1
Q
What are the 2 types of sensory cells in the inner ear, and what is their role?
A
- inner and outer hair cells
- inner is critical sensory cell
- outer amplifies signal
2
Q
What makes hearing loss an ideal condition to be targeted by SC therapy?
A
- huge pop (approx 250 mil) have substantial hearing loss in their lifetime
- age related (20% of total pop)
- 90% sensorineural (to do w/ hair cells and neurons)
- small no.s needed –> normal ears have around 16,000 hair cells and 30-40,000 neurons, so more realistic to replace them all
- no drug treatment, only therapy is hearing aids or cochlear implants (ie. palliative)
3
Q
Why were auditory SCs isolated from a 9-10wk old fetal cochlear?
A
- better model for humans than another species
- just before onset of terminal differentiation –> after this progenitors not easily available, ie. these cells are for life
4
Q
What was found from exploring culture condition to promote growth of auditory progenitor cells?
A
- serum free conditions support culturing of hFASCs
- if only IGF then do not proliferate
- best results w/ all these factors (bFGF, IGF, EGF, OSCFM), expand culture can split and expand again
5
Q
What was the result of a neuralising protocol for hair cells?
A
- induced bipolar cells that displayed potassium delayed rectifiers and VG sodium currents
- remain quite immature, but have enough properties to show differentiating in right way
- after inducing hair cell differentiation, cells displayed inward potassium and calcium currents = characteristics of hair cells
6
Q
What is the proliferative capacity of hFASCs?
A
- more like multipotent than pluripotent SCs
- can’t keep running forever, will run out
- limited capacity not an ideal property, but gives info to explore other SCs
7
Q
What happens in auditory dev when FGF3/10 KO?
A
- otic placode not specified
- one comes from NT and one from mesoderm, come together and make otic placode
8
Q
What happens when cells exposed to FGF3/10?
A
- formation of colonies +ve for otic placode
- ideally want to show co exp of markers in same cell, seen w/ eg. Sox2 and Pax8
9
Q
What did microarray analysis of FGF induced pops show?
A
- larger the set of markers to define a signature the better, found 40 genes primarily exp in otic placode
- looked to see if exp differently to what would happen randomly
- as control used similar signature for pluripotency, highly exp in initial ESC pop
10
Q
What 2 types of colony were induced when FGF induced, and what did they have in common?
A
- otic epithelial progenitors (hOEPs)
- otic neuroprogenitors (hONPs)
- share same markers
11
Q
How could hair cell and neuronal phenotypes be gen from hESC-OEPs and hESC-ONPs?
A
- manually purify cells and differentiate them
- OEPs could prod hair cell like cells
- ONPs could prod neuronal like cells, but not hair cells, more committed than OEPS
12
Q
How could this benefit cochlear implants?
A
- tested ability of cells to work in vivo in disease model
- concentrated on working w/ ONPs, as less clinical need to replace hair cells as have these implants, so neuronal cells more important
- cochlear electrode stimulates directly the spiral ganglion neurons
- cochlear implant relies of presence of neurons
- less people have affected neurons, but have greater need for treatment
13
Q
What animal was used as a model for auditory neuropathy, and why?
A
- gerbil
- frequency range closer than eg. mice/rats (much higher pitch)
14
Q
How were otic neuroprogenitors induced from hESCs?
A
- harvested by trypsinisation
- FGF3 and 10 induced and enrichment for hONP
- expanded in OSCFM
- pop continues doubling over gens
15
Q
How was it known that transplanted cells connect centrally w/ the cochlear nucleus?
A
- see fibers growing out of cochlear and going to brain
- normal neuron will contact cell and make high fidelity big synapsis
- connecting w/ neurons and brain stem
- want to check cells functional and in right place but ALSO make right anatomical connections
16
Q
What is the origin of the auditory brainstem response (ABR)?
A
- gen by auditory nerve and subsequent structure w/in auditory brainstem
17
Q
How is human hearing (and gerbil) tested in a clinical setting?
A
- ABR
- if brain can hear sound see typical wave pattern
18
Q
Was functional recovery of transplanted animals seen?
A
- after drug animal becomes profoundly death
- 10 weeks after transplant see recovery of system
- repop of spiral ganglion
19
Q
What are the assoc challenges of cell therapies for the inner ear?
A
- biological safety (tumorigenesis)
- safe gen and manufacture –> efficient yields, suitable methods for cell purification, GMP culture systems and protocols (can’t use other animal products), gen stability, epigenetic changes
- delivery
- good understanding of host/donor interaction
- combo w/ other techs (CI)
20
Q
How does a cochlear implantation differ in gerbils, comp to humans?
A
- can’t be as large
- not outside the ear
- electrode stimulates electrical impulses to stim cochlear to hear
21
Q
How successful were implants for animals w/ hair cell loss?
A
- function for weeks in chronically implanted animals, and in some cases even months
- need neurons and electrode
22
Q
What is now being explored for treating hearing loss?
A
- exploring combined use of SCs and cochlear implants to prod functional ear
23
Q
In summary what did this experiment show could be achieved in relation to hearing loss?
A
- inner ear progenitors can be derived from hESCs by using a protocol that resembles normal dev
- these progenitors have the capacity to differentiate into sensory hair cell-like cells and neurons in vitro
- they have the pot to induce functional recovery in vivo
24
Q
What is the role of landscape and attractors in Waddington’s model?
A
- cells roll down a hill and make decisions
- in a flat plane representing all states, every state has certain free energy and some more stable than others
- so hills are unstable states and basins are stable
- depressions known as attractors
- likelihood of a cell making a decision dep on height diff between locations
25
How were these landscapes shown from gene reg networks?
- set up a series of kinetic equations to work out what happens if set up system w/ diff levels of GATA1 and PU.1
- so based on mathematical analysis can show there are several stable positions, corresponding to diff fates of differentiated and the undifferentiated cell
26
What is the diff in growth of normal and culture adapted H7 hESCs?
- culture adapted able to grow better
27
How was the diff between normal and culture adapted H7 hESCs studied, and what was found?
- studied transcriptome of undifferentiated cells, before they spontaneously differentiate
- substates of hESCs defined by SSEA3 (antigen)
- adaptation to culture traps the cells in their cell state
- clonogenic assay (ESCs will form colonies, not efficiently but still form, but differentiated cells wont)
- SSEA3+ formed colonies but SSEA3- did not
- in normal cells they turn of SSEA3- and differentiate
- in adapted cells there may be some kind of barrier trapping them from differentiating
28
What is the significance of the SC basin of attraction?
- multiple substates exist
- when in basin are SCs
- as move up hill get to a point where no longer express SSEA3 and differentiate
* DIAG*
29
Are substates in SC compartment lineage based?
- can move between states
| - ie. endoderm, ectoderm, mesoderm
30
What happens when treat NTERA2 embryonal carcinoma (EC) cells w/ retinoic acid, what questions did this raise?
- if treat w/ RA all differentiate
- if all the same why are they not all neurons?
- when do they make decisions?
- differentiate for some time after RA added
31
What was the result of labelling single EC cells treated w/ RA?
- colonies formed are predominantly neural or non-neural
- v few were mixed and even fewer had 5% neurons
- so must’ve decided whether they will eventually be a neuron before differentiation
32
What was the result if postpone adding RA for 48 hrs?
- get a more mixed pop of cells
| - suggests substates w/in SC compartment
33
What did single cell transcriptomic data reveal about GATA6 exp in SCs?
- revealed GATA6 heterogeneity
- mostly clustered where exp Oct4 and Nanog, suggests SCs
- GATA6 not exp in undifferentiated SCs
- these cells exp GATA6 could represent subset that are biased to making eg. endoderm
34
How was GATA6 exp further investigated in SCs?
- used reporter line w/ GFP introd into ATG site in 1 of the GATA6 genes
- so can easily monitor exp of GATA6 as fluorescence
35
How was GATA6 exp investigated w/ flow cytometry analysis?
- look at exp of other markers
- small group of cells SSEA3+ AND GATA6+
- 3-6+ represent differentiated cells
- tests if 3+6L/H are stem cells
- 3+6L make colonies as well as 3+6- and 3+6H make them relatively well
36
Can GATA6+ cell interconvert between GATA6+/- substates w/in the SC compartment?
- yes
37
What lineage bias do GATA6+ cells show?
- bias towards the endodermal lineage
38
What evidence is there for GATA6+ cells still being SCs?
- clonogenic assay, but this is inefficient
- took single cells from pops and let them form a colony, tend to differentiate
- if markers of SCs present then cell colony came from was most likely a SC
- so can convert back to SC so are SCs, but biased
39
Can GATA6+ cells be trapped by cross-antagonism, how?
- used reporter line (MIXL1)
- subsets can be controlled under defined culture conditions
- from MIXL1 +ve pop can pick cells and grow, these revert back to SCs
40
What diff injuries can occur to arteries?
- physical: could be due to stent or turbulent flow (instead of lamina)
- chemical: could be high concs of cholesterol (particularly when oxidised) or nicotine (upregulates pro-apoptotic signalling pathways)
- biological: could be bacteria, virus etc., 5-6 weeks after pneumonia particularly susceptible
41
Can the endothelium regen once its damaged?
- in many people doesn’t regen (in some people it does)
42
How significant a disease is CHD?
- commonest cause of death globally
- WHO estimates 7.6 mil/year
- major economic burden on healthcare systems
43
What is the consequence of atherosclerotic plaques?
- limits area blood can flow, thickened area very acentric (often grows from 1 side)
- cells move from layer to layer to preserve barrier layer
- when plaque restricts blood flow can break, platelets adhere and can cause ischemia
44
How is CHD traditionally treated?
- PCI (percutaneous coronary intervention) *ie. via skin
| - 2 mil procedures/year worldwide
45
What is the limitation of PCIs?
- >90% req stent implantation
| - restenosis is a major limitation --> when push plaque back can get restenosis which is thickening inside plaque
46
What is stenosis?
- inhibition of blood throw by plaque
47
How was the problem of restenosis tackled?
- put drugs on metal stents = drug eluting stents
| - see blood flow maintained for over 2 years (provided stent placed correctly)
48
Why is there growing concern over the safety of drug eluting stents?
- increased stent thrombosis lead to diminished efficacy
49
What is stent thrombosis?
- serious condition w/ adverse clinical outcomes
- need prolonged anti platelet therapy
- don't want metal exposed to blood, as causes clots
- due to stents being so effiecient
50
What pro healing alts were there to stents?
- VEGF elution
- Oestradiol loaded stent
- use of integrin binding peptides
- -> all these had quite limited success
- EPC capture stents
51
How do EPC capture stents work?
- capture SCs in blood and adhere to stent, forming protective layer, stent had CD34 Abs bound (but doesn't just bind SCs)
52
What is a limitation of EPC capture stents?
- relies on ability to mobilise functional circulating EPCs, older patients who are more likely to be affected by CHD have less SCs --> mobilisation inversely related to risk factors, lots of interindividual variation, no standardisation
53
What is the Sheffield pro-healing stent?
- stent pre coated w/ endothelial cells before implant
- human trophoblast derived endovascular cells (HTEC)
- derived from hESCs
- similar to native cytotrophoblast EV cells
- important in implantation of embryo in the uterus
54
Whats are the properties of HTECs?
- immune activity --> soluble HLA G
- anti-inflammatory
- promote vessel growth
- not SMCs --> if have endothelium then good repair mech, so don't want SMCs to be present and form plaques
- take up Ac DiL low density lipoprotein
- express VWF
55
How was the Sheffield pro-healing stent developed?
- develop EC from SCs (human and mouse)
- characterisation of EC
Loading of cells onto stents
- cells able to grow on metal
Implantation of coated stents into experimental models
56
What experimental model was used for the Sheffield pro-healing stent and why?
- pig, as arteries similar size
57
What was the limitations of the pig implants of the Sheffield pro-healing stent?
- if cells on spring, then have to thread t/ body and deploy spring, will cells still be there?
58
Were cells left on the stent after implantation?
- labelled w/ indium for tracking --> label there, but can we be sure cells are?
- 2.7% after 7 days --> no.s are similar to when drugs used
59
What did SEM of 1 hr explanted arteries show?
- are the hTEC seeded the ones we put there, or ones from blood that adhered?
- after 3 days the bare metal stent is now covered, hTEC seeded formed cobbled appearance (typically what the endothelium looks like)
60
What was the stent coverage w/ HTEC like over time?
- signif diff w/ control until 7 days
| - disappointing that lumen is similar, would expect if SCs working that treated pigs would have a larger lumen
61
Overall, what are the good things about the Sheffield pro-healing stent?
- safe
| - does not increase neointimal thickening
62
What are future directions for study of the Sheffield pro-healing stent?
- mechanism, currently not understood
- needs to be tested in humans
- could we freeze stents w/ cells on, so adhere better?
63
What is heart failure and when does this occur?
- permanent cardiac muscle loss
| - occurs after heart attack
64
How could we potentially "mend broken hearts"?
- regen of cardiac muscle w/ pluripotent SCs --> heart failure
65
Can adult SCs from bone marrow/heart be used for cardiac SC therapy?
- secrete beneficial paracrine factors, but do not engraft in infarcted heart --> don't lead to regen of cardiomyocytes in vivo/vitro
66
Can pluripotent SCs be used for cardiac SC therapy?
- give rise to cardiomyocytes that engraft LT in animal models, beat in synchrony w/ heart and secrete beneficial paracrine factors
- LT cardiomyocyte engraftment partially regens injured heart, which is hypothesised to bring clinical benefits
67
What is a problem w/ use of iPSCs for cardiac SC therapy?
- would not be able to be cultured quick enough in the instance of a heart attack
68
Do we need cells, or just GFs, for cardiac therapy?
- a reason we may need cells, is that when heart infarcts becomes weak, so may need cells to provide structural support
69
What remaining questions and challenges are there regarding cardiac SC therapy?
- which approach to use? ESCs, iPS, cell delivery, or small mols, quantity of cell
- didn’t keep pigs for long time to see if survived
- beat w/ particular rhythm, sometimes unique to person so would have to characterise carefully to make sure didn’t lead to arrhythmias
BMS6056 - Stem Cell Biology
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