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Stem Cells Flashcards

1
Q

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

A

Asymmetric self-renewal- cells associated with tissues

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

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2
Q

Briefly summarise the the different regulation of stem cells

A

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

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3
Q

What is meant by totipotent?

A

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

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4
Q

What is meant by pluripotent?

A

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

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5
Q

What is meant by mutipotent?

A

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

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6
Q

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

A

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)

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7
Q

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

A

Is it positive for alkaline phosphatase

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8
Q

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

A 
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
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9
Q

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

A

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

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10
Q

How is stem cell division different from normal cell division?

A

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

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11
Q

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

A

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

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12
Q

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

A

Quiescence

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13
Q

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

A

Mof proteins
Cyclin Ds
Lamins

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14
Q

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

A

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

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15
Q

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

A

Oct4
Sox2
Nanog

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16
Q

Describe Oct4

A

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

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17
Q

Describe Sox2

A

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

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18
Q

Describe nanog

A

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

19
Q

Describe LIF

A

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

20
Q

Describe skin stem cells

A

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

21
Q

Briefly describe neurogenesis in drosophila

A

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

22
Q

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

A

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.

23
Q

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

A

Basal complex/GMC- prospero, numb, Miranda

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

24
Q

Briefly describe asymmetric cell divisions in the cerebral cortex

A

Symmetric planar division
Asymmetric planar division
Apical-basal division

25
How are different types of neurons made from one neuroblast?
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
26
Where to haematopoietic stem cells come from?
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
27
What is EHT?
Endothelial to haematopoietic transition | Not cell division but specialisation from an adherent cell type to a round non-adherent cell type (progenitor cells)
28
Where does the expansion of the haematopoietic system occur in foetuses?
Foetal liver after which it is moved to the bone marrow
29
What is the difference between long term and short term HSCs?
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
30
What is the paradox surrounding haematopoietic stem cells?
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
31
Describe leukaemia
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
32
Briefly describe stem cell transplant
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
33
What is the importance of stromal cells for HSCs?
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
34
Describe multi potential bone marrow stromal cells
Tri-lineage potential following in vivo transplantation Bone, fat and cartilage A type of mesenchymal stem cells
35
What is the mesenchyme?
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
36
Describe the uses of mesenchymal stem cells
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
37
What are the therapeutic implications of the immunosuppressive effects of MSC?
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?
38
What are the Yamanaka factors?
The growth factors needed to transform adult somatic cells into pluripotent stem cells Oct4, Sox2, Klf4, C-myc
39
What are the advantages of induced pluripotent stem cells?
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.
40
Briefly describe why each Yamanaka factor is used
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
41
Are there better ways of stem cell reprogramming?
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
42
Why reprogram different cells into iPSC?
Less invasive methods than skin fibroblasts Disease modelling- reprogramming from diseased cells and using gene correction Drug screening to reduce disease phenotype
43
What are the advantages of understanding the molecular mechanisms of reprogramming?
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

SCD Chaz (5 decks)

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