RESUMEN
Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.
Asunto(s)
Supervivencia de Injerto/fisiología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/citología , Bazo/citología , Animales , Antígenos CD34/metabolismo , Femenino , Citometría de Flujo/métodos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inyecciones , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Bazo/metabolismo , Quimera por Trasplante , Trasplante HeterólogoRESUMEN
Among the genetic factors playing a key role in the etiology of intellectual disabilities (IDs) and autism spectrum disorders (ASDs), several encode RNA-binding proteins (RBPs). In this study, we deciphered the molecular and cellular bases of ID-ASD in a patient followed from birth to the age of 21, in whom we identified a de novo CSDE1 (Cold Shock Domain-containing E1) nonsense variation. CSDE1 encodes an RBP that regulates multiple cellular pathways by monitoring the translation and abundance of target transcripts. Analyses performed on the patient's primary fibroblasts showed that the identified CSDE1 variation leads to haploinsufficiency. We identified through RNA-seq assays the Wnt/ß-catenin signaling and cellular adhesion as two major deregulated pathways. These results were further confirmed by functional studies involving Wnt-specific luciferase and substrate adhesion assays. Additional data support a disease model involving APC Down-Regulated-1 (APCDD1) and cadherin-2 (CDH2), two components of the Wnt/ß-catenin pathway, CDH2 being also pivotal for cellular adhesion. Our study, which relies on both the deep phenotyping and long-term follow-up of a patient with CSDE1 haploinsufficiency and on ex vivo studies, sheds new light on the CSDE1-dependent deregulated pathways in ID-ASD.
Asunto(s)
Trastorno del Espectro Autista , Proteínas de Unión al ADN , Discapacidad Intelectual , Proteínas de Unión al ARN , Vía de Señalización Wnt , Adolescente , Trastorno del Espectro Autista/genética , Adhesión Celular/genética , Niño , Preescolar , Proteínas de Unión al ADN/genética , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/genética , Proteínas de Unión al ARN/genética , Adulto Joven , beta Catenina/genéticaRESUMEN
INTRODUCTION: Inflammatory Breast Cancer (IBC) is the most aggressive form of breast carcinoma characterized by rapid onset of inflammatory signs and its molecular fingerprint has not yet been elucidated. OBJECTIVES: The objective of this study was to detect both gene expression levels and alternate RNA splice variants specific for IBC. METHODS: W e performed splice-sensitive array profiling using Affymetrix Exon Array and quantitative RT-PCR analyses in 177 IBC compared to 183 non-IBC. We also assessed the prognostic value of the identified candidate genes and splice variants. RESULTS: A 5-splice signature (HSPA8, RPL10, RPL4, DIDO1 and EVL) was able to distinguish IBC from non-IBC tumors (p<10-7). This splice signature was associated with poor metastasis-free survival in hormone receptor-negative non-IBC (p=0.02), but had no prognostic value in IBC. PAM analysis of dysregulated genes in IBC compared to non-IBC identified a 10-gene signature highly predictive of IBC phenotype and conferring a poor prognosis in non-IBC. The genes most commonly upregulated in IBC were 3 hemoglobin genes able to reliably discriminate IBC from non-IBC (p<10-4). Hb protein expression in epithelial breast tumor cells was confirmed by immunohistochemistry. CONCLUSION: IBC has a specific spliced transcript profile and is characterized by hemoglobin gene overexpression that should be investigated in further functional studies.
RESUMEN
OBJECTIVES: The first-passage adherent human bone marrow fibroblast-like cell population corresponds, in terms of phenotype and three-lineage differentiation capacity (assayed in bulk culture), to commonly termed "mesenchymal stem cells". Here we determine the proportion of high proliferative capacity multipotent cells present in this population in order to estimate the proportion of cells that can or cannot be considered as stem and progenitor cells. MATERIAL AND METHODS: The single-cell cultures were established starting from human bone marrow-derived first-passage fibroblast-like cells and the proliferating clones were either transferred to secondary cultures to evaluate their further clonogenicity, or split into three wells to assess differentiation into each of the three different lineages. RESULTS: The analysis of 197 single-cell cultures from three different bone marrow donors shows that onlyâ¼40% of so-called "mesenchymal stem cells" exhibit multipotency and are capable of sustained clonogenicity in secondary cultures. CONCLUSION: Even in the first ex vivo passage under favorable conditions the majority (â¼60%) of so-called "mesenchymal stem cells" are not multipotent and thus do not represent a stem cell entity.
Asunto(s)
Células Madre Mesenquimatosas/citología , Antígenos CD/análisis , Células de la Médula Ósea/clasificación , Adhesión Celular , División Celular , Linaje de la Célula , Autorrenovación de las Células , Separación Celular , Células Cultivadas , Células Clonales/citología , Ensayo de Unidades Formadoras de Colonias , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Análisis de la Célula Individual , Células del Estroma/citologíaRESUMEN
Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/ß-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/ß-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNßcat mice displaying a constitutive activation of Wnt/ß-catenin signaling in basal cells. K5ΔNßcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNßcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and ß-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.
Asunto(s)
Células Madre Neoplásicas/patología , Neoplasias de la Mama Triple Negativas/patología , Proteína p53 Supresora de Tumor/deficiencia , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Eliminación de Gen , Ratones , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Proteína p53 Supresora de Tumor/genética , beta Catenina/metabolismoRESUMEN
TAF15 (formerly TAFII68) is a member of the FET (FUS, EWS, TAF15) family of RNA- and DNA-binding proteins whose genes are frequently translocated in sarcomas. By performing global gene expression profiling, we found that TAF15 knockdown affects the expression of a large subset of genes, of which a significant percentage is involved in cell cycle and cell death. In agreement, TAF15 depletion had a growth-inhibitory effect and resulted in increased apoptosis. Among the TAF15-regulated genes, targets of microRNAs (miRNAs) generated from the onco-miR-17 locus were overrepresented, with CDKN1A/p21 being the top miRNAs-targeted gene. Interestingly, the levels of onco-miR-17 locus coded miRNAs (miR-17-5p and miR-20a) were decreased upon TAF15 depletion and shown to affect the post-transcriptional regulation of TAF15-dependent genes, such as CDKN1A/p21. Thus, our results demonstrate that TAF15 is required to regulate gene expression of cell cycle regulatory genes post-transcriptionally through a pathway involving miRNAs. The findings that high TAF15 levels are needed for rapid cellular proliferation and that endogenous TAF15 levels decrease during differentiation strongly suggest that TAF15 is a key regulator of maintaining a highly proliferative rate of cellular homeostasis.