Questions

Question 1

what are the 4 reprogramming factors used?

Answer 1

oct4, sox2, klf4, c-myc

Question 2

how do the reprogramming factors work?

Answer 2

activating downstream transcription factors which then unwind and demethylate pluripotency genes and methylate differentiation genes

Question 3

what are 5 ways of reprogramming somatic cells into iPSC

Answer 3

sendia (non integrating virus), adenovirus, lenti or retrovirus, mRNA, protein

Question 4

what is the efficiency of integrative reprogramming of iPSC?

Answer 4

0.1-1%

Question 5

what is the definition of a stem cell?

Answer 5

• a cell that can self renew when it divides into a daughter cell to produce a cell that is an exact copy. And also produce cell that is specified down a specific lineage

Question 6

within stem cell division, which DNA strand does the stem daughter take?

Answer 6

the template

Question 7

what do the three germ layers form?

Answer 7

ectoderm (neural and keratinocytes), mesoderm (muscle, blood system, fibroblasts), endoderm enterocytes (gut and pancreas)

Question 8

what 3 tissues dont regenerate in humans?

Answer 8

retina, eye, CNS and heart (a bit)

Question 9

what does pluripotent mean?

Answer 9

all three germ layers

Question 10

what does culture adaptation mean?

Answer 10

selecting stem cells in vitro that increase proliferative potential

Question 11

explain how acetylation and methylation works?

Answer 11

methylation of promoters suppresses expression of genes, methylation of histones can be suppressive or activating, acetylation of histones upregulates genes.

Question 12

what did gurdon do?

Answer 12

he removed the nucleus from a somatic cell and put it in an inucleated xenopus egg and it was reprogrammed.

Question 13

how can you test pluripotency?

Answer 13

in vitro differentiation into three germ layers, implant into SCID mouse and look at teratoma formation, can you remove pluripotency factors and the cells remain iPSC

Question 14

what is wadding tons genetic landscape?

Answer 14

the hill with the cell doing into one of the two valleys

Question 15

why are zebrafish a good model organism for tissue repair?

Answer 15

• transparent so can do realtime fluorescent labelling
• they have amazing regenerative abilities: can regenerate the heart, kidneys, CNS, eye, fins
• good for genetics
• vertebrate
• cheap

Question 16

why can zebrafish regenerate the retina and humans can’t?

Answer 16

their muller cells behave as adult stem cells but in humans they divide once and then form a square

Question 17

explain how the environment is important for regeneration?

Answer 17

the environment needs to be conducive to regeneration. For example in PNS repair the macrophages remove growth inhibiting myelin and the ECM is remodelled.

Question 18

what three models are good for regeneration studies?

Answer 18

zebrafish, salamander, mouse

Question 19

what are hox genes?

Answer 19

the are transcription factors that activate the expression of multiple TFs that give portions of the body their tissue components.

Question 20

what are some interesting papers in the area that you’ve read on the area of stem cell research (outside the department)? (3)

Answer 20

1. Guillot: use transplantation of mesenchymal stem cells that express a normal collagen type 1 molecule. Inject into babies before immune competency. Take normal MSC from normal babies amniotic fluid and injected into mice with the disease model increased bone strength and reduced bone fracturing.
2. Using resident fibrocartilage stem cells to treat fibrocartilage disease and injury. Sclerotin to block WNT signalling and upregulate their action. They were first to discover these cells and also found that patients suffering from firbocatrilage disease may have increased WNT in their superficial zone niche. (next step- different joint)
3. Idea that progenitors, rather than stem cells, support the tissue. The chance of dividing asymmetrically vs symmetrically is sufficient- can never get rid of this progenitor- if mutated will be there forever. (more lineage tracing, model what happens in disease- would stem cell kick into action)

Question 21

Describe the process of treating osteogenesis imperfecta.

Answer 21

It is a disease that results from mutation in collagen type 1 in unborn babies. Guillot transplants Mesenchymal stem cells from normal unborn babies and transplants into unborn affected babies before they are immune competent Inject into blood stream. They then form bone progenitors that express correct ECM molecule. Normal babies.

Question 22

Describe the process of research treatment into fibrocartilage disease and injury and the next step.

Answer 22

• the group found a fibrocartilage stem cells int he mandibular joint that can produce cartilage and bone (EDU Injection into joint). They showed that WNT signalling was tightly controlled across this niche, activating on WNT signalling within the Superficial zone cause premature differentiation of the FSCSs and depletion of the stem cell pool. They showed that in a rabbit disease model of, cartilage can regenerate at a greater rate when in the presence of WNT inhibitor-sclerostin. They also found in humans suffering from fibrocartilage disease may have increase wnt signalling in their superficial zone.

Question 23

in terms of the slow-cycling stem cell/progentiro theory, how could this be tested?

Answer 23

linear tracing of progenitors- activate temporally and look at the increase in number over time- does it fit model? what about in injury?

Question 24

what are the names of the three authors that wrote the papers that you’re interested in?

Answer 24

• Catherine Becker and Thomas Becker

- Rambukkana

Question 25

Describe Thomas Becker’s paper and what can be done next.

Answer 25

Regeneration of the CNS within zebrafish. Becker’s paper looked at a subpopulation of neural progenitor cells called PMN, within the first 2 days post-fert give rise to neurons and then switch to oligodendrocyte. These cells are thought to be the cells from which ependyma-radial glial cells derive from. ERG cells are the adult CNS stem cell. It is thought that the plasticity between the oligodendrocyte and and neural production cell types may be plastic in development and in adults. They used the similarity as a model to show that the innate immune system may have a profound role in CNS regeneration. They showed that after 2dpf, the pMNs could revert and produce neurons in regeneration. but they didn’t show the functional contribution. They also had problems with getting the transgene to work after regen so had to use staining to show that these cells were contributing to the regen neurons (shame). They showed that microglial and macrophages are recruited to the site of injury significantly in the larval injury and that when they suppressed the immune response they saw a decreased regeneration rate. Then they showed that ablating neurons , rather than lesion, was sufficient to trigger microgia/macrophage recruitment and regeneration in the larval (when the adapted immune hadn’t developed yet).
I think a lot more needs to be done to show that the regeneration process is similar. I think you could look at what transcription factors that are up or down regulated are binding to- the coiling of the DNA (ATAC seq) and use TaDa to see how the expression profile changes!

Question 26

what are the issues that you have with the paper?

Answer 26

• just not wholly convinced about the transferability of the larval regeneration to the adult.
• yes they showed that both recruit the immune system and that dopamine can enhance the regeneration process, but i don’t think this is that convincing. Could still be different pathways. The fact that one takes a month and the other a few days shows they are different. Would like further proof of the signalling pathways, serotonin works the same way too?
• are the ERG cells being activated in the same way as pMN, same signals etc?
• would be cool to see some real-time pMN differentiation.
• maybe some real-time down reg of oligo markers and upregl of neuronal markers.
• couldn’t you just have adults that have all their immune system suppressed and inject medium from activated macrophages, if proteins?

Question 27

describe the Rambukkana paper. why is it interesting.

Answer 27

This paper showed that the leprosy bacteria have so evolved to take advantage of the plasticity of the schwann cell, which it reprogrammes into a mesenchymal stem cell and then differentiates into muscle, adipocytes, smooth muscle and infects these tissues in the body. And can also infect macrophages this way etc (non stem cell stuff).
what is it about the stem cell that makes them so amenable to this, what does the bacteria do? could this be an alternative system of reprogramming, takahashi suggest that the reason retro vs CCP reprogramming works better is via a toll receptor (immune activation) How does the bacteria instigate the epigenetic changes?

Question 28

what are the next steps for the Rambukkana paper?

Answer 28

• can it have the same effect on similar cells- the precept seems to be plastic cell type and neural crest derived.
• how does it upregulate the regulators such as ERK (how this works isn’t actually known yet anyway)
• can you take away the harmful bits of the bacteria and have the same reprogramming effect?

Question 29

briefly explain the liver paper from the institute

Answer 29

using recombinant laminins instead of matrigel because more defined and not animal component, and can mature the hepatocytes which hasn’t yet been achieved.

Question 30

why is serum free approach good?

Answer 30

because more defined, less risk of fungus etc or production of animal protein derived proteins. Better for translational medicine.

Question 31

What is crispr cas9, why is it good and why is it bad?

Answer 31

it is a bacteria defence mechanism against viral attacks. It involve integrating the viral DNA into the genome so that is can be later attacked. This can be used for genome editing. Can produce a guide RNA which the cas9 will use to cleave DNA at this area in the genome. However, due to the bacteria not wanting to be too specific, the cas9 system has many off-target effects.

Question 32

briefly explain the wound healing stuff you know?

Answer 32

that connexins can make vessels leaky, increasing the inflammatory response in chronic lesions, they should be down regulated but they aren’t. They change the ECM and migration and reepithelialisation etc. but nexagon can prevent this from happening- works really well.