RESUMEN
We produced transgenic medaka fish lines that mimicked the expression of the GAP43 gene. Fish lines with the proximal 2-kilobase (kb) 5'-untranslated region (UTR) as the expression promoter specifically expressed enhanced green fluorescent protein (EGFP) in neural tissues, such as the brain, spinal cord, and peripheral nerves, and its expression decreased with growth, but persisted until adulthood. A functional analysis of the promoter using partially deleted UTRs revealed that functions related to neural tissue-specific promoter activity were widely distributed in the region upstream of the proximal 400-b. Furthermore, the distal half of the 2-kb UTR contributed to expression throughout the brain, while the region 400-b upstream of the proximal 600-b was strongly associated with expression in specific areas, such as the telencephalon. In addition, a region from 957 to 557b upstream of the translation initiation site was important for the long-term maintenance of promoter activity into adulthood. Among the transcription factors with recognition sequences in this region, Sp1 and CREB1 have been suggested to play important roles in the GAP43 promoter expression characteristics, such as strong expression in the telencephalon and long-term maintenance of expression.
Asunto(s)
Oryzias , Animales , Oryzias/metabolismo , Animales Modificados Genéticamente/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Médula Espinal/metabolismoRESUMEN
Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.
Asunto(s)
Proteínas de Caenorhabditis elegans , Semaforinas , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular , Expresión Génica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/genética , Semaforinas/genética , Semaforinas/metabolismoRESUMEN
Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.
Asunto(s)
Pérdida de Heterocigocidad , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Oryzias/genética , Animales , Animales Modificados Genéticamente , Carcinogénesis , Línea Celular , Daño del ADN , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Femenino , Regulación de la Expresión Génica , Hígado/patología , Masculino , Mutagénesis , Mutación , Proteínas Recombinantes , TranscriptomaRESUMEN
Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.
Asunto(s)
Línea Celular , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Xerodermia Pigmentosa/metabolismo , Animales , Reprogramación Celular , Fibroblastos/metabolismo , Fibroblastos/fisiología , Humanos , Japón , Ratones , Xerodermia Pigmentosa/genéticaRESUMEN
Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.
Asunto(s)
Trasplante de Médula Ósea , Hipofosfatasia/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Trasplante de Médula Ósea/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Lactante , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Trasplante Homólogo/métodos , Resultado del TratamientoRESUMEN
Non-neuronal acetylcholine (ACh) is predicted to function as a local cell signaling molecule. However, the physiological significance of the synthesis of non-neuronal ACh in the intestine remains unclear. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids showed a noticeable capacity for ACh synthesis that was sensitive to a potent inhibitor of choline acetyltransferase. Imaging MS revealed endogenous ACh localized in the epithelial layer in mouse small intestinal epithelium in vivo, suggesting that there are non-neuronal resources of ACh. Treatment of organoids with carbachol downregulated the growth of organoids and the expression of marker genes for epithelial cells. On the other hand, antagonists for mAChRs enhanced the growth and differentiation of organoids, indicating the involvement of mAChRs in regulating the proliferation and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice.
Asunto(s)
Acetilcolina/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Organoides/efectos de los fármacos , Receptores Acoplados a Proteínas G/fisiología , Células Madre/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Agonistas Colinérgicos/farmacología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Organoides/citología , Organoides/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Madre/citología , Células Madre/metabolismoRESUMEN
Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.
Asunto(s)
Hormona Liberadora de Gonadotropina/fisiología , Preferencia en el Apareamiento Animal , Neuronas/fisiología , Oryzias/fisiología , Ácido Pirrolidona Carboxílico/análogos & derivados , Reconocimiento en Psicología , Percepción Visual , Animales , Femenino , Masculino , Mutación , Oryzias/genética , Factores SexualesRESUMEN
Bone marrow (BM) transplantation (BMT) is one of the treatment strategies for congenital metabolic disease, but leukemia secondary to intensive cytoreductive treatment is a major concern. Besides BM cells, mesenchymal stem cells (MSC) are also used for transplantation. An 8-month-old girl with hypophosphatasia underwent transplantation of haploidentical BM cells followed by two transplants of MSC obtained from her father to facilitate osteogenesis. Fludarabine(Flu)/cyclophosphamide (CPA)/anti-thymocyte globulin were used for myeloablative conditioning, but the patient developed therapy-related leukemia harboring t(9;22)(q34;q11.2); minor BCR-ABL (t-leukemia with Ph) at the age of 32 months. At the age of 40 months she underwent a second BM and third MSC transplant from the same donor. Thereafter, she achieved complete histological and molecular remission. The present case suggests that the combination of cytotoxic agents (Flu/CPA) and MSC led to t-leukemia with Ph as a consequence of chromosome instability and suppression of host anti-tumor immunity.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Hipofosfatasia/terapia , Leucemia Mielógena Crónica BCR-ABL Positiva/etiología , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Purgación de la Médula Ósea/efectos adversos , Preescolar , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Femenino , Estudios de Seguimiento , Humanos , Lactante , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Agonistas Mieloablativos/administración & dosificación , Agonistas Mieloablativos/efectos adversos , Retratamiento , Vidarabina/administración & dosificación , Vidarabina/efectos adversos , Vidarabina/análogos & derivadosRESUMEN
The mechanical features of individual cells have been regarded as unique indicators of their states, which could constantly change in accordance with cellular events and diseases. Particularly, cancer progression was characterized by the disruption and/or reorganization of actin filaments causing mechanical changes. Thus, mechanical characterization of cells could become an effective cytotechnological approach for early detection of cancer. To develop mechanical cytotechnology, it would be necessary to clarify the mechanical properties in various cell adhesion states. In this study, we investigated the surface mechanical behavior of cancer and normal cells in the adherent and suspended states using atomic force microscopy. Adherent normal stromal cells showed high surface stiffness due to developed actin cap structures on their apical surface, whereas cancer cells did not have developed filamentous actin structures, and their surface stiffness was low. Upon cell detachment from the substrate, filamentous actin structures of adherent normal stromal cells reorganized to the cortical region and their surface stiffness decreased consequently however, the stiffness of suspended normal cells remained higher than that of cancer cells. These suspended state actin structures were similar, regardless of the cell type. Furthermore, the mechanical responses of the cancer and normal stromal cells to perturbation of the actin cytoskeleton were different, suggesting distinct regulatory mechanisms for actin cytoskeleton in cancer and normal cells in both adherent and suspended states. Therefore, cancer cells possess specific mechanical and actin cytoskeleton features different from normal stromal cells.
Asunto(s)
Actinas/metabolismo , Neoplasias/patología , Células del Estroma/citología , Animales , Fenómenos Biomecánicos , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Citoesqueleto/metabolismo , Humanos , Masculino , Microscopía de Fuerza Atómica , Ratas , Ratas Endogámicas F344RESUMEN
OBJECTIVE: Infrared laser-evoked gene operator is a new microscopic method optimized to heat cells in living organisms without causing photochemical damage. By combining the promoter system for the heat shock response, infrared laser-evoked gene operator enables laser-mediated gene induction in targeted cells. We applied this method to the vascular system in zebrafish embryos and demonstrated its usability to investigate mechanisms of vascular morphogenesis in vivo. APPROACH AND RESULTS: We used double-transgenic zebrafish with fli1:nEGFP to identify the endothelial cells, and with hsp:mCherry to carry out single-cell labeling. Optimizing the irradiation conditions, we finally succeeded in inducing the expression of the mCherry gene in single targeted endothelial cells, at a maximum efficiency rate of 60%. In addition, we indicated that this system could be used for laser ablation under certain conditions. To evaluate infrared laser-evoked gene operator, we applied this system to the endothelial cells of the first intersegmental arteries, and captured images of the connection between the vascular systems of the brain and spinal cord. CONCLUSIONS: Our results suggest that the infrared laser-evoked gene operator system will contribute to the elucidation of the mechanisms underlying vascular morphogenesis by controlling spatiotemporal gene activation in single endothelial cells, by labeling or deleting individual vessels in living embryos.
Asunto(s)
Vasos Sanguíneos/embriología , Células Endoteliales/efectos de la radiación , Respuesta al Choque Térmico , Rayos Infrarrojos , Activación Transcripcional , Animales , Animales Modificados Genéticamente , Expresión Génica , Rayos Láser , Modelos Animales , Neovascularización Fisiológica/genética , Sensibilidad y Especificidad , Pez CebraRESUMEN
Intracellular transport is spatiotemporally controlled by microtubule-dependent motor proteins, including kinesins. In order to elucidate the mechanisms controlling kinesin expression, it is important to analyze their genomic regulatory regions. In this study, we cloned the neuronal tissue-specific kinesin in medaka fish and generated transgenic fish which mimic endogenous neuronal kinesin expression in order to elucidate the mechanisms which regulate kinesin expression. Searches for medaka neuronal orthologues by RT-PCR identified a candidate gene expressed only in neuronal tissues. Using BAC clones, we determined the cDNA sequence and the gene structure of the candidate neuronal kinesin. Evolutionary analysis indicated that the candidate gene encoded medaka KIF5Aa. The endogenous medaka orthologue was found to be expressed only in the nervous system, including the brain and spinal cord, while expression of KIF5Ab was not exclusive to neuronal tissues. Transgenic (Tg) medaka that expressed EGFP under the control of the 6.9 kbp 5' and 1.9kbp 3' flanking regions of the KIF5Aa gene showed characteristic expression throughout the nervous system, including the brain, spinal cord, olfactory pit, eye and cranial nerve. Immunohistological analysis showed that EGFP expression in Tg fish co-localized with expression of HuC/D, a neuronal marker. These results demonstrate that the 6.9 kbp 5' and 1.9 kbp 3' flanking regions of medaka KIF5Aa have neuronal-specific promoter activity mimicking endogenous expression of medaka KIF5Ab. This transgenic fish strain will be useful for further functional analysis of the effects of these regulatory regions on gene expression.
Asunto(s)
Encéfalo/metabolismo , Cinesinas/metabolismo , Neuronas/metabolismo , Oryzias/metabolismo , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Clonación Molecular , Expresión Génica , Cinesinas/genética , Oryzias/genética , Regiones Promotoras GenéticasRESUMEN
G protein-coupled receptors are critical regulators of diverse developmental processes such as oocyte maturation, fertilization, gastrulation, and organogenesis. To further study the molecular mechanisms underlying these processes, we cloned and characterized the orphan leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6), a stem cell marker in mammalian hair follicles, in medaka fish, Oryzias latipes. To examine the expression pattern of lgr6, we performed whole-mount in situ hybridization (WISH) during embryogenesis. The expression of lgr6 was first detected as a band in the anterior part of the posterior brain vesicle in 0.5-1 day post fertilization (dpf) embryos. This band disappeared by 2 dpf, but new signals appeared in the otic vesicles bordering the original band and also detected in the nasal placode and posterior lateral line primordia. At later stages (3-5 dpf), lgr6 was widely expressed in the brain, otic vesicle, neuromasts, root of the pectoral fin, cranial cartilage, and gut. Then, we conducted more detailed expression analysis of lgr6 in adult gut using WISH and immunohistochemical staining. Lgr6-positive cells were detected in the crypt-like proliferative zone and in parts of the villus. We also performed RT-PCR of mRNAs from different tissues. The lgr6 mRNA was found highest in the kidney and gill. The transcript was also present in the brain, heart, liver, spleen, intestine, skeletal muscle, testis, and ovary, similar to that of mammalian LGR6. These results suggest that medaka lgr6 plays an important role in organ development during embryogenesis and serves as a good molecular marker for future studies of postembryonic organ-specific development in mammals.
Asunto(s)
Proteínas de Peces/metabolismo , Organogénesis , Oryzias/embriología , Oryzias/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Sistema de la Línea Lateral/citología , Sistema de la Línea Lateral/metabolismo , Datos de Secuencia Molecular , Oryzias/metabolismo , Receptores Acoplados a Proteínas G/genética , Alineación de SecuenciaRESUMEN
The mechanical properties of cells are unique indicators of their states and functions. Though, it is difficult to recognize the degrees of mechanical properties, due to small size of the cell and broad distribution of the mechanical properties. Here, we developed a simple virtual reality system for presenting the mechanical properties of cells and their dispersion using a haptic device and a PC. This system simulates atomic force microscopy (AFM) nanoindentation experiments for floating cells in virtual environments. An operator can virtually position the AFM spherical probe over a round cell with the haptic handle on the PC monitor and feel the force interaction. The Young's modulus of mesenchymal stem cells and HEK293 cells in the floating state was measured by AFM. The distribution of the Young's modulus of these cells was broad, and the distribution complied with a log-normal pattern. To represent the mechanical properties together with the cell variance, we used log-normal distribution-dependent random number determined by the mode and variance values of the Young's modulus of these cells. The represented Young's modulus was determined for each touching event of the probe surface and the cell object, and the haptic device-generating force was calculated using a Hertz model corresponding to the indentation depth and the fixed Young's modulus value. Using this system, we can feel the mechanical properties and their dispersion in each cell type in real time. This system will help us not only recognize the degrees of mechanical properties of diverse cells but also share them with others.
Asunto(s)
Biofisica/métodos , Algoritmos , Gráficos por Computador , Simulación por Computador , Computadores , Técnicas Citológicas , Módulo de Elasticidad , Diseño de Equipo , Células HEK293 , Humanos , Células Madre Mesenquimatosas/citología , Microscopía de Fuerza Atómica/métodos , Microscopía por Video/métodos , Modelos Estadísticos , Estrés Mecánico , Propiedades de Superficie , Temperatura , Factores de Tiempo , Interfaz Usuario-ComputadorRESUMEN
Feline McDonough Sarcoma (FMS)-like tyrosine kinase 4 (FLT4) is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. We generated a transgenic medaka fish in which the lymphatic vessels and some blood vessels are visible in vivo by transferring the promoter of medaka flt4 driving the expression of enhanced green fluorescent protein (EGFP) using a see-through medaka line. To do this, we identified and cloned medaka flt4 and generated a construct in which the promoter was the 4-kb region upstream of the translation initiation site. The fluorescent signal of EGFP could be observed with little background, and the expression pattern correlated well with that of flt4 determined by whole-mount RNA in situ hybridization. Because a see-through medaka line is transparent until adult, the model is useful for visualizing the lymphatic vessels not only in embryo and fry but also in adult. This model will be a useful tool for analyzing lymphatic development.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Linfangiogénesis/genética , Vasos Linfáticos/anatomía & histología , Oryzias/crecimiento & desarrollo , Oryzias/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Oryzias/metabolismo , Filogenia , Alineación de Secuencia , Receptor 3 de Factores de Crecimiento Endotelial Vascular/química , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (AMSCs) have been used clinically for tissue regeneration; however, their proliferation/differentiation potentials are limited. Recently, induced pluripotent stem cells (iPSCs), known to have nearly unlimited potential to proliferate and differentiate into cells of all three germ layers, have gained wide interest in regenerative medicine. Here, we generated iPSCs from frozen-stocked AMSCs and BMSCs and examined their biological characteristics by comparative analyses. Although the iPSCs were more challenging to generate from the BMSCs than the AMSCs, both iPSC populations expressed pluripotent markers, such as stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumour-related antigens (TRAs) TRA-1-60 and TRA-1-81, OCT3/4 and NANOG. Furthermore, both cell populations differentiated well into three germ layer-derived cells, both in vitro and in vivo. These results indicate that iPSCs derived from frozen AMSCs/BMSCs exhibit equally acceptable iPSC characteristics and have potential in clinical applications as an alternative source of autogenous stem cells.
Asunto(s)
Tejido Adiposo/citología , Células de la Médula Ósea/citología , Separación Celular/métodos , Criopreservación/métodos , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Adipogénesis , Antígenos de Superficie/metabolismo , Forma de la Célula , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Citometría de Flujo , Congelación , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Teratoma/patologíaRESUMEN
The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.
Asunto(s)
Colágeno Tipo II/metabolismo , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Oryzias/genética , Secuencia de Aminoácidos , Aletas de Animales/citología , Aletas de Animales/metabolismo , Animales , Mapeo Cromosómico , Clonación Molecular , Colágeno Tipo II/genética , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Proteínas de Peces/genética , Sitios Genéticos , Humanos , Datos de Secuencia Molecular , Oryzias/embriología , Oryzias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , SinteníaRESUMEN
Mesenchymal stem cells (MSCs) have been extensively investigated for their applications in regenerative medicine. Successful use of MSCs in cell-based therapies will rely on the ability to effectively identify their properties and functions with a relatively non-destructive methodology. In this study, we measured the surface stiffness and thickness of rat MSCs with atomic force microscopy and clarified their relation at a single-cell level. The role of the perinuclear actin cap in regulating the thickness, stiffness, and proliferative activity of these cells was also determined by using several actin cytoskeleton-modifying reagents. This study has helped elucidate a possible link between the physical properties and the physiological function of the MSCs, and the corresponding regulatory role of the actin cytoskeleton.
Asunto(s)
Actinas/ultraestructura , Proliferación Celular , Fenómenos Mecánicos , Células Madre Mesenquimatosas/fisiología , Células Madre Mesenquimatosas/ultraestructura , Animales , Células Cultivadas , Citoesqueleto/ultraestructura , Masculino , Microscopía de Fuerza Atómica , Ratas , Ratas Endogámicas F344 , Fibras de Estrés/ultraestructuraRESUMEN
The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/citología , Células del Estroma/citología , Diente/citología , Animales , Diferenciación Celular/genética , Células Cultivadas , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Factor 4 Similar a Kruppel , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Células del Estroma/metabolismoRESUMEN
Heat shock protein promoters (hsp promoters) are powerful tools for investigating gene functions, as the expression of targeted genes can be controlled simply by heating. However, there have been no reports of the utilization of an endogeneous medaka (Oryzias latipes) hsp promoter to induce exogenous gene expression in medaka. We identified and cloned a functional medaka hsp promoter (olphsp70.1) and verified its ability to act as an inducible promoter both in vitro and in vivo. The hsp promoter efficiently induced exogenous gene expression in cultured cells, developing embryos, and also in adult fishes. When used to control the expression of Venus, a variant of yellow fluorescent protein, in transgenic medaka, the hsp promoter was functional in all tissues except for the gonads of adults. These results indicate that the medaka hsp promoter can be a powerful tool for inducing exogenous gene expression and investigating gene functions both in vitro and in vivo in medaka.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/metabolismo , Oryzias/genética , Oryzias/metabolismo , Regiones Promotoras Genéticas , Animales , Animales Modificados Genéticamente , Línea Celular , Clonación Molecular , Embrión no Mamífero , Proteínas de Choque Térmico/genética , Luciferasas/genética , Luciferasas/metabolismoRESUMEN
Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single-cell gene induction using an infrared laser-evoked gene operator (IR-LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR-LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR-LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.