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BMS6056 - Stem Cell Biology > Research Talks > Flashcards

Research Talks Flashcards

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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
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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)
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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
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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
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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
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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
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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

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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

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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
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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
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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
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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
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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)

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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
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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
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16
Q

What is the origin of the auditory brainstem response (ABR)?

A
  • gen by auditory nerve and subsequent structure w/in auditory brainstem
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17
Q

How is human hearing (and gerbil) tested in a clinical setting?

A
  • ABR

- if brain can hear sound see typical wave pattern

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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
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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)
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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
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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
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22
Q

What is now being explored for treating hearing loss?

A
  • exploring combined use of SCs and cochlear implants to prod functional ear
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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
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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
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25
Q

How were these landscapes shown from gene reg networks?

A
  • 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
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26
Q

What is the diff in growth of normal and culture adapted H7 hESCs?

A
  • culture adapted able to grow better
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27
Q

How was the diff between normal and culture adapted H7 hESCs studied, and what was found?

A
  • 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
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28
Q

What is the significance of the SC basin of attraction?

A
  • 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
Q

Are substates in SC compartment lineage based?

A
  • can move between states

- ie. endoderm, ectoderm, mesoderm

30
Q

What happens when treat NTERA2 embryonal carcinoma (EC) cells w/ retinoic acid, what questions did this raise?

A
  • 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
Q

What was the result of labelling single EC cells treated w/ RA?

A
  • 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
Q

What was the result if postpone adding RA for 48 hrs?

A
  • get a more mixed pop of cells

- suggests substates w/in SC compartment

33
Q

What did single cell transcriptomic data reveal about GATA6 exp in SCs?

A
  • 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
Q

How was GATA6 exp further investigated in SCs?

A
  • 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
Q

How was GATA6 exp investigated w/ flow cytometry analysis?

A
  • 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
Q

Can GATA6+ cell interconvert between GATA6+/- substates w/in the SC compartment?

A
  • yes
37
Q

What lineage bias do GATA6+ cells show?

A
  • bias towards the endodermal lineage
38
Q

What evidence is there for GATA6+ cells still being SCs?

A
  • 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
Q

Can GATA6+ cells be trapped by cross-antagonism, how?

A
  • 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
Q

What diff injuries can occur to arteries?

A
  • 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
Q

Can the endothelium regen once its damaged?

A
  • in many people doesn’t regen (in some people it does)
42
Q

How significant a disease is CHD?

A
  • commonest cause of death globally
  • WHO estimates 7.6 mil/year
  • major economic burden on healthcare systems
43
Q

What is the consequence of atherosclerotic plaques?

A
  • 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
Q

How is CHD traditionally treated?

A
  • PCI (percutaneous coronary intervention) *ie. via skin

- 2 mil procedures/year worldwide

45
Q

What is the limitation of PCIs?

A
  • > 90% req stent implantation

- restenosis is a major limitation –> when push plaque back can get restenosis which is thickening inside plaque

46
Q

What is stenosis?

A
  • inhibition of blood throw by plaque
47
Q

How was the problem of restenosis tackled?

A
  • put drugs on metal stents = drug eluting stents

- see blood flow maintained for over 2 years (provided stent placed correctly)

48
Q

Why is there growing concern over the safety of drug eluting stents?

A
  • increased stent thrombosis lead to diminished efficacy
49
Q

What is stent thrombosis?

A
  • 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
Q

What pro healing alts were there to stents?

A
  • VEGF elution
  • Oestradiol loaded stent
  • use of integrin binding peptides
  • -> all these had quite limited success
  • EPC capture stents
51
Q

How do EPC capture stents work?

A
  • capture SCs in blood and adhere to stent, forming protective layer, stent had CD34 Abs bound (but doesn’t just bind SCs)
52
Q

What is a limitation of EPC capture stents?

A
  • 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
Q

What is the Sheffield pro-healing stent?

A
  • 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
Q

Whats are the properties of HTECs?

A
  • 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
Q

How was the Sheffield pro-healing stent developed?

A
  • 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
Q

What experimental model was used for the Sheffield pro-healing stent and why?

A
  • pig, as arteries similar size
57
Q

What was the limitations of the pig implants of the Sheffield pro-healing stent?

A
  • if cells on spring, then have to thread t/ body and deploy spring, will cells still be there?
58
Q

Were cells left on the stent after implantation?

A
  • 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
Q

What did SEM of 1 hr explanted arteries show?

A
  • 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
Q

What was the stent coverage w/ HTEC like over time?

A
  • 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
Q

Overall, what are the good things about the Sheffield pro-healing stent?

A
  • safe

- does not increase neointimal thickening

62
Q

What are future directions for study of the Sheffield pro-healing stent?

A
  • mechanism, currently not understood
  • needs to be tested in humans
  • could we freeze stents w/ cells on, so adhere better?
63
Q

What is heart failure and when does this occur?

A
  • permanent cardiac muscle loss

- occurs after heart attack

64
Q

How could we potentially “mend broken hearts”?

A
  • regen of cardiac muscle w/ pluripotent SCs –> heart failure
65
Q

Can adult SCs from bone marrow/heart be used for cardiac SC therapy?

A
  • secrete beneficial paracrine factors, but do not engraft in infarcted heart –> don’t lead to regen of cardiomyocytes in vivo/vitro
66
Q

Can pluripotent SCs be used for cardiac SC therapy?

A
  • 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
Q

What is a problem w/ use of iPSCs for cardiac SC therapy?

A
  • would not be able to be cultured quick enough in the instance of a heart attack
68
Q

Do we need cells, or just GFs, for cardiac therapy?

A
  • a reason we may need cells, is that when heart infarcts becomes weak, so may need cells to provide structural support
69
Q

What remaining questions and challenges are there regarding cardiac SC therapy?

A
  • 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 (3 decks)

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