Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Péter Balogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 5 GENOMIC AND OTHER CELL TRACING APPROACHES, REPROGRAMMING TÁMOP-4.1.2-08/1/A-2009-0011 Animal cloning • • • • • • • • 1952: 1963: 1986: 1996: 2000: 2001: 2003: 2005: Tadpole Carp Mice Sheep Monkey Cattle, cat Rat, horse, mule Dog • 2008: Human TÁMOP-4.1.2-08/1/A-2009-0011 Stem Cell Potential Type Description Examples Totipotent Cells develop into a new individual Cells of 1-4 days old embryos Pluripotent Cells form any cell type Some cells of blastocyst (5-14 days old) Multipotent Differentiated cells, but Fetal tissue, cord blood, can form other tissues and adult stem cells TÁMOP-4.1.2-08/1/A-2009-0011 Origin of stem cells and reprogramming Zygote Epiblast Late embryo/ Blastocyst (post-implantation) early foetus Fate decision Adult Skin Fate decision Inner cell mass Epiblast Central nervous system Primordial germ cells Bone marrow Embryonic stem cells Totipotent Pluripotent Epiblast stem cells Pluripotent Embryonic germ cells Adult stem cells Pluripotent Multipotent or unipotent Induced pluripotent stem cells Pluripotent Other + Oct4, Sox2, Klf4, Myc TÁMOP-4.1.2-08/1/A-2009-0011 Conventional Sources of Stem Cells 1 Somatic stem 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 TÁMOP-4.1.2-08/1/A-2009-0011 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 Somatic Cell Nuclear Transfer TÁMOP-4.1.2-08/1/A-2009-0011 • Challenging: In cloned cell lines, about 4% of genes function abnormally, owing to 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 TÁMOP-4.1.2-08/1/A-2009-0011 Micromanipulation equipment • Inverted microscope (fluorescent) • CO2 incubator • Thermal / heatable stage • Holding pipette (inner diameter 10 µm) • Injection pipette ( inner diameter 7 µm) Chromosome removal (‘Enucleation’) TÁMOP-4.1.2-08/1/A-2009-0011 • Chemical enucleation: using specific inhibitors • Mechanical enucleation: 1 The egg is immobilized on the holding pipette with the chromosome–spindle. 2 The zona pellucida is penetrated by the injection pipette and the injection pipette is pushed against the chromosome– spindle complex for aspiration. 3 Aspiration of the chromosome–spindle complex. 4 Complete removal of the chromosome–spindle complex and exit of injection pipette. TÁMOP-4.1.2-08/1/A-2009-0011 Nuclear injection • Electrofusion • Microinjection: 1 Penetration of the egg’s zona pellucida by the injection pipette. 2 Aspiration of small amount of cytoplasm to facilitate re-sealing of the egg’s plasma membrane. TÁMOP-4.1.2-08/1/A-2009-0011 Egg activation • Mammalian eggs are arrested in metaphase stage during ovulation. • SCNT is unable to reinitiate / trigger the cell cycle, PLCζ enzyme is missing, resulting in abolished Ca2+ influx. • During egg activation Ca2+ rise is essential, which can be evoked by strontium chloride (SrCl2). • SrCl2 treatment is more effective than EtOH or ionophores. • One hour after nuclear injection happened, egg activation can be performed in specialized conditions. TÁMOP-4.1.2-08/1/A-2009-0011 Blastocysts and ESC colony formation • ESCs can be derived from eight cell embryos or from morula stage, however the most efficien scenario, when blastocysts are used. • By the 5th or 6th day after plating, an inner cell mass outgrowth is usually observed. • For the culture of ESC cells feeder cells are essential. • Four to five days later ESC colonies should appear at the side of culture TÁMOP-4.1.2-08/1/A-2009-0011 Stem cell characterization I • 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 genes on and off at the right time for the processes of cell TÁMOP-4.1.2-08/1/A-2009-0011 Stem cell characterization II • 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. TÁMOP-4.1.2-08/1/A-2009-0011 Stem cell markers I • Oct4: octamer-binding transcription factor 4 homeodomain tr. molecule is coded by POU5F1 gene and marks ES cells and undifferentiated, maternal factor active in oocyte and in embryos. • Sox2: or SRY (sex determining region Y)-box 2 HMG factor act as a transcriptional activator after forming a protein complex with other proteins (Oct4, Pax6). Essential for iPSc formation. • SSEA3/4: stage specific embryonic antigens are of five to six monosaccharides attached to a ceramide lipid tail. Their presence rapidly increasing during differentiation. SSEA-3 and SSEA-4 were recently shown not to be essential for the maintenance of hESC pluripotency TÁMOP-4.1.2-08/1/A-2009-0011 Stem cell markers II • TRA-1-60, TRA-1-81: tumor rejection antigens widely used markers for stem cell characterization. They can recognize a keratan-sulfated proteoglycan (KSPG) in neuraminidase-sensitive and neuraminidaseinsensitive fashion. • Alkaline phosphatase is a hydrolase enzyme, it is also essential to identify stem cells and verify their functionality. Cell tracing in stem cell biology: non genomic TÁMOP-4.1.2-08/1/A-2009-0011 • BrdU (bromodeoxyuridine) incorporation • Fluorescent dyes: – CM-DiI – CFSE – Hoechst 33342 – PKH26 TÁMOP-4.1.2-08/1/A-2009-0011 Cell tracing in stem cell biology: genomic I 1 GFP • 27 kDa protein (isolated originally from jellyfish) • popular reporter system in tissue after cloning gene of interest • different GFP variant 2 Lac-Z • lac operon gene from E. coli • histochemical reporter using X-gal substrate TÁMOP-4.1.2-08/1/A-2009-0011 Cell tracing in stem cell biology: genomic II 3 Y chromosome marker • The detection is a relatively simple process compared to gene cloning and expression based methods (GFP, LacZ) • FISH analysis • High labeling efficiency • Widely used stem cell transplantation approaches (cardiac, intestine disease) TÁMOP-4.1.2-08/1/A-2009-0011 In vivo imaging for cell tracing • New development of time lapse and twophoton microscopy gave boost for live cell imaging including cell tracing. • 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 kinetics can be TÁMOP-4.1.2-08/1/A-2009-0011 Cell tracing in stem cell biology z t1 x y t2 Automated image analyses and statistical analyses tn Migration Proliferation Single-cell fate analyses Cell-shape change TÁMOP-4.1.2-08/1/A-2009-0011 Reprogramming Somatic cells can be dedifferentiated into stem cells, so called induced pluripotent stem (iPS) cells using certain aprroaches. • • • • Cell-fusion based Nuclear extract based Transfection of pluripotent factors Somatic cell nuclear transfer TÁMOP-4.1.2-08/1/A-2009-0011 Molecular mechanisms of self-renewal Lif PI3K Grb2 Jak Akt MAPK STAT3 Tbx3 Klf4 Nanog Sox2 Oct3/4 Cdx Gata4 2 Prevention of differentation Cell-cycle regulation S G2 G1 M b-Myb c-Myc Genes involved in reprogramming TÁMOP-4.1.2-08/1/A-2009-0011 • Nanog:The nanog cDNA consists of 2184 nucleotides (nt) and contains a single open reading frame encoding a poly-peptide of 305 amino acids. – the role in pluripotency of both inner cell mass (ICM) and embryonic stem (ES) cells – the ability to maintain ES cell self-renewal. • Klf4: Krüppel-like factor, interact with CREB transcription factor, expressed in ESC and MSCs. • Lin28: a cytoplasmic mRNA binding protein, binds to IGF-2 mRNA, enhance the efficiency of the formation of iPSc from human fibroblasts, marker of undifferentiated human embryonic stem cells, able to bind let-7 miRNA and inhibit it. • Oct4: see previously • Sox2: see previously TÁMOP-4.1.2-08/1/A-2009-0011 Telomerase activity I • Telomeres are ribonucleoprotein heterochromatic structures at the ends of chromosomes that protect them from degradation and from being detected as double-strand DNA breaks. • When Dolly was cloned using SCNT, reliable question was raised about the age of her cells? Telomere was shorter, by approximately 20%, when compared with agematched controls. • After some conflicting results concluded that shortened telomeres of somatic donor cells could be indeed re-elongated during reprogramming, although the degree of TÁMOP-4.1.2-08/1/A-2009-0011 Telomerase activity II Telomers in iPS cells • High levels of Tert (reverse transcriptase component of telomerase) and high telomerase activity were described in iPS cells. • iPS reprogramming of normal cells (mice and human) results telomerase activation and restoration of telomeres, like setting the clock, to a length and chromatin state that is similar to that found in ES cells. • Telomerase activation during iPS reprogramming is associated with upregulation of TERT, but also TERC (Tel. Associated rNA component) become activated. Moreover OCT4 and NANOG bind to the TERC gene reg. element, which may explain why TÁMOP-4.1.2-08/1/A-2009-0011 Summary • ES and iPS cell fates can be monitored with a branch of fluorescent vital dyes (non-genomic/ genomic) using in vivo imaging techniques. • Restoration of pluripotency factors and self-renewal specific genes (Oct4, Sox2, Klf4) can exhibit the reprogramming for iPS cells. • iPS cell generation can be good candidates for regenerative medicine, however there are still several