Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices.
- MeSH
- buněčná a tkáňová terapie MeSH
- kadheriny genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky * MeSH
- obchod MeSH
- vitronektin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cell (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular 'raw material' needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.
- MeSH
- biokompatibilní potahované materiály farmakologie MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- extracelulární matrix - proteiny metabolismus MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- myši MeSH
- nervové kmenové buňky cytologie účinky léků metabolismus MeSH
- neurony cytologie účinky léků metabolismus MeSH
- peptidy farmakologie MeSH
- pluripotentní kmenové buňky cytologie účinky léků metabolismus MeSH
- proliferace buněk účinky léků MeSH
- vitronektin farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
BACKGROUND: Vitronectin is an abundant plasma glycoprotein identified also as a part of extracellular matrix. Vitronectin is substantially enriched at sites of injured, fibrosing, inflamed, and tumor tissues where it is believed to be involved in wound healing and tissue remodeling. Little is known about the mechanism of vitronectin localization into the damaged tissues. METHODOLOGY/PRINCIPAL FINDINGS: 2E12 antibody has been described to bind a subset of late apoptotic cells. Using immunoisolation followed by mass spectrometry, we identified the antigen recognized by 2E12 antibody as vitronectin. Based on flow cytometry, we described that vitronectin binds to the late apoptotic and necrotic cells in cell cultures in vitro as well as in murine thymus and spleen in vivo. Confocal microscopy revealed that vitronectin binds to an intracellular cytoplasmic structure after the membrane rupture. CONCLUSIONS/SIGNIFICANCE: We propose that vitronectin could serve as a marker of membrane disruption in necrosis and apoptosis for flow cytometry analysis. Moreover, we suggest that vitronectin binding to dead cells may represent one of the mechanisms of vitronectin incorporation into the injured tissues.
- MeSH
- apoptóza fyziologie MeSH
- buněčné linie MeSH
- erytrocyty cytologie metabolismus MeSH
- hmotnostní spektrometrie MeSH
- Jurkat buňky MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- lidé MeSH
- myši MeSH
- nekróza MeSH
- průtoková cytometrie MeSH
- slezina cytologie metabolismus MeSH
- thymus cytologie metabolismus MeSH
- vazba proteinů MeSH
- vitronektin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The piezoelectric sensor (quartz crystal microbalance, QCM) was used to monitor cell adhesion in real time. Two cell lines, rat epithelial cells (WB F344) and lung melanoma cells (B16F10) were used. The cells were adhered and grown on the gold surface of the sensor pre-coated with adsorbed layer of extracellular matrix proteins as vitronectin and laminin. The process of cell attachment and spreading on the gold surface was continuously monitored and displayed by changes of the resonant frequency Deltaf and resistance DeltaR values of the piezoelectric resonators. The initial phase of cell attachment and spreading induced a decrease of frequency and increase of resistance relating viscoelastic properties of the cell monolayer on the sensing surface. The steady-state of both shifts was achieved after a few hours. The presence and state of cells on the surface was confirmed by fluorescent microscopy. The obtained results demonstrate that the piezoelectric sensor is suitable for studies of the cell adhesion processes. Thus obtained cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment.
- MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- buněčná adheze fyziologie MeSH
- buněčné linie MeSH
- financování organizované MeSH
- krysa rodu rattus MeSH
- nádorové buněčné linie MeSH
- vitronektin MeSH
- zlato MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- infarkt myokardu patofyziologie MeSH
- matrixové metaloproteinasy fyziologie MeSH
- myši MeSH
- vitronektin analýza fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- kongresy MeSH
- MeSH
- 1-fosfatidylinositol-3-kinasa genetika imunologie MeSH
- aktivace enzymů genetika imunologie MeSH
- analýza buněčné migrace metody využití MeSH
- buňky HT-29 cytologie imunologie MeSH
- cytokiny genetika imunologie MeSH
- extracelulární matrix genetika imunologie MeSH
- faktor stimulující štěpení genetika imunologie MeSH
- insulinu podobný růstový faktor I genetika imunologie MeSH
- integriny genetika imunologie MeSH
- kadheriny genetika imunologie MeSH
- kateniny genetika imunologie MeSH
- lidé MeSH
- MAP kinasový signální systém genetika imunologie MeSH
- membránové glykoproteiny genetika imunologie MeSH
- nádorové buňky kultivované cytologie imunologie MeSH
- pohyb buněk genetika imunologie MeSH
- receptory buněčného povrchu genetika imunologie MeSH
- vitronektin genetika imunologie MeSH
- Check Tag
- lidé MeSH
