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1.
Stem Cells Transl Med ; 13(9): 898-911, 2024 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-39042522

RESUMEN

Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Inmunoterapia/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Línea Celular , Neoplasias/terapia , Neoplasias/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Flujo de Trabajo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo
2.
Stem Cells Dev ; 20(3): 485-93, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20536317

RESUMEN

In the early mammalian embryo, lineage separation of and subsequent crosstalk between the trophectoderm (TE) and inner cell mass (ICM) are required to support further development. Previous studies have shown that the homeobox transcription factor Cdx2 is required for TE differentiation and that lack of Cdx2 expression causes death of embryos at the peri-implantation stage. In this study, we effectively eliminated Cdx2 transcripts by microinjection of siRNA into embryos and evaluated the effect on efficiency of deriving embryonic stem cells (ESCs). By this approach, we successfully created nonviable embryos similar to reported knockout embryos. Accordingly, the efficiency of ESC derivation dropped from 19.1% in control blastocysts to 2% in Cdx2-deficient blastocysts, indicating loss of pluripotency in the ICM. Strikingly, when 8-cell stage embryos were cultured under ESC culture conditions before lineage separation, fully functional pluripotent stem cell lines were obtained, with efficiency even greater than that for control embryos. These results demonstrate that Cdx2 plays an essential role within the microenvironment created by the TE to support ICM pluripotency but that the ESC culture system, with mouse embryonic fibroblasts, could rescue the pluripotent cell population for efficient ESC derivation.


Asunto(s)
Masa Celular Interna del Blastocisto/citología , Ectodermo/citología , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/deficiencia , Animales , Masa Celular Interna del Blastocisto/metabolismo , Factor de Transcripción CDX2 , Células Cultivadas , Ectodermo/metabolismo , Células Madre Embrionarias/trasplante , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Microinyecciones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/trasplante , Embarazo , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Teratoma/patología , Factores de Transcripción/genética , Transcripción Genética
3.
Development ; 137(24): 4159-69, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21098565

RESUMEN

The separation of the first two lineages - trophectoderm (TE) and inner cell mass (ICM) - is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a 'switch' regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Factor de Transcripción CDX2 , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Proteínas de Homeodominio/genética , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Potencial de la Membrana Mitocondrial/genética , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Microscopía Electrónica de Transmisión , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Oocitos/citología , Embarazo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética
4.
Biochem Biophys Res Commun ; 335(3): 810-8, 2005 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-16105659

RESUMEN

The mechanism of blood-brain barrier breakdown in the complex pathogenesis of cerebral malaria is not well understood. In this study, primary cultures of porcine brain capillary endothelial cells (PBCEC) were used as in vitro model. Membrane-associated malaria antigens obtained from lysed Plasmodium falciparum schizont-infected erythrocytes stimulated human peripheral blood mononuclear cells (PBMC) to secrete tumor necrosis factor alpha. In co-cultivation with the brain endothelial cell model, the malaria-activated PBMC stimulated the expression of E-selectin and ICAM-1 on the PBCEC. Using electric cell-substrate impedance sensing, we detected a significant decrease of endothelial barrier function within 4h of incubation with the malaria-activated PBMC. Correspondingly, immunocytochemical studies showed the disruption of tight junctional complexes. Combination of biochemical and biophysical techniques provides a promising tool to study changes in the blood-brain barrier function associated with cerebral malaria. Moreover, it is shown that the porcine endothelial model is able to respond to human inflammatory cells.


Asunto(s)
Barrera Hematoencefálica , Plasmodium falciparum/fisiología , Animales , Antígenos de Protozoos/inmunología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Plasmodium falciparum/inmunología , Porcinos , Factor de Necrosis Tumoral alfa/farmacología
5.
Biosens Bioelectron ; 19(6): 583-94, 2004 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-14683642

RESUMEN

Apoptosis is a strictly regulated and genetically encoded cell 'suicide' that may be triggered by cytokines, depletion of growth factors or certain chemicals. It is morphologically characterized by severe alterations in cell shape like cell shrinkage and disintegration of cell-cell contacts. We applied a non-invasive electrochemical technique referred to as electric cell-substrate impedance sensing (ECIS) in order to monitor the apoptosis-induced changes in cell shape in an integral and quantitative fashion with a time resolution in the order of minutes. In ECIS the cells are grown directly on the surface of small gold-film electrodes (d = 2 mm). From readings of the electrical impedance of the cell-covered electrode, performed with non-invasive, low amplitude sensing voltages, it is possible to deduce alterations in cell-cell and cell-substrate contacts. To improve the sensitivity of this impedance assay we used endothelial cells derived from cerebral micro-vessels as cellular model systems since these are well known to express electrically tight intercellular junctions. Apoptosis was induced by cycloheximide (CHX) and verified by biochemical and cytological assays. The time course of cell shape changes was followed with unprecedented time resolution by impedance readings at 1 kHz and correlated with biochemical parameters. From impedance readings along a broad frequency range of 1-10(6) Hz we could assign the observed impedance changes to alterations on the subcellular level. We observed that disassembly of barrier-forming tight junctions precedes changes in cell-substrate contacts and correlates strongly with the time course of protease activation.


Asunto(s)
Apoptosis/fisiología , Técnicas Biosensibles/instrumentación , Técnicas de Cultivo de Célula/instrumentación , Electroquímica/instrumentación , Células Endoteliales/citología , Células Endoteliales/fisiología , Animales , Apoptosis/efectos de los fármacos , Técnicas Biosensibles/métodos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Cicloheximida/farmacología , Impedancia Eléctrica , Electroquímica/métodos , Electrodos , Células Endoteliales/efectos de los fármacos , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
6.
Brain Res ; 981(1-2): 30-40, 2003 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12885423

RESUMEN

Cultured cerebral capillary endothelial cells are often used as a functional in vitro model of the blood-brain barrier (BBB) to determine drug uptake or to study barrier properties. Usually serum is supplemented to these cultures for cell proliferation. Here, we demonstrate the effect of serum and the serum-derived factors lysophosphatidic acid (LPA) and vascular endothelial growth factor (VEGF) on the barrier properties of cultured porcine brain capillary endothelial cells (PBCEC). Serum prevents tight junction formation of confluent PBCEC monolayers and moreover, opens already established tight junctions shown by decreasing transendothelial electrical resistances (TER). These effects are highly polarised with serum almost exclusively acting from the basolateral side of the cell culture. Immunocytochemistry of PBCEC revealed a delocalisation of the cell border lining tight junction proteins ZO-1, occludin and claudin-5 when serum was added. A serum fraction of 67 kDa was isolated by size-exclusion chromatography, identified as albumin and found to cause a serum-like decrease of the TER. However, fatty acid-free serum albumin does not develop this barrier weakening effect, indicating that small protein-bound factors might be responsible. For instance, serum-bound LPA demonstrated a TER-decreasing effect as well, but in contrast to serum mainly when added to the apical side of PBCEC. Addition of VEGF caused a serum-like decrease of the TER with the same polar effect; however, VEGF will be denatured by heat and could thus not be the heat-sensitive factor. Thus, we hypothesise that serum contains a variety of factors which weaken the tightness of a PBCEC monolayer from the apical side as expected but also from the basolateral side. Although the structure of the 67 kDa factor could not be analysed, this finding is of importance for in vitro models not only of the blood-brain barrier mostly using serum-containing media.


Asunto(s)
Permeabilidad Capilar/fisiología , Endotelio Vascular/metabolismo , Plasma/metabolismo , Animales , Barrera Hematoencefálica/fisiología , Encéfalo/citología , Encéfalo/ultraestructura , División Celular/fisiología , Células Cultivadas , Cromatografía en Gel , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Inmunohistoquímica , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Ocludina , Fosfoproteínas/metabolismo , Porcinos , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Proteína de la Zonula Occludens-1
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