Induced Pluripotent Stem Cells Flashcards
What are 3 ways to generate pluripotent cells?
- Nuclear transfer → differentiated somatic nucleus inserted into a secondary oocyte. It is difficult and inefficient – however it is a good way of reprogramming nuclei.
- Cell fusion → Fusion of a differentiated with an undifferentiated cell. Creates a heterocarrion. The differentiated cells nuclear chromatin undergoes epigenetic modification and turns off genes associated with differentiation, and embryonic gene expression ensures – this is even older than nuclear transfer.
- Genetic/Epigenetic manipulation → eg) induced pluripotent stem cells. Produced by reprogramming or differentiated cells to a state similar to an embryonic stem cell. This is the most favoured way.
How did Yamanaka discovery the 4 factors needed to induce pluripotency?
Takahashi and Yamanaka, 2006:
• Took factors assumed might be involved in the down-regulation of differentiation genes and upregulation of pluripotency genes.
• Reduced 100s of potential factors down to 24, and then in the end to 4.
• Retroviral transfection of a combination of these 4 factors induce mouse embryonic fibroblasts to become pluripotent cells:
− Oct-4 → maintenance of pluripotency
− Sox-2 → maintenance of pluripotency
− c-Myc → involved in cancer cell proliferation
− Klf-4 → zinc finger transcription factor
- Yamanaka’s assay was for colonies (we know that stem cells form smooth, rounded colonies)
- Colonies using these 4 factors were the same as colonies formed using a group of 10 factors.
- He then took each of the 4 factors out individually, and colony formation was reduced → so you need all of them together.
- Success however was still only
How did Yamanaka generate iPS cells using the 4 factors?
- Take differentiated cells with a reporter. Original experiments used Fbx15 but this is not a good ES like reporter and many partially reprogrammed cells express it. Nanog promoter driver GFP transgene used → cells will express GFP when Nanog upregulated
- Culture with retrovirus containing constucts for Oct-4, Sox-2, C-myc and Klf-4
- After a few weeks, see pluripotent cells forming (low success rate, 0.01-0.1%). Identified by morphology or Nanog-GFP reporter
- Select the pluripotent cells and look for markers of pluripotency (Oct-4, Nanog, Sox-2, SSEA1 (or SSEA 3/4 in human)
- Test these cells for differentiation capacity in vitro – ability to form chimeras and teratomas.
What are the roles of the 4 factors in iPS cells?
-myc → proliferation factor – immortalizes the cells. Upregulates p53, however downregulation of p53 increases efficiency of reprogramming.
• Klf-4 → along with c-Myc opens up the chromatin, blocks apoptosis and supports self renewal. Also upregulates sox-2 and nanog.
• Oct-4 → essential transcription factor in maintain pluripotent state. Represses differentiation genes
• Sox-2 → cofactor for Oct-4
What are factors involved in extended molecular rearrangement to get iPS cells?
- Suppression of somatic gene expression
- DNA methylation erasure/re-writing -→ remove the H3K27me3/change them to H3K4me3
- New histone modifications
- Metabolic shift to glycolysis – ES cells rely on glycolys rather then Krebs cycle
- Reactivation of the second X chromosome (mouse) → human X is not reactivated during reprogramming, perhaps because human ES cells are more equivalent to the epiblast stage, where X inactivation has already occurred
- Induction of endogenous pluripotency loci → stop relying on retroviral transgenes and start expressing them themselves
- Autophagy → digests all cellular organelles not needed
What are factors involved during passaging to get iPS cells?
- Telomere lengthening – happens to a greater extent in some cell lines than others
- Selection
What are factors involved in improved efficiency of reprogramming?
- Vitamin C → may increase efficiency by overcoming senescence in differentiated cells, and protecting against free radicals
- Histone deacetylase inhibition
- Factor stoichiometry – what proportion of each factor you have
- Oxygen tension → stem cells grow better at 3-5% rather than 20%
How were the first human ES cells reprogrammed?
• Found to be possible using the 4 Yamanaka factors in 2007
• Fibroblastsa also reprogrammed using lentiviral vectors containing Oct-4. Nanog, Sox-2 and Lin28
− Lin28 is an evolutionary conserved RNA binding protein
− Facilitates expression of Oct-4 by binding to its RNA and possibly increasing its translation
What are characteristics of iPS cells
- ES-like gene expression pattern and can be maintained in medium with LIF and BMP (murine) or Activin and FGF (human) → iPS cells have a more closely related genotype to ES cells than the somatic cells from which they were derived
- Reactivation of the second X chromosome (mouse)
- ES-like histone and DNA methylation pattern → suggesting similar epigenetic regulation
- Formation of teratomas and chimeras
- Transmission to the germ line in mice (functional gametes) – ultimate test of pluripotency
What new iPS technologies can help efficiency?
- Avoid using c-myc (mitogen) → use n-myc instead
- Avoid insertion of multiple retroviral vectors → use polycistronic vectors instead (one vector carrying all the genes)
- Use the least amount of reprogramming factors possible → select cells already expressing some of the necessary reprogramming factors, then you need to add less.
• Eg Neural cells already express Sox-2, c-myc and Klf-4 → so only need to insert oct-4 for reprogramming - Retrovirus may integrate into an area of the genome containing an important gene and cause a mutation → use a non-integrating adenovirus instead. Sendai is the most efficient.
• Work by Hochedlinger et al - Use of plasmids (no virus and non-integrating) → although expression of the factors is transient and success rate much lower (1:1000), still managed to get the ES cells.
- Use of removable, transposon flanked genes
• one study used the Cre/LoxP recombination system to excise integrated transgenes after transfection.
• However, removal of multiple transposons is labour-intensive
• Still leaves residual potential mutagenic vector sequences - Transfection of synthetic mRNA or ptoein for the factors, rather than genes → avoids any interference with the DNA
• Membrane permeable versions of the Yamanaka factors were generated by adding a host basic amino acid sequence from HIV called TAT
• Repeated treatments are needed and this is expensive
• Gene expression and DNA methylation similar to hESCs
• Differentiated into all 3 germ layers and form teratomas
• However, take longer and efficiency lower
• Synthetic RNA now available, but again expensive and requires repeat treatments - Kinase inhibitors to improve efficiency → ERK and GSK maintain ESC state
- Use of small molecules → histone deacetylase inhibitors, methylation modifiers etc.. to increase efficiency
- microRNAs → miR203 upregulates genes involved in pluripotency
What are the problems associated with iPS cells?
- Reactivation of c-myc in differentiated tissue of resulting chimeras leads to tumours
- Epigenetic pattern is not identical to ES cells (there is increased DNA methylation) and there are abnormal transcriptional activations in certain chromosome regions (12 in mice)
• Lister et al, 2011 → comparison of iPS methylation sites with ES and adult cells
• Reprogramming errors common
• Large megabase sections of DNA, often at telomeres and centromeres, incorrectly methylated
• Many differences common to all iPS cells and are inherited with division
• DNA methylation in pluripotent cells unstable - Virally reprogrammed cells gain mutations
- Promoter occupancy by 4 factors is not identical to ES cells → TFs in the iPS cells are binding a slightly different cohort of promoters, leading to slightly different transcriptome
- Telomere length → telomeres are to some extent lengthened, but not to the full extent
- Gene expression not identical
• Overexpression of a lot of pluripotency factors, particularly c-myc and Klf-4
• Many genes have dosage effects, bad news if you have too much of them - iPS cells have epigenetic memory → remember the differentiated cell type they were from, and have a bias to give progenitors of that cell type. Differentiate better into the cells of the lineage they came from.
• Kim et al, 2010:
− Blood derived iPSCs gave more haematopoietic colonies than fibroblast derived iPSCs
− ES cells derived from nuclear transfer of fibroblast cells gave more haematopoetic differentiation than fibroblast derived iPSCs
− Both ntESCs and ES cells derived from fertilized embryos gave better differentiation than reprogrammed iPScells.
− Fibroblast derived iPSC gave more bone marrow colonies than blood derived iPSCs
− Fibroblast derived iPSCs gave more bone marrow colonies than ntESCs - Reprogramming causes mutation even without viral vectors → many in genes involved with cell proliferation, cancer and development. However, growing the cells in culture seems to select against some genetic abnormalities.
What was the murine proof of principle experiment that suggested iPS cells could be used to correct genetic defects?
Hanna et al, 2007
• Fibrobalsts form skin of a transgenic mouse carrying the human sickle cell anaemia gene
− Reprogrammed
− Genetic defect corrected
− Differentiated them back to haematogenic progenitors
− Injected them back into mouse
− 4 weeks later the iPS cells had begun producing normal blood cells, and the nice were no longer anemic
What were the first iPS cells generated from a patient?
DImos et al, 2008
• iPS cells generated form a patient with ALS
− Skin biopsy taken from 82 year old women with ALS
− Fibroblasts reprogrammed
− iPS cells induced to develop into motor neurons
− Shows that old cells and disease cells can still be programmed to the stem cell state
What is the problem with iPS cells generated from patients?
However, iPS cells from diseased cells keep their genetically programmed defect:
• Skin fibroblasts from patient with spinal muscular atrophy
• Reprogrammed to iPS
• Differentiated into neurons
• Neurons retain the disease phenotype (mutation in survival motor neuron 1) and much reduced incidence of neuron formation.
➢ This can still give the potential for studying the disease in vitro, and developing the potential for testing drugs which may correct the neural phenotype
Why do we not have to always reprogramme cells back to the ES like state?
- We many not always need to reprogramme cells back to the ES like state → can have direct transdifferentiation to defined lineages
- TF overexpression or removal can lead to cells changing fate, without any increase in differentiation potential
Marro et al, 2011
• Direct lineage conversion of hepatocytes to neurons
• Overexpression of 3 neural TFs
• Induced cells display functional action potentials
• However, still don’t know if the cells remain stably reprogrammed when introduced factors our diluted out (eg, with non-integrating viral vectors).
