lecture 5: modelling of human disease with pluripotent cells Flashcards Preview

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Flashcards in lecture 5: modelling of human disease with pluripotent cells Deck (32):
1

What are properties of pluripotent stem cells?

  • 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

What are the two types of human pluripotent stem cells?

  • 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

What is a newly developed route for deriving pluripotent SCs?

  • 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

What are embryonic stem cells?

  • 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

How are stem cells used as laboratory tools? 

  • 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

  What is another experimental species from which stem cells are derived and why was this important?

  • 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

What is the importance of iPS cells?

  • induced pluripotent stem cells provide a new approach to tissue matching for transplantation and powerful research tools 

8

What is somatic cell nuclear transfer and patient specific therapy? 

  • 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

What was the approach of Takahashi and Yamanaka to stem cells?

  • reductionist
  • reprogramming to pluripotency 
  • induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors 

10

What are induced pluripotent stem cells?

  • 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

What are the applications of iPSC?

  • 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

How can stem cells be used to model non-cell autonomous disorders?

  • efforts beginning 
  • amyelotrophic lateral sclerosis 
  • interaction between astrocytes and motorneurons 

13

What are the implications of iPSC for functional genomics? 

  • 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

Why is it important that stem cells can be used to study CNS development?

  • 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

What are the advantages of iPSC?

  • no ethical issues around provenance (other ethical issues)
  • facile access to starting material 
  • technology for reprogramming widely accessible 

16

How can iPSC be used genetic research tools?

  • 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

How are iPSC made?

  • 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

What is the challenge of 'Making the Right Stuff'?

  • variations in differentiation capacity of Both ES and iPS cells in culture 

19

What is a comparison of differentiation potential in pluripotent cells?

  • 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 
  •  

20

What is the issue of variability in differentiation potential of cell lines?

  • 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

How can we get and why do we need purification of end cells for analysis?

  • 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

How can we get around individual variation? 

  • 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

What is TALE?

  • new technology 
  • transcription factor activation like effector 
  • TALE endonucleases enable facile genetic manipulation of human pluripotent stem cells 

24

What is CRISPRS?

  • 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

What are examples of recent disease modelling studies? 

  • rapidly growing field
  • alzheimer's 
  • huntington's
  • rett syndrome
  • lesch-nyhan syndrome 
  • parkinson's 
  • spinal muscular dystrophy 

26

What are issues with iPSC epigenetics?

  • 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

What are genetic lesions in iPSC?

  • 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

Are ES cell lines stable?

  • 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

 What is the gold standard of stem cells?

  • 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

What are some further challenges in disease modelling and some possible answers to these challenges?

  • 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

What are some future applications of pluripotent stem cells? 

  • 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

Summary of induced pluripotent stem cells 

  • 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