RESUMEN
Male infertility is responsible for approximately half of all cases of reproductive issues. Spermatogenesis originates in a small pool of spermatogonial stem cells (SSCs), which are of interest for therapy of infertility but remain not well defined in humans. Using multiparametric analysis of the side population (SP) phenotype and the α-6 integrin, THY1, and ß-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype ß-2 microglobulin (ß-2M)-SPα-6+THY1+, which is highly enriched in stem cells. By analyzing the expression signatures of this SSC-enriched population along with other germinal progenitors, we established an exhaustive transcriptome of human spermatogenesis. Transcriptome profiling of the human ß-2M-SPα-6+THY1+ population and comparison with the profile of mouse undifferentiated spermatogonia provide insights into the molecular networks and key transcriptional regulators regulating human SSCs, including the basic-helix-loop-helix (bHLH) transcriptional repressor HES1, which we show to be implicated in maintenance of SSCs in vitro.
Asunto(s)
Células Madre Germinales Adultas , Espermatogénesis , Animales , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Espermatogénesis/genética , Espermatogonias/metabolismo , Células Madre/metabolismo , Testículo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events.
Asunto(s)
Células Madre Germinales Adultas/metabolismo , Técnicas de Reprogramación Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre Germinales Adultas/efectos de los fármacos , Animales , Hipoxia de la Célula , Linaje de la Célula , Células Cultivadas , Reprogramación Celular/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Proteína-1 Reguladora de Fusión/genética , Proteína-1 Reguladora de Fusión/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Células Madre Pluripotentes Inducidas/patología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre Embrionarias de Ratones/efectos de los fármacos , Factor 3 de Transcripción de Unión a Octámeros/genética , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción SOXB1/genética , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In adults, stem cells are responsible for the maintenance of many actively renewing tissues, such as haematopoietic, skin, gut and germinal tissues. These stem cells can self-renew or be committed to becoming progenitors. Stem-cell commitment is thought to be irreversible but in male and female Drosophila melanogaster, it was shown recently that differentiating germ cells can revert to functional stem cells that can restore germinal lineage. Whether progenitors are also able to generate stem cells in mammals remains unknown. Here we show that purified mouse spermatogonial progenitors committed to differentiation can generate functional germinal stem cells that can repopulate germ-cell-depleted testes when transplanted into adult mice. We found that GDNF, a key regulator of the stem-cell niche, and FGF2 are able to reprogram in vitro spermatogonial progenitors for reverse differentiation. This study supports the emerging concept that the stem-cell identity is not restricted in adults to a definite pool of cells that self-renew, but that stemness could be acquired by differentiating progenitors after tissue injury and throughout life.
Asunto(s)
Desdiferenciación Celular/genética , Linaje de la Célula/genética , Células Germinativas/metabolismo , Espermatogonias/metabolismo , Células Madre/metabolismo , Animales , Desdiferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Germinativas/citología , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regeneración/efectos de los fármacos , Regeneración/fisiología , Espermatogonias/citología , Trasplante de Células Madre/métodos , Células Madre/citología , Testículo/citología , Testículo/metabolismoAsunto(s)
Células de la Médula Ósea/citología , Espermatogénesis , Células Madre/citología , Animales , Células de la Médula Ósea/efectos de los fármacos , Busulfano/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Espermatogénesis/efectos de los fármacos , Células Madre/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacosRESUMEN
The goal of our study was to identify a subset of genes commonly expressed in Side Populations (SP), isolated by Hoechst staining followed by flow cytometry, from adult mouse bone marrow, male adult germinal cells, muscle primary culture, and mesenchymal cells. These SP cells have been proposed to be a "stem-like" population and are used here as a "model" that may reveal mechanisms which would be relevant for a better understanding of stem cell properties. Transcriptional profiles for SP and the more differentiated non-SP cells isolated from the four tissues were compared by hybridization on microarray using a common external reference. Among the 503 genes differentially expressed, which discriminate SP and non-SP cells in all the tissues, the genes upregulated in SP cells are implicated in the quiescent status of the cells, the maintenance of their pluripotency and the capacity to undergo asymmetric division. These genes may be responsible for the decision for self-renewal of these cells, whereas the repression of lineage-affiliated genes in SP cells could be responsible for their undifferentiated state. These genes, acting in concert, may be the key players that mediate the mechanisms that control stem cell functions, and our results suggest that we have identified common "stemness functions" of these "stem-like" cells.
Asunto(s)
Células de la Médula Ósea/clasificación , Células de la Médula Ósea/metabolismo , Perfilación de la Expresión Génica , Centro Germinal/metabolismo , Mesodermo/metabolismo , Células Musculares/metabolismo , Células Madre/metabolismo , Animales , Células de la Médula Ósea/citología , Línea Celular , Separación Celular , Células Cultivadas , Centro Germinal/citología , Masculino , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Células Musculares/citología , Células Madre/citologíaRESUMEN
Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelial cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having "side population" phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.
Asunto(s)
Células Endoteliales/citología , Antígeno Lewis X/biosíntesis , Neuronas/citología , Células Madre/citología , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Endoteliales/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/fisiología , Fenotipo , Células Madre/fisiologíaRESUMEN
Testis is one of the organs with the most telomerase activity in the adult. This activity protects chromosomes from telomere attrition and ensures the transmission of full-length chromosomes to progeny. Little is known about telomerase activity during adult germ cell differentiation, however. We demonstrate here that the telomerase activity of adult mouse testis resides in the alpha6-integrin-positive Side Population containing spermatogonia and enriched in spermatogonial stem cells. The telomerase activity of these cells fell upon entry into meiosis and during the subsequent spermiogenesis. In addition, the telomerase activity of cells in various stages of differentiation was unaffected by aging and, notably, remained high in the alpha6-integrin-positive Side Population.
Asunto(s)
Integrina alfa6/metabolismo , Espermatogonias/enzimología , Células Madre/enzimología , Telomerasa/metabolismo , Testículo/citología , Factores de Edad , Envejecimiento/fisiología , Animales , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Espermatogénesis/fisiología , Tetraspanina 29 , Antígenos Thy-1/metabolismoRESUMEN
BACKGROUND: Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. METHODS: Hoechst 33342 staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. RESULTS: Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst 33342 staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst 33342 red fluorescence pattern. CONCLUSIONS: Hoechst 33342 staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis.
Asunto(s)
Bencimidazoles/química , Citometría de Flujo/métodos , Meiosis , Fármacos Sensibilizantes a Radiaciones/química , Espermatogénesis/fisiología , Testículo/citología , Animales , Rayos gamma , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Espermatocitos/metabolismo , Espermatocitos/efectos de la radiación , Espermatogénesis/efectos de la radiación , Coloración y Etiquetado , Testículo/efectos de la radiaciónRESUMEN
Stem cells in various somatic tissues (bone marrow, skeletal muscle) can be identified by the 'Side Population' marker based on Hoechst 33342 efflux. We show that mouse testicular cells also display a 'Side Population' that express Bcrp1 mRNA, the ABC transporter responsible for Hoechst efflux in hematopoietic cells. Inhibition of Hoechst efflux by specific BCRP1 inhibitor Ko143 show that germinal 'Side Population' phenotype is dependent on BCRP1 activity. Analysis of two well-defined models of altered spermatogenesis (W/Wv mutants and cryptorchid male mice) and RNA expression studies of differentiation markers demonstrate that germinal 'Side Population' contains spermatogonial cells. In addition, alpha 6-integrin and Stra8 germinal stem cell markers, are expressed in the 'Side Population'. In vivo repopulation assay clearly establishes that testis 'Side Population' in adult mice is highly enriched in male germ stem cells.