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1.
Mol Cell ; 65(1): 176-190, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28017587

RESUMO

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Criptocromos/metabolismo , Células-Tronco Embrionárias/metabolismo , Animais , Comportamento Animal , Criptocromos/química , Criptocromos/deficiência , Criptocromos/genética , Genótipo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Atividade Motora , Mutação , Células NIH 3T3 , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fenótipo , Fosforilação , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção
2.
Nagoya J Med Sci ; 74(1-2): 157-65, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22515122

RESUMO

Induced pluripotent stem cells (iPSCs) have been directly generated from fibroblast cultures though retrovirus- or lentivirus-mediated ectopic overexpression of only a few defined transcriptional factors. This remarkable achievement has greatly enhanced our ability to explore the causes of, and potential cures for, many genetic diseases, and strengthened the promise of regenerative medicine. In fact, to date, many kinds of somatic cells from different tissues have exhibited a capacity for reprogramming toward an embryonic stem cell-like state, but major bottlenecks in iPSC derivation and therapeutic use remain, including low reprogramming efficiencies and the tumorigenesis of the generated iPSC. Here, we successfully generated miR-302s-induced pluripotent stem cells (mirPS cells) from human embryonic kidney (HEK) 293 cells via transfection of the miR-302s expression vector. We also determined the optimal culture conditions to generate mirPS on feeder cells, which included the use of serum-free N2B27 medium. The mirPS cells generated by our improved conditions showed the expression of pluripotent marker genes such as OCT3/4, NANOG, and SOX2 under growth conditions via reverse transcription-PCR, whereas no expression of these genes was observed in HEK293 cells. On the other hand, under differentiation conditions, mirPS cells formed ball-shaped structures (embryoid bodies), and showed the ability to differentiate into three germ layers (ectoderm, mesoderm, and endoderm) in vitro. The results suggested that our generated mirPS cells are actually functional as a cell resource to apply to regenerative medicine, and mirPS cells are suitable materials to clarify the mechanism underlying the reprogramming from somatic cells.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , Transfecção , Animais , Diferenciação Celular , Técnicas de Cocultura , Meios de Cultura Livres de Soro , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , MicroRNAs/genética , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Tempo , Transfecção/métodos
3.
J Pharm Health Care Sci ; 8(1): 24, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36180937

RESUMO

BACKGROUND: Although automated dispensing robots have been implemented for medication dispensing in Japan, their effect is yet to be fully investigated. In this study, we evaluated the effect of automated dispensing robots and collaborative work with pharmacy support staff on medication dispensing. METHODS: A robotic dispensing system integrating the following three components was established: (1) automated dispensing robot (Drug Station®), which is operated by pharmacy support staff, (2) automated dispensing robot for powdered medicine (Mini DimeRo®), and (3) bar-coded medication dispensing support system with personal digital assistance (Hp-PORIMS®). Subsequently, we evaluated the incidences of dispensing errors and dispensing times before and after introducing the robotic dispensing system. Dispensing errors were classified into two categories, namely prevented dispensing errors and unprevented dispensing errors. The incidence of dispensing errors was calculated as follows: incidence of dispensing errors = total number of dispensing errors/total number of medication orders in each prescription. RESULTS: After introducing the robotic dispensing system, the total incidence of prevented dispensing errors was significantly reduced (0.204% [324/158,548] to 0.044% [50/114,111], p < 0.001). The total incidence of unprevented dispensing errors was significantly reduced (0.015% [24/158,548] to 0.002% [2/114,111], p < 0.001). The number of cases of wrong strength and wrong drug, which can seriously impact a patient's health, reduced to almost zero. The median dispensing time of pharmacists per prescription was significantly reduced (from 60 to 23 s, p < 0.001). CONCLUSIONS: The robotic dispensing system enabled the process of medication dispensing by pharmacist to be partially and safely shared with automated dispensing robots and pharmacy support staff. Therefore, clinical care for patients by pharmacists could be enhanced by ensuring quality and safety of medication.

4.
Cell Biol Int ; 35(4): 325-34, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21029049

RESUMO

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.


Assuntos
Glândulas Suprarrenais/citologia , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular Tumoral/citologia , Células Cromafins/citologia , Animais , Linhagem Celular Tumoral/metabolismo , Proliferação de Células , Células Cultivadas , Células Cromafins/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Neurogênese , Neurônios/citologia
5.
Nagoya J Med Sci ; 82(2): 217-224, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32581402

RESUMO

In a novel regenerative cell-based treatment developed by us for the patients with stress urinary incontinence, autologous adipose-derived stem cells (ASCs) are injected into the periurethral region and the external urethral sphincter. Since the candidates for this treatment included prostate cancer patients after radical prostatectomy, we investigated the effects of ASCs on prostate cancer cell proliferation in vitro and in vivo to confirm the feasibility of our therapeutic approach. The LNCaP (human prostate cancer cell line) cells and ASCs were co-cultured, and prostate-specific antigen (PSA) concentration in their culture medium supernatant was measured at 48 and 96 h. The PSA concentration significantly decreased in the coculture medium supernatant as compared to the culture medium with LNCaP cells alone. On the contrary, PSA concentrations in the culture medium of LNCaP cells were not affected by supplementation with ASC culture supernatant. After subcutaneous transplantation of LNCaP cells, with or without ASCs, in immunodeficient mice, tumor growth was compared. The growth of LNCaP xenograft tumor in immunodeficient mice was significantly suppressed by ASC addition. These results indicated that ASCs inhibit prostate cancer cell growth, without no proliferative effect on prostate cancer cells.


Assuntos
Proliferação de Células , Neoplasias da Próstata/patologia , Transplante de Células-Tronco , Células-Tronco , Incontinência Urinária por Estresse/terapia , Tecido Adiposo/citologia , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Células PC-3 , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Regeneração , Uretra
6.
Sci Rep ; 10(1): 7656, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376945

RESUMO

Transplantation of autologous human induced pluripotent stem cell-derived retinal pigment epithelial (hiPSC-RPE) sheets is a promising therapy for age-related macular degeneration (AMD). As melanin content is a representative feature of healthy RPE, we used polarization-sensitive optical coherence tomography (PS-OCT) to estimate the relative melanin content of RPE in diseased and non-diseased area, and in human iPSC-RPE sheets in vitro and in vivo by evaluating the randomness of polarization (entropy). Two aged Japanese women, one with neovascular AMD that underwent transplantation of an autologous hiPSC-RPE cell sheet and another with binocular dry AMD, were selected for this study. Entropy value was minimal in cells containing no melanin, whereas that of human RPE and hiPSC-RPE sheets was high. En face entropy of the cultured hiPSC-RPE sheet was compared with its grey-scale photo and its values were found to be inversely correlated with the extent of absence of pigmentation in vitro. En face entropy maps were compared to colour fundus photographs, fundus autofluorescence images, and fluorescein angiography images from patients. Entropy values of intact and defective RPEs and of iPSC-RPE transplant areas were determined in vivo using PS-OCT B-scan images. PS-OCT was found to be applicable in the estimation of relative melanin content of cultured and transplanted RPEs in regenerative medicine.


Assuntos
Biomarcadores , Células-Tronco Pluripotentes Induzidas/citologia , Melaninas/metabolismo , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/metabolismo , Tomografia de Coerência Óptica , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cultura de Células , Diferenciação Celular , Feminino , Angiofluoresceinografia , Células HEK293 , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Epitélio Pigmentado da Retina/citologia , Tomografia de Coerência Óptica/métodos
7.
Sci Rep ; 10(1): 19513, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33177585

RESUMO

Imaging of melanin in the eye is important as the melanin is structurally associated with some ocular diseases, such as age-related macular degeneration. Although optical coherence tomography (OCT) cannot distinguish tissues containing the melanin from other tissues intrinsically, polarization-sensitive OCT (PS-OCT) can detect the melanin through spatial depolarization of the backscattered light from the melanin granules. Entropy is one of the depolarization metrics that can be used to detect malanin granules in PS-OCT and valuable quantitative information on ocular tissue abnormalities can be retrived by correlating entropy with the melanin concentration. In this study, we investigate a relationship between the melanin concentration and some depolarization metrics including the entropy, and show that the entropy is linearly proportional to the melanin concentration in double logarithmic scale when noise bias is corrected for the entropy. In addition, we also confirm that the entropy does not depend on the incident state of polarization using the experimental data, which is one of important attributes that depolarization metrics should have. The dependence on the incident state of polarization is also analyzed for other depolarization metrics.


Assuntos
Melaninas/análise , Tomografia de Coerência Óptica/métodos , Benchmarking , Simulação por Computador , Técnicas de Diagnóstico Oftalmológico/instrumentação , Entropia , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador/métodos , Epitélio Pigmentado da Retina/diagnóstico por imagem , Suspensões/química , Tomografia de Coerência Óptica/instrumentação
8.
J Clin Med ; 9(7)2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32668747

RESUMO

Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient's blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.

9.
J Tissue Eng Regen Med ; 13(3): 459-468, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30644171

RESUMO

The ability to move cells and tissues from bench to bedside is an essential aspect of regenerative medicine. In this study, we propose a simple and static shipping system to deliver tissue-engineered cell sheets. Notably, this system is electronic-device-free and simplified to minimize the number of packing and opening steps involved. Shipping conditions were optimized, and application and verification of the system were performed using human iPS cell-derived or fetal retinal pigment epithelium (RPE) cell sheets. The temperature of the compartments within the insulated container was stable at various conditions, and filling up the cell vessel with medium effectively prevented turbulence-induced mechanical damage to the RPE cell sheets. Furthermore, no abnormal changes were observed in RPE morphology, transepithelial electrical resistance, or mRNA expression after transit by train and car. Taken together, our simple shipping system has the potential to minimize the costs and human error associated with bench to bedside tissue transfer. This specially designed regenerative tissue shipping system, validated for use in this field, can be used without any special training. This study provides a procedure for easily sharing engineered tissues with the goal of promoting collaboration between laboratories and hospitals and enhancing patient care.


Assuntos
Células Epiteliais/fisiologia , Preservação Biológica , Epitélio Pigmentado da Retina/fisiologia , Meios de Transporte , Linhagem Celular , Meios de Cultura , Humanos , Reprodutibilidade dos Testes , Reologia , Estresse Mecânico , Temperatura
10.
PLoS One ; 14(3): e0212369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30865653

RESUMO

Regenerative medicine has received a lot of attention as a novel strategy for injuries and diseases that are difficult to cure using current techniques. Cell production, which is vital for regenerative medicine, has undergone remarkable progress via breakthroughs in developmental biology and tissue engineering; currently, cell production requires numerous experimental operators performing manual, small-scale cell cultures. Other major obstacles for cell production and regenerative medicine include the variable quality of products based on the experimental procedure, the skills of operators, the level of labor required for production, and costs. Technological developments are required to overcome this, including automation instead of manual culture. Age-related macular regeneration (AMD) is a refractory ocular disease that causes severe deterioration in central vision due to senescence in the retinal pigment epithelium (RPE). Recently, we performed an autologous transplantation of induced pluripotent stem (iPS) cell-derived RPE cell sheets and started clinical research on allografts from RPE cell suspensions differentiated from iPS cells. The use of regenerative therapies for AMD using iPS cell-derived RPE is expected to become more widespread. In the present study, human iPS cell-derived RPE cells were cultured to form RPE cell sheets using equipment with a closed culture module. The quality of the automated cultured RPE cell sheets was confirmed by comparing their morphological and biological properties with those of manually generated RPE cell sheets. As a result, machine-cultured RPE sheets displayed the same quality as manually cultured RPE sheets, showing that iPS cell-derived RPE cell sheets were successfully cultured by an automated process.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Medicina Regenerativa/métodos , Epitélio Pigmentado da Retina/citologia , Automação Laboratorial , Técnicas de Cultura de Células/métodos , Células Cultivadas , Proteínas do Olho/metabolismo , Estudos de Viabilidade , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Degeneração Macular/terapia , Fatores de Crescimento Neural/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Serpinas/metabolismo , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
J Neurosci Res ; 86(8): 1694-710, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18293415

RESUMO

To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation.


Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Diferenciação Celular/fisiologia , Células Cromafins/citologia , Fator Neurotrófico Ciliar/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Neurônios/citologia , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/fisiologia , Animais , Morte Celular/fisiologia , Linhagem Celular Transformada , Células Cultivadas , Células Cromafins/fisiologia , Humanos , Fator de Crescimento Neural/fisiologia , Neurônios/fisiologia , Ratos , Temperatura , Fatores de Tempo
12.
Neurosci Lett ; 438(1): 42-7, 2008 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-18455310

RESUMO

We recently established adrenal medullary cell line tsAM5D, which was immortalized by use of a temperature-sensitive mutant of the oncogene simian virus 40 large T-antigen. In the present study, when co-treated with glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), tsAM5D cells proliferated at the permissive temperature (33 degrees C) for the T-antigen expression and differentiated into neuron-like cells at the nonpermissive temperature (39 degrees C). Interestingly, in GDNF/CNTF-treated cultures, the addition of pan-specific transforming growth factor (TGF)-beta-neutralizing antibody did not affect the cell proliferation at 33 degrees C, but significantly reduced the survival of neuronally differentiated cells at 39 degrees C. Using real-time RT-PCR for analysis of GDNF/CNTF-treated cells, we found that the expression of mRNAs for TGF-beta1, TGF-beta2, and TGF-beta3 was up-regulated by the temperature shift. These results suggest that autocrine TGF-beta signaling is necessary for the survival of GDNF/CNTF-differentiated tsAM5D cells upon the temperature shift.


Assuntos
Medula Suprarrenal/crescimento & desenvolvimento , Medula Suprarrenal/metabolismo , Comunicação Autócrina/fisiologia , Células Cromafins/metabolismo , Neurônios/metabolismo , Fator de Crescimento Transformador beta/genética , Medula Suprarrenal/citologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Transformada , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Temperatura , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/genética , Regulação para Cima/genética
13.
Stem Cell Reports ; 10(3): 1059-1074, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29503091

RESUMO

Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets.


Assuntos
Células-Tronco Embrionárias/patologia , Retina/patologia , Degeneração Retiniana/patologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Células Fotorreceptoras/patologia , Transplante de Células-Tronco/métodos
14.
Curr Eye Res ; 41(4): 558-68, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-25880804

RESUMO

PURPOSE: To establish a practical research tool for studying the pathogenesis of retinal ganglion cell (RGC) diseases, we optimized culture procedures to induce neurite outgrowth from three-dimensional self-organizing optic vesicles (3D-retinas) differentiated in vitro from mouse and human embryonic stem cells (ESCs). MATERIALS AND METHODS: The developing 3D-retinas isolated at various time points were placed on Matrigel-coated plates and cultured in media on the basis of the 3D-retinal culture or the retinal organotypic culture protocol. The number, length, and morphology of the neurites in each culture condition were compared. RESULTS: First, we confirmed that Venus-positive cells were double-labeled with a RGC marker, Brn3a, in the 3D-retina differentiated from Fstl4::Venus mouse ESCs, indicating specific RGC-subtype differentiation. Second, Venus-positive neurites grown from these RGC subsets were positive for beta-III tubulin and SMI312 by immunohistochemistry. Enhanced neurite outgrowth was observed in the B27-supplemented Neurobasal-A medium on Matrigel-coated plates from the optic vesicles isolated after 14 days of differentiation from mouse ESCs. For the differentiated RGCs from human ESCs, we obtained neurite extension of >4 mm by modifying Matrigel coating and the culture medium from the mouse RGC culture. CONCLUSION: We successfully optimized the culture conditions to enhance lengthy and high-frequency neurite outgrowth in mouse and human models. The procedure would be useful for not only developmental studies of RGCs, including maintenance and projection, but also clinical, pathological, and pharmacological studies of human RGC diseases.


Assuntos
Células-Tronco Embrionárias Humanas/citologia , Degeneração Neural/terapia , Neuritos/patologia , Crescimento Neuronal/fisiologia , Células Ganglionares da Retina/patologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Degeneração Neural/patologia
15.
Cancer Res ; 71(8): 2938-48, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21349947

RESUMO

The basic helix-loop-helix transcription factor NeuroD1 has been implicated in the neurogenesis and early differentiation of pancreatic endocrine cells. However, its function in relation to cancer has been poorly examined. In this study, we found that NeuroD1 is involved in the tumorigenesis of neuroblastoma. NeuroD1 was strongly expressed in a hyperplastic region comprising neuroblasts in the celiac sympathetic ganglion of 2-week-old MYCN transgenic (Tg) mice and was consistently expressed in the subsequently generated neuroblastoma tissue. NeuroD1 knockdown by short hairpin RNA (shRNA) resulted in motility inhibition of the human neuroblastoma cell lines, and this effect was reversed by shRNA-resistant NeuroD1. The motility inhibition by NeuroD1 knockdown was associated with induction of Slit2 expression, and knockdown of Slit2 could restore cell motility. Consistent with this finding, shRNA-resistant NeuroD1 suppressed Slit2 expression. NeuroD1 directly bound to the first and second E-box of the Slit2 promoter region. Moreover, we found that the growth of tumor spheres, established from neuroblastoma cell lines in MYCN Tg mice, was suppressed by NeuroD1 suppression. The functions identified for NeuroD1 in cell motility and tumor sphere growth may suggest a link between NeuroD1 and the tumorigenesis of neuroblastoma. Indeed, tumor formation of tumor sphere-derived cells was significantly suppressed by NeuroD1 knockdown. These data are relevant to the clinical features of human neuroblastoma: high NeuroD1 expression was closely associated with poor prognosis. Our findings establish the critical role of the neuronal differentiation factor NeuroD1 in neuroblastoma as well as its functional relationship with the neuronal repellent factor Slit2.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Processos de Crescimento Celular/fisiologia , Movimento Celular/fisiologia , Regulação para Baixo , Gânglios Simpáticos/metabolismo , Gânglios Simpáticos/patologia , Amplificação de Genes , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Oncogênicas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Esferoides Celulares
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