L4 Differentiation Flashcards
What triggers differentiation and what happens when it is triggered?
The removal of extrinsic conditions triggers differentiation.
Cells clump together to form cell aggregates called embryoid bodies.
What are the pros and cons of embryoid bodies?
Pros: They are cheap to produce and generate the three germ layers.
Cons: It is difficult to reproduce aggregation in a reproducible way and you must wait a number of days before the bodies are formed and must be collected.
What are Cystic, Bright Cavity and Dark Cavity best at?
Cystic = best at producing endoderm.
Bright Cavity = good production of the three germ layers and best organised. Closer to real embryos.
Dark Cavity = good production of the three germ layers.
What are the methods used to ensure mouse and human embryoid bodies are grown from a single cell?
1) The Hanging Drop Method. This is used for mES cells.
On a dish you have a drop containing a single mES cell. You then turn the dish upside down. The drop does not fall off the dish and the cell is no longer in contact with the plastic surface of the dish. Each drop will turn into an embryoid body.
2) Controlled Aggregation. This is used for hES cells.
Droplets containing hES cells are placed in inverted pyramidal wells. You can put a set number of cells in each of the wells.
What is directed differentiation and what are the important variables involved?
You use directed differentiation if you want to develop a particular cell type/tissue. This is achieved using exogenous growth factors.
1) Concentration of growth factors.
2) Time at which the factors are added.
3) Combination of factors added.
4) Substrate the cells grow on.
What are the methods used to sort cell cultures of differentiated cells?
- FACS (sorting for specific markers).
- Density gradients (use a media where the different cells will float at different levels).
- Selectable markers such as GFP and antibiotic resistance.
What did Li et al (1998) do and discover?
They managed to generate purified nebula precursors from ES cells by using lineage selection.
They did this by fusing the Sox2 gene with LacZ (allows you to trace the expression of Sox2). They managed to show that additional FGF2 causes proliferation of cells that express Sox2.
What did D’Amour et al (2006) do?
They managed to produce pancreatic hormone-expressing endocrine cells from hES cells by utilising what they knew about the development of these cell in vitro.
However, only a small percentage of insulin-producing cells were obtained. They were not glucose-responsive and did not maintain expression of key beta-cell markers.
Describe the breakthroughs Kroon et al (2008) made with pancreatic stem cells.
They managed to modify D’Amour et al’s method and improve the quality of the cells that were being produced.
They placed the pancreatic endoderm they had derived from mES cells back into the mice and then the cells mature for 3 months. They saw that these cells had grafted and produced mini-pancreases that had multiple cell types and produced insulin
When they did this with diabetic mice, the mice were able to control glucose levels. When the grafted cells were removed these effects were reversed.
What was the big flaw in Kroon et al (2008)’s experiement?
15% of the ES cell grafts developed tumours.
What were the two surface markers Kelly et al (2011) discovered and why was this important?
- CD142 found in the pancreatic endoderm.
- CD200 found in the endocrine (mature cell type).
This was important because you can now use these markers to tell if the cells are too mature to be transplanted and if they are capable of making mature pancreatic tissue at all.
Ideally, you want cells that are positive for CD142 and negative for CD200.
What did Kelly et al (2011) eventually manage to achieve with these new cell markers?
They managed to derive the right population of cells and remove the chances of tumourigenesis.
What are organoids and when were they useful?
They are highly organised tissue with more than one lineage and are functionally relevant.
This method has been successful with eyes and brain organoids were used to study the effects of the Zika virus.
