primitive HSCs from bone marrow. Several methods have been available including the commonly used technique of isolating HSCs based on a specific cell-surface phenotype. As surface marker expression is dynamic and may fluctuate depending on the proliferative or activation state of the cell, our laboratory has established a unique functional in vivo assay (the 2-day homing assay) to isolate murine HSCs. This protocol selects for HSCs on the basis of their ability to home to bone marrow and yields a population that can reconstitute the murine hematopoietic system with the transplantation of a single cell. In contrast to other methods that use specific cell-surface antigens to acquire HSCs, our functional assay aids in obtaining a primitive HSC that exhibits both hematopoietic and epithelial engraftment capabilities. The 2-day homing protocol involves harvesting whole bone marrow and performing a physical separation method (elutriation) to acquire a fraction of small-sized cells (fraction 25). Fraction 25 cells are then depleted of later progenitors and differentiated hematopoietic cells, labeled with a fluorescent tracking dye and transplanted into lethally irradiated recipient mice. Two days after transplantation, the bone marrow is harvested from the primary recipient, and HSCs that have homed to the bone marrow are collected by fluorescence-activated cell sorting. In addition to the traditional 2-day homing protocol, we have included in this chapter our recently developed method of using density gradient centrifugation to replace the elutriation step that also selects for a primitive HSC.
Asunto(s)
Células Madre Hematopoyéticas/citología , Animales , Separación Celular , Trasplante de Células Madre Hematopoyéticas , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separation of whole bone marrow into small-sized cells by counter-flow elutriation. We sought to provide the scientific community with an alternate approach to acquire our stem cells by replacing elutriation with the use of density-gradient centrifugation. METHODS: The elutriated fraction 25 population was characterized based on density using a discontinuous gradient. The long-term reconstituting potential of whole bone marrow cells collected at each density interface was determined by subjecting the fractions to the two-day homing protocol, transplanting them into lethally irradiated recipient mice, and assessing peripheral blood chimerism. We also investigated the ability of high-density bone marrow cells isolated in conjunction with the ALDH protocol to repopulate the hematopoietic system of myeloablated recipients. RESULTS: Bone marrow cells collected at the high-density interface of 1.081/1.087 g/mL (fraction 3) had the capacity for homing to marrow and the ability to provide long-term hematopoietic reconstitution. Fraction three lineage-depleted ALDH-bright cells could also engraft and provide long-term hematopoiesis at limiting dilutions. CONCLUSIONS: Density-gradient centrifugation can be used in conjunction with either of our stem cell isolation protocols to obtain cells with long-term reconstitution ability. We anticipate that this strategy will encourage and enable investigators to study the biology of HSCs isolated using functional characteristics.
Asunto(s)
Células de la Médula Ósea/citología , Separación Celular/métodos , Células Madre Hematopoyéticas/citología , Aldehído Deshidrogenasa/inmunología , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/efectos de la radiación , Centrifugación por Gradiente de Densidad/métodos , Activación Enzimática , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Tasa de Supervivencia , Acondicionamiento Pretrasplante , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
AIMS: The transgenic enhanced green fluorescent protein (EGFP) expressing 'green' mouse (C57BL/6-TgN(ACTbEGFP)1Osb) is a widely used tool in stem cell research, where the ubiquitous nature of EGFP expression is critical to track the fate of single or small groups of transplanted haematopoietic stem cells (HSC). Our aim was to investigate this assumed ubiquitous expression by performing a detailed histological survey of EGFP expression in these mice. METHODS: Fluorescent microscopy of frozen tissue sections was used to perform a detailed histological survey of the pattern of EGFP expression in these mice. Flow cytometry was also used to determine the expression pattern in blood and bone marrow. RESULTS: Three patterns of EGFP expression were noted. In most tissues there was an apparently stochastic variegation of the transgene, with individual cell types demonstrating highly variable rates of EGFP expression. Certain specific cell types such as pancreatic ductal epithelium, cerebral cortical neurones and glial cells and glomerular mesangial cells consistently lacked EGFP expression, while others, including pancreatic islet cells, expressed EGFP only at extremely low levels, barely distinguishable from background. Lastly, in the colon and stomach the pattern of EGFP expression was suggestive of clonal inactivation. Only cardiac and skeletal muscle showed near ubiquitous expression. CONCLUSIONS: These findings raise questions regarding the 'ubiquitous' expression of EGFP in these transgenic mice and suggest caution in relying overly on EGFP alone as an infallible marker of donor cell origin.
Asunto(s)
Investigación Biomédica/métodos , Linaje de la Célula , Proteínas Fluorescentes Verdes/biosíntesis , Modelos Animales , Células Madre , Animales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Microscopía FluorescenteRESUMEN
Purification to homogeneity for a rare stem cell (SC) population by both function and phenotype is a prerequisite to determine if SCs can change their fate (plasticity). Since cell fate determination has been suggested by both external environmental cues and intrinsic gene regulation, plasticity should be studied using both influences. Different frequencies of marrow SC plasticity may be attributed to either different isolation technologies or different developmental stage SCs with more or less multipotentiality. Tissue-specific SCs may reside in marrow, or alternatively, primitive marrow SC may respond directly to regenerative signals by migration to injury sites and repairing the damaged tissue. It is important to dissect the relationship between primitive/tissue-specific SCs and regenerative signals.
Asunto(s)
Células Madre/citología , Células Madre/fisiología , Diferenciación Celular , Fusión Celular , Mapeo Cromosómico , Células Epiteliales/citología , Células Epiteliales/fisiología , Técnicas de Transferencia de Gen , Marcadores Genéticos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , ARN Mensajero/genética , Donantes de TejidosRESUMEN
OBJECTIVES: The purpose of this study was to investigate cell fates and long-term repopulating potential of a primitive hematopoietic stem cell (HSC) population (i.e., FR25Lin(-) cells) in vitro. MATERIALS AND METHODS: FR25Lin(-) cells were isolated by elutriation and cell sorting and cultured with a combination of cytokines for 7 days. Utilizing the membrane dye PKH-26, cultured cells were separated into two subsets based on their proliferation rates and assayed for progenitors and HSC. RESULTS: Fresh FR25Lin(-) cells were mostly quiescent; however, some of this population entered cell cycle after cytokine exposure reaching a peak 4 to 5 days after culture. Two subsets of cultured cells were isolated: 1) cells that had divided several times (PKH(dull) cells) and 2) cells that remained undivided or divided only once or twice (PKH(bright) cells). The PKH(dull) cells accounted for 94% of total viable cells in culture after 5 days. The PKH(dull) subset contained all the multi-potential in vivo progenitors (CFU-S) and 10 times more committed progenitors (CFU-C). Quantitative analysis of HSC engraftment from the PKH(bright) subset demonstrated stem cell maintenance. For the PKH(dull) subset, on day 5, HSC numbers increased. By day 7, increased differentiation in the PKH(dull) population supports expanding differentiation divisions. CONCLUSIONS: Our primitive HSC population underwent different types of cell divisions stimulated by cytokines, resulting in subsets with different self-renewal and differentiation potentials. This in vitro/in vivo model provides a useful tool for studies of early events during HSC self-renewal and differentiation.
Asunto(s)
Diferenciación Celular , División Celular , Células Madre Hematopoyéticas/citología , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Cruzamientos Genéticos , Sinergismo Farmacológico , Femenino , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-6/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Quimera por Radiación , Proteínas Recombinantes/farmacología , Factor de Células Madre/farmacología , Trombopoyetina/farmacologíaRESUMEN
It has recently been shown that specific lineage-depleted murine hematopoietic stem cells that home to the bone marrow 2 days after transplantation of ablated primary recipients are capable of long-term engraftment and repopulation of secondary recipients. We were interested in determining whether the rate at which the ablating radiation dose was delivered to the mice affected the homing of lineage-depleted stem cells to the bone marrow and/or sites of tissue damage. Fractionated, lineage-depleted donor marrow cells were isolated and labeled with the membrane dye PKH26. Recipient mice were lethally irradiated with 11 Gy ionizing radiation using varying dose rates and were immediately injected with PKH26-labeled progenitor stem cells. With the exception of the lowest dose-rate group, all irradiated mice had an approximately fivefold (P = 0.014 to 0.025) reduction in stem cell homing to the bone marrow compared to unirradiated control animals. A fivefold reduction of stem cell homing to the spleen compared to unirradiated animals was also observed, though this was not statistically significant for any dose-rate group (P = 0.072 to 0.233). This difference in homing could not be explained by increased stem cell apoptosis/necrosis or non-marrow tissue homing to the intestine, lung or liver. We show that the dose rate at which a lethal dose of total-body radiation is delivered does not augment hematopoietic progenitor stem cell homing to the bone marrow, spleen or sites of early radiation-mediated tissue damage at either 2 or 5 days postirradiation/transplantation. The observation that greater homing was seen in unirradiated control mice calls into question the concept that adequate bone marrow stem cell homing requires radiation-induced "space" to be made in the marrow, certainly for the enriched early progenitor hematopoietic stem cells used for this set of experiments. Further experiments will be needed to determine whether these homed cells are as capable of giving rise to long-term engraftment/repopulation of the marrow of secondary recipients as they are in irradiated recipients.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Animales , Movimiento Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Tolerancia a Radiación , Irradiación Corporal TotalRESUMEN
The development of induced pluripotent stem cell (iPSC) technology has generated enthusiasm about the therapeutic potential of these cells for treating a variety of diseases. However, the evidence that they actually will be clinically useful is limited. Here, we discuss the potential therapeutic applications of iPSCs for treating cancer and other diseases and highlight the current barriers restricting their use.
Asunto(s)
Neoplasias/terapia , Células Madre Pluripotentes/citología , Trasplante de Células Madre , Células Madre Adultas/citología , Animales , Neoplasias Hematológicas/terapia , Humanos , Investigación Biomédica TraslacionalRESUMEN
Internal tandem duplication (ITD) mutations within the FMS-like tyrosine kinase-3 (FLT3) render the receptor constitutively active driving proliferation and survival in leukemic blasts. Expression of FLT3-ITD from the endogenous promoter in a murine knockin model results in progenitor expansion and a myeloproliferative neoplasm. In this study, we show that this expansion begins with overproliferation within a compartment of normally quiescent long-term hematopoietic stem cells (LT-HSCs), which become rapidly depleted. This depletion is reversible upon treatment with the small molecule inhibitor Sorafenib, which also ablates the disease. Although the normal LT-HSC has been defined as FLT3(-) by flow cytometric detection, we demonstrate that FLT3 is capable of playing a role within this compartment by examining the effects of constitutively activated FLT3-ITD. This indicates an important link between stem cell quiescence/homeostasis and myeloproliferative disease while also giving novel insight into the emergence of FLT3-ITD mutations in the evolution of leukemic transformation.
Asunto(s)
Neoplasias de la Médula Ósea/patología , Duplicación de Gen , Técnicas de Sustitución del Gen , Células Madre Hematopoyéticas/patología , Homeostasis , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Neoplasias de la Médula Ósea/genética , Compartimento Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Duplicación de Gen/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/genética , Masculino , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Niacinamida/análogos & derivados , Niacinamida/farmacología , Fenotipo , Compuestos de Fenilurea/farmacología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células de Población Lateral/efectos de los fármacos , Células de Población Lateral/metabolismo , Células de Población Lateral/patología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Sorafenib , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses, without causing immediate cytotoxicity, produce an antitumor "memory" response, including inhibition of subpopulations of cancer stem-like cells. These effects are accompanied by sustained decreases in genomewide promoter DNA methylation, gene reexpression, and antitumor changes in key cellular regulatory pathways. Low-dose decitabine and azacitidine may have broad applicability for cancer management.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Leucemia/genética , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Daño del ADN , Decitabina , Silenciador del Gen , Humanos , Leucemia/tratamiento farmacológico , Leucemia/patología , Ratones , Datos de Secuencia Molecular , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Transducción de Señal , Células Tumorales CultivadasRESUMEN
BACKGROUND: Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)(+) transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+) BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+) cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+) and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner. CONCLUSIONS/SIGNIFICANCE: These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.
Asunto(s)
Vasos Sanguíneos/citología , Células de la Médula Ósea/citología , Hipoxia/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Pulmón/irrigación sanguínea , Animales , Western Blotting , Trasplante de Médula Ósea , Quimiotaxis , Dependovirus/genética , Femenino , Vectores Genéticos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía FluorescenteRESUMEN
OBJECTIVE: To investigate the mechanism of glycosylphosphatidylinositol (GPI) anchor deficiency in Burkitt lymphoma cell lines. METHODS: We identified a large GPI anchor protein deficient population in three different Burkitt lymphoma cell lines through proaerolysin treatment of the cells and flow cytometry analysis using a proaerolysin variant (FLAER). The mechanism of GPI anchor protein deficiency was studied by DNA gene sequencing, a cell-free assay to investigate the GPI anchor biosynthetic pathway, microarray analysis, and quantitative real-time polymerase chain reaction. RESULTS: Burkitt lymphoma cell lines harbor large populations of FLAER(neg) cells, which are resistant to proaerolysin. In all three cell lines, silencing of a gene involved in an early step in GPI-anchor biosynthesis was responsible for the lack of GPI-anchored proteins on the cell surface. Quantitative polymerase chain reaction and microarray analysis demonstrate that the level of mRNA for PIGL and PIGY is lower in the FLAER(neg) Ramos cells and that mRNA levels of PIGY are reduced in the Akata and Daudi cells. Hypermethylation of these genes was associated with the low levels of mRNA and treatment of the cells with 5-aza-2' deoxycytidine restored cell surface GPI-anchored proteins to the FLAER(neg) cells. CONCLUSION: GPI-anchored protein deficiency in Burkitt lymphoma cells is not due to a genetic mutation (e.g., PIGA); rather, the lack of GPI-anchored proteins results from transcriptional silencing of PIGL and PIGY.
Asunto(s)
Linfoma de Burkitt/metabolismo , Silenciador del Gen , Glicosilfosfatidilinositoles/biosíntesis , Linfoma de Burkitt/genética , Línea Celular Tumoral , Citometría de Flujo , Glicosilfosfatidilinositoles/deficiencia , Glicosilfosfatidilinositoles/genética , Sistema Hematopoyético/citología , Humanos , Inmunohistoquímica , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
What controls the inherent differences between fetal and adult hematopoietic stem cells (HSCs)? In this issue of Cell, Kim et al. (2007) demonstrate in mice that the endodermal transcription factor Sox17 is required for the maintenance of fetal and neonatal but not adult HSCs.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Células Madre Fetales/citología , Células Madre Fetales/metabolismo , Proteínas HMGB/deficiencia , Proteínas HMGB/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Proteínas HMGB/fisiología , Ratones , Factores de Transcripción SOXF , Factores de Transcripción/fisiologíaRESUMEN
A low-oxygenic niche in bone marrow limits reactive oxygen species (ROS) production, thus providing long-term protection for hematopoietic stem cells (HSCs) from ROS stress. Although many approaches have been used to enrich HSCs, none has been designed to isolate primitive HSCs located within the low-oxygenic niche due to difficulties of direct physical access. Here we show that an early HSC population that might reside in the niche can be functionally isolated by taking advantage of the relative intracellular ROS activity. Many attributes of primitive HSCs in the low-oxygenic osteoblastic niche, such as quiescence, and calcium receptor, N-cadherin, Notch1, and p21 are higher in the ROS(low) population. Intriguingly, the ROS(low) population has a higher self-renewal potential. In contrast, significant HSC exhaustion in the ROS(high) population was observed following serial transplantation, and expression of activated p38 mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) was higher in this population. Importantly, treatment with an antioxidant, a p38 inhibitor, or rapamycin was able to restore HSC function in the ROS(high) population. Thus, more potent HSCs associated with the low-oxygenic niche can be isolated by selecting for the low level of ROS expression. The ROS-related signaling pathways together with specific characteristics of niche HSCs may serve as targets for beneficial therapies.
Asunto(s)
Técnicas Citológicas/métodos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Oxígeno , Especies Reactivas de Oxígeno , Animales , Western Blotting , Trasplante de Médula Ósea , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Femenino , Citometría de Flujo , Fluoresceínas , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/metabolismo , Serina-Treonina Quinasas TOR , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a self-renewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.
Asunto(s)
Activinas/farmacología , Proteínas Morfogenéticas Óseas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Subunidades beta de Inhibinas/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Proteínas Wnt/metabolismo , Aneuploidia , Animales , Proteínas Morfogenéticas Óseas/genética , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Embrión de Mamíferos/citología , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Pluripotentes/citología , Transducción de Señal/efectos de los fármacos , Trasplante de Células Madre/efectos adversos , Teratoma/etiología , Trasplante Heterólogo , Proteínas Wnt/genéticaRESUMEN
The continuing interest in the biology of stem cells is enhanced by new discoveries surrounding developmental plasticity of both embryonic and adult stem cells. Adoptive transfer of concepts and definitions from the hematopoietic system to other tissue stem cells suggests inclusion of characteristics such as ability to self-renew and differentiate to functionally reconstitute a tissue/organ of origin. How adequate and accurate are these definitions? Within the great unknown of how these cells function, modulate their gene expression patterns and respond to extrinsic signals, it is apparent that there are numerous levels of stemness. We may envision a scale of developmental flexibility. At one end of the scale are positioned the embryonic stem cells, and at the other end are positioned partially-differentiated, differentiation restricted (committed) tissue/organ stem cells. There is evidence that some stem cells in the adult are pluripotent, thus positioned close to the embryonic end of the stem scale. It is uncertain yet to what extent stem cells can move back and forth along the stem scale.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Plasticidad Neuronal/fisiología , Células Madre/fisiología , Antígenos CD/análisis , Ciclo Celular , Diferenciación Celular , División Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Fenotipo , Células Madre/citologíaRESUMEN
PURPOSE: Using various nonphysiological tissue injury/repair models numerous studies have demonstrated the capacity of bone marrow derived cells to contribute to the repopulation of epithelial tissues following damage. To investigate whether this phenomenon might also occur during periods of physiological tissue degeneration/regeneration we compared the ability of bone marrow derived cells to rejuvenate the prostate gland in mice that were castrated and then later treated with dihydrotestosterone vs mice with prostate epithelium that had been damaged by lytic virus infection. MATERIALS AND METHODS: Using allogenic bone marrow grafts from female donor transgenic mice expressing green fluorescent protein transplanted into lethally irradiated males we were able to assess the contributions of bone marrow derived cells to recovery of the prostatic epithelium in 2 distinct systems, including 1) surgical castration followed 1 week later by dihydrotestosterone replacement and 2) intraprostatic viral injection. Eight to 10-week-old male C57/Bl6 mice were distributed among bone marrow donor-->recipient/prostate injury groups, including 5 with C57/Bl6-->C57/Bl6/no injury, 3 with green fluorescent protein-->C57/Bl6/no injury, 3 with green fluorescent protein-->C57/Bl6/vehicle injection, 4 with green fluorescent protein-->C57/Bl6/virus injection and 3 each with green fluorescent protein-->C57/Bl6/castration without and with dihydrotestosterone, respectively. Prostate tissues were harvested 3 weeks after dihydrotestosterone replacement or 14 days following intraprostatic viral injection. Prostate tissue immunofluorescence was performed with antibodies against the epithelial marker cytokeratin 5/8, the hematopoietic marker CD45 and green fluorescent protein. RESULTS: Mice that sustained prostate injury from vaccinia virus infection with concomitant severe inflammation and glandular disruption showed evidence of bone marrow derived cell reconstitution of prostate epithelium, that is approximately 4% of all green fluorescent protein positive cells in the epithelial compartment 14 days after injury expressed cytokeratin 5/8, similar to the proportion of green fluorescent protein positive cells in the prostate that no longer expressed the hematopoietic marker CD45. When prostatic degeneration/regeneration was triggered by androgen deprivation and reintroduction, no green fluorescent protein positive prostate epithelial cells were detected. CONCLUSIONS: These findings are consistent with a requirement for inflammation associated architectural destruction for the bone marrow derived cell contribution to the regeneration of prostate epithelium.