Embryonic Stem Cells Flashcards
Stem cell
A stem cell is a cell which can self-renew and differentiate
-anyone of these cells has potential to differentiate (if differentiate no way to tell that stem cell)
Stem cell characteristics
- Rare cells
- Slow cell cycle (infrequently)
- Symmetric or asymmetric cell division (on avergae there will be one new cell and one that differentiates)
- Unspecialised
Two Categories of Stem Cells
Pluripotent Stem Cells (have capacity to differentiate into all cell types)
- ES cells (embryonic stem cells)
- EG cells (embryonic germ cells)
Somatic Stem Cells
-Foetal (found during development, transient)
-Adult
(these are all considered the same in this course)
Production of ESC and EGC (diagram in notes slide 5, look at!!)
- fertilized egg, get 2 cell division
- early blastocyst (2 cell types sometime 3), trophoblast (ectoderm, exembryotic cells?? allow to attach to uterus wall), inner cell mass (rise rise to primitive embryo and epiblast, in embryo not stem cells change every couple days)
- > can take out cells and culture embryotic stem cells, can put back into blastocyst and will continue development from when they were, will contribute to every tisse in adult body (except exembryotic tissues of trophoblast), why call pluripotent, cannot make whole embryo but make every cell type in adult and some exembryotic
- egg cylinder (ectoplacentral cone, epiblast)
- gastrulating embryo (germ cells, embryonic ectoderm, mesoderm)
- > EGC same as stem cells but derived from cells that will give rise to germ cells, generated from prymodial germ cells (transient population of cells that not stem cells, but if take them out will make cell lines same as embryotic germ cells)
- > EGC almost every function of ESC
Embryonic Stem Cells
- Derived from ICM
- Immortal
- Pluripotent (can put back in embryo & continue dev)
- In vitro
- In vivo
- In vitro
- From several species (incl.human & mouse)
(there was extra in notes about cancer, taritoma and telomeres but couldn’t understand prof)
Derivation of hES cells ((diagram in notes slide 7)
cultured blastocyst-> isolated inner cell mass -> first plating (cultured inner cell mass and irradiated mouse fibroblast feeder cells) – 9-15days –> cultured inner cell mass dissociated -> SEcond plating to establish colonies (and replated onto new feeder cells) – 7-10days –> Established cultures (hES cells)
Culture of ES Cells (mouse and human)
* focus on mouse
Mouse ES -Media containing serum ->Feeder cells ->LIF (lycemia inhibitor factor): molecule, cytocyn, binds to receptor and complex with co receptor and acts together in series of phosphoralation events with result in activation of TF (STAT3 dimerizes, binds to promoter, activates genes and keeps cells undifferentiated, at same time activation of ERK pathway does opposite and promotes differentiation) -Serum-free media ->LIF ->BMP4 (diagram slide 8!!!) Human ES -On feeder cells ->Fibroblast growth factor 2 -On Matrigel™ ->Feeder conditioned media ->FGF2
Key Transcription Factors (important!)
-pluripotentcy is maintained by a network of TFs that regulate each other and regulate other genes that maintain pluripotent state
Oct4
-Dosage critical
-important TF, expressed in oocytes in every stage until blastocyst and in every stage after blastocyst in spiblast portion?
-KO don’t get inner mass cells persisting, if more also harmful (will differentiate too much or too little, goldy lock amount)
-essential for ESC and inner cell mass
Nanog
- Sufficient to maintain mES
- ability to keep ESC undifferentiated (internal youth maintained)
Sox2
- Primes cells for neural differentiation
- KO embryos will not develop into blastocytes
- crucial for pluripotent cells in vitro and in vivo
- embryos die early no blastocysts, if over expression cells will not self renew and not differentiate
ES cell differntiation
- outside of colony see cells start to spread out a little more (start of differentiation)
- can make neuronal cells, mesoderm ect
- Embryoid bodies or monolayer culture
- Growth factors/Retinoic acid
- still don’t know everything, can’t fully make differntiate what we want, still do own thing
ES cells can contribute to all tissues in vivo
Teratomas (tumors)
-Contain derivatives of all germ layers
Chimeras (embryo made up from more than one other individuals, eg black and white mouse cells, patches of each colour of fur)
-Cells colonise all three germ layers
-Can contribute to the germline (if don’t make germ cells not fully pluripotent, this does make germ cells, so will be passed down to future generation)
-Useful in the production of transgenic or “knockout”mice
Creating a mouse knockout (important, on exam!)
Diagram slide 12 and 13
- ES cells are transfected with a targeting vector (mutated BMP7 with neor gene insertion)
- Selection of transfected colonies (small # cells get DNA repair, homologous recombination, get one allell mutanted other not, select heterozygous ESC by their neomycin resistance)
- Screening of correctly targeted clones (make sure are what think they are)
- Blastocyst injection and transfer (inject into blastocyst, and inject blasocyst into uterus)
- formation of chimeric mice (brown and white fur)
- breed chimeric to wild type (some will have germ cells (made from ESC), half carry mutation half will not)
- get WT BMP7+/BMP7+, and two heterozygote BMP7+/BMP7- (genome of ESC, other half WT, mate these two)
- get WT, heterozygote and homozygote (BMP7-/BMP7-, will never be born bc BMP7 crutial for dev, kidneys, eye, cause bleeding)
- Knockout mice are used to work out the function of genes
- Similar strategy to overexpress a gene of interest
- Knock-in: replace a normal gene with a version which has a mutation OR with a reporter gene (EGFP/βgeo)
Problems with conventional gene targeting approach
- Homologous recombination is relatively inefficient
- Many clones need to be screened to find correct event (plasmids tend to be huge, difficult to handle, time consuming)
- Targeting vectors often very large and difficult to construct
- Screening strategies can be complicated and time-consuming
Alternative technologies for genome editing
- Emerged after 2001 with ability to target proteins to specific DNA sequences
- DNA can be cut by targeting a nuclease
- DNA repair is via either HR or NHEJ
- > HR (homologuous recombination) allows better targeting (if cut DNA repair much more efficient)
- > NHEJ is error prone so can introduce mutations (ligase grabs two ends of DNA and puts together, lots of mutants, 3 nucleotids get frame shift cuts protein short, don’t get proper protein
- Very fast pace in progress
Meganucleases
- Like restriction enzymes but with very long recognition sites
- Site choice limited, can be engineered to change recognition site
- change sequence specificity that will bind to target, make target DNA want
- not sure how exact it is
Zinc-finger nucleases
- Target sequences are restricted (can be tailored to certain sequences but need C’s and G’s in DNA)
- Specificity
- Synthesis difficult (high cost)
- synthetic molecules made up from DNA bind motifs have specific preferences for different sequences (make sequence you want)
- diagram slide 17
