lecture 5: modelling of human disease with pluripotent cells Flashcards
1
Q
What are properties of pluripotent stem cells?
A
- grow indefinitely in vitro
- maintain normal genetic makeup
- cloned lines capable of differentiation into a wide range of somatic and extraembryonic tissues in vivo and in vitro-at high frequency and under a range of conditions
- capable of colonising all tissues including germ line after blastocyst injection to give chimeric offspring
2
Q
What are the two types of human pluripotent stem cells?
A
- embryonic stem cells
- derived around 1998
- directly from pluripotent cells from the embryo
- induced pluripotent stem cells
- taking an epithelial cell
- adding yamanaka factors in vitro
- reprogramme back to an state resembling an ES cell
3
Q
What is a newly developed route for deriving pluripotent SCs?
A
- nuclear transfer/transplantation
- employs cloning technology to make a cloned embryo from an existing individual
- process is interrupted and stem cells derived
- many species of mammal have now been cloned
- a cloned kitten costs $50K US
- Human SCNT: multiple refinements to the procedure enabled ES generation from a small number of oocutes
- cloning: a powerful tool to study cellular reprogramming and the gold standard
- remains pretty inefficient but is very powerful
- some evidence that these were closer to ES cells than iPS cells
4
Q
What are embryonic stem cells?
A
- derived from spare embryos before specialised tissue of the body begin to form
- can multiply indefinitely in laboratory cultures
- retain the ability of embryonic cells to turn into any type of tissue
- nov 98: human embryonic stem cells discovered
- 2012 - first human trials of human embryonic stem cell therapeutics
- em
5
Q
How are stem cells used as laboratory tools?
A
- designer mice for research
- nobel prize in medicine 2007
- gene knockout technology has enabled a revolution in mammalian genetics, development, physiology and the study of disease
6
Q
What is another experimental species from which stem cells are derived and why was this important?
A
- embryonic stem cells from rat
- chimeric rat pups made from embryonic stem cells
- germline competent embryonic stem cells derived from rat blastocysts
- workers have tried for 20 years to make rat embryonic stem cells
- rats are widely used in physiology and pharmacology and drug discovery
- until now there have been no tools to make specific modifications n the rat genome to create disease models, like we can in mouse (nobel prize 2007)
- new discoveries about embryonic stem cell growth regulation (ES cell self renewal as a default pathway) to make rat ES cells for the first time
- an important new tool for basic research and drug discovery
7
Q
What is the importance of iPS cells?
A
- induced pluripotent stem cells provide a new approach to tissue matching for transplantation and powerful research tools
8
Q
What is somatic cell nuclear transfer and patient specific therapy?
A
- cloning is a very inefficient process so took quite a while to develop this for stem cell development
- obtaining oocytes is an invasive procedure
9
Q
What was the approach of Takahashi and Yamanaka to stem cells?
A
- reductionist
- reprogramming to pluripotency
- induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors
10
Q
What are induced pluripotent stem cells?
A
- iPSC
- somatic cells “reprogrammed” by viral transfection
- ES-specific transgenes inroduced into host cells Oct-4, Sox2, Klf-4, c-Myc
- subset of cells: ES-like colonies = iPS
- avoids use of embryos
11
Q
What are the applications of iPSC?
A
- research: disease modelling
- therapy: tissue matching
- pluripotent stem cells have important applications in biomedical research
- basic studies of early human development and its disorders-birth defects, childhood cancers
- functional genomics in human cells
- discovery of novel factors controlling tissue regeneration and repair
- in vitro models for drug discovery and toxicology
- e.g. modelling the Q-T syndrome with human iPSC
- congenital type 2 LQTS: model for LQT caused by heart failure, cardiac hypertrophy or drugs
12
Q
How can stem cells be used to model non-cell autonomous disorders?
A
- efforts beginning
- amyelotrophic lateral sclerosis
- interaction between astrocytes and motorneurons
13
Q
What are the implications of iPSC for functional genomics?
A
- allows us to take infromation from monogetic, complex or GWA study traits and actually test this in cells
- better understand role of genes in disease
- maybe just need to look at biomarkers
- there are differences between mice and humans
- we can make targeted genetic modifications in human ES cells to create disease models
- we can study the effects of the mutations on development and physiology of specific cell types
- we can use the differentiated cells to develop and screen new medicines
14
Q
Why is it important that stem cells can be used to study CNS development?
A
- cortical structures in vitro from human ES cells, Eiraku et al. Cell stem cell 3: 519,2008
- human cerebral cortex in a dish
- human cortical development differs significantly from other mammals
- ES and iPS cells can be used to model human cortical development
- Schizophrenia, autism and epilepsy are disorders of brain development
- iPSC from patients with these diseases can be used to recapitulate key events in pathogenesis
- schizophrenia susceptibility genes expressed in network in foetal cortex
- integration of neural progenitors from human ES cells into mouse cerebral cortex: implications for brain repair in childhood
15
Q
What are the advantages of iPSC?
A
- no ethical issues around provenance (other ethical issues)
- facile access to starting material
- technology for reprogramming widely accessible
16
Q
How can iPSC be used genetic research tools?
A
- enable in vitro modelling of complex multigenic diseases
- enable examination of effects of single gene mutation on different genetic backgrounds and examination of the effect of modifier genes unlike ESC
- enable examination of individual variations in tissue regeneration and repair pathways and development of biomarkers for clinical outcomes in regenerative medicine
17
Q
How are iPSC made?
A
- Yamanaka first used retroviral vectors that delivered them into the genome
- all sorts of problems associated with it
- big stumbling block for gene therapy
- whole range of different ways around this
- most prominent perhaps episomal vectors that don’t integrate but float around in the cytoplasm and express the reprogramming factors but ultimately disappear
- problems now mostly about efficiency
- need to make sure we don’t damage the genome
18
Q
What is the challenge of ‘Making the Right Stuff’?
A
- variations in differentiation capacity of Both ES and iPS cells in culture
19
Q
What is a comparison of differentiation potential in pluripotent cells?
A
- ES/iPS cells
- non-directed EB differentiation (16 days, 2-5 replicates) - basically a way of making the cells spontaneously go down a whole lot of differentiation pathways at once
- expression profiling for 500 lineage marker genes
- quantification of expression differences versus reference
- gene set enrichment analysis for lineage marker genes
- lineage scorecard estimate of differentiation propensities
- kind of all over the shop
- hard if you have a patient whose cells are not good at making a particular cell type because will make it hard to do systematic studies
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20
Q
What is the issue of variability in differentiation potential of cell lines?
A
- variation in differentiation potential may require isolation and testing of multiple clones
- variation probably relates not to gene expression in pluripotnet state but rather to its stability - what is important is how cells exist in pluripotent state
- can look at differences in DNA methylation, transcriptional variation, ability to differentiate into particular cell lines, level of variabilty/noise
21
Q
How can we get and why do we need purification of end cells for analysis?
A
- when we look at differentiation from a bunch of cell lines we want to be comparing apples to apples - not to oranges
- although we describe this differentiation process as though it is really well controlled, in fact most of the differntiation protocols give you a mix of different stuff at the end
- you don’t want that mix to be varying from patient to patient and confusing your readout
- flow cytometry is one way of purifying the cells for analysis
- reporter cell lines:
- a reporter gene e.g. a fluorescent protein has been targeted to a locus of a specific gene that is switched on at a very specific stage of development
- e.g. a neuron specific gene
- powerful way of making a homogenous culture of cells at a particular stage in development
22
Q
How can we get around individual variation?
A
- targeted genetic manipulation to yield isogenic cell lines with wild type or mutant genotype provides powerful controls for analysis of disease phenotype
- homologous recombination
- now much faster
- take a patient with a disease, correct a gene in one cell line and compare that to the original cell line
- much tighter readout of phenotype in theory
23
Q
What is TALE?
A
- new technology
- transcription factor activation like effector
- TALE endonucleases enable facile genetic manipulation of human pluripotent stem cells
24
Q
What is CRISPRS?
A
- Clustered, regularly interspaced, short palindromic repeat
- simpler to make than TALENS and provide high efficiency gene targeting
- off target effects are still a consideration
25
Q
What are examples of recent disease modelling studies?
A
- rapidly growing field
- alzheimer’s
- huntington’s
- rett syndrome
- lesch-nyhan syndrome
- parkinson’s
- spinal muscular dystrophy
26
Q
What are issues with iPSC epigenetics?
A
- erasure of epigenome during reprogramming may erase important features of disease susceptibility
- memory of somatic tissue of origin due to imperfect reprogramming - may be erased after long term cultivation
- e.g. taking a blood cell, still having blood cell markers
27
Q
What are genetic lesions in iPSC?
A
- lesions that are introduced adventitiously
- function significance of mutations not always clear
- chromosomal rearrangements in later stages similar in ES and iPSC
- there are aberrations of somatic origin, those introduced during reprogramming, and those acquired in culture
- changes that we observe are consistently very similar to changes seen in cancer
- therefore need to keep in mind that there is some degree of loss of integrity of stem cell genome and epigenome in vitro and this may influence the phenotype/behaviour of cells
28
Q
Are ES cell lines stable?
A
- most ES cell lines are remarkably stable
- ~75% will have no changes even after hundreds of generations in culture
- bad news is we have no clue why the other 25% go off the rails
29
Q
What is the gold standard of stem cells?
A
- ES cell
- however ES and iPSC have:
- similar patterns of gene expression in the pluripotent state
- similar patterns of DNA methylation and histone modification
- similar susceptibility to genetic change during long term culture
- variation in capacity for differentiation into specific lineages
- As we better understand iPSC it is likely that they will produce a viable alternative to ES cells
30
Q
What are some further challenges in disease modelling and some possible answers to these challenges?
A
- non cell autonomous disorders: organ in a dish
- production of fully mature functional cells: better cell culture technoogy to mimic in vivo environment
- long time span to development of pathology, disorders of ageing: artificial acceleration of the ageing process
31
Q
What are some future applications of pluripotent stem cells?
A
- making germ cells: offspring from oocytes derived from in vitro primordial germ cell-like cells in mice
- differentiation of human iPS cells into gametes
- new possibilities for research on human germ line-infertility, early development
- but significant ethical questions over fertilisation and embryo production using IPS-cell derived gametes
- with IPS cells gametes could be created from individuals of any age, living or dead
- we could potentially make germline modifications in human
- monsters
- chimera: an organism comprised of cells of two or more unique genetic background
- contribution of human embryonic stem cells to mouse blastocysts
- formation of chimera via inoculation of ES cells into preimplantation animal embryo
- object to assess development capacity or response to embryonic environment or create organs for transplantation
- outcome is an animal or embryo or foetus with substantial human cell contribution to many tissues
- unlikely to work where host species differs signficantly in developmental terms for humans; minor degree of chimerism is not very informative
- high levels of chimerism controversial from an ethical point of view e.g. human cell into monkey blastocyst
- but there are some important uses for this technology
- e.g. humanised blood an immune system in mice
- production of organs from iPSC in vio via interspecies chimera
- rat pancrease in mouse chimera formed blastocyst injection of rat iPSC into Pdx-/- mouse
- human glial mouse chimeric brain
- human glial progenitors show very substatial functional engraftment
- mice show improved learning ability
- mouse grafts had no such effect
32
Q
Summary of induced pluripotent stem cells
A
- powerful tools for research in genomics, disease modelling and drug discovery
- banks of iPSC made from cord blood or other tissue will provide a resource for transplantation in the future
- many obstacles must still be overcome
