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1.
Stem Cell Res ; 73: 103239, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37922745

RESUMEN

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.


Asunto(s)
Fibrilación Atrial , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Diferenciación Celular , Línea Celular , Plásmidos
2.
Stem Cell Res ; 71: 103189, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37660554

RESUMEN

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.


Asunto(s)
Cardiomiopatías , Células Madre Pluripotentes Inducidas , Humanos , Prealbúmina/genética , Leucocitos Mononucleares , Mutación/genética , Cardiomiopatías/genética
3.
Stem Cell Res ; 63: 102847, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35772296

RESUMEN

Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.


Asunto(s)
Células Madre Pluripotentes Inducidas , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Nucleótidos/metabolismo
4.
Stem Cell Res ; 60: 102717, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35231795

RESUMEN

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.


Asunto(s)
Células Madre Pluripotentes Inducidas , Diferenciación Celular , Línea Celular , Fibroblastos , Estratos Germinativos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino
5.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-34638840

RESUMEN

BACKGROUND: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). METHODS: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. RESULTS: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. CONCLUSIONS: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.


Asunto(s)
Atrofia Geográfica , Células Madre Pluripotentes Inducidas , Epitelio Pigmentado de la Retina , Animales , Antígenos de Diferenciación/biosíntesis , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Atrofia Geográfica/metabolismo , Atrofia Geográfica/patología , Atrofia Geográfica/cirugía , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/trasplante , Porcinos
6.
Stem Cell Res ; 25: 1-5, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29246570

RESUMEN

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Nefritis Hereditaria/metabolismo , Adulto , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Exones/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Mutación/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Plásmidos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
7.
Stem Cell Res ; 25: 291-295, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29150092

RESUMEN

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Nefritis Hereditaria/genética , Adulto , Línea Celular , Células Cultivadas , Reprogramación Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Cuerpos Embrioides/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Factor 4 Similar a Kruppel , Masculino , Mutación , Nefritis Hereditaria/metabolismo
8.
Stem Cell Res ; 20: 105-114, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28334554

RESUMEN

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.


Asunto(s)
Bancos de Muestras Biológicas , Células Madre Pluripotentes Inducidas/citología , Línea Celular , Criopreservación , Europa (Continente) , Humanos
9.
J Biol Chem ; 289(4): 2084-98, 2014 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-24311783

RESUMEN

Induced pluripotent stem cells (iPSCs) maintain during the first few culture passages a set of epigenetic marks and metabolites characteristic of their somatic cell of origin, a concept defined as epigenetic donor memory. These residual somatic features are lost over time after extensive culture passaging. Therefore, epigenetic donor memory may be responsible for the higher differentiation efficiency toward the tissue of origin observed in low passage iPSCs versus high passage iPSC or iPSCs derived from a different tissue source. Remarkably, there are no studies on the relevance of microRNA (miRNA) memory following reprogramming, despite the established role of these molecules in the context of pluripotency and differentiation. Using hematopoietic progenitors cells as a model, we demonstrated that miRNAs play a central role in somatic memory retention in iPSCs. Moreover, the comparison of the miRNA expression profiles among iPSCs from different sources allowed for the detection of a set of candidate miRNAs responsible for the higher differentiation efficiency rates toward blood progenitors observed in low passage iPSCs. Combining bioinformatic predictive algorithms with biological target validation, we identified miR-155 as a key player for the in vitro differentiation of iPSC toward hematopoietic progenitors. In summary, this study reveals that during the initial passages following reprogramming, iPSCs maintained the expression of a miRNA set exclusive to the original somatic population. Hence the use of these miRNAs might hold a direct application toward our understanding of the differentiation process of iPSCs toward hematopoietic progenitor cells.


Asunto(s)
Diferenciación Celular , Epigénesis Genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/biosíntesis , Perfilación de la Expresión Génica/métodos , Células Madre Hematopoyéticas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Especificidad de Órganos
10.
J Cell Sci ; 126(Pt 22): 5116-31, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-24046443

RESUMEN

Adult muscle stem cells and their committed myogenic precursors, commonly referred to as the satellite cell population, are involved in both muscle growth after birth and regeneration after damage. It has been previously proposed that, under these circumstances, satellite cells first become activated, divide and differentiate, and only later fuse to the existing myofiber through M-cadherin-mediated intercellular interactions. Our data show that satellite cells fuse with the myofiber concomitantly to cell division, and only when the nuclei of the daughter cells are inside the myofiber, do they complete the process of differentiation. Here we demonstrate that M-cadherin plays an important role in cell-to-cell recognition and fusion, and is crucial for cell division activation. Treatment of satellite cells with M-cadherin in vitro stimulates cell division, whereas addition of anti-M-cadherin antibodies reduces the cell division rate. Our results suggest an alternative model for the contribution of satellite cells to muscle development, which might be useful in understanding muscle regeneration, as well as muscle-related dystrophies.


Asunto(s)
Cadherinas/metabolismo , Comunicación Celular/genética , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Células Satélite del Músculo Esquelético/citología , Animales , Anticuerpos , Cadherinas/administración & dosificación , Cadherinas/antagonistas & inhibidores , Diferenciación Celular/genética , División Celular/genética , Distrofina/genética , Humanos , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Células Madre/citología , Células Madre/metabolismo , Xenopus
11.
Cell Rep ; 3(4): 1005-11, 2013 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-23545500

RESUMEN

Transcription-factor-induced reprogramming of somatic cells to pluripotency is a very inefficient process, probably due to the existence of important epigenetic barriers that are imposed during differentiation and that contribute to preserving cell identity. In an effort to decipher the molecular nature of these barriers, we followed a genome-wide approach, in which we identified macrohistone variants (macroH2A) as highly expressed in human somatic cells but downregulated after reprogramming to pluripotency, as well as strongly induced during differentiation. Knockdown of macrohistone variants in human keratinocytes increased the efficiency of reprogramming to pluripotency, whereas overexpression had opposite effects. Genome-wide occupancy profiles show that in human keratinocytes, macroH2A.1 preferentially occupies genes that are expressed at low levels and are marked with H3K27me3, including pluripotency-related genes and bivalent developmental regulators. The presence of macroH2A.1 at these genes prevents the regain of H3K4me2 during reprogramming, imposing an additional layer of repression that preserves cell identity.


Asunto(s)
Reprogramación Celular , Histonas/metabolismo , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Histonas/antagonistas & inhibidores , Histonas/genética , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Mutación , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo
12.
Nat Cell Biol ; 13(12): 1443-9, 2011 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-22020439

RESUMEN

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.


Asunto(s)
Elementos E-Box/fisiología , Regulación Leucémica de la Expresión Génica/fisiología , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Diferenciación Celular/genética , Células HL-60 , Homeostasis/genética , Humanos , Leucemia Promielocítica Aguda/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo
13.
Cell Stem Cell ; 9(3): 233-46, 2011 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-21885019

RESUMEN

Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation.


Asunto(s)
Células Madre Adultas/metabolismo , Proliferación Celular , Queratinocitos/metabolismo , Proteínas Represoras/metabolismo , Proteína SUMO-1/metabolismo , Adulto , Células Madre Adultas/patología , Diferenciación Celular/genética , Células Cultivadas , Senescencia Celular/genética , Ensamble y Desensamble de Cromatina , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Epidermis/patología , Prepucio/patología , Perfilación de la Expresión Génica , Humanos , Recién Nacido , Queratinocitos/patología , Ligasas , Masculino , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas
14.
EMBO J ; 30(17): 3635-46, 2011 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-21811233

RESUMEN

Jarid2 is required for the genomic recruitment of the polycomb repressive complex-2 (PRC2) in embryonic stem cells. However, its specific role during late development and adult tissues remains largely uncharacterized. Here, we show that deletion of Jarid2 in mouse epidermis reduces the proliferation and potentiates the differentiation of postnatal epidermal progenitors, without affecting epidermal development. In neonatal epidermis, Jarid2 deficiency reduces H3K27 trimethylation, a chromatin repressive mark, in epidermal differentiation genes previously shown to be targets of the PRC2. However, in adult epidermis Jarid2 depletion does not affect interfollicular epidermal differentiation but results in delayed hair follicle (HF) cycling as a consequence of decreased proliferation of HF stem cells and their progeny. We conclude that Jarid2 is required for the scheduled proliferation of epidermal stem and progenitor cells necessary to maintain epidermal homeostasis.


Asunto(s)
Diferenciación Celular , Células Epidérmicas , Queratinocitos/citología , Proteínas del Tejido Nervioso/metabolismo , Células Madre/citología , Animales , Células Cultivadas , Epidermis/metabolismo , Folículo Piloso/metabolismo , Histonas/metabolismo , Humanos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Complejo Represivo Polycomb 2 , Células Madre/metabolismo
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