Stem Cells

Question 1

What are the circumstances that you would find asymmetric or symmetric stem cells?

Answer 1

Asymmetric self-renewal- cells associated with tissues

Symmetric self-renewal- transient stem cells involved in early development

Question 2

Briefly summarise the the different regulation of stem cells

Answer 2

Extrinsic- neighbouring cells, orientation, morphogens, growth factors, cytokines
Intrinsic- growth factor and cytokines receptors, transcriptional regulators, signal pathway, cell survival

Question 3

What is meant by totipotent?

Answer 3

The zygote is totipotent up to the 8 cell stage, it has the potential to give rise to any and Al human cells

Question 4

What is meant by pluripotent?

Answer 4

They can give rise to all tissue types, ie. the embryo proper
They cannot give rise to extra-embryonic structures- trophectoderm

Question 5

What is meant by mutipotent?

Answer 5

They give rise to a limited range of cells within a tissue type
Their daughter cells become the progenitors of cell lines

Question 6

What ability must a stem cell have to be classified as pluripotent?

Answer 6

The ability to give rise to the three germ layers- endoderm (eg. Neurones of the brain), mesoderm (eg. Red blood cell) and ectoderm (eg. Pancreatic cell)

Question 7

What stain would you use to test for pluripotency in vitro?

Answer 7

Is it positive for alkaline phosphatase

Question 8

What would you plate stem cells with do that they keep their pluripotency?

Answer 8

Irritated feeder layer
Up to day 13 mouse embryo
Remove heads and internal organs
Treat with trypsin and plate cells into a dish
They form embryonic fibroblasts
They're irradiated to stop growth

Question 9

What would pluripotent stems show grown in vitro and in vivo?

Answer 9

In vitro- Differentiation is triggered when frien in suspension and embryoid bodies form
In vivo- teratoma formed when injected into a nude mouse

Question 10

How is stem cell division different from normal cell division?

Answer 10

Normal cell division gives rise to two non-identical daughter cells of the same type
Stem cell division gives rise to one use identical daughter cell in asymmetric division and two identical daughter cells in symmetric division

Question 11

What in the niche signals to the stem cell to differentiate?

Answer 11

The niche cells and the ECM influence what type of cell is produced- secreted or cell surface factors control cell renewal, maintenance and survival
The micro environment is important to the differentiation profile of the SC

Question 12

When a cell is not differentiating it is said to be in…?

Answer 12

Quiescence

Question 13

What proteins are important in Stem cell differentiation in the niche?

Answer 13

Mof proteins
Cyclin Ds
Lamins

Question 14

What are some physiochemical factors found in the stem cell microenvironment that affect it?

Answer 14

pH
Oxygen tension
Ionic strength (Ca in particular)
Metabolites and ATP

Question 15

What is the ‘holy trinity’ of factors that control stem cell differentiation?

Answer 15

Oct4
Sox2
Nanog

Question 16

Describe Oct4

Answer 16

Pou domain transcription factor
Maintains pluripotency
Tightly regulated and associated with a number of target genes implicated with the maintenance of pluripotency
-regulatory elements in target genes in close vicinity of Sox2
Down regulation leads to differentiation

Question 17

Describe Sox2

Answer 17

SRY box 2
Member of the HMG domain DNA-BP family- transcription factor
Prevents stem cell differentiation by co-regulation with Oct4 on a lot of pluripotency associated genes
Up regulation leads to differentiation

Question 18

Describe nanog

Answer 18

Unique homeobox transcription factor involved in the self renewal of undifferentiated ESC
Down stream effectors of signals of LIF and BMP
Elevated levels excludes the inclusion of LIF in the feeder layer

Question 19

Describe LIF

Answer 19

Leukaemia inhibitory factor
Interleukin and cytokine family
Essential for maintaining pluripotency in vitro
Binds to heterodimeric LIF receptor and gp130 on cell membrane
Activation of Jak/Stat pathway
Activated Stat3 maintains pluripotency

Question 20

Describe skin stem cells

Answer 20

2-3 found in the Bulge environment in the hair shaft

When division is required Noggin and Wnt signalling co-ordinate to block out the effect of Wnt inhibitors and BMP

Question 21

Briefly describe neurogenesis in drosophila

Answer 21

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 22

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

Answer 22

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 23

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

Answer 23

Basal complex/GMC- prospero, numb, Miranda

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

Question 24

Briefly describe asymmetric cell divisions in the cerebral cortex

Answer 24

Symmetric planar division
Asymmetric planar division
Apical-basal division

Question 25

How are different types of neurons made from one neuroblast?

Answer 25

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 26

Where to haematopoietic stem cells come from?

Answer 26

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 27

What is EHT?

Answer 27

Endothelial to haematopoietic transition

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

Question 28

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

Answer 28

Foetal liver after which it is moved to the bone marrow

Question 29

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

Answer 29

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 30

What is the paradox surrounding haematopoietic stem cells?

Answer 30

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 31

Describe leukaemia

Answer 31

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 32

Briefly describe stem cell transplant

Answer 32

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 33

What is the importance of stromal cells for HSCs?

Answer 33

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 34

Describe multi potential bone marrow stromal cells

Answer 34

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

Question 35

What is the mesenchyme?

Answer 35

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 36

Describe the uses of mesenchymal stem cells

Answer 36

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 37

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

Answer 37

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 38

What are the Yamanaka factors?

Answer 38

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

Question 39

What are the advantages of induced pluripotent stem cells?

Answer 39

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 40

Briefly describe why each Yamanaka factor is used

Answer 40

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 41

Are there better ways of stem cell reprogramming?

Answer 41

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 42

Why reprogram different cells into iPSC?

Answer 42

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

Question 43

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

Answer 43

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