RESUMO
Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neuroblastoma , Doença de Parkinson , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Optogenética , Doença de Parkinson/genéticaRESUMO
Metastatic prostate cancers have a high mortality rate. KiSS1 was originally identified as a metastasis suppressor gene in metastatic melanoma and breast cancer, but its role in prostate cancer has been contradictory. This study was therefore undertaken to investigate the effects of KiSS1 overexpression on the growth and migration of human metastatic prostate cancer cells. We first tested the effect of KiSS1 overexpression on the growth and migration of DU145 human metastatic prostate cancer cells in vitro. DU145 cells were infected with the culture medium of 293T cells, which produce lentivirus particles containing KiSS1. A 2.5-fold increase in proliferation of KiSS1-overexpressing cancer cells was observed, and these cells formed tumor spheroids about 3 times larger than the vector control group. qPCR and immunoblotting revealed the association between increased cell growth and regulation of the PI3K/Akt and cell cycle genes, and also that increases in ß-catenin and CD133 contribute to tumor aggregation. KiSS1 overexpression resulted in upregulation of the ß-arrestin1/2 and Raf-MEK-ERK-NF-κB pathways via KiSS1R. Moreover, the migration and invasion of KiSS1-overexpressing cells were determined to be faster than the control group, along with 1.6-fold increased metastatic colonization of the KiSS1-overexpressing cancer cells. These were associated to the regulation of EMT gene expressions, such as E-cadherin and N-cadherin, and the upregulation of MMP9. In a xenograft mouse model inoculated with DU145 cells infected GFP or KiSS1 via a lentiviral vector, KiSS1 statistically significantly increased the tumor growth, with upregulation of PCNA and Ki-67 in the tumor tissues. In addition, KiSS1 increased the angiogenic capacity by upregulating VEGF-A and CD31, both in vitro and in vivo. Taken together, our results indicate that KiSS1 not only induces prostate cancer proliferation, but also promotes metastasis by increasing the migration, invasion, and angiogenesis of malignant cells.
Assuntos
Kisspeptinas , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Kisspeptinas/genética , Kisspeptinas/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following in vivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders.
Assuntos
Células-Tronco Pluripotentes , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular , Distrofina , Humanos , Camundongos , Camundongos Endogâmicos mdx , Desenvolvimento Muscular , Músculo Esquelético , Mioblastos , Transplante de Células-TroncoRESUMO
Understanding the signaling pathways that regulate the final differentiation of human myoblasts is essential for successful cell transplantation and drug screening for the treatment of muscular dystrophy. In an effort to improve myotube formation from hiPSC-derived myoblasts, we validated a collection of 13 small molecules in a newly established in vitro screening platform for the assessment of myotube formation. The analysis of myotube formation as measured by the fusion index showed that the combinational inhibition of the TGFß signaling with NOTCH signaling enhances the ability of multi-nucleated myotube production. Combinational treatment of inhibitors for TGFß and NOTCH signaling pathways improved myotube formation in a dose-dependent manner. This effect was achieved by inhibiting the combinatorial mechanism of signaling. The combination treatment of small molecules effective in inducing multinucleated myotubes was validated in healthy human primary myoblasts. In addition, it was also applied to DMD patient iPSC-derived myoblasts to enhance the generation of multinucleated myotubes.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Mioblastos/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Células Cultivadas , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.
Assuntos
Células-Tronco Embrionárias/citologia , Organoides/citologia , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Técnicas de Transferência Nuclear , Organoides/metabolismo , SuínosRESUMO
Although the effects of stem cells expressing anticancer genes on tumor growth have been demonstrated by many researchers in various types of cancer, relatively few studies have investigated their inhibitory effects on cancer metastasis. In the present study, we examined the inhibitory effects of cytosine deaminase (CD)/5fluorocytosine (5FC) and interferonß (IFNß) using genetically engineered neural stem cells (hNSCs) in a cellular and metastasis model of renal cell carcinoma (RCC). The CD/5FC method has the advantage of minimizing damage to normal tissues since it selectively targets cancer cells by the CD gene, which converts prodrug 5FC to the drug 5fluorouracil. Moreover, we used hNSCs as a tool to effectively deliver the anticancer genes to the tumor site. These stem cells are known to possess tumortropism because of chemoattractant factors expressed in cancer cells. Therefore, we ascertained the expression of these factors in A498 cells, a cell line of RCC, and identified the A498specific migration ability of hNSCs. We also confirmed that the proliferation of A498 cells was significantly reduced by therapeutic hNSCs in the presence of 5FC. Furthermore, we established an A498 metastasis model. In the animal experiment, the weight of the lungs increased in response to cancer metastasis, but was normalized by hNSCs expressing CD and/or IFNß genes, while the incidence of liver metastasis was suppressed by the hNSCs. Overall, the results of this study demonstrate that stem cells expressing anticancer genes have the potential for use as an alternative to conventional therapy for metastatic cancer.
Assuntos
Carcinoma de Células Renais/terapia , Citosina Desaminase/genética , Interferon beta/genética , Neoplasias Hepáticas/terapia , Células-Tronco Neurais/citologia , Transplante de Células-Tronco/métodos , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citosina Desaminase/metabolismo , Feminino , Engenharia Genética , Humanos , Interferon beta/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Metástase Neoplásica , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Muscular dystrophies are a group of genetic muscle disorders that cause progressive muscle weakness and degeneration. Within this group, Duchenne muscular dystrophy (DMD) is the most common and one of the most severe. DMD is an X chromosome linked disease that occurs to 1 in 3500 to 1 in 5000 boys. The cause of DMD is a mutation in the dystrophin gene, whose encoded protein provides both structural support and cell signaling capabilities. So far, there are very limited therapeutic options available and there is no cure for this disease. In this review, we discuss the existing cell therapy research, especially stem cell-based, which utilize myoblasts, satellite cells, bone marrow cells, mesoangioblasts and CD133+ cells. Finally, we focus on human pluripotent stem cells (hPSCs) which hold great potential in treating DMD. hPSCs can be used for autologous transplantation after being specified to a myogenic lineage. Over the last few years, there has been a rapid development of isolation, as well as differentiation, techniques in order to achieve effective transplantation results of myogenic cells specified from hPSCs. In this review, we summarize the current methods of hPSCs myogenic commitment/differentiation, and describe the current status of hPSC-derived myogenic cell transplantation.
Assuntos
Distrofia Muscular de Duchenne/terapia , Mioblastos/citologia , Células-Tronco Pluripotentes/citologia , Transplante de Células-Tronco/métodos , Diferenciação Celular/fisiologia , Humanos , Mioblastos/transplanteRESUMO
Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.
Assuntos
Quimiocina CCL2/genética , Quimiocina CXCL1/genética , Doenças Genéticas Inatas , Células-Tronco Pluripotentes Induzidas/metabolismo , Células de Schwann/metabolismo , Adulto , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Células Cultivadas , Reprogramação Celular , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocinas , Células-Tronco Embrionárias/patologia , Feminino , Edição de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Predisposição Genética para Doença/genética , Genética Humana , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Ratos , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/patologia , TransplanteRESUMO
Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-ß inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.
Assuntos
Diferenciação Celular/fisiologia , Glioblastoma/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios/patologia , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Glioblastoma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Suínos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Peripheral nerve injuries often lead to incomplete recovery and contribute to significant disability to approximately 360,000 people in the USA each year. Stem cell therapy holds significant promise for peripheral nerve regeneration, but maintenance of stem cell viability and differentiation potential in vivo are still major obstacles for translation. Using a made-in-house 96-well vertical electrical stimulation (ES) platform, we investigated the effects of different stimulating pulse frequency, duration and field direction on human neural crest stem cell (NCSC) differentiation. We observed dendritic morphology with enhanced neuronal differentiation for NCSCs cultured on cathodes subject to 20â¯Hz, 100µs pulse at a potential gradient of 200â¯mV/mm. We further evaluated the effect of a novel cell-based therapy featuring optimized pulsatile ES of NCSCs for in vivo transplantation following peripheral nerve regeneration. 15 mm critical-sized sciatic nerve injuries were generated with subsequent surgical repair in sixty athymic nude rats. Injured animals were randomly assigned into five groups (N = 12 per group): blank control, ES, NCSC, NCSC + ES, and autologous nerve graft. The optimized ES was applied immediately after surgical repair for 1 h in ES and NCSC + ES groups. Recovery was assessed by behavioral (CatWalk gait analysis), wet muscle-mass, histomorphometric, and immunohistochemical analyses at either 6 or 12 weeks after surgery (N = 6 per group). Gastrocnemius muscle wet mass measurements in ES + NCSC group were comparable to autologous nerve transplantation and significantly higher than other groups (p < 0.05). Quantitative histomorphometric analysis and catwalk gait analysis showed similar improvements by ES on NCSCs (p < 0.05). A higher number of viable NCSCs was shown via immunochemical analysis, with higher Schwann cell (SC) differentiation in the NCSC + ES group compared to the NCSC group (p < 0.05). Overall, ES on NCSC transplantation significantly enhanced nerve regeneration after injury and repair, and was comparable to autograft treatment. Thus, ES can be a potent alternative to biochemical and physical cues for modulating stem cell survival and differentiation. This novel cell-based intervention presents an effective and safe approach for improved outcomes after peripheral nerve repair.
Assuntos
Regeneração Nervosa/fisiologia , Crista Neural/citologia , Traumatismos dos Nervos Periféricos/terapia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Estimulação Elétrica/métodos , Humanos , Imuno-Histoquímica , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Ratos , Transplante de Células-Tronco/métodosRESUMO
Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and in vitro fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60⯵M). After 42â¯h of IVM, the 30⯵M LPA-treated group showed a significant (P <0.05) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30⯵M LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30⯵M LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30⯵M LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen, PCNA) in the oocytes and cumulus cells of the individuals in the 30⯵M LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (uPAR) in cumulus cells was significantly increased in the 30⯵M LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30⯵M LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.
Assuntos
Células do Cúmulo/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Lisofosfolipídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Suínos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa , Partenogênese/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/deficiência , Glicoesfingolipídeos/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , 1-Desoxinojirimicina/análogos & derivados , Linhagem Celular Tumoral , Glucosilceramidase/genética , Humanos , Multimerização ProteicaRESUMO
The neural crest (NC) is a transient population of multipotent cells giving rise to the peripheral nervous system, skin pigmentation, heart, and facial mesenchyme. The broad cell fate potential of NC makes it an attractive cell fate to derive from human pluripotent stem cells (hPSCs) for exploring embryonic development, modeling disease, and generating cells for transplantation. Here, we discuss recent publications and methods for efficiently differentiating hPSCs into NC. We also provide methods to direct NC into two different terminal fates: melanocytes and sensory neurons.
Assuntos
Técnicas de Cultura de Células/métodos , Crista Neural/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Smad/antagonistas & inibidores , Proteínas Wnt/metabolismo , Animais , Contagem de Células , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Humanos , Melanócitos/citologia , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células Receptoras Sensoriais/citologia , Proteínas Smad/metabolismoRESUMO
A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases. Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.
Assuntos
Reprogramação Celular/fisiologia , Técnicas Citológicas/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/virologia , Vírus Sendai/fisiologia , HumanosRESUMO
Realizing the therapeutic potential of human induced pluripotent stem (iPS) cells will require robust, precise and safe strategies for genetic modification, as cell therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify human iPS cells at 'safe harbor' sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. We demonstrate that â¼10% of integrations of a lentivirally encoded ß-globin transgene in ß-thalassemia-patient iPS cell clones meet our safe harbor criteria and permit high-level ß-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. This approach, combining bioinformatics and functional analyses, should be broadly applicable to introducing therapeutic or suicide genes into patient-specific iPS cells for use in cell therapy.
Assuntos
Engenharia Genética/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transgenes/genética , Globinas beta/metabolismo , Talassemia beta/terapia , Animais , Diferenciação Celular , Linhagem Celular , Células Eritroides/citologia , Feminino , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Dados de Sequência Molecular , Transgenes/fisiologia , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/metabolismoAssuntos
Pesquisa Biomédica/métodos , Doença , Células-Tronco Pluripotentes Induzidas/citologia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Animais , Pesquisa Biomédica/tendências , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Medicina de Precisão/métodos , Medicina de Precisão/tendências , Medicina Regenerativa/tendências , Transplante de Células-Tronco/tendênciasRESUMO
Embryonic stem cells (ESCs) are established from blastocysts and give rise to various types of cells and tissues. In the present study, we assessed the osteogenic potential of ESCs using in vitro culture conditions and in vivo differentiation in tooth sockets. An ESC-derived embryoid body (EB) was formed and subsequently induced to an osteogenic lineage. The differentiated EB cells exhibited increased expression of various osteogenic markers as determined by real-time PCR analysis. Likewise, the differentiated EB-derived cells had enhanced alkaline phosphatase activity and calcium accumulation, as determined by cytochemical methods. For in vivo transplantation, mixtures of ESCs and hydroxyapatite/ tricalcium phosphate particles or EBs alone were transplanted into female rat tooth sockets. After 12 weeks, we observed formation of osteogenic structure in the tooth sockets without evidence of teratomas. These data suggest that pluripotent ESCs can serve as an alternative source for the reconstruction of craniofacial structures, as well as for further applications.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Osteogênese/fisiologia , Células-Tronco Pluripotentes/fisiologia , Transplante de Células-Tronco , Alvéolo Dental/citologia , Animais , Biomarcadores/metabolismo , Fosfatos de Cálcio/metabolismo , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Feminino , Hidroxiapatitas/metabolismo , Células-Tronco Pluripotentes/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Crista Neural/citologia , Crista Neural/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Embrião de Galinha , Humanos , CamundongosRESUMO
Murine embryonic stem cells (mESC) readily form embryoid bodies (EBs) that exhibit hematopoietic differentiation. Methods based on EB formation or ESC coculture with murine bone marrow stromal cell lines have revealed pathways of both primitive and definitive hematopoietic differentiation progressing from primitive mesoderm via hemangioblasts to endothelium and hematopoietic stem and progenitor cells. The addition of specific hematopoietic growth factors and morphogens to these cultures enhances the generation of neutrophils, macrophages, megakaryocyte/platelets, and hemoglobinized mature red cells. In addition, selective culture systems have been developed to support differentiation into mature T lymphocytes, natural killer cells, B cells, and dendritic cells. In most cases, culture systems have been developed that support equivalent differentiation of various human ESC (hESC). The major obstacle to translation of ESC hematopoietic cultures to clinical relevance has been the general inability to produce hematopoietic stem cells (HSC) that can engraft adult, irradiated recipients. In this context, the pattern of ES hematopoietic development mirrors the yolk sac phase of hematopoiesis that precedes the appearance of engraftable HSC in the aorta-gonad-mesonephros region. Genetic manipulation of mESC hematopoietic progeny by upregulation of HOXB4 or STAT5 has led to greatly enhanced long- or short-term multilineage hematopoietic engraftment, suggesting that genetic or epigenetic manipulation of these pathways may lead to functional HSC generation from hESC.