RESUMEN
Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.
Asunto(s)
Diferenciación Celular , Hemangioblastos , Células Madre Hematopoyéticas , Factor de Células Madre , Trombopoyetina , Animales , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Ratones , Trombopoyetina/metabolismo , Factor de Células Madre/metabolismo , Hemangioblastos/metabolismo , Hemangioblastos/citología , Células Endoteliales/metabolismo , Células Endoteliales/citología , Transducción de Señal , Hematopoyesis/fisiología , Desarrollo Embrionario , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Hígado/embriología , Hígado/metabolismo , Hígado/citologíaRESUMEN
Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.
Asunto(s)
Riñón/citología , Riñón/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Animales , Diferenciación Celular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Nefronas , Organogénesis/genética , Organogénesis/fisiología , Organoides/citología , TranscriptomaRESUMEN
Intermediate progenitors of both excitatory and inhibitory neurons, which can replenish neurons in the adult brain, were recently identified. However, the generation of intermediate progenitors of GABAergic inhibitory neurons (IPGNs) has not been studied in detail. Here, we characterized the spatiotemporal distribution of IPGNs in mouse cerebral cortex. IPGNs generated neurons during both embryonic and postnatal stages, but the embryonic IPGNs were more proliferative. Our lineage tracing analyses showed that the embryonically proliferating IPGNs tended to localize to the superficial layers rather than the deep cortical layers at 3 weeks after birth. We also found that embryonic IPGNs derived from the medial and caudal ganglionic eminence (CGE) but more than half of the embryonic IPGNs were derived from the CGE and broadly distributed in the cerebral cortex. Taken together, our data indicate that the broadly located IPGNs during embryonic and postnatal stages exhibit a different proliferative property and layer distribution.
RESUMEN
Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. We previously reported NEPHRIN and SD abnormalities in the podocytes of kidney organoids generated from patient-derived induced pluripotent stem cells (iPSCs) with an NPHS1 missense mutation (E725D). However, the mechanisms underlying the disease may vary depending on the mutations involved, and thus generation of iPSCs from multiple patients is warranted. Here we established iPSCs from two additional patients with different NPHS1 mutations and examined the podocyte abnormalities in kidney organoids derived from these cells. One patient had truncating mutations, and NEPHRIN was undetectable in the resulting organoids. The other patient had a missense mutation (R460Q), and the mutant NEPHRIN in the organoids failed to accumulate on the podocyte surface to form SD precursors. However, the same mutant protein behaved normally when overexpressed in heterologous cells, suggesting that NEPHRIN localization is cell context-dependent. The localization of another SD-associated protein, PODOCIN, was impaired in both types of mutant organoids in a cell domain-specific manner. Thus, the new iPSC lines and resultant kidney organoids will be useful resources for dissecting the disease mechanisms, as well as for drug development for therapies.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/metabolismo , Síndrome Nefrótico/fisiopatología , Organoides/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón , Masculino , Proteínas de la Membrana/genética , Proteínas Mutantes/genética , Mutación MissenseRESUMEN
Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Animales , Animales Recién Nacidos , Linaje de la Célula , Regulación hacia Abajo , Riñón/citología , Riñón/embriología , Glomérulos Renales/citología , Glomérulos Renales/embriología , Glomérulos Renales/crecimiento & desarrollo , Glomérulos Renales/metabolismo , Trasplante de Riñón , Túbulos Renales/citología , Túbulos Renales/embriología , Túbulos Renales/crecimiento & desarrollo , Túbulos Renales/metabolismo , Ratones , Podocitos/citología , Podocitos/metabolismo , RNA-Seq , Análisis de la Célula Individual , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Proteome analysis using human serum is a technological advancement that will enable the discovery of novel biomarkers and biomarker patterns of various human diseases. Although proteome analysis using serum has potential in disease prevention, early diagnosis and treatment of diseases, and evaluation of pharmacotherapies, this technology is still in its infancy. Thus, we sought to develop an advanced method of conducting proteome analysis on human serum. In this study, we report the development of the semi-comprehensive protein analytical technique, which involves the systematic use of iTRAQ labeling, HPLC, nano-LC and MS. We compared the composition of the serum proteome in males and females using this technique and detected gender-based differences in serum protein composition. This technology will enable the generation of databases that may ultimately lead to the discovery of specific biomarkers or biomarker patterns of various diseases.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Suero/metabolismo , Caracteres Sexuales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Fraccionamiento Químico , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Cromatografía de Fase Inversa , Femenino , Humanos , Masculino , Nanotecnología , Péptidos/química , Péptidos/metabolismo , Reproducibilidad de los Resultados , Programas Informáticos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Núcleo Celular , Metilación de ADN , Células Madre Embrionarias , Epigénesis Genética , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Células Cultivadas , Cromosomas/metabolismo , Cromosomas/ultraestructura , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo , Genes Reporteros , Humanos , Ratones , Ratones Noqueados , Señales de Localización Nuclear , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized clinically by progressive ptosis, dysphagia and limb weakness, and by unique intranuclear inclusions in the skeletal muscle fibers. The disease is caused by the expansion of a 10-alanine stretch to 12-17 alanine residues in the poly(A)-binding protein, nuclear 1 (PABPN1; PABP2). While PABPN1 is a major component of the inclusions in OPMD, the exact cause of the disease is unknown. To elucidate the molecular mechanism and to construct a useful model for therapeutic trials, we have generated transgenic mice expressing the hPABPN1. Transgenic mice lines expressing a normal hPABPN1 with 10-alanine stretch did not reveal myopathic changes, whereas lines expressing high levels of expanded hPABPN1 with a 13-alanine stretch showed an apparent myopathy phenotype, especially in old age. Pathological studies in the latter mice disclosed intranuclear inclusions consisting of aggregated mutant hPABPN1 product. Furthermore, some TUNEL positive nuclei were shown around degenerating fibers and a cluster of it in the lesion in necrotic muscle fibers. Interestingly, the degree of myopathic changes was more prominent in the eyelid and pharyngeal muscles. Further, muscle weakness in the limbs was apparent as shown by the fatigability test. Nuclear inclusions seemed to develop gradually with aging, at least after 1 week of age, in model mouse muscles. We established the first transgenic mouse model of OPMD by expressing mutated PABPN1, and our model mice appear to have more dramatic alternations in myofiber viability.