Exploring mental health using stem cells Flashcards
What are IPSCs?
Induced Pluripotent Stem Cells
- capacity to generate different cell types: foetal and adult bodies
- > nervous system, heart and circulatory systems, muscles
What are pluripotent cells?
Cells with capacity to generate different cell types
- foetal and adult bodies
- > nervous system, heart and circulatory systems, muscles
Which cells have an ephemeral (temporary) pluripotent property?
Inner cell mass cells, in the blastocyst
- these cells are incredibly ephemeral
- > their pluripotency only lasts a few days and never reappears during the entire life cycle
- > hard to study
Which cells have a permanent pluripotent property?
Embryonic stem cells
- derived directly from inner cell mass
- permanent pluripotency
- they can give rise to all different cell types that make up the body
Who initiated the discovery of the biological basis for pluripotency?
What was his/her experiment?
John Gordon
> Used enucleated egg (nucleus destroyed with radiation) containing pluripotent cells of Frog A
> Implanted nucleus of Frog B into egg -> egg with transplanted nucleus
> Egg develops into tadpole -> clone of Frog B
-> pluripotent constructed cell
- > Nucleus, even from fully differentiated cell, had all info. to generate an organism
- > There are factors in the enucleated egg that, once combined, tell the nucleus that it has to start behaving as a pluripotent cell
=> Existence of factors in the cytoplasm of pluripotent cells that dictate pluripotency
Who found the biological basis for pluripotency which led to the discovery of iPS cells?
What are the Yamanaka factors?
Shinya Yamanaka
> Suggested the factors must be gene products = proteins
-> List of 24 potential pluripotency factors
Takahashi and Yamanaka (2006, 2007)
> Step 1: Have an assay for pluripotency: a way to recognise he’d produced pluripotency in cell that weren’t pluripotent
- pluripotent cells always seem to have the Fbx15 gene active -> a reporter for pluripotency (if cells turn blue they’ve become pluripotent)
- transfection of fibroblasts with the 24 pluripotency factors
-> within these 24 factors are the important ones leading to pluripotency
> Step 2:
- Repeated experiment several times, leaving a factor out each time
- factors needed to induce pluripotency vs. factors not needed
> Step 3:
- Repeated experiment with the 10 remaining factors, sequentially leaving one out at a time
=> Yamanaka factors: Oct3/4, Sox2, KIf4, c-Myc
- if he uses these 4 factors, he gets blue cells, believed to be pluripotent ; leaving out 2 of them, he doesn’t get blue colonies
- > all of the 4 remaining factors are needed to generate pluripotent cells
- > these 4 factors are sufficient on their own
> Step 4:
- generate embryoid bodies with 3 germ layers: ectoderm, mesoderm, endoderm
- it is still a hypothesis that the blue cells are pluripotent
> Step 5:
- take fibroblasts that had been transduced with the 4 factors, grow them into embryoid bodies and show each germ layer is represented in the embryoid bodies
- histological sections of mice confirmed the cells had contributed to creation of all different body tissues = iPSCs
- fibroblasts transduced with the 4 factors, injected in blastocyst, significantly contributed to the formation of germ line -> made it possible to produce mice that were entirely derived from these
=> Generate a whole line of mice derived from those transduced fibroblasts which were indeed pluripotent
What happened after Takahashi and Yamanka’s 2006 experiment on mouse (skin) fibroblasts?
> 2007: Takahashi and colleagues: human (skin fibroblasts)
> Onwards: Yamanaka’s procedure accepted worldwide
- changes to protocol
How was the generation of IPSC lines used for the study of childhood disorders?
iPS cells can be derived from any cell type in the body:
- blood cell sample
- urine sample
- hair sample -> suitable for the study of childhood disorders (e.g. autism)
Hair keratinocytes
- reprogramming with Yakamana factors (Oct3/4, Sox2, KIf4, c-Myc)
- > iPS cell colonies
How to make iPS cells to create neurons?
- Collect sample: blood cells, urine, hair
- Reprograming using Yakamana genes (Oct3/4, Sox2, KIf4, C-Myc)
- You get iPS cell colonies
- iPS cell line
- used in the study of brain development disorders - Neural ‘rosettes’ - progenitor cells
- Neurons
Which genes are the Yakamana factors?
- Oct3/4
- Sox2
- KIf4
- c-Myc
What is the cultured neuralisation timeline?
> Day 1: iPS cell lines
- SMAD signalling pathway inhibition to induce the iPS cell lines to produce neuroepithelium
- > add SMAD inhibitors to the culture of these induced pluripotent cells
> Day 21: neural ‘rosettes’
- progenitor cells that can do tissue histogenesis (compared to NSCs found in other contexts)
- proper polarised neuroepithelium -> cells are trying to make a two-dimensional neural tube
= two-dimensional cell culture (in vitro)
> Day 28 to 35: young neurons
> Day 35 to 53: mature neurons
What is reassuring about the cultured neuralisation and iPSCs technology?
It is a slow process (53 days to get mature neurons)
- reassuring because it reproduces the timing and the differentiation processes that seem to underly human neural development
=> iPSCs allow us to look at human neural development in a cultural dish
What is histogenesis?
Processes that begin with the generation of neuroblasts, to the morphological and biochemical differentiation of mature neurons in the cortical plate
What is specific about the histogenesis of iPSCs?
- If you allow cell development, the iPS cells try and undergo proper histogenesis
- iPS cells have this capacity for histogenesis remarkably larger than any other neural developing systems in vitro
Why does the cultured in vitro differentiation process and neural development ceases after cerebral tissue is developed?
Lack of blood supply which does not exist in the culture dish
