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1.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804138

RESUMEN

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor ß1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Condrogénesis/genética , Receptores del Factor Estimulante de Colonias/genética , Factor de Crecimiento Transformador beta1/genética , Animales , Cartílago/crecimiento & desarrollo , Diferenciación Celular/genética , Proliferación Celular/genética , Condrocitos/citología , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Ratones
2.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33803024

RESUMEN

Microglia are resident immune cells of the central nervous system and play critical roles during the development, homeostasis, and pathologies of the brain. Originated from yolk sac erythromyeloid progenitors, microglia immigrate into the embryonic brain parenchyma to undergo final postnatal differentiation and maturation driven by distinct chemokines, cytokines, and growth factors. Among them, TGFß1 is an important regulator of microglial functions, mediating homeostasis, anti-inflammation, and triggering the expression of microglial homeostatic signature genes. Since microglia studies are mainly based on rodent cells and the isolation of homeostatic microglia from human tissue is challenging, human-induced pluripotent stem cells have been successfully differentiated into microglia-like cells recently. However, employed differentiation protocols strongly vary regarding used cytokines and growth factors, culture conditions, time span, and cell yield. Moreover, the incomplete differentiation of human microglia can hamper the similarity to primary human microglia and dramatically influence the outcome of follow-up studies with these differentiated cells. This review summarizes the current knowledge of the molecular mechanisms driving rodent microglia differentiation in vivo, further compares published differentiation protocols, and highlights the potential of TGFß as an essential maturation factor.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Microglía/citología , Animales , Uniones Célula-Matriz/metabolismo , Humanos , Microglía/metabolismo , Modelos Biológicos , Factor de Crecimiento Transformador beta/metabolismo
3.
Stem Cell Reports ; 16(4): 940-953, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33852884

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation.


Asunto(s)
/patología , Colon/patología , Mucosa Intestinal/patología , Organoides/patología , Línea Celular , Colon/virología , Células Epiteliales/virología , Humanos , Células Madre Pluripotentes Inducidas/citología , Inflamación/virología , Mucosa Intestinal/virología , Modelos Biológicos , Organoides/citología , Organoides/virología , Replicación Viral/fisiología
4.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800815

RESUMEN

In the last decade, different research groups in the academic setting have developed induced pluripotent stem cell-based protocols to generate three-dimensional, multicellular, neural organoids. Their use to model brain biology, early neural development, and human diseases has provided new insights into the pathophysiology of neuropsychiatric and neurological disorders, including microcephaly, autism, Parkinson's disease, and Alzheimer's disease. However, the adoption of organoid technology for large-scale drug screening in the industry has been hampered by challenges with reproducibility, scalability, and translatability to human disease. Potential technical solutions to expand their use in drug discovery pipelines include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create isogenic models, single-cell RNA sequencing to characterize the model at a cellular level, and machine learning to analyze complex data sets. In addition, high-content imaging, automated liquid handling, and standardized assays represent other valuable tools toward this goal. Though several open issues still hamper the full implementation of the organoid technology outside academia, rapid progress in this field will help to prompt its translation toward large-scale drug screening for neurological disorders.


Asunto(s)
Descubrimiento de Drogas/métodos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Organoides/efectos de los fármacos , Animales , Automatización , Encéfalo/citología , Sistemas CRISPR-Cas , Técnicas de Cultivo de Célula , Colágeno , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos/métodos , Industria Farmacéutica/organización & administración , Predicción , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/citología , Laminina , Aprendizaje Automático , Microscopía/métodos , Enfermedades del Sistema Nervioso/patología , Proteoglicanos , RNA-Seq , Reproducibilidad de los Resultados , Análisis de la Célula Individual
5.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33810153

RESUMEN

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.


Asunto(s)
Supervivencia de Injerto/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Inhibidores de Proteínas Quinasas/farmacología , Trasplante de Células Madre , Quinasas Asociadas a rho/antagonistas & inhibidores , Amidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Macaca fascicularis , Piridinas/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo
7.
Science ; 371(6535): 1245-1248, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33737484

RESUMEN

Mosaic mutations can be used to track cell lineages in humans. We used cell cloning to analyze embryonic cell lineages in two living individuals and a postmortem human specimen. Of 10 reconstructed postzygotic divisions, none resulted in balanced contributions of daughter lineages to tissues. In both living individuals, one of two lineages from the first cleavage was dominant across tissues, with 90% frequency in blood. We propose that the efficiency of DNA repair contributes to lineage imbalance. Allocation of lineages in postmortem brain correlated with anterior-posterior axis, associating lineage history with cell fate choices in embryos. We establish a minimally invasive framework for defining cell lineages in any living individual, which paves the way for studying their relevance in health and disease.


Asunto(s)
Blastómeros/citología , División Celular , Linaje de la Célula , Desarrollo Embrionario , Adulto , Anciano , Blastocisto/citología , Células Sanguíneas , Diferenciación Celular , Línea Celular , Reparación del ADN , Femenino , Feto/citología , Variación Genética , Genoma Humano , Humanos , Mutación INDEL , Células Madre Pluripotentes Inducidas/citología , Masculino , Células-Madre Neurales/citología , Polimorfismo de Nucleótido Simple
8.
Methods Mol Biol ; 2269: 25-33, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687669

RESUMEN

In an increasingly geriatric population, in which elderly people frequently face chronic diseases and degenerative conditions, cell therapies as part of novel regenerative medicine approaches are of great interest. Even though today's cell therapies mostly rely on adult stem cells like the mesenchymal stem cells or primary somatic cells, pluripotent stem cells represent an enormously versatile cell model to explore possible new avenues in the field of regenerative medicine due to their capacity to grow indefinitely and to differentiate into the desired cell types. The discovery of reprogramming somatic cells into induced pluripotent stem cells augmented the pool of applicable cell entities so that researchers nowadays can resort to embryonic stem cells, but also to a plethora of patient- and disease-specific induced pluripotent stem cells. The ease of targeted genome engineering is an additional benefit that allows using pluripotent stem cells for disease modeling, drug discovery, and the development of cell therapies. However, the task is still demanding as the generation of subpopulations and a sufficient cell maturation for some cell entities have yet to be achieved. Likewise, even though for some applications the cells of interest can be produced in the large-scale dimensions and purity that are required for clinical purposes, proper integration, and function in the host tissue remain challenging. Nonetheless, the immense progress that has been made over the last decades warrants the prominent role of pluripotent stem cells in regenerative medicine as in vitro models to broaden our knowledge of disease onset/progression and treatment as well as in vivo as a substitution of damaged/aged tissue.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Pluripotentes/metabolismo , Medicina Regenerativa , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Células Madre Pluripotentes/citología
9.
Methods Mol Biol ; 2269: 233-244, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687683

RESUMEN

We describe the protocol for the efficient in vitro differentiation of human neural stem cells (NSCs) from human-induced pluripotent stem cells (iPS cells). NSCs differentiate via neural epithelial progenitors enabling the analysis of early neuronal development. They represent neural progenitor cells, which are capable of differentiating into neurons and glia.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Neuroglía/metabolismo , Neuronas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neuroglía/citología , Neuronas/citología
10.
Methods Mol Biol ; 2269: 245-254, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687684

RESUMEN

Peripheral nerves have a limited ability to regenerate and current clinical approaches involving microsurgery give suboptimal recovery. Engineered tissues using aligned cellular collagen hydrogels can be used as in vitro models through the incorporation of human Schwann cells. However, primary human Schwann cells are difficult to obtain and can be challenging to culture. The ability to generate Schwann cells from human-induced pluripotent stem cells (hiPSCs) provides a more reliable cell source for modeling peripheral nerve tissue. Here, we describe protocols for generating hiPSC-derived Schwann cells and incorporating them into 3D engineered tissue culture models for peripheral nerve research.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Regeneración Nerviosa , Nervios Periféricos/metabolismo , Células de Schwann/metabolismo , Ingeniería de Tejidos , Humanos , Células Madre Pluripotentes Inducidas/citología , Nervios Periféricos/citología
11.
Nat Genet ; 53(3): 304-312, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33664506

RESUMEN

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.


Asunto(s)
Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/fisiología , Células Madre Pluripotentes Inducidas/citología , Sitios de Carácter Cuantitativo , Transcriptoma , Diferenciación Celular/genética , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Neurogénesis/genética , Estrés Oxidativo/efectos de los fármacos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Rotenona/toxicidad , Análisis de Secuencia de ARN , Análisis de la Célula Individual
12.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669419

RESUMEN

We have established an immune cell therapy with immortalized induced pluripotent stem-cell-derived myeloid lines (iPS-ML). The benefits of using iPS-ML are the infinite proliferative capacity and ease of genetic modification. In this study, we introduced 4-1BBL gene to iPS-ML (iPS-ML-41BBL). The analysis of the cell-surface molecules showed that the expression of CD86 was upregulated in iPS-ML-41BBL more than that in control iPS-ML. Cytokine array analysis was performed using supernatants of the spleen cells that were cocultured with iPS-ML or iPS-ML-41BBL. Multiple cytokines that are beneficial to cancer immunotherapy were upregulated. Peritoneal injections of iPS-ML-41BBL inhibited tumor growth of peritoneally disseminated mouse melanoma and prolonged survival of mice compared to that of iPS-ML. Furthermore, the numbers of antigen-specific CD8+ T cells were significantly increased in the spleen and tumor tissues treated with epitope peptide-pulsed iPS-ML-41BBL compared to those treated with control iPS-ML. The number of CXCR6-positive T cells were increased in the tumor tissues after treatment with iPS-ML-41BBL compared to that with control iPS-ML. These results suggest that iPS-ML-41BBL could activate antigen-specific T cells and promote their infiltration into the tumor tissues. Thus, iPS-ML-41BBL may be a candidate for future immune cell therapy aiming to change immunological "cold tumor" to "hot tumor".


Asunto(s)
Ligando 4-1BB/metabolismo , Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Células Madre Pluripotentes Inducidas/citología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/terapia , Células Mieloides/metabolismo , Células Mieloides/trasplante , Neoplasias Cutáneas/terapia , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CXCR6/metabolismo , Neoplasias Cutáneas/patología , Resultado del Tratamiento
13.
Nat Genet ; 53(3): 313-321, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33664507

RESUMEN

Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.


Asunto(s)
Variación Genética , Células Madre Pluripotentes Inducidas/fisiología , Sitios de Carácter Cuantitativo , Síndrome de Bardet-Biedl/genética , Canales de Calcio/genética , Línea Celular , Ataxia Cerebelosa/genética , Metilación de ADN , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Polimorfismo de Nucleótido Simple , Proteínas/genética , Enfermedades Raras/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARN , Secuenciación Completa del Genoma
14.
J Vis Exp ; (168)2021 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-33645553

RESUMEN

Many human neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.


Asunto(s)
Células Sanguíneas/citología , Neuronas/citología , Anticuerpos/química , Técnicas de Cultivo de Célula , Desdiferenciación Celular , Diferenciación Celular/fisiología , Separación Celular , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Glicoproteínas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neuronas/ultraestructura
15.
J Vis Exp ; (168)2021 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-33645588

RESUMEN

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.


Asunto(s)
Bioensayo/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Neuroblastoma/patología , Fagocitosis , Animales , Muerte Celular , Línea Celular Tumoral , Análisis de Datos , Colorantes Fluorescentes/química , Células Madre Embrionarias Humanas/citología , Humanos , Concentración de Iones de Hidrógeno , Células Madre Pluripotentes Inducidas/citología , Control de Calidad , Reproducibilidad de los Resultados , Imagen de Lapso de Tiempo
16.
J Vis Exp ; (168)2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33645570

RESUMEN

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient's blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Leucocitos Mononucleares/citología , Miocitos Cardíacos/citología , Diferenciación Celular , Proliferación Celular , Reprogramación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Madre Pluripotentes Inducidas/citología , Técnicas de Placa-Clamp , Proteínas Wnt/metabolismo
17.
Mol Med Rep ; 23(4)2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33655330

RESUMEN

Osteoporosis is a disease characterized by the degeneration of bone structure and decreased bone mass. Induced pluripotent stem cell­derived mesenchymal stem cells (iPSC­MSCs) have multiple advantages that make them ideal seed cells for bone regeneration, including high­level proliferation, multi­differentiation potential and favorable immune compatibility. Distal­less homeobox (DLX)3, an important member of the DLX family, serves a crucial role in osteogenic differentiation and bone formation. The present study aimed to evaluate the effects of DLX3 on the proliferation and osteogenic differentiation of human iPSC­MSCs. iPSC­MSCs were induced from iPSCs, and identified via flow cytometry. Alkaline phosphatase (ALP), Von Kossa, Oil Red O and Alcian blue staining methods were used to evaluate the osteogenic, adipogenic and chondrogenic differentiation of iPSC­MSCs. DLX3 overexpression plasmids were constructed and transfected into iPSC­MSCs to generate iPSC­MSC­DLX3. iPSC­MSC­GFP was used as the control. Reverse transcription­quantitative PCR (RT­qPCR) and western blotting were performed to measure the expression of DLX3 2 days after transfection. Subsequently, cell proliferation was assessed using a Cell Counting Kit­8 assay on days 1, 3, 5 and 7. RT­qPCR and western blotting were used to analyze osteogenic­related gene and protein expression levels on day 7. ALP activity and mineralized nodules were assessed via ALP staining on day 14. Statistical analysis was performed using an unpaired Student's t­test. Flow cytometry results demonstrated that iPSC­MSCs were positive for CD73, CD90 and CD105, but negative for CD34 and CD45. iPSC­MSC­DLX3 had significantly lower proliferation compared with iPSC­MSC­GFP on days 5 and 7 (P<0.05). mRNA expression levels of osteogenic markers, such as ALP, osteopenia (OPN), osteocalcin (OCN) and Collagen Type I (COL­1), were significantly increased in iPSC­MSC­DLX3 compared with iPSC­MSC­GFP on day 7 (P<0.05). Similarly, the protein expression levels of ALP, OCN, OPN and COL­1 were significantly increased in iPSC­MSC­DLX3 compared with iPSC­MSC­GFP on day 7 (P<0.05). The number of mineralized nodules in iPSC­MSC­DLX3 was increased compared with that in iPSC­MSC­GFP on day 14 (P<0.05). Thus, the present study demonstrated that DLX3 serves a negative role in proliferation, but a positive role in the osteogenic differentiation of iPSC­MSCs. This may provide novel insight for treating osteoporosis.


Asunto(s)
Desarrollo Óseo/genética , Diferenciación Celular/genética , Proteínas de Homeodominio/genética , Células Madre Pluripotentes Inducidas/citología , Osteogénesis/genética , Factores de Transcripción/genética , Adipogénesis/genética , Fosfatasa Alcalina/genética , Regeneración Ósea/genética , Calcificación Fisiológica/genética , Proliferación Celular/genética , Condrogénesis/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo
18.
Nat Protoc ; 16(4): 2213-2256, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33772245

RESUMEN

Tissue-like structures from human pluripotent stem cells containing multiple cell types are transforming our ability to model and understand human development and disease. Here we describe a protocol to generate cardiomyocytes (CMs), cardiac fibroblasts (CFs) and cardiac endothelial cells (ECs), the three principal cell types in the heart, from human induced pluripotent stem cells (hiPSCs) and combine them in three-dimensional (3D) cardiac microtissues (MTs). We include details of how to differentiate, isolate, cryopreserve and thaw the component cells and how to construct and analyze the MTs. The protocol supports hiPSC-CM maturation and allows replacement of one or more of the three heart cell types in the MTs with isogenic variants bearing disease mutations. Differentiation of each cell type takes ~30 d, while MT formation and maturation requires another 20 d. No specialist equipment is needed and the method is inexpensive, requiring just 5,000 cells per MT.


Asunto(s)
Corazón/fisiología , Células Madre Pluripotentes Inducidas/citología , Ingeniería de Tejidos/métodos , Diferenciación Celular , Fenómenos Electrofisiológicos , Humanos , Modelos Biológicos , Miocitos Cardíacos/citología , Andamios del Tejido/química
19.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670616

RESUMEN

Arrhythmogenic Right Ventricular cardiomyopathy (ARVC) is an inherited cardiac muscle disease linked to genetic deficiency in components of the desmosomes. The disease is characterized by progressive fibro-fatty replacement of the right ventricle, which acts as a substrate for arrhythmias and sudden cardiac death. The molecular mechanisms underpinning ARVC are largely unknown. Here we propose a mathematical model for investigating the molecular dynamics underlying heart remodeling and the loss of cardiac myocytes identity during ARVC. Our methodology is based on three computational models: firstly, in the context of the Wnt pathway, we examined two different competition mechanisms between ß-catenin and Plakoglobin (PG) and their role in the expression of adipogenic program. Secondly, we investigated the role of RhoA-ROCK pathway in ARVC pathogenesis, and thirdly we analyzed the interplay between Wnt and RhoA-ROCK pathways in the context of the ARVC phenotype. We conclude with the following remark: both Wnt/ß-catenin and RhoA-ROCK pathways must be inactive for a significant increase of PPARγ expression, suggesting that a crosstalk mechanism might be responsible for mediating ARVC pathogenesis.


Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Adipogénesis/genética , Algoritmos , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/metabolismo , Displasia Ventricular Derecha Arritmogénica/patología , Células Cultivadas , Simulación por Computador , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Modelos Teóricos , PPAR gamma/genética , PPAR gamma/metabolismo , gamma Catenina/metabolismo
20.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672998

RESUMEN

With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Reposicionamiento de Medicamentos/métodos , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neuronas/química , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Neuronas/efectos de los fármacos
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