My project

Question 1

How are ‘pericytes’ isolated from patients?

Answer 1

• in the delevalle paper, muscle biopsies were taken from skeletal muscle.
• they are dissected very finely.
• fragments of interstitial tissue containing small vessels are put into a dish with megacell etc and cultured for a week
  after the outgrowth of fibrobast like cells, small round ‘refractive’ cells that have bad adhesion. These cells are collected via pipetting

Question 2

how common is DMD?

Answer 2

1 in 3500

Question 3

what is the role of dystrophin protein?

Answer 3

it acts within a dystrophin associated protein complex (DAPC) which stabilises the sarcolemma during muscle contraction. It does this by linking the sarcomere to the extracellular ECM (laminin). An absence leads to contraction induced muscle degeneration.

Question 4

what is exon skipping and how does it work?

Answer 4

it involves administering an antisense oligonucleotide that is complimentary to the exon containing the mutation, which hides it from the splicing machinery, forming a truncated but functional protein. It doesn’t work with large deletions or regulatory mutations.

Question 5

what is the read through technique?

Answer 5

administer a small molecule that is able to induce a conformational chance in the mRNA structure, allowing the ribosome to skip over a mutation-induced stop codon with a single amino acid. This produces a full length protein.

Question 6

what is a gene replacement therapy?

Answer 6

use either a vector or a cell to replace the mutated gene.

Question 7

why does the size of dystrophin hinder vector candidates?

Answer 7

it surpasses the cloning capacity of the convetional vectors such as Adenoassociated vector and lentiviruses

Question 8

why is a plasmid not feasible?

Answer 8

can’t be delivered to the muscle as easily- needs to be electroplated into muscle.

Question 9

why is a DYS HAC a good ex vivo gene therapy approach?

Answer 9

they have an infinite long capacity, have a centromere, tellers, and replication origin. This means that the copy number within proliferating cells is tightly regulated.

Question 10

when has a DYS hac been used successfully before?

Answer 10

correct a murine mesoangioblasts.

Question 11

in mice, where are mesoangioblasts found?

Answer 11

embryonic dorsal aorta

Question 12

how many cells are required for systemic treatment?

Answer 12

1 trillion or more

Question 13

how did dellavalle identify that the human counterpart of mesoangioblasts were pericytes?

Answer 13

They studied the gene expression of the ‘interstitial refractive cells’ and found that they expressed pericyte markers (AP, SMA, NG2), not endothelial or myogenic markers.

Question 14

how are pericytes and satellite cells positioned?

Answer 14

satellite cells are under the basal lamina of the muscle fibre basal lamina and persecutes are position beneath the endothelial basal lamina

Question 15

how was pericytes ability to differentiate in0 myogenic cells shown in vitro?

Answer 15

the ALP+ cells were cocultured with mouse myogenic cells, and they fused to form hybrid myotubes and when exposed to muscle-differentiation medium.

Question 16

how was the myogenic potential of parasites shown in vivo?

Answer 16

they injected human pericytes into mice intraarterially and they colonised downstream muscles

Question 17

how did dellevalle demonstrated that human parasites could be used in dystrophin replacement therapy?

Answer 17

they injected human parasites into mdx mice and they were able to fuse to myotubes and stimulated dystrophin positive myofibres

Question 18

what is the concept behind the reprogramming aspects of the project?

Answer 18

• notch pathway inhibition leads to premature myogenic differentiation and depletion of the muscle progenitor population.
• activation of notch inhibits MyoD-mediated myogenesis.
• DLL4 is expressed by developing endothelium with PDGF-BB which recruits pericytes from the surrounding mesenchyme.
• this gave rise to the theory that, during development, myoblasts may be recruited by the developing endothelium and converted into pericytes.
• they then mimicked this using murine myoblasts and treating them with DLL4 and PDGF-BB.

Question 19

why was the expression of Pax3 important?

Answer 19

because capellari treated pax 3 + cells from E11.5 cells.

Question 20

how did they show that these treated cells were pericytes?

Answer 20

they looked at the upregulation of pericyte markers and the downregulation of MyoD and Myf5.
- these cells were able to stabilise endothelial cells

Question 21

How did capillari demonstrate that treatment in vivo could convert myoblasts to pericytes?

Answer 21

• they used cre and loxp do induce NICD expression in MyoD expressing cells- severely reduced skeletal muscle differentiation and increased expression of parasite markers in vessel associated cells

Question 22

how has the ability of mesoangioblasts to cross the endothelial wall been shown?

Answer 22

murine mesoangioblasts have been used to treat a mouse model of limb gurdle muscular dystrophy via intra-arterial injection and recapitulation of the diseased muscle with normal muscle.

Question 23

what are pericytes?

Answer 23

they support via direct contact and paracrine signalling to endothelial cells- capillaries.

Question 24

what is gibson assembly?

Answer 24

it works by having two fragments that you want to join, you anneal primers to the end of one so that it is complimentary to the fragment you wish to anneal. AN exonuclease then chews back the 5’ end. The fragments anneal, the polymerase retranscribes back.

Question 25

suggest problems that you’ve had and how you have overcome them.

Answer 25

PCRs: first try again with same reagents to see if it was you, then change the reagents.

immunostaining and for the assay: importance of having negative and positive controls. For example by supervisor said that having the treated and treated would be sufficient. However, then we wouldn’t know whether the technicalities were working. So we should use a cell that we know can migrate.

Question 26

what is Jam-A and how can you block it?

Answer 26

it is a junctional adhesion molecules and you can use an antibody to block it.

Question 27

how does TADA work?

Answer 27

it is an in vivo tissue specific transcriptional profile technique. You use the gal4 UAS system in fish. A promoter for a tissue-specific marker driving Gal4. Then you have a UAS site upstream of a reporter gene, followed by transcription factor fused to DAM, without an IRES in-between. In eukaryotic cells, ribosomes can translate bicistronic regions lacking an IRES at low levels, which is needed because DAM is toxic. So Dam will methylate then you digest the unmethylated regions. and add adaptors to the regions that are methylated, sequence these and you know where your transcription

Question 28

how do muscles contract?

Answer 28

there is a calcium influx into the muscle due to the opening of ion channels. These bind to troponin which uncovers the myosin head binding site of the actin filaments. hydrolysed ATP from the previous stroke, releases the inorganic phosphate and this causes the myosin head to undertake a power stroke. A new ATP molecules then binds and releases the head from the actin. it is hydrolysed and the head goes into a high energy conformation and then rebinds. then the cycle repeats

Question 29

describe how muscle development occurs

Answer 29

the paraxial mesoderm is formed via the upregulation of WNT activity and the downregulation of BMP signalling. The paraxial mesoderm then gives rise to somites. This forms the dermomyotome dorsally. Pax 3 cells migrate to form ventral myocytes that expand along the axis of the embryo. Then pax 3 pax 7 cells migrate in waves. The first forming the primary myotome, the next the secondary myotome and the latter the quiescent satellite cells. The pax 7 and pax 3 activate might and MyoD expression which then instigate the expression of myogenin and myosin heavy chain.

Question 30

what is the problem with the project in terms of pericytes?

Answer 30

The are taken from biopsies in specific arbitrary manner in the delavalle papers. This paper also found that the expression of parasite markers was varied in these cells. so they are a heterogeneous and could differ in their migratory properties and myogenicity. If we could understand more about the pericyte then we could understand what it is that gives it migratory properites etc.

Question 31

if the repogramming doesn’t work, which signals could we expose them to?

Answer 31

TGF-beta has been shown to act in recruiting pericytes

Question 32

briefly explain the evidence for the use of pericytes in stem cell therapy?

Answer 32

human pericytes have been isolated and shown to differentiate spontaneously in vitro into myotubes when cocultured. and when injected into SCID mice, (male cells into female mice then qPCR for y chromosome). They then also injected human pericytes into mdx mice and found that they could produce dystrophin+ fibres and improve functional recovery of the limbs.

Question 33

what is the evidence behind capillari’s paper?

Answer 33

notch inhibition (DLL1) leads to premature myogenic differentiation and notch over expression (DLL1) prevents myogenesis. 
DLL4 and DLL1. PDGF-BB is expressed in embryonic endothelial cells and is involved in parasite recruitment. SO they hypothesized that these two signals may be able to rep gramme myoblasts into pericytes.

Question 34

what stage of myoblasts did they use in caperllari?

Answer 34

E11.5, Pax 3+

Question 35

describe the three ways that they demonstrated that myoblasts could be reprogrammed by endothelial cell signals.

Answer 35

1. they cocultured with HUVECs and they found that myoblasts upregulated parasite genes (AP)
2. they treated with the two signals and found that they upregulated pericyte markers and were able to support HUVEC vascular structures in a stabilisation assay
3. they drove the expression notch via a MyoD promoter and showed that myogenesis was inhibited and an increase in cells expressing parasite markers around vasculature.

Question 36

briefly, how is this reprogramming thought to work?

Answer 36

DLL4 can work alone, PDGF-BB cant’t but can enhance the effects of notch. DLL4 signalling is thought to prevent Myf5 activating myogenin expression by inhibiting the expression of might coactivators and upregulated ID3 which sequester Myf5. This is though to prevent progression of the myogenic lineage and also in some way activate the pericyte programme.

Question 37

describe the three different medias used in the caron protocol.

Answer 37

1. the skeletal muscle induction medium contains a GSK inhibitor which promotes WNT and an BMP inhibitor, alk 3 and 4 inhibitor. also has EGF in and 5% HS.
2. the myoblast medium has many growth factors: IGF, EGF, HGH, FGF, insulin and 5% HS.
3. the differentiation medium and no growth factors, a necrosis inhibitor and insulin.

Question 38

how many myoblasts and myotubes on average do you get from one iPSC?

Answer 38

2,000 myoblasts but can be expanded without decreasing myogencity, and 700 myotubes, 35% differentiation rate.

Question 39

what is the cell tracker label which i found?

Answer 39

CFDA