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1.
Cell ; 136(3): 411-9, 2009 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-19203577

RESUMEN

The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.


Asunto(s)
Células Madre Adultas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Células Madre Embrionarias/metabolismo , Células Germinativas/citología , Factor 4 Similar a Kruppel , Antígeno Lewis X/metabolismo , Ratones , Miocitos Cardíacos/citología
2.
Bioorg Med Chem Lett ; 30(16): 127347, 2020 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-32631546

RESUMEN

Human pluripotent stem cells (hPSCs) are a useful cell source for regenerative medicine. Despite having a potential of hPSCs for cell-based therapy, there is a need for a selective human pluripotency sensor for monitoring of live hPSCs. Here, we report the discovery of a novel pluripotency sensor (SHI5) from BODIPY-based library by high-throughput cell-based screening and describe the use of SHI5 to identify and isolate human embryonic stem cells and human induced pluripotent stem cells. We demonstrate that SHI5-based assay can be applied to live cells that gain pluripotency in the reprogramming process without any effect on their viability. We also show that SHI5 is internalized through a clathrin-mediated endocytosis pathway. These findings suggest that SHI5 can be an attractive sensor for pluripotency cells during reprogramming. Taken together, SHI5-based screening for hPSCs opens probably unlimited possibilities of detection probe for hPSC therapy via assures their safety issue.


Asunto(s)
Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular , Humanos , Estructura Molecular
3.
Proc Natl Acad Sci U S A ; 114(11): E2243-E2252, 2017 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-28246330

RESUMEN

Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.


Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Factores de Transcripción/genética , Animales , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Muerte Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Análisis por Conglomerados , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Perfilación de la Expresión Génica , Humanos , Ratones , Mutación , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Estrés Oxidativo , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/ultraestructura , Factores de Transcripción/metabolismo , Transcriptoma , Proteínas tau/genética , Proteínas tau/metabolismo
4.
EMBO J ; 34(23): 2971-83, 2015 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-26497893

RESUMEN

The generation of patient-specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4-mediated direct reprogramming, using anchorage-independent growth to ensure high purity. Homogeneous iOPCs exhibit typical small-bipolar morphology, maintain their self-renewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild-type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self-renewing iOPCs and yields insights for the in-depth study of demyelination and regenerative medicine.


Asunto(s)
Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/fisiología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Células Madre/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/citología , Inmunohistoquímica , Cariotipo , Masculino , Ratones , Ratones SCID , Factor 3 de Transcripción de Unión a Octámeros/genética , Oligodendroglía/citología , Ratas , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/genética , Trasplante de Células Madre , Células Madre/citología , Células Madre/fisiología
5.
Cells Tissues Organs ; 207(1): 1-14, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31189166

RESUMEN

The number of human embryology collections is very limited worldwide. Some of these comprise the Carnegie Collection, Kyoto Collection, and the Blechschmidt Collection. One further embryonic collection is the Hinrichsen Collection of the Ruhr University Bochum, Germany, which also contains very well-preserved embryos/fetuses, along with approximately 16,000 histological sections. The digitization of this collection is indispensable to enable conservation of the collection for the future and to provide a large group of embryologists, researchers, and physicians access to these histological slides. A small selection of these scans is available at the website of the Digital Embryology Consortium [https://-human-embryology.org/wiki/Main_Page].


Asunto(s)
Embrión de Mamíferos/patología , Feto/diagnóstico por imagen , Imagenología Tridimensional , Imagen por Resonancia Magnética , Embrión de Mamíferos/anomalías , Femenino , Feto/anomalías , Humanos , Recién Nacido , Masculino
6.
Nature ; 495(7442): 516-9, 2013 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-23503660

RESUMEN

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.


Asunto(s)
Células Madre Pluripotentes/metabolismo , Factor de Transcripción TFIID/metabolismo , Transcripción Genética , Animales , Reprogramación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Masculino , Ratones , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Factores Asociados con la Proteína de Unión a TATA/deficiencia , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/deficiencia , Factor de Transcripción TFIID/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
Transfus Med Hemother ; 44(3): 143-150, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28626365

RESUMEN

BACKGROUND: The ex vivo generation of human hematopoietic stem cells (HSCs) with long-term repopulating capacity and multi-lineage differentiation potential represents the holy grail of hematopoiesis research. In principle, human induced pluripotent stem cells (hiPSCs) provide the tool for both studying molecular mechanisms of hematopoietic development and the ex vivo production of 'true' HSCs for transplantation purposes and lineage-specific cells, e.g. red blood cells, for transfusion purposes. CD43-expressing cells have been reported as the first hematopoietic cells during development, but whether or not these possess multilineage differentiation and long-term engraftment potential is incompletely understood. METHODS: We performed ex vivo generation of hematopoietic cells from hiPSCs using an embryoid body(EB)-based, xeno-product-free differentiation protocol. We investigated the multilineage differentiation potential of different FACS-sorted CD43-expressing cell subsets by colony-forming assays in semisolid media. Further, erythroid differentiation was investigated in more detail using established protocols. RESULTS: By using CD43, we were able to measure hematopoietic induction efficiency during hiPSC-derived EB differentiation. Further, we determined CD43+ cells as the cell population of origin for in vitro erythropoiesis. Furthermore, colony formation demonstrates that the multipotent hematopoietic stem and progenitor cell fraction is particulary enriched in the CD43hi CD45+ population.

8.
Histochem Cell Biol ; 146(6): 647-650, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27695942

RESUMEN

Anatomy as a descriptive topic of research and instruction in medicine has been increasingly influenced by discoveries in molecular cell and developmental biology and most recently the advent of human induced pluripotent stem cells and organoids. We summarize here how anatomy has been influenced by developmental and stem cell biologists, and how in vitro modelling of the three-dimensional body environment is emerging to understand structure and function of cells during differentiation processes in development and disease.


Asunto(s)
Anatomía/métodos , Organoides/citología , Células Madre Pluripotentes/citología , Anatomía/tendencias , Animales , Diferenciación Celular , Humanos , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo
9.
Haematologica ; 100(1): 32-41, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25326431

RESUMEN

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.


Asunto(s)
Diferenciación Celular , Células Eritroides/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Biomarcadores/metabolismo , Metilación de ADN , Epigenómica , Células Eritroides/metabolismo , Sangre Fetal/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
10.
Nature ; 461(7264): 649-3, 2009 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-19718018

RESUMEN

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.


Asunto(s)
Desdiferenciación Celular , Reprogramación Celular , Feto/citología , Neuronas/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Biomarcadores/análisis , Diferenciación Celular , Línea Celular , Metilación de ADN , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Perfilación de la Expresión Génica , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Humanos , Factor 4 Similar a Kruppel , Ratones , Neuronas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética
11.
Proc Natl Acad Sci U S A ; 109(40): 16196-201, 2012 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-22991473

RESUMEN

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.


Asunto(s)
Reprogramación Celular/fisiología , Metilación de ADN/genética , Epigénesis Genética/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Línea Celular , Reprogramación Celular/genética , Islas de CpG/genética , Epigénesis Genética/genética , Epigenómica , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Humanos , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN
12.
PLoS Biol ; 9(7): e1001099, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21765802

RESUMEN

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH⁻/⁻ mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH⁻/⁻-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH⁻/⁻ iPS cell lines, we aggregated FAH⁻/⁻-iPS cells with tetraploid embryos and obtained entirely FAH⁻/⁻-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAH⁻/⁻ mice. Then, we transduced FAH cDNA into the FAH⁻/⁻-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.


Asunto(s)
Fibroblastos/efectos de los fármacos , Prueba de Complementación Genética/métodos , Terapia Genética/métodos , Vectores Genéticos/farmacología , Hidrolasas , Células Madre Pluripotentes Inducidas , Lentivirus , Tirosinemias , Animales , Supervivencia Celular , Células Cultivadas , Cromosomas/química , Ciclohexanonas/farmacología , Modelos Animales de Enfermedad , Femenino , Feto , Fibroblastos/citología , Humanos , Hidrolasas/deficiencia , Hidrolasas/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/fisiología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Nitrobenzoatos/farmacología , Embarazo , Regiones Promotoras Genéticas , Virus Formadores de Foco en el Bazo/química , Virus Formadores de Foco en el Bazo/genética , Tetraploidía , Tirosinemias/genética , Tirosinemias/metabolismo , Tirosinemias/patología , Tirosinemias/terapia
13.
Nature ; 454(7204): 646-50, 2008 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-18594515

RESUMEN

Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.


Asunto(s)
Células Madre Adultas/citología , Reprogramación Celular , Neuronas/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Quimera , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Perfilación de la Expresión Génica , Genes myc/genética , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Proteínas de Homeodominio/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN no Traducido , Factores de Transcripción SOXB1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transducción Genética
14.
Bio Protoc ; 14(9): e4984, 2024 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-38737507

RESUMEN

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

15.
Cell Stem Cell ; 31(5): 676-693.e10, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38626772

RESUMEN

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.


Asunto(s)
Demencia Frontotemporal , Neuronas , Osteopontina , Proteínas tau , Osteopontina/metabolismo , Osteopontina/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Demencia Frontotemporal/metabolismo , Humanos , Neuronas/metabolismo , Neuronas/patología , Animales , Proteínas tau/metabolismo , Ratones , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Microglía/metabolismo , Microglía/patología , Mutación/genética
16.
Mol Med ; 19: 399-408, 2013 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-24408113

RESUMEN

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1-3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of ß-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.


Asunto(s)
Eritropoyetina/farmacología , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Neurogénesis , Animales , Proliferación Celular , Supervivencia Celular , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/fisiología , Oligodendroglía/fisiología , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Potenciales Sinápticos
17.
Gastroenterology ; 142(4): 907-17, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22245845

RESUMEN

BACKGROUND & AIMS: Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. Hematopoietic and muscle stem cells can be more efficiently reprogrammed than differentiated blood or muscle cells, yet similar findings have not been shown in other primary organ systems. Moreover, molecular characteristics of the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. We analyzed the effect of differentiation stage of freshly isolated, mouse liver cells on the reprogramming efficiency. METHODS: Liver progenitor cell (LPC)-enriched cell fractions were isolated from adult (6-8 wk) and fetal (embryonic day 14.5) livers of mice and reprogrammed to become induced pluripotent stem (iPS) cells. Different transcription factors were expressed in liver cells, and markers of pluripotency were examined, along with the ability of iPS cells to differentiate, in vitro and in vivo, into different germ layers. RESULTS: Fetal and adult LPCs had significantly greater reprogramming efficiency after transduction with 3 or 4 reprogramming factors. Transduction efficiency-corrected reprogramming rates of fetal LPCs were 275-fold higher, compared with unsorted fetal liver cells, when 3 reprogramming factors were transduced. The increased reprogramming efficiency of LPCs, compared with differentiated liver cells, occurred independently of proliferation rates, but was associated with endogenous expression of reprogramming factors (Klf4 and c-Myc) and BAF (Brg1/Brm associated factor)-complex members Baf155 and Brg1, which mediate epigenetic changes during reprogramming. Knockdown of BAF complex members negated the increased reprogramming efficiency of LPCs, compared with non-LPCs. CONCLUSIONS: LPCs have intrinsic, cell proliferation-independent characteristics resulting in an increased reprogramming capacity compared to differentiated liver cells.


Asunto(s)
Diferenciación Celular , ADN Helicasas/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , ADN Helicasas/genética , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Genotipo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Hígado/embriología , Ratones , Proteínas Nucleares/genética , Fenotipo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Factores de Tiempo , Factores de Transcripción/genética , Transducción Genética , Transfección
18.
Ann Anat ; 247: 152050, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36693546

RESUMEN

The pure transfer of face-to-face teaching to a digital learning environment can be accompanied by a significant reduction in the physiological arousal of students, which in turn can be associated with passivity during the learning process, often linked to insufficient levels of concentration and engagement in the course work. Therefore, the aim of this study was to investigate whether students' psychobiological stress responses can be enhanced in the context of anatomical online learning and how increased physiological parameters correlate with characteristics of learning experiences in a digital learning environment. Healthy first-year medical students (n = 104) experienced a regular practical course in Microscopic Anatomy either in face-to-face learning, in passive online learning or in an interaction-enhanced version of online learning. Compared to passive online learning, students engaged in the interaction-enhanced version of online learning displayed a significantly reduced Heart Rate Variability (P 0.001, partial η2 = 0.381) along with a strong increase in salivary cortisol (P 0.001, partial η2 = 0.179) and salivary alpha-amylase activity (P 0.001, partial η2 = 0.195). These results demonstrated that the physiological arousal of students engaged in online learning can be enhanced via interactive teaching methods and pointed towards clear correlations between higher physiological responses and elementary criteria of learning experience such as engagement and attention.


Asunto(s)
Educación a Distancia , Estudiantes de Medicina , Humanos , Educación a Distancia/métodos , Aprendizaje , Curriculum , Nivel de Alerta , Enseñanza
19.
Stem Cells Int ; 2023: 9246825, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38020204

RESUMEN

Large numbers of Calpain 3 (CAPN3) mutations cause recessive forms of limb-girdle muscular dystrophy (LGMD2A/LGMDR1) with selective atrophy of the proximal limb muscles. We have generated induced pluripotent stem cells (iPSC) from a patient with two mutations in exon 3 and exon 4 at the calpain 3 locus (W130C, 550delA). Two different strategies to rescue these mutations are devised: (i) on the level of LGMD2A-iPSC, we combined CRISPR/Cas9 genome targeting with a FACS and Tet transactivator-based biallelic selection strategy, which resulted in a new functional chimeric exon 3-4 without the two CAPN3 mutations. (ii) On the level of LGMD2A-iPSC-derived CD82+/Pax7+ myogenic progenitor cells, we demonstrate CRISPR/Cas9 mediated rescue of the highly prevalent exon 4 CAPN3 mutation. The first strategy specifically provides isogenic LGMD2A corrected iPSC for disease modelling, and the second strategy can be further elaborated for potential translational approaches.

20.
Elife ; 122023 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-37963071

RESUMEN

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.


Humans contains around 650 skeletal muscles which allow the body to move around and maintain its posture. Skeletal muscles are made up of individual cells that bundle together into highly organized structures. If this group of muscles fail to develop correctly in the embryo and/or fetus, this can lead to muscular disorders that can make it painful and difficult to move. One way to better understand how skeletal muscles are formed, and how this process can go wrong, is to grow them in the laboratory. This can be achieved using induced pluripotent stem cells (iPSCs), human adult cells that have been 'reprogrammed' to behave like cells in the embryo that can develop in to almost any cell in the body. The iPSCs can then be converted into specific cell types in the laboratory, including the cells that make up skeletal muscle. Here, Mavrommatis et al. created a protocol for developing iPSCs into three-dimensional organoids which resemble how cells of the skeletal muscle look and arrange themselves in the fetus. To form the skeletal muscle organoid, Mavrommatis et al. treated iPSCs that were growing in a three-dimensional environment with various factors that are found early on in development. This caused the iPSCs to organize themselves in to embryonic and fetal structures that will eventually give rise to the parts of the body that contain skeletal muscle, such as the limbs. Within the organoid were cells that produced Pax7, a protein commonly found in myogenic progenitors that specifically mature into skeletal muscle cells in the fetus. Pax 7 is also present in 'satellite cells' that help to regrow damaged skeletal muscle in adults. Indeed, Mavrommatis et al. found that the myogenic progenitors produced by the organoid were able to regenerate muscle when transplanted in to adult mice. These findings suggest that this organoid protocol can generate cells that will give rise to skeletal muscle. In the future, these lab-grown progenitors could potentially be created from cells isolated from patients and used to repair muscle injuries. The organoid model could also provide new insights in to how skeletal muscles develop in the fetus, and how genetic mutations linked with muscular disorders disrupt this process.


Asunto(s)
Músculo Esquelético , Células Satélite del Músculo Esquelético , Humanos , Músculo Esquelético/metabolismo , Diferenciación Celular , Feto/metabolismo , Células Satélite del Músculo Esquelético/fisiología , Desarrollo de Músculos/fisiología , Factor de Transcripción PAX7/metabolismo
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