B.18 iPSCs Autism Flashcards
Pluripotent Stem Cells
-cell with capacity to differentiate into tissues of three germ layers, endoderm, ectoderm and mesoderm
Multipotent Stem Cells
- cell with capacity to diffirentiate into limited range of cell types, most common stem cells in adult tissue (bone marrow stem cells)
Totipotent Stem Cells
-able to generate extra embryonic tissue as well as three germ layers
Mouse Developmental Pluripotency
> 2cell stage > 4 cell stage > Blastula >Blastocyst > implantation
- derived from zygotes which divides by cleavage plain devision- generate a ball of cells and builds Blastocyst
- Blastocusts is build of ectoderm and inner cell mass (epiblast which containes pluripotent stem cells)
- mouse embryonic stem cells can be generated by isloating the cell mas and growing it in cell culture
- Epiblast stem cells are also pluripontent but primed and derived from post implantation epiblast
- epiblast stem cells do not have a germline competency!
- Human Pluripotent stem cells similar to mouse and derived from blastocyst mass, probably no germline comptetency
Naive vs Primed
Naive:
Epiblast Cells of pre implantation blastocysts
Primed:
Epiblast cells post implantation
Somatic Cell Nuclear transfer
cloning
> somatic cell from body >remove nucleus >implant nucleus into denucleated egg =egg is able to reprogramm somatic nucleus -> if reimplanted live animals can be derived (frog, sheep)
-Takahashi and Yamanaka Factors: possible to convert adult cells into an ES like state without eggs -> luripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
Differentiation
- requires downregulation of pluripotency gene
-24 factors had been identified to reverse differentiation (Yamanaka) - when these where introduced iPS morphology appeared
-4 crucial factors identified
-10 factors shown to be more efficient than all 24
-germline compentecnty of those cells discovered when introduced back into blastocyst (mice)
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Application in disease modeling
- pre-clinical studies/ cellular phenotyping
- cellular therapies
ASD definition
- developmental disorder
- typical onset around 30 months of age
- verbal communication issues
- interaction problems
- limited imitation of others
- lack of shared play
- absent or non verbal communication
ASD genetic info
- highly heterogenous
- no single gene mutation
- high heritability (between 60 and 90 %)
- identifiable genetic causes around 25%- like chromosomal rearrangement and copy number variants
- SHANK3 most common single gene identified but accounts for very small proprtion of cases
Why iPSCs in ASD
- ASD genetically determined -> abnormalities can be captured in cells
- early age of onset in ASD -> iPSCs also early developmental stage
- ASD results from embryonic neurodevelopmental events
- human cells- can study early human development -> not possible in mice
Theories Of Pathenogenesis in ASD 1.
deregulated neurogenisis:
- this is a robust clinical observation
indicators for this are: enlarged head circumference due to enlarged brain probably due to excess neuron production)
-correlates with neocortical disorganisation in ASD patients
Can be studied in rosettes using iPSCs
- rosettes are developmental signiture or neuroprogenitors in stem cell culture of embryonic stem cells
Theories Of Pathogenisis in ASD 2
defect in glutamatergic system
-> synapses can be generated in culture with functional electrical activity
Models
- Compare healthy to patient specific
i. e.: patient derived iPSCs generate defective neuron which can be compared to healthy controls
- > downside is different genetic backround for sample and control
2.genome editing
-take iPSCs from affected individual and correct it
OR knock in mutation into control sample
-> it cannot be certain that individual with knock in would have gotten diease
Models Pro and Cons
Patient specific iPSCs vs unrelated controls:
Pro
-disease validity
-technically easier to generate
-chromosomal abnormalities can be studied more easily
Con
-influence of genetic background
-copy number variances can entail more than one gene
Genome Edited PSCs vs isogenic controls:
Pro
- no issue with genetic background
-‘cleaner’ genetic alterations can be studied
Con
-disease validity when creating mutation in control line
