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Genomic and other cell tracing approaches , reprogramming

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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Genomic and other cell tracing approaches , reprogramming

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. PéterBalogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 5 Genomic and othercelltracingapproaches, reprogramming

  3. Animalcloning • 1952: Tadpole • 1963: Carp • 1986: Mice • 1996: Sheep • 2000: Monkey • 2001: Cattle, cat • 2003: Rat, horse, mule • 2005: Dog • 2008: Human

  4. Stem Cell Potential

  5. Origin of stem cells and reprogramming Zygote Blastocyst Epiblast (post-implantation) Lateembryo/ earlyfoetus Adult Skin Fate decision Fate decision Centralnervous system Innercellmass Epiblast Primordial germcells Bonemarrow Embryonic stemcells Epiblast stemcells Embryonic germcells Adult stemcells Other Totipotent Pluripotent Pluripotent Pluripotent Multipotentor unipotent + Oct4, Sox2, Klf4, Myc Inducedpluripotent stemcells Pluripotent

  6. Conventional Sources of Stem Cells • 1 Somaticstem cells • Harvested from mature organs or tissues (bone marrow) • Multipotent, may be tissue specific, pluripotent? • Many established clinical uses • 2 Embryonic stem cell • Derived from ICM of blastocyst • Pluripotent, differentiate to all cell lineages • Encumbered by technical and ethical issues • May be induced from adult tissues

  7. Origins of ES Cell Lines • 1 Excess IVF embryos • 2 Therapeutic Cloning (somatic cell nuclear transfer) • Donor oocyte+somatic cell nucleus • Cells have characteristics of nuclear donor • Lines representing different diseases • Individualized lines: non-immunogenic to donor

  8. Somatic Cell Nuclear Transfer • Challenging: In cloned cell lines, about 4% of genes function abnormally, owingto departures from normal activation or expression of certain genes-Imprinting, methylation state • Limited success: ~25 percent of nuclear transfers led to a blastocyst; 35 percent of blastocysts led to establishment of cell lines

  9. Micromanipulationequipment • Invertedmicroscope (fluorescent) • CO2incubator • Thermal / heatablestage • Holding pipette (innerdiameter 10 µm) • Injectionpipette ( innerdiameter 7 µm)

  10. Chromosomeremoval(‘Enucleation’) • Chemicalenucleation: usingspecificinhibitors • Mechanicalenucleation: • 1The egg is immobilized on the holding pipette with the chromosome–spindle. • 2The zonapellucida is penetrated by the injection pipette and the injection pipette is pushed against the chromosome–spindle complex for aspiration. • 3Aspiration of the chromosome–spindle complex. • 4Complete removal of the chromosome–spindle complex and exit of injection pipette. • 5Release of chromosome–spindle complex.

  11. Nuclearinjection • Electrofusion • Microinjection: • 1Penetration of the egg’s zonapellucida by the injection pipette. • 2Aspiration of small amount of cytoplasm to facilitate re-sealing of the egg’s plasma membrane.

  12. Eggactivation • Mammalianeggsarearrestedinmetaphasestageduringovulation. • SCNT is unabletoreinitiate/ triggerthecellcycle, PLCζenzyme is missing, resultinginabolishedCa2+influx. • Duringeggactivation Ca2+rise is essential, whichcan be evokedbystrontiumchloride (SrCl2). • SrCl2treatment is more effectivethanEtOHorionophores. • Onehourafternuclearinjectionhappened, eggactivationcan be performedinspecializedconditions.

  13. Blastocysts and ESC colonyformation • ESCscan be derivedfromeightcellembryosorfrommorulastage, howeverthe most efficienscenario, whenblastocystsareused. • By the 5th or 6th day after plating, an inner cell massoutgrowth is usuallyobserved. • Fortheculture of ESC cellsfeedercellsareessential. • Four to five days later ESC colonies should appearattheside of culturedishes.

  14. StemcellcharacterizationI • Characterization: test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells • Growing and subculturing the stem cells for many months microscope inspection for the healthy and undifferentiated of cells. • Using specific techniques to determine the presence of surface markers that are found only on undifferentiated cells • Presence of Oct4 a transcription factor, which helps turn geneson and off at the right time for the processes of cell differentiation and embryonic development.

  15. StemcellcharacterizationII • Determining whether the cells can be subcultured after freezing, thawing, and replating • Testing whether the human embryonic stem cells are pluripotent by: • allowing the cells to differentiate spontaneously in cell culture • manipulating the cells so they will differentiate to form specific cell types • injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma • Teratomas typically contain a mixture of many differentiated or partly differentiated cell types • An indication that the embryonic stem cells are capable of differentiating into multiple cell types. • When embryoid bodies are formed they begin to differentiate spontaneously/They can form muscle cells/nerve cell/another types.

  16. StemcellmarkersI • Oct4:octamer-bindingtranscriptionfactor 4 homeodomaintr. molecule is codedby POU5F1 gene and marks ES cells and undifferentiated, maternalfactoractiveinoocyteandinembryos. • Sox2:or SRY (sex determiningregion Y)-box 2 HMG factor act as a transcriptional activatorafter forming a protein complex with other proteins (Oct4, Pax6). EssentialforiPScformation. • SSEA3/4:stagespecificembryonicantigensareof five to six monosaccharides attached to a ceramidelipidtail. Theirpresencerapidlyincreasingduringdifferentiation. SSEA-3 and SSEA-4 were recently shown not to be essential for the maintenance of hESCpluripotency

  17. StemcellmarkersII • TRA-1-60, TRA-1-81: tumor rejectionantigenswidelyusedmarkersforstemcellcharacterization. Theycanrecognizea keratan-sulfatedproteoglycan (KSPG) inneuraminidase-sensitive and neuraminidase-insensitivefashion. • Alkalinephosphataseis a hydrolaseenzyme, it is alsoessentialtoidentifystemcells and verifytheirfunctionality.

  18. Celltracinginstemcellbiology:non genomic • BrdU(bromodeoxyuridine) incorporation • Fluorescentdyes: • CM-DiI • CFSE • Hoechst 33342 • PKH26

  19. Celltracinginstemcellbiology: genomicI • 1 GFP • 27 kDa protein (isolatedoriginallyfromjellyfish) • popularreportersystemintissueaftercloninggene of interest • different GFP variant • 2 Lac-Z • lacoperongenefromE. coli • histochemicalreporterusingX-galsubstrate

  20. Celltracinginstemcellbiology: genomicII • 3 Y chromosome marker • The detection is a relativelysimpleprocesscomparedtogenecloning and expressionbasedmethods (GFP, LacZ) • FISH analysis • Highlabelingefficiency • Widelyusedstemcelltransplantationapproaches (cardiac-, intestinedisease)

  21. In vivo imagingforcelltracing • New development of timelapse and two-photonmicroscopygaveboostforlivecellimagingincludingcelltracing. • Stem cells can be imaged at various time points and locations to generate time-lapse movies, and automated image analysis and statistical analyses are used to quantify the dynamic cells’ behaviour. • Together with cell migration, changes in cell shape and changes in proliferation kineticscan be monitored.

  22. Celltracinginstemcellbiology z t1 x Automated image analyses and statisticalanalyses y t2 tn Proliferation Cell-shapechange Migration Single-cellfateanalyses

  23. Reprogramming • Somaticcellscan be dedifferentiatedintostemcells, socalledinducedpluripotentstem (iPS) cellsusingcertainaprroaches. • Cell-fusionbased • Nuclearextractbased • Transfection of pluripotentfactors • Somaticcellnucleartransfer

  24. Molecular mechanisms ofself-renewal Lif PI3K Grb2 Jak Akt MAPK STAT3 Cell-cycle regulation Tbx3 Klf4 S G2 Nanog Sox2 b-Myb c-Myc Oct3/4 G1 M Cdx2 Gata4 Prevention of differentation

  25. Genesinvolvedinreprogramming • Nanog:The nanogcDNAconsistsof 2184 nucleotides (nt) and containsa single open reading frameencoding a poly-peptide of 305 amino acids. • theroleinpluripotencyof both inner cell mass (ICM) and embryonic stem(ES)cells • the ability to maintain ES cell self-renewal. • Klf4:Krüppel-likefactor, interactwith CREB transcriptionfactor, expressedin ESC and MSCs. • Lin28: a cytoplasmicmRNAbinding protein, binds to IGF-2 mRNA, enhance the efficiency of the formation of iPSc from human fibroblasts, marker of undifferentiated human embryonic stem cells, abletobind let-7 miRNA and inhibitit. • Oct4:seepreviously • Sox2:seepreviously

  26. TelomeraseactivityI • Telomeres are ribonucleoprotein heterochromatic structuresat the ends of chromosomes that protect them fromdegradation and from being detected as double-strand DNA breaks. • When Dolly wasclonedusing SCNT, reliablequestionwasraisedabouttheage of hercells? Telomerewas shorter, by approximately 20%, when comparedwithage-matchedcontrols. • Aftersomeconflictingresultsconcludedthatshortenedtelomeresof somatic donor cells could be indeed re-elongatedduring reprogramming, although the degree of elongationwas quite variable, underscoring the complexity of telomerelength control.

  27. TelomeraseactivityIITelomersiniPScells • Highlevels of Tert (reversetranscriptasecomponentoftelomerase) and high telomerase activity were describediniPScells. • iPSreprogramming of normalcells (mice and human) resultstelomeraseactivation and restoration of telomeres, likesettingtheclock, to a length and chromatinstatethat is similartothatfoundin ES cells. • TelomeraseactivationduringiPS reprogramming is associated with upregulation of TERT, butalso TERC (Tel. AssociatedrNAcomponent) becomeactivated. MoreoverOCT4 and NANOG bind to the TERC gene reg. element, which may explain why these components are upregulated in iPS cells

  28. Summary • ES and iPScellfatescan be monitoredwith a branch of fluorescentvitaldyes (non-genomic/ genomic) usingin vivo imagingtechniques. • Restoration of pluripotencyfactors and self-renewalspecificgenes (Oct4, Sox2, Klf4) canexhibitthereprogrammingforiPScells. • iPScellgenerationcan be goodcandidatesforregenerativemedicine, howevertherearestillseveralunansweredquestions/ concernsinreprogramming.

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