Tissue stem cells

Question 1

What potency are postimplantation epiblast ?

Answer 1

pluripotent

Question 2

what potency are adult stem cells

Answer 2

multipotent

Question 3

what are the function of adult stem cells

Answer 3

long term hemeostasis and repair, replenish the cells

lost through normal wear-and-tear and are ready to rapidly expand upon injury

Question 4

when does multipotent progenitors exist

Answer 4

towards the end of gastrulation

Question 5

what do multipotent progenitors do

Answer 5

they receive signals from surrounding tissues and start to differentiate into a more restricted progenitor or terminally differentiated cell types

Question 6

compare self-renew ability between tissue stem cells and progenitors

Answer 6

TSC can self renew without losing potential. Progenitor cannot, it is highly proliferative but potential decreases over time and become more and more specialised

Question 7

when does adult stem cells appear

Answer 7

appear in fetal stem cells at around the time of birth

Question 8

function of hematopoietic stem cells

Answer 8

it can give rise to all cells

Question 9

what can replenish hematopoietic stem cell after irradiation

Answer 9

bone marrow transplant

Question 10

do all adult tissue have resident stem cells?

Answer 10

no eg. pancreas, n patients with type I diabetes, where the immune system wrongly targets the beta cells of pancreas that
produce insulin in response to elevated blood sugar levels. In the absence of these cells, the pancreas is not able to produce insulin and blood sugar levels
are left unchecked leading to high blood sugar (hyperglycaemia)

Question 11

name another organ with no resident stem cells

Answer 11

brain, can lead to neurodegenerative diseases resulting in loss of function due to neuronal dysfunction

Question 12

what are transit amplifying cells

Answer 12

an undifferentiated population in transition between SCs and differentiated cells

Question 13

How do you identify tissue stem cells

Answer 13

by label retaining assay, giving post-natal mice labelled nucleotide analogue (IdU) which incorporates DNA in all proliferating cells and some number of cells were found to retain label and designated as label retaining cells

Question 14

what cells can Label retaining assay identify

Answer 14

stem cells in intrafollicular epidermis giving rise to stratified epithelium of skin , neural stem cells at SVZ and dentate gyrus, satellite cells (muscles stem cells, precursor of muscle cells) in muscles

Question 15

What is the function of paneth cells

Answer 15

support stem cells

Question 16

what can be observed in label retaining assay in small intestine

Answer 16

paneth cells are retained and there is constant turnover of intestinal stem cells that continuously divide and are retained as stem cells or TA cells

Question 17

how do stem cells maintain undifferentiated

Answer 17

by staying in stem cell niche. Niche provides a specific microenvironment composed of extracellular matrix and various support cell types which provides the right biochemical and physical environment to maintain stem cells undifferentiated

Question 18

what is key to keep stem cells undifferentiated

Answer 18

cell position, Differentiation often occurs as cells leave the niche and those
move away from the signals the stem cells need.

Question 19

How does paneth cells affect stem cell differentiation

Answer 19

paneth cells, fibroblast, myofibroblast, smooth muscle cells and endothelial cells located in the crypt base produce signalling molecules to activate Wnt signalling and inhibit BMP signalling. cells move away from crypt base and paneth cells and decrease Wnt signalling and increase BMP signalling, diving stem cell differentiation

Question 20

Function of stem cell niche

Answer 20

to recreate stem cell niche to expand TSC in vitro, study the pathological expansion of stem cell pool through dysregulation of niche or signals emanating from niche

Question 21

how do you capture progenitor cells in vitro

Answer 21

immortalising the cell type of interest by forcing expression of oncogene, yielding mixed cultures where differentiated and undifferentiated cells are mingled, progenitors can be maintained in a homogenous self-renewal state, preserving the capacity to re-enter development and differentiation

Question 22

what cells can be captured in vitro ?

Answer 22

embryonic stem cells, epiblast stem cells, neural stem cells and neuroepithelium stem cells

Question 23

what similarities do NS cells and NES cells have

Answer 23

they both grow in same culture condition- basal medium supplemented with EGF and bFGF

Question 24

Where are NES cells derived from

Answer 24

from human fetal tissue during short window of time in development (week 5-7)

Question 25

What do NES cells differentiate into

Answer 25

hindbrain-specific cell types

Question 26

Where are NS cells derived from

Answer 26

from mouse and human fetal cortex at later stage (E16.5 in mouse, week>7 in human)

Question 27

What can NS cells give rise to

Answer 27

neurons, astrocytes, retaining characteristics of radial glial cells

Question 28

Why are adult stem cells hard to grow in vitro?

Answer 28

ASC reply heavily on complex niche to direct between self-renewal and differentiation,

Question 29

What are organoids

Answer 29

miniature organ-like structures that grow in 3D and contain stem cells and differentiated cell progeny. arranged in a manner very reminiscent of tissue of origin and can be propagated

Question 30

How are organoids derived ?

Answer 30

Lgr5 is a protein expressed by the intestinal stem cells (also called Crypt base columnar cells). These cells can be purified and isolated. Under the right culture conditions organoids can be derived. The organoids self-assemble into mini-guts, with Lgr5 positive cells, interspersed with Paneth cells, giving rise to differentiated cells of the villus.

Question 31

How do you find adult stem cells ?

Answer 31

label retaining assay, lineage tracing, cell isolation and serial transplantation, molecular profiling of stem cells,

Question 32

what is the methodology behind label retaining?

Answer 32

a short of pulse of nucleotide analogue given to tissue, nucleotide incorporated into DNA. If cells are highly proliferative, labelled nucleotide will be diluted over time and if cells proliferate slowly, label will be retained

Question 33

What are the limitations of label retaining

Answer 33

only useful to identify slowly proliferating cells, If the stem/progenitor cell proliferates very rarely, it will be difficult for the cell to be labelled in the first place, no information on potential

Question 34

What limitations do lineage tracing has

Answer 34

- Dyes dilute over time and not all systems are readily accessible for injection

Question 35

What do cell isolation and serial transplantation of HSCs do

Answer 35

- HSCs are extracted from bone marrow and purified by means of combination of cell surface markers - HSCs can be isolated from second individual and transplant onto third -> serial transplantations (prove self-renewal) to reconstitute entire blood lineage

Question 36

What does molecular profiling tells us

Answer 36

can start designing strategies for locating the different cell types within tissues, isolating them for in vitro experiments or using lineage tracing to study what types of cells they can give rise to, identify growth factors or matrix proteins they might produce, and look for signs of signalling pathway activity in the tissue stem cells themselves.

Question 37

How can you find out if it is a progenitor or a stem cell?

Answer 37

Generic lineage trace. If it is a stem cell, undifferentiated descendants of that labelled cell will still be present over a long period of time. If cell in progenitor, descendants will not be present as undifferentiated cells but only as differentiated progeny.

Question 38

How can you start to determine what culture condition might allow you to grow organoids from this putative stem cell? 

Answer 38

Look at what genes (and therefore likely proteins) are expressed in the stem cell and surrounding cells. Analyse pathways which might be active. The important growth factors and activated pathways might give you a clue what to add to the growth medium for your organoids.

Question 39

Cancer is thought to arise from mutated tissue stem cells rather than terminally differentiated cells. Can you think why?

Answer 39

Stem cells already have many of the properties that characterise cancer cells (cell cycle, telomerase activity, etc). Therefore the threshold for these cells to become cancerous is lower than that of a terminally differentiated cell, which would have to acquire many more mutations.

Question 40

4. Name 3 things that the niche can provide stem cells with.

Answer 40

- signals to keep the SC undifferentiated - signals to push the SC to differentiate at the right time - the right physical/mechanical environment