Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity

Comparison of the transcriptomes of pluripotent embryonic stem cells, multipotent adult progenitor cells and lineage restricted mesenchymal stem cells identified a unique gene expression profile of multipotent adult progenitor cells

Neuroectoderm differentiation of multipotent and pluripotent stem cells and their role in an animal model of stroke

Neurologische aandoeningen, zoals een beroerte en neurodegeneratieve ziektes, resulteren in onomkeerbare hersenschade waarvoor tot op vandaag nog geen genezende therapie voor bestaat. Meer en meer aandacht wordt geschonken aan celvervangende therapie voor de behandeling van neurologische aandoeningen. Uit deze studies blijkt dat stam/progenitorcellen kunnen migreren in hersenweefsel, na intracerebrale of intraveneuse transplantatie. In een aantal studies is functionele verbetering geobserveerd na transplantatie van stam/progenitorcellen. Echter, in veel gevallen, dragen de stamcellen niet zelf bij tot de regeneratie van het beschadigde weefsel, maar ze ondersteunen de rekrutering van endogene vasculaire en/of neurale cellen naar de ischemische lesie en/of ondersteunen neurale plasticiteit om neurale circuits te herstellen. In deze thesis wilden we het effect evalueren van exogene stamcellen in een diermodel met een beroerte. De algemene doelstelling van deze thesis was te achterhalen of transplantatie van NSC gederiveerd van pluripotente stamcellen het functioneel gedrag van de dieren na stroke zou verbeteren; en indien zo, via welk mechanisme. 1. Differentiatie van multi- en pluripotente stamcellen naar neuroectodermRat MAPC, voor het eerst beschreven door ons labo in 2002, werden gedifferentieerd door middel van drie protocols: vorming van EB, op PA6 stromale feeders en als monolayer. Na enkele aanpassingen van het monolayer protocol, vonden we een significante daling van Oct4 expressie en een stijging in Sox2 en Pax6 expressie. Echter, genen, specifiek ge-exprimeerd in primitief endoderm die ook tot expressie komen in niet gedifferentieerde rMAPC, bleven tot expressie komen in de gedifferentieerde cellen. Daarom dat genetische selectiemethodes nodig zullen zijn om een populatie te isoleren van neurale progenitorcellen. We evalueerden ook een tweede stamcel populatie, pluripotente mESC en miPSC. mESC en miPS werden gedifferentieerd met het monolayer protocol naar radiale-gliale stamcellen, die Pax6, Blbp en Glast tot expressie brachten. Deze konden vervolgens verder gedifferentieerd worden naar mature astrocyten en in mindere mate naar neuronen. 2. Evaluatie van de effeciëntie van stamcellabeling met verschillende ijzer-oxide partikels. Eén van de klinisch meest relevante niet-invasieve beeldvormingstechnieken om stamcellen in vivo te volgen is magnetische resonante beeldvorming (MRI) waarbij cellen gelabeled worden met een magnetisch contrast agens voor implantatie. We evalueerden de labelingsefficiëntie van verschillende cellijnen (rMAPC, mESC, mMSC, mESC-NSC, MEF) met verschillende (ultra)-small superparamagnetische ijzer-oxide partikels ((U)SPIOs; Resovist, Endorem and Sinerem). Fantoomstudies en ijzerkwantificatie toonden aan dat rMAPC, mMSC, mESC-NSC, MEF het best gelabeld worden door Resovist, terwijl mESC beter gelabeled worden met Endorem. Bovendien evalueerden we de mogelijke schadelijke effecten die ijzerpartikels kunnen hebben op het stamcelfenotype. We bestudeerden de proliferatiecapaciteit, Oct4 mRNA expressieniveaus, celoppervlakte fenotype en differentiatiecapaciteit van de verschillende celpopulaties met en zonder (U)SPIO labeling. In geen enkele conditie zagen we significante verschillen in het fenotype of functioneel gedrag van de cellen. Een uitzondering is de vertraagde proliferatie van rMAPC die gelabeled waren met Endorem of Sinerem. Als proof of principle hebben we Resovist gelabelde rMAPC getransplanteerd in een rat beroerte model om aan te tonen dat migratie van de cellen te zien was met MRI, wat ook bevestigd werd met histologie. 3. Vergelijking van verschillende celpopulaties getransplanteerd in een diermodel met stroke, en evaluatie van de invloed ervan op het funstionele herstel.We vergeleken het effect van transplantatie van mESC-NSC, mMSC en MEF in een fotothrombotisch beroerte model in C57/Bl6 muizen, om het functionaal deficiet te herstellen. Om het effect van de getransplanteerde cellen te evalueren op het functioneel gedrag van de muizen, testten we hoelang ze op een versnellende rotarod konden lopen. Tijd gespendeerd op de rotarod werd gemeten voor stroke en op d1, d13, d20 en d27 na stroke. Bij muizen die normale saline (NS) geïnjecteerd kregen, was er een spontane verbetering te zien in hun tijd om op de rotarod te blijven, maar op d27 bleef de tijd die ze op de rotarod bleven singificant lager dan voor stroke. Transplantatie met MEF gaf geen significante verbetering van het gedrag in vergelijking met de NS groep. In tegenstelling, tijd op de rotarod in muizen die getransplanteerd werden met mESC-NSC verbeterde significant in vergelijking met NS en MEF groep op d13 en d27. Dieren getransplanteerd met mMSC toonden ook een significante verbetering op de rotarod. We hebben nagekeken of de verschillende celpopulaties naar de stroke migreren met behulp van MRI en histologie. Op de MRI konden we een hypointense contrast zone afkomstig van de mESC-NSC en MEF gelabeled met Resovist zien tussen de locatie van injectie en de beroerte regio, wat bevestigd werd met histologie (75% en 100% van MEF of mESC-NSC behandelde dieren respectievelijk). Een dergelijke migratie was niet te zien voor mMSC behandelde dieren. Een fractie van de GFP+ mESC-NSC in het Corpus Callosum co-labelde met antistoffen tegen Gfap en NeuN, wat suggereert dat sommige van de NSC differentieerden. Deze studie is de eerste studie die verschillende celpopulaties vergelijkt als potentiële behandeling voor beroerte: we vonden dat funstionele verbetering te zien was wanneer mMSC en mESC-NSC werden getransplanteerd. Aangezien mMSC niet migreren en geen neuronen en astrocyten vormen, waar dit wel het geval is voor mESC-NSC, is het hoogst waarschijnlijk dat de functionele verbetering via trofische effecten en niet via celvervanging gebeurt door beide celtypes.status: publishe

The Notch signaling system is present in the postnatal pituitary: marked expression and regulatory activity in the newly discovered 'side population'

Recently, we discovered in the adult anterior pituitary a subset of cells with 'side population' (SP) phenotype, enriched for expression of stem/progenitor cell-associated factors like Sca1, and of Notch1 and Hes1, components of the classically developmental Notch pathway. In the present study, we elaborated the expression of the Notch signaling system in the postnatal pituitary, and examined its functional significance within the SP compartment. Using RT-PCR, we detected in the anterior pituitary of adult mouse the expression of all 4 vertebrate Notch receptors, as well as of Hes1, 5 and 6, key downstream targets and effectors of Notch. All Notch receptors, Hes1 and Hes5 were measured at higher mRNA levels in the Sca1(high) SP than in the main population (MP) of differentiated hormonal cells. In contrast, Hes6, known as an inhibitor of Hes1, was more abundant in the MP. Cells with SP phenotype, enriched for Sca1(high) expression, were detected throughout postnatal life. Their proportion was higher in immature mice, but did not change from adult (8-week-old) to much older age (1-year-old). Notch pathway expression was higher in the Sca1(high) SP than in the MP at all postnatal ages analyzed. Functional implication of Notch signaling in the SP was investigated in re-aggregate cultures of adult mouse anterior pituitary cells. Treatment with the gamma-secretase inhibitor DAPT downregulated Notch activity and reduced the proportion of SP cells. Activation of Notch signaling with the conserved DSL motif of Notch ligands, or with a soluble ligand, caused a rise in SP cell number, at least in part due to a proliferative effect. The SP also expanded in proportion when aggregates were treated with LIF, bFGF and EGF, again at least partly accounted for by a mitogenic action. These intrapituitary growth factors all activated Notch signaling, and DAPT abrogated the expansion of the SP by bFGF and LIF, thus exposing a possible crosstalk. In conclusion, we show that the Notch pathway, typically situated in embryogenesis, is also present and active in the postnatal pituitary, that it is particularly expressed within the SP independent of age, and that it plays a role in the regulation of SP abundance. Whether our data indicate that Notch regulates renewal and fate decisions of putative stem/progenitor cells within the pituitary SP as found in other tissues, remains open for further exploration.status: publishe

Visualizing Stem Cells by MRI Labels: the Effect of Paramagnetic Iron Oxide Particles on the Stem Cell Phenotype

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Characterization of the Inflammatory Response in a Photothrombotic Stroke Model by MRI: Implications for Stem Cell Transplantation

PURPOSE: The aim of this study was to evaluate the specificity of magnetic resonance imaging (MRI) contrast in a photothrombotic (PT) stroke model with and without engraftment of superparamagnetic iron oxide (SPIO)-labeled stem cells. PROCEDURES: We monitored animals with PT stroke versus animals with PT stroke and stem cell engraftment by T(2)/T(2)*w MRI 4-8 h and 2, 4, 6/7 and 14 days after PT induction. Results were correlated with immunohistochemistry. RESULTS: T(2)*w MRI images showed hypointense contrast due to the accumulation of inflammatory cells and corresponding iron accumulation and glial scar formation in the border zone of the lesion, similar as what was observed for SPIO-labeled cells. Histological analysis was thus indispensable to distinguish between labeled transplanted cells and immune cells. CONCLUSION: These results raise caution regarding the non-invasive monitoring of SPIO-labeled transplanted stem cells by MRI in models that result in a strong inflammatory response.status: publishe

PEGylation of phospholipids improves their intermembrane exchange rate

The polar headgroup of dimyristoylphosphatidylethanolamine (DMPE) was modified by attaching a hydrophilic polymer-amino acid complex and the effects of this modification on the non-protein-mediated transfer features of the lipid determined. The first step in the organic synthesis protocol was the conversion of alpha,omega-dicarboxy poly(ethylene glycol) into its anhydride form, followed by attachment of the molecule to the amino group of DMPE. In the second step, the remaining free -COOH group on the polymer terminus was activated consecutively with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysuccinimide, and then coupled to tryptophan. The purified conjugate was mixed with dipentadecanoylphosphatidylglyceroI (1/9 molar ratio) and sonicated to produce small unilamellar vesicles. These vesicles (donors) were then mixed with dipentadecanoylphosphatidylglyceroI magnetoliposomes (acceptors) and the transfer kinetics of the modified lipid followed, using high-gradient magnetophoresis as the fractionation technique. The transfer behavior of non-modified DMPE was also examined. The first-order kinetic plots, corrected for back exchange lipid movement, showed that hydrophilization of DMPE dramatically improved its transfer capacity. These findings are explained by considering the thermodynamical consequences of the exchange process. The possibility of using biomolecule-derivatized phospholipids to trigger physiological effects in biological cells is briefly discussed

Effects of MRI Contrast Agents on the Stem cell Phenotype

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell based therapies. To follow the fate of grafted cells in vivo, a number of non-invasive imaging approaches have been developed. Magnetic Resonance Imaging (MRI) is a high resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of 3 different stem cell populations (mouse Embryonic Stem Cells, rat Multipotent Adult Progenitor Cells and mouse Mesenchymal Stem Cells) with three different (ultra) small superparamagnetic iron oxide (U)SPIOs particles (Resovist(R), Endore(R), Sinerem(R)) was compared. Labeling efficiency with Resovist(R) and Endorem(R) differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI, did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.status: publishe

MAPC culture conditions support the derivation of cells with nascent hypoblast features from bone marrow and blastocysts

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Expression and endothelial-like and hepatocyte-like differentiation for mMAPC-1, mMAPC-2, mClone-3, rMAPC, and rClone-2

The levels of () mRNA in mouse (m, left) and rat (r, right) clones compared with those of mRNA. The mouse clones are also compared with mESCs. ΔCT is difference in threshold cycles calculated as Oct4 CT - Gapdh CT. ND, not detected. Endothelial-like differentiation. mRNA levels of endothelial markers in mouse (left panel) and rat (right panel) clones before and after differentiation, measured at day 9 in two independent differentiations of each clone. Levels are compared with those in universal mouse RNA and rat spleen RNA, respectively. Left panel: blue diamonds, Pecam (×10) (values shown were scaled by the factors in brackets); pink squares, Lyve1; orange triangles, vWF. Right panel: blue diamonds, VE-Cad (×100); pink squares, Flt-1; orange triangles, Flk-1; turquoise crosses, vWF (×100). Hepatocyte-like differentiation. mRNA levels of hepatocyte markers in mouse and rat clones before and after differentiation. Levels are compared with levels in mouse hepatocytes and rat liver, respectively. Two representative differentiations measured at day 18 are shown. Left panel: blue diamonds, F2 (×100); pink squares, Tat, (×10); green triangles, Afp (×10); turquoise crosses, Ttr (×10). Right panel: blue diamonds, Afp (×100); pink squares, Alb, (×10); orange triangles, Tat (×100); turquoise crosses, Ttr (×10). See text for abbreviations.<p><b>Copyright information:</b></p><p>Taken from "Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity"</p><p>http://genomebiology.com/2007/8/8/R163</p><p>Genome Biology 2007;8(8):R163-R163.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC2374994.</p><p></p