Stem Cells N, H, M, iPSc

Question 1

Briefly describe neurogenesis in drosophila

Answer 1

Neuroblasts are the neural stem cells
They delaminate from the neuroepithelia
They undergo asymmetric cell division into ganglion mother cells (GMC) that are the precursors to neurons

Question 2

Describe the difference between cell division in the neuroepithelium and in the division that occurs in GMCs in Drosophila

Answer 2

Symmetric division in the epithelium has a vertical metaphase axis, and the proteins that gather at the apical and basal sides are divided equally, so the cells multiply out horizontally.
Asymmetric division has a horizontal metaphase axis that leaves each cell with a different population of proteins when the daughter cells divide vertically.

Question 3

Name proteins that are found and aren’t found in the GMC after asymmetric division of a neuroblast

Answer 3

Basal complex/GMC- prospero, numb, Miranda

Apical complex/neuroblasts- bazooka, inscuteable, Par3, Par6, aPK-C

Question 4

Briefly describe asymmetric cell divisions in the cerebral cortex

Answer 4

Symmetric planar division
Asymmetric planar division
Apical-basal division

Question 5

How are different types of neurons made from one neuroblast?

Answer 5

A temporal code of transcription factors expressed in the neuroblast as development progresses
Each GMC inherits a different combination which determines the type of neuron produced
Also intermediate neural precursors (INP) have a temporal code of transcription factors with the age of birth
This give a 2D matrix of possible neurons
Later born neurons migrate more superficially

Question 6

Where to haematopoietic stem cells come from?

Answer 6

Originally develop in blood islands
The first permanent ones are generated in the aorta-gonad-mesonephros (AGM) region; at the ventral wall of the dorsal aorta

Question 7

What is EHT?

Answer 7

Endothelial to haematopoietic transition

Not cell division but specialisation from an adherent cell type to a round non-adherent cell type (progenitor cells)

Question 8

Where does the expansion of the haematopoietic system occur in foetuses?

Answer 8

Foetal liver after which it is moved to the bone marrow

Question 9

What is the difference between long term and short term HSCs?

Answer 9

Both can reconstitute all blood lineages in an irradiated mouse- both equally pluripotent
Serial transplantation is only possible with LT-HSC
LT-HSC are able to fully self renew and maintain the cell pool
ST-HSCs are not able to do this as their differentiation outcompetes self renewal

Question 10

What is the paradox surrounding haematopoietic stem cells?

Answer 10

Approx. 200,000,000,000 new cells per day but the HSCs are mostly quiescent
This is because high proliferation occurs at the progenitor stage

Question 11

Describe leukaemia

Answer 11

Rapidly proliferating immature blood cells that do not sufficiently differentiate so there is an over production of non-functional blood cells
Can treat by inducing differentiation using retinoic acid (ATRA) in acute promyelocytic leukaemia
Targeted therapy- disturb cell survival signalling pathway or antibodies marking cells for the immune system
Transplantation

Question 12

Briefly describe stem cell transplant

Answer 12

G-CSF is given to the donor this causes the HSCs to go into the blood stream
This is caused due to the inhibition of the interaction of the HSCs with osteoblasts and is thought to be regulated by neurons and monocytes

Question 13

What is the importance of stromal cells for HSCs?

Answer 13

Provide stem cell niche
Also provide support and structural scaffold to the parenchyma like cells of tissues
Multipotential bone marrow stromal cells form a spindle-shape adherent cells in culture and act as a feeder layer for HSC in vitro

Question 14

Describe multi potential bone marrow stromal cells

Answer 14

Tri-lineage potential following in vivo transplantation
Bone, fat and cartilage
A type of mesenchymal stem cells

Question 15

What is the mesenchyme?

Answer 15

Multipotent Stem cells that arise from the mesoderm
Can become bone, cartilage, muscle, marrow, tendon/ligament, adipose tissue, connective tissue or components of the circulatory system- have a perivascular location

Question 16

Describe the uses of mesenchymal stem cells

Answer 16

Intravenous injection to sites of damage to trigger tissue repair
Direct tissue repair- differentiation of stem cells into target tissue type and engraftment - not reliable
Direct tissue repair- production of tissue progenitor cells that differentiate to target cells
Indications of immunomodulation- inhibits dendritic cells so there is reduced natural killer cell activity
Reduced production of inflammatory cytokines eg. TNF

Question 17

What are the therapeutic implications of the immunosuppressive effects of MSC?

Answer 17

Can be used to treat autoimmunity eg. MS, rheumatoid arthritis and type 1 diabetes
Reducing T cell activity
May be useful in preventing graft vs host disease- no difference in survival rate but reduce inflammation- mechanism of action? Heterogeneity?

Question 18

What are the Yamanaka factors?

Answer 18

The growth factors needed to transform adult somatic cells into pluripotent stem cells
Oct4, Sox2, Klf4, C-myc

Question 19

What are the advantages of induced pluripotent stem cells?

Answer 19

Taken from patient- no rejection
Fewer ethical issues
Same properties as embryonic stem cells
Larger potential than mesenchymal stem cells
Can be used in patient specific cell therapy, drug screening, human disease models etc.

Question 20

Briefly describe why each Yamanaka factor is used

Answer 20

Oct4- homeodomain factor encoded by the Pou5f1 gene. Is required for the formation of the inner cell mass in early embryogenesis- levels must be tightly regulated
Sox2- required for ICM and epiblast formation in early embryogenesis. Together with Oct4 (and nanog) are master regulators of pluripotency by regulating the transcription of genes involved in ESC identity
Klf4- zinc finger containing domain. Associated with the inhibition of cell proliferation by controlling the expression of cell cycle regulators
Can active Cdh1 and repress p53
C-myc- proto-oncogene, a transcription factor that targets genes associated with an active chromatin signature, activates transcription by regulation Pol2 pause release
It up-regulates genes involved in DNA replication and cell division and down regulates cell adhesion genes

Question 21

Are there better ways of stem cell reprogramming?

Answer 21

Instead of a retrovirus that integrates into the genome use one tha does not intergrate
Use the mRNA of the Yamanaka factors instead of the cDNA
Use the proteins of the Yamanaka factors
Use chemicals
As another combination of genes based on stochastic gene expression such as SNEL (Sox2, nanog, Edrrb, lin28)
Uses small molecules like histone deacetylases with the Yamanaka factors to assist with epigenetic modification

Question 22

Why reprogram different cells into iPSC?

Answer 22

Less invasive methods than skin fibroblasts
Disease modelling- reprogramming from diseased cells and using gene correction
Drug screening to reduce disease phenotype

Question 23

What are the advantages of understanding the molecular mechanisms of reprogramming?

Answer 23

Understand the transcriptional changes that take place during reprogramming
Early- oct4, Lin28,Estrb, Sal4, laminA/C and B
Intermediate- Gdf3
Late- Sox2, telomerase (candidate regulators)
Understand the processes needed
Epigenetic changes- less methylation and more acetylation as the cell is reprogrammed