1
Q
What was the 1st use of the term SCs?
A
- Haeckel (1868) –> built on Darwin’s theory, cells diverge in same way species have
- termed stammbaum = tree of life
- then stamzelle = stem cell
2
Q
How did the use of the term SCs develop and become wider?
A
- Borevi (1892) proposed that SCs are not only initial cells, but also those between the fertilised egg and committed germ cells
- Hacker (1892) started applying the term stem cells to cyclops embryo cell undergo asymmetric divisions
- Pappenheim (1905) found stem cells were present in hematopoiesis
3
Q
What is a SC?
A
- cells that have the potential to gen diff specialised tissue (differentiation) as well as copies of themselves (self replication)
- DIAG*
4
Q
What diff criteria are used to classify SCs?
A
- by age of development
- by tissue of origin
- by their potential to prod diff cell types
- how SCs are used as therapies
5
Q
How is age of development used to classify SCs?
A
- embryonic or adult
6
Q
How is tissue of origin used to classify SCs?
A
- neural SCs, hematopoietic (blood), umbilical cord etc.
7
Q
How is potential to prod diff cell types to classify SCs?
A
- totipotent = all cell types of human body, inc trophoblast
- pluripotent = derivates from the 3 germ layers (ie. ESCs), can become any cells apart from those in trophoblast
- multipotent = diff cell types from a tissue or organ (neural, blood, renal etc.)
- unipotent = differentiate into only a single cell type (ie. muscle satellite cells)
8
Q
How does the rate at which SCs divide change during their lifetime?
A
- divide slowly
- then when needed to act divide quickly
- when in fast cell cycle known as transit amplifying cells
- after divided then differentiate and prod post mitotic progeny
9
Q
What diff strategies are there for use of SCs as therapies?
A
- allogenic
- autologous
- recruitment of endogenous SCs from the same tissue
10
Q
How is an allogenic approach used for SC therapies?
A
- SCs derived from a diff donor and expanded in the lab, can be used to treat a large pop of patients
- eg. ESCs, cord blood cells
- allogenic as outside the initial person
11
Q
How can an autologous approach used for SC therapies?
A
- SCs to be transplanted are derived from the same patient and reprogrammed to be pluripotent
- eg. auto transplant from bone marrow or prod iPSCs
- this is a patient specific approach
12
Q
What factors must be induced to gen iPS cells?
A
- SOX2, OCT4, MYC, KLF4
13
Q
How can recruitment of endogenous SCs be used for SC therapy?
A
- can recruit from the same tissue
- theoretically poss to use medicines to ‘awaken’ endogenous SCs in damages tissues
14
Q
Apart from direct therapeutic apps what can SCs also provide?
A
- excellent models to screen for new drugs –> important to test on human models
- models to study genetic conditions (especially iPSCs) –> can take cells from patient w/ genetic mutation, create pluripotent SCs and create model of particular tissue affected to help understand the biology of particular mutation in particular tissue
- models combining the former 2 = pharmacogenomics –> understand how can use drugs to treat mutations by compensating for phenotype, needs to be in patient specific manner to some extent
- insight into fundamental biological problems
15
Q
Is regenerative medicine a new concept? examples
A
- bone marrow transplantation (mid 1950s)
- corneal grafts –> one of 1st types of transplant surgery successfully performed, in early 1900s
- skin grafts for burns victims (developed significantly in 2nd world war)
- 1st successful kidney transplant in 1954
16
Q
What experiments and trials should be performed before SCs can be routinely used for therapies?
A
- efficacy
- safety
- purity and controlled manufactured process
17
Q
What is the importance of cancer SCs for anti-cancer therapies?
A
- used to treat cancer cells by stopping proliferation and shrinking size of tumours, but as tumours are gen by SCs, results in tumour regrowing, as SCs not affected
- but if destroy SC then tumour loses ability to gen new cell and does result in tumour degrading
- DIAG*
18
Q
What Nobel Prizes have been awarded in Physiology and Medicine relating to SCs?
A
- 2007: awarded jointly to Carpecchi, Evans and Smithies for discoveries of principle for introducing specific gene mods in mice by the use of ESCs
- 2010: awarded to Edwards for development of in vitro fertilisation
- 2012: Gurdon and Yamanaka for discovery that mature cells can be reprogrammed to become pluripotent
19
Q
How have teratoma studies provided SC insights over the years?
A
- initially studied in strain 129 mice
- in the 60s showed complexity of tissues in tumours can be originated to a single cell –> embryonic carcinomas (EC) are SCs
- EC cells resemble pluripotent ECs
- can input ECs into blastocyst and will contribute to all tissues, thus pluripotent
20
Q
When do EC cells grow better?
A
- if have a layer of feeder cells
21
Q
What tissues in the body can ESCs form?
A
- all the tissues in the body
- ie. ectoderm, mesoderm, endoderm and germ cells
22
Q
From where are ESCs derived?
A
- ICM of blastocysts
23
Q
What are the properties of ESCs?
A
- non transformed
- indefinite proliferative potential, high amp capacity
- stable diploid karyotype
- clonogenic, so can originate a culture from a single cell
- pluripotent so can gen all fetal and adult cell types in vitro, in vivo and in teratoma cells
- incorp in chimaeras
- germline transmission in chimeras
- permissive to genetic manipulation
24
Q
What properties of ESCs are harder to achieve in hESCs?
A
- stable diploid karyotype
- clonogenicity
- hard to assess pluripotency, as can’t test all types
- incorp into chimeras impractical and ethical issues, but demonstrated in a recent paper
- germline transmission in chimeras not practical or ethical
25
How were transgenic animals created?
- mESCs injected into blastocyst will incorp into all embryonic cell types, if genetically manipulated could gen a transgenic animal
- visualised w/ beta galactosidase as a marker
26
What happens when ESCs are injected into a competent adult isogenic host?
- form teratomas
| - diff structures inc gut-like, neural epithelium, bone, cartilage, striated muscle and glomeruli like
27
What extrinsic factors are there for self renewal?
- LIF (leukaemia inhibitory factor) or feeder layers
- once withdrawn prolif continues but differentiation induced
- initially LIF comes from feeder layers
28
What is the LIF pathway?
* DIAG*
- LIF binds to LIF receptor, of which gp130 is a part (co-receptor)
- this activates the JAK pathway, which upregulates STAT3, which is critical to maintain these cells pluripotency
- LIF also acts on SHP-2 pathway
29
What is the consequence on cell fate due to LIF also acting on SHP-2?
- not an even balance and in the absence of LIF, ESCs tend to differentiate
- but if LIF present then STAT3 signalling cascade activated and balance tipped in favour of cell renewal
30
What is the result when LIF is exp in serum free conditions in mESCs?
- LIF alone is insufficient to maintain pluripotency and block neural differentiation
31
What did Ying et al (2003) show?
- LIF and BMP4 (/2) req to sustain self renewal and pluripotency
- BMPs via Smads induce Id genes that block entry into neural lineages
- at the same time LIF/Stat3 inhibit BMPs from inducing mesoderm/endoderm
- LIF and BMPs use competing actions to co-operate to sustain self-renewal
32
How do mESC and hESC colonies differ?
- mESCs grew forming small colonies that tend to project out
| - hESCs usually flat w/ well defined edges
33
How do markers exp differ between mESCs and hESCs?
- SSEA-1 marker only in mESCs
| → SSEA-4 only in hESCs
34
What pathways for self renewal are conserved between mESCs and hESCs?
- Stat3 signalling
- Nanog
- Oct-Sox
- FGF signalling
- TGFβ signalling
- BMPR1α
- microRNAs
- methylation, eg. X-inactivation
- cell cycle (eg. Rb)
- Igf2-H19
35
What are some known diffs between self renewal in mice and humans?
- LIFR-gp130 --> as hESCs not dep on LIF made their iso more difficult
- req for activin/nodal signalling
- FGF signalling --> critical for iso hESCs
- cell cycle rates and cell death
- Rex1, variable exp in hESC line
- surface antigens (SSEA, TRA)
36
How do ES cell phenotypes differ between humans and mice?
- hESCs / mESCs
- SSEA1- / SSEA1+
- SSEA3+ / SSEA3-
- SSEA4+ / SSEA4-
- TRA-1-60+ / TRA-1-60-
- GCTM2+ / GCTM2-
- Thy1+ / Thy1-
- MHC+ / MHC-
- ALP+ / ALP+
- Nanog+ / Nanog-
37
What are human ESCs more equivalent to in mice, how was this found?
- iso cells from mouse epiblast (in egg cylinder stage) and found v similar to hESCs
- so more equivalent to EpiSCs than mESCs
- so diffs observed largely due to a shift in timing
38
Are mESCs and hESCs primed or naive?
- hESCs primed
- mESCs naive as less dev
- EpiSCs are primed
39
How does the differentiative potency change t/ dev?
*DIAG*
40
How was the ground state of human naive pluripotency captured?
- can either derive new cells or push established line back to become naive
- approach was deleting or activating enhancers (as they differ during differentiation), so can tell whether naive or primed
- ad human ES cell line and exposed to mix of cofactors, removed and alt diff factors until got to pool of 8 (NHSM), this changes pluripotent primed cell into more naive cell
- iso cells from human blastocyst and converted primed to naive cells by exposing to these factors
41
Why was it challenging to capture the ground state of human naive pluripotency?
- as dev into primed state so quickly
42
How was the role of enhancers shown to be important in hESCs?
- used flow cytometry reporter assay, comp cell no.s to intensity of fluorescence
- for differentiated hESCs, none +ve for Oct4
- if del proximal enhancer in naive hESCs, then increased fluorescence, so must be using distal enhancer
- when del distal enhancer then lose cells
- opp is true of primed hESCs
- shows primed and naive cell types control Oct4 exp via these 2 enhancers
43
How was robust gen of cross species chimeric humanised mice achieved?
- microinjection of naive human iPSCs into mouse morulas
- prod chimeras
- were incorp into mature tissues
44
What are the 2 stages of pluripotency?
1) A naive or ground state (ICM like)
| 2) A primed state (epiblastic like)
45
How does doubling time differ between naive and primed cells?
- reduced in naive
| - increased in primed
46
How do X chroms differ between naive and primed cells?
- active in naive
| - inactive in primed
47
How does ability for single cell cloning differ between naive and primed cells?
- poss in naive
| - poor in primed
48
How does enhancer use differ between naive and primed cells?
- naive use Oct4 distal enhancer
| - primed use Oct4 proximal enhancer
49
How does ICM integration differ between naive and primed cells?
- poss in naive
| - low in primed
50
How does factors dependent on differ between naive and primed cells?
- naive are LIF dep
| - primed are activin/FGF dep
51
How does interaction between naive and primed cells occur in mice?
*DIAG*
52
How does interaction between naive and primed cells occur in humans?
*DIAG*
53
Why are naive human pluripotent ESCs important?
- fundamental understanding of pluripotency
- easier to mod genetically (more efficient HR)
- have provided confirmation of human mouse chimerism (humanised organs for transplant, eg. heart, w/o having to worry about chance of rejection?)
54
How was comparability of the many hESC lines investigated?
- the international stem cell initiative
- comp diff lines and grew in comparable way, analysing similarities and diffs
- core of elements that is v similar, and many pathways conserved, but are diffs too
- if looking for a cell line to do a particular job, then some may be more suitable than others
55
Why do mESC lines tend to be reasonably similar?
- inbred strain, so little variability
56
How does capacity to differentiate differ between hESC lines?
- differing efficiencies and efficacy
57
What does the stage of pluripotency imply?
- the ability of a cell to self renew and gen lineages from the 3 germ layers
58
What confers the ability of a cell to self renew?
- by a set of TFs, whose exp is carefully balanced to achieve the right balance
59
How does the mouse epiblast dev?
- 1st stage = ball of cells (morula)
- in early blastocyst 2 main cell types, ICM and trophoblast (becomes placenta)
- ICM forms epiblast, which then forms cylindrical epiblast in mouse and formation of primitive endoderm --> forms membranes around embryo (VE and PE)
60
What is the battlefield model of pluripotency?
- group of factors conflicting between pluripotency TFs that seek to to direct ESC differentiation to opposing lineages
61
What do Nanog, Oct4 and Sox2 define?
- Nanog important for endoderm
- Oct4 for mesoderm
- Sox2 for ectoderm
62
What prot doms are in Nanog, Oct4 and Sox2?
* DIAG*
- green are DNA BDs
- Nanog is typical homeodomain TF
- Oct4 has POU dom
- Sox2 can interact w/ Oct4 via TAD dom
63
How do relative levels of Oct4 influence ESC fate?
- if steady level of exp then pluripotent ESC
- if downreg then trophoectoderm
- if upreg then extra embryonic endoderm (if earlier) and mesoderm (if later)
* DIAG*
64
What is Oct3/4 and how is exp critical?
- Oct3/4 = POU TF = Pou5f1
- essential for pluripotent potential of ICM in vivo --> w/o Oct3/4 the embryo (inner cells) failed to acquire the potential to prod diff lineages and only prod extra embryonic trophoectoderm
65
Why is continuous Oct4 function necessary to maintain pluripotency in ESCs?
- otherwise results in trophoblast
66
How do Oct3/4 and Cdx2 interact, and what is the importance of this interaction?
- reciprocal repression loop determines trophectoderm differentiation
- decreasing Oct3/4 results in increased Cdx2
- forced increase in Cdx2 results in trophoblast (and decreased Oct3/4)
- Oct3/4 and Cdx2 appear to bind in a complex that inhibits their indiv transcriptional activity
67
What is the importance of Sox2, and how was this shown?
- multipotent cell lineages in early mouse dev dep on Sox2 function
- embryos where Sox2 del failed to gen an epiblast
- both factors (Sox2 and Oct4) are req in lineage leading to epiblast formation, and in their absence trophectoderm is formed
- in null mice blastocysts show defective ICM dev in culture
- Sox2 KO mutant embryos lack an epiblast
68
How was Nanog identified as a pluripotency factor by an in silico screen?
- used digital differential display technique
- selected highly exp genes in ESC pop (as if important then likely exp at high levels), and looked for their exp in other tissues
- some highly exp genes also in other tissues, so not specific
- but Nanog was specific
- proved was a pluripotency gene by forcing exp from a constitutive promoter, to see if it could cause pluripotency w/o LIF --> it could
69
How was Nanog identified as a pluripotency factor by a functional screen?
- exp genes exp in ESCs, in cells to see what combo conferred ESC properties and morphology
- used reporter line w/ alkaline phosphatase (marker of pluripotency)
- looked for genes upreg and becoming more ESC like
- repeated w/ narrower selection of genes until could purify
- put this cDNA seq into cells mutated for the LIF1 receptors, and remained pluripotent w/o being able to detect LIF1 signalling
70
How was it shown that Nanog is essential for self renewal?
- novel homeodom prot v transient exp in embryo
- Nanog -/- has no pluripotent ectoderm, instead forms visceral/parietal endoderm
- increased Nanog can overcome req for LIF (and BMP in serum)
- Nanog -/- ESCs and ICM lose pluripotency and differentiate into extraembryonic endoderm
71
How do diff pluripotency factors endow ESCs w/ multilineage potential?
* DIAG*
- shows primary roles of each pluripotency factors
- interaction between 4 keeps cell pluripotent
72
How was transcription levels of Oct4 and Nanog monitored, and what were the findings?
- gen ESC lines that can report on exp of Oct4 and/or Nanog t/ knock in of fluorescence genes --> so red when exp Oct4, green when exp Nanog and yellow when co-exp
- control is fibroblast, so no exp
- Oct4 gives fairly homogenous exp
- Nanog levels differ in pop where all +ve for Oct4
73
How was the functional implication of Nanog levels differing in a pop studied?
- FACS sorted into GFP+ and GFP- lines and cultured for 6 days
- +ve pop gens a -ve pop
- -ve pop starts to gen a +ve pop
- so not set into 2 diff pathways, still pluripotent
74
How does FACS work?
- pop of cells labelled w/ diff markers
- put into system causing precise flow of cells
- so have tiny droplets which each contain a cell
- pass in front of laser, which differentially activates/charges cells exp certain fluorescence, then sorted into +/- lines
75
What does the model that Nanog may act as a rheostat providing variable resistance to differentiation mean?
- initially can switch between +ve and -ve (ie. transient)
| - when get to point of commitment, -ve cells become very -ve and differentiate
76
What is the Waddington landscape?
- explained differentiation as cell moving to stable state of less energy
- if in crater at top of hill then takes energy to get out but then v easily moves down, can go to valley a or b (diff fates)
* DIAG*
77
How is heterogeneity useful in a Waddington landscape?
- to facilitate prod of diff subsets (ie. which valley goes to), by moving into diff positions, so cell closer to left more likely to go to fate A etc.
78
How can cell surface markers be used to look at Waddington landscapes?
- SSEA3 tends to be marker of v early cells, exp stably in centre of crater
- as start moving up get exp of other substrates and loses SSEA3, but still +ve for TRA1-60
- so if SSEA3- and TRA1-60+ then becoming more differentiated and closer to commitment
79
Why is heterogeneity important?
- cells from diff states may preferentially dev into particular lineages
80
What options do cells have in terms of their fate?
- can either self renew, differentiate or die
81
What can factors preventing differentiation or death impact on?
- could have direct effect on self renewal
82
What is the consequence of impacting on self renewal (genetic stability)?
- cells cultured in vivo could dev genetic abnormalities
- mutations that favour self renewal would tend to be selected by the culture
- although detrimental for therapeutic app, this could provide valuable insight into genes that control prolif and self renewal
- gain of chromosomes 12, 17 and 1 seem to be clear hotspots of mutations that confer competitive advantage
- seen no gain of genes from 4, means not that important or that so important that any changes are fatal so not beneficial
83
How can differentiation of ESCs be triggered, and how can this happen?
- removal of extrinsic conditions for self renewal
- happens in many occasions by cells aggregating
- forms ‘embryoid bodies’, resemble gastrulation and early embryonic dev in vivo
84
What are the properties of EBs?
- highly heterogeneous
| - initially obtained from embryonic carcinoma cells
85
How does organised gastrulation in EBs occur?
- resemble to a point early embryo
- Wnt signalling mediates self organisation and axis formation in EBs
- active Wnt turns on lacZ reporter, can see where activity is in embryo
86
How does presence of Wnt or DKK1 in media influence EB dev?
- if Wnt in media then early dev of EBs
| - if DKK1 in media then late dev in Wnt responsive areas
87
What are the advs of EBs?
- cheap to prod
| - gen 3 germ layers
88
What are the disadvs of EBs?
- difficult to control aggregation in a reproducible way (shape, size)
- no. of days before they are collected
89
What diff EBs prod what parts in hESCs?
- cystic EBs best at prod endoderm
- bright cavity EBs good at prod 3 germ layers, best organised and closer to real embryos
- dark cavity EBs have good prod of the 3 germ layers
90
How can EBs be prod in a more controlled manner?
1) hanging drop method (works well for mice), can get 1 cell per 10ul of media, plate on petri dish then turn upside down, creates single embryo body of typical size and shape
2) controlled aggregation (ie. tissue culture plates w/ special geometry), in each well put fixed no. of cells in fixed vol, so can control size/shape etc, forms at bottom, get v homogenous pop
91
How can directed differentiation be induced?
- either using EBs or plating cells as monolayers
- GFs: important variables inc conc, when added and which combo
- substrate cells grown on (eg. plastic, laminin pushes cells towards particular differentiation)
92
Why is it necessary to isolate the desired cell type from a culture?
- culture conditions could selectively gen the cell type of interest, but the most common outcome is a mix of cells, w/ desired cell type contaminated by others
- rarely get 100% desired cells, so need to purify
93
How can the desired cell type be isolated from a culture?
- FACS to sort for specific cell markers --> at an intermediate progenitor stage or at final cell type (when fully differentiated)
- density gradients --> less common now, but often used for hematopoietic cells, put cells in tube w/ media w/ certain viscosity, to create gradient of density, cells w/ diff shapes/properties tend to separate when centrifuged
- insert selectable markers --> good for research, but unsuitable for clinical app
94
What happens if use activin or Wnt to block ectoderm formation?
- Wnt early in dev, so get prod of mesoderm
| - activin exp later, so get prod of endoderm
95
As well as being a pluripotency factor, what is an important role of Sox2?
- drives endodermal differentiation
96
How were purified neural progenitor cells purified from ESCs by lineage selection?
- replaced allele of Sox2 gene w/ cassette inc LacZ reporter or neomycin resistance cassette
- G418 used as antibiotic
- enrichment of Sox2 +ve cells after G418 selection
- on differentiation: in selected culture relatively pure culture of neurons
- in this pop can look for activation of FGF2, and in these cells had prolif of Sox2 cells
97
How were ESCs used to cure a man on TID?
- transplant of pancreatic islets allowed patient to become insulin independent
98
How does dev of islet cells and pancreas occur in mice?
- diff markers exp at diff times, allows detection of diff stages
- Foxa2 and Sox17 exp initially in foregut patterning
- Pdx1 exp during pancreas specification
- Ptf1a exp during budding
- Ngn3 exp during branching
- Ins and Glc exp during beta and alpha cell dev
99
What protocol did D'Amour et al create to study GFs, what were the findings?
- exposed cell to diff series of GFs and see how gene exp changed
- concentrated on pancreatic endoderm precursor, as mature enough to be useful but not too mature
- released C-peptide in response to multiple secretory stimuli
- but only a small % of insulin exp cells obtained
- not glucose responsive and don’t process proinsulin well
- did not maintain exp of key beta cell markers
- more like fetal beta cells
- common problem w/ maturation in vitro, as hard to demonstrate whole process
100
How is insulin prod in the body?
- prod as proinsulin, then C-peptide released outside cell, can measure this to get idea of rest of insulin
101
How did Kroon et al improve the protocol of D'Amour et al?
- just because 2 markers exp at same time doesn't mean exp by same cell types
- wanted to check if cells worked in vivo
- morphology and marker profile of grafts
- cells working in vivo, as did co-exp markers
- then in disease model
- but >15% of grafts developed tumours, tumours more likely to be prod by contaminated cells
- but able to reduce to 0%
102
What were the relevance of the results of Kroon et al?
- obtaining the right molecular profile of differentiated cells is not enough
- evidence has to be provided that cells are functional (ideally in vitro and in vivo)
- data in vivo is more powerful if tested in a model of disease
103
How did Kelly et al study cell surface markers to isolate pancreatic cell types derived from hESCs?
- screening to identify markers
- focussed on CD142 in pancreatic endoderm and CD200 as endocrine marker
- then looked for other markers to indicate cell type
- in non sorted grafts around half prod teratomas
- in separated cells, when CD142 enriched transplanted 5/12 failed to engraft and/survive --> but of the successful graft non formed teratomas
104
What is ViaCyte researching?
- testing cell therapy in the clinic
105
What are organoides (ie. the future of 3D differentiation)
- conceptually diff from EBs
- targeting more complex, late differentiation into organs
- achieve good tissue architecture and good differentiation
106
What have organoides been used to study?
- studying Zika virus and how infection works
107
What are the diff fates of cells in Waddingtons epigenetic landscape model?
- normal dev: cell follows slope into valley, representing differentiated state
- pluripotent reprogramming: can move back up hill and move to diff differentiated state
- direct conversion: moves between diff valleys w/o going up hill
108
How was direct conversion of SCs found?
- from neoplastic lesions, go t/ transdifferentiation, to prod diff lineages
109
What were the 2 possibilities for what could happen to genes during differentiation (Gurdon 60/70s)?
- as gene exp changes and many downreg, is genetic material lost OR are genes repressed/silenced (therefore reversible)
110
What early experiments did Gurdon carry out in the 60/70s?
- cellular reprogramming initially explored by SCNT
- took intestinal cells of mature albino frog and transferred their nucleus to enucleated egg of same frog type and induced blastocyst formation
- then implanted into adult green frog (diff strain), that frog had progeny that was all mature albino
111
What did early experiments by Gurdon in the 60/70s show?
- that genetic material present in mature nucleus was intact and can be reset
112
How was the 1st cloning of a mammal achieved?
- Wilmut et al 1997
- used breast of sheep to take nuclei from, implanted into enucleated egg and into black faced sheep (showed coming from original line)
- prod Dolly, had a reasonably healthy life of approx 7 years, had her own offspring
- but issues at end of life poss related to epigenetics
113
How can patient specific SC therapy be carried out?
- take cells from patient (biopsy) and perform SCNT
- develop to blastocyst stage
- used in vitro to dev SCs which can differentiate into the desired tissue and be transplanted back into patient
114
How could reproductive cloning be carried out in humans (in theory)?
- take cells from patient (biopsy), SCNT, develop to blastocyst stage
- blastocyst implanted into uterus of surrogate mother
115
Has SCNT been carried out in humans?
- 2 papers by Hwang claimed to (2004/5)
| - but both retracted, as fabrication of data and ethical issues
116
What did Tachibana et al (2013) study?
- hESCs derived by SCNT
- in vitro
- but this technology was overtaken by other advances in the field (ie. Takahashi & Yamanaka)
117
What did Takahashi & Yamanaka (2006) aim to do, and why was this idea rejected?
- understand factors which needed to be present in cells to confer pluripotency
- but too many factors so rejected
118
How did Takahashi & Yamanaka (2006) start develop an assay to read pluripotency (ie. what gene was used)?
- knew Fbx15 exp in ESCs and early embryos, but not req to maintain pluripotency and mouse dev (can knock down and not affect pluripotency)
- created knock in assay of Fbx15
- ESCs homozygous for knock in v resistant to high doses of G418 (antibiotic)
- but somatic cells derived from this are very sensitive to normal levels of G418
- assumed any activation of Fbx15P cells will lead to resistance to G418
119
How many candidate genes did Takahashi & Yamanaka (2006) select as having a pivotal role in maintaining ESC identity and how was this narrowed down?
- 24
- introd each candidate separately into engineered Fbx15βgeo/βgeo MEFs --> no survival in G418
- when introduced all 24 together, gen 22 G418 resistant clones
- 12 clones selected, 5 looked v similar to ESCs
- RT-PCR to show clones and looked for exp of ESC markers
- withdrew individual factors to determine which are necessary to form G418 resistant colonies
120
Which of the candidate genes were found to be req for induction of pluripotent SCs (Takahashi & Yamanaka, 2006)
- Oct3/4, Sox-2, c-Myc and Klf4 req to induce pluripotent SCs from MEFs or adult fibroblasts but only at low freq
- Nanog was dispensable
121
What did Takahashi & Yamanaka (2006) do after identifying OSKM factors?
- microarray analysis of ESCs, iPSCs and Fbx15βgeo/βgeo MEFs --> identified genes commonly upreg in ESCs and iPSC, but there were diffs
* * iPSCs were not identical to ESCs
- showed cells w/ OSKM could form teratomas (derivatives from all 3 germ layers)
122
What did Takahashi & Yamanaka (2006) find loss of Sox2 resulted in?
- only undifferentiated cells (which could not differentiate)
123
How did Takahashi & Yamanaka (2006) repeat their experiment for further validation?
- repeated w/ adult fibroblasts from tail tips (TTFs) --> repeated characterisation, also injected clones into blastocyst and found cells contributed to all 3 germ layers
124
How have others built on Takahashi & Yamanaka's (2006) experiment?
- slightly alt protocol
- obtained germline transmission
- and also derived human iPSCs
125
How do the Yamanaka and Thomson factors compare?
- Yamanaka: OSKM
| - Thomson: Oct4, Sox2, Nanog, Lin28
126
What was a problem people had w/ Yamanaka's factors?
- c-Myc is an oncogene
127
What diff way of reprogramming a cell are there?
- SCNT: rapid (<5 hrs), but now mostly replaced by other methods
- cell fusion w/ pluripotent SC: rapid (<48 hrs), only useful in specific research situations
- TF expression: eg. OSKM, slow (> 10 days)
- small mol exposure: slow (> 10 days)
128
What happens to cells when OSKM factors are introduced?
- stochastic phase, some become senescent, some acquire diff fates, some die off, but a few become primed to enter intermediate stage and set up intrinsic factors of pluripotent cells (this is the rate limiting step)
- then enter deterministic phase where exp pluripotent factors, shows only need transient OSKM exp, don’t need them exogenously exp once entered this stage
129
What current methods are there to reprogramme factor deliver to make iPSCs?
- integrating virus
- non-integrating vector
- excisable vector
- protein
- small mol replacements
130
Which of these methods are best for making iPSCs?
- non integrating or protein
131
Why is sickle cell anaemia such a prevalent disease?
- heterozygotes have greater malaria resistance
132
How did Hanna et al (2007) use iPSCs to treat a sickle cell anaemia mouse model?
- harvested tail tip fibroblasts
- infect w/ OSKM
- homozygous mutated mice derived iPS clones
- corrected β sickle mutation in iPSCs by specific gene targeting
- fifferentiate into EBs
- transplant corrected hematopoietic progenitors and put back into irradiated mouse to correct phenotype
133
What did Hanna et al's (2007) experiment show?
- showed could take iPSCs from organism w/ genetic disease, correct them and transplant them back in to correct phenotype
134
What is an app of producing human iPSCs to study human disease?
- to prod cells w/ disease to use for testing drugs
135
What are some of the issues w/ hESCs?
- genomic instability --> more prone to mutations
- need continual supply of high quality embryos to set up line
- potential for tumour formation (eg. teratomas)
- questions regarding functional differentiation
- problem of immune rejection
- ethical issues
136
Are ESCs and iPSCs completely equivalent?
* DIAG*
- big overlap but also diffs
- can have bad quality pluripotent SCs
137
What are the advantages of human iPSCs?
- no req for administration of immunosuppressive drugs
- opportunity to repair defect by HR
- opportunity to repeatedly differentiate into desired cell type for continued therapy
- less ethical issues
138
What is the main issue w/ using human iPSCs for patient specific therapy?
- reprogramming event may trigger unwanted mutations which are difficult to assess, so for many clinical apps research begins w/ ESCs
- also would be v expensive --> in region of $3 mil per patient
139
How could the process of reprogramming trigger changes in the antigen profile of cells?
- shown that if take ESCs from same or diff strain and put back into mouse then not attacked by IS, as recognised
- but iPSCs can be immunogenic, attacked even in same strain of animal, producing immune response
- abnormal overexp of some prots contributed directly to immunogenicity of cells
- but much more research done in this field --> shown to be true in certain cases
* may not be such a big issue after all
140
How direct differentiation be obtained from 1 phenotype to another?
- can directly convert fibroblasts to functional neurons by application of defined factors
- don’t need to go back to pluripotent SC 1st
- mature neurons, which can fire AP
- pool of 5 factors needed
141
What needs to be studied before human iPSCs can be used therapeutically?
- are all iPSCs the same?
- need to dev robust and reliable differentiation protocols for human iPSCs
- what is the relative efficiency of the diff differentiation methods
- how will future iPSCs be screened for quality?
142
What are the advantages of human iPSCs?
- no req for administration of immunosuppressive drugs
- opportunity to repair defect by HR
- opportunity to repeatedly differentiate into desired cell type for continued therapy
- less ethical issues
143
What is the main issue w/ using human iPSCs for patient specific therapy?
- reprogramming event may trigger unwanted mutations which are difficult to assess, so for many clinical apps research begins w/ ESCs
- also would be v expensive --> in region of $3 mil per patient
144
How could the process of reprogramming trigger changes in the antigen profile of cells?
- shown that if take ESCs from same or diff strain and put back into mouse then not attacked by IS, as recognised
- but iPSCs can be immunogenic, attacked even in same strain of animal, producing immune response
- abnormal overexp of some prots contributed directly to immunogenicity of cells
- but much more research done in this field --> shown to be true in certain cases
* may not be such a big issue after all
145
How direct differentiation be obtained from 1 phenotype to another?
- can directly convert fibroblasts to functional neurons by application of defined factors
- don’t need to go back to pluripotent SC 1st
- mature neurons, which can fire AP
- pool of 5 factors needed
146
What needs to be studied before human iPSCs can be used therapeutically?
- are all iPSCs the same?
- need to dev robust and reliable differentiation protocols for human iPSCs
- what is the relative efficiency of the diff differentiation methods
- how will future iPSCs be screened for quality?
147
What is the history of mesenchymal SCs?
- originally hypothesised that cells derived from the bone marrow were involved in injury repair
- in 60/70s described as adherent, fibroblastic-like colonies from monolayer cultures of bone marrow, thymus and spleen
- these fibroblastic-like colonies were named CFU-F (colony forming units-fibroblast)
- showed to be able to differentiate into bone, cartilage and adipose tissue
148
How was the true identity of mesenchymal SCs demonstrated?
- when transplants of clonal bone marrow MSCs prod bone in vivo
149
Are all MSCs SCs, why?
- mounting evidence indicates that the adherent cell pop iso from bone marrow (and other tissues) are highly heterogeneous and may consist of several subpops
- so not all these cells fulfill SC criteria
150
What is the criteria a cell must fulfil to be considered a SC?
- unlimited proliferative capacity and ability to prod multiple lineages
151
What does MSC stand for?
- originally mesenchymal SCs, but found not all were SCs
- so proposed they should be called mesenchymal stromal cells, and mesenchymal SCs only used for those which meet the criteria
- but MSC applies to both, so used interchangeably
152
What are the minimum criteria to define MSCs?
- remain plastic adherent under standard culture conditions
- exp CD105, CD73 and CD90
Lack exp of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR --> CD45 and CD34 most important, as haematopoietic cells tend to be +ve
- differentiate into osteoblasts, adipocytes and chondrocytes in vitro
153
What is the lineage potential of MSCs?
- can prod osteocytes, adipocytes, chondrocytes and myocytes
154
Do the origins of MSCs vary?
- yes
| - tissues in head/neck come from neural crest --> migrates out and forms these tissues
155
Where can MSCs be iso from
- initially iso from bone marrow
- but now also purified from multiple tissues, inc adipose tissue, placenta, dental pulp, synovial mem, peripheral blood, periodontal ligament, endometrium, umbilical cord (UC) and umbilical cord blood (UCB)
156
In what tissues are MSCs present, and what does this suggest?
- evidence suggested MSCs may be present in virtually any vascularised tissues t/o whole body
- their presence may be related to cells in periphery of blood vessels
- suggests maybe we should think about cells as the properties assoc w/ tissues, rather than as a whole?
157
How was a perivascular origin for MSCs in multiple human organs uncovered?
- screened multiple adult and fetal tissues by immunofluorescence (in muscle, pancreas, placenta, lungs, skin etc.)
- in all tissues detected NG2+ and CD146+ cells surrounding small blood vessels
- iso cells from blood vessel walls using FACS
- selected for CD146+ and selected out CD56 (myogenic cells), CD45 (HSCs), CD34 (endothelial and HSCs)
- good proliferate capacity, not immortal but could expand for 3-40 cultures and maintain exp of markers t/ passaging
- transplanted into SCID mice, cells differentiated into muscle and bone
- in vitro prod cartilage, adipose tissue and bone --> not pluripotent, as don't get all lineages (would prod teratomas if truly pluripotent)
- iso cells +ve for CD73
- MSCs CD markers are exp by cells in vivo
158
What are SCID mice?
- immunosuppressed, ie. don't recognise transplants as foreign
159
What diff clinical uses for MSCs are there?
- for cell replacement
- trophic, paracrine effect
- immunomodulation
- anti-cancer tools
160
How are MSCs being used for cell replacement?
- attractive as potential for autologous transplants
- several clinical trials underway
- mainly in orthopaedics to replace bone and cartilage --> inducing into other lineages is more difficult
- also used to replace muscle in cardiac infarction
161
Where are MSCs typically derived from for clinical use?
- traditionally obtained from bone marrow, but adipose tissue increasingly becoming source material
162
How can MSCs be utilised clinically for their trophic, paracrine effect?
- MSCs secrete factors that help healing and repair
| - when culture MSC will prod mols such as IL-2 and TGFβ/PGE-2
163
How can MSCs be used for immunomodulation?
- can affect IS t/ secreted factors and contact mediated interaction
- role in chronic inflammation, so their app can cause problems
164
Why do MSCs have potential as anti-cancer tools?
- have inherent tropism to migrate to tumours
- nature of interaction not fully understood, but related to PAR-1 receptor
- tumour creates microenv, prod proteinases, eg. MMP-1, starts digesting ec matrix as tumour grows
- MMP-1 could mediate MSC migration t/ activation of PAR-1 receptor
165
What are gliomas?
- highly aggressive tumours, prod by glia cells, particularly in brain
166
How were human bone marrow derived MSCs used for the treatment of gliomas?
- gliomas induced in mice by U87 xenograft in right brain frontal lobe
- hMSCs labelled and injected into carotid artery
- w/in 7 days cells located in tumour
- control showed wasn’t just mechanical trapping, ie. injected on LHS and found still localised to tumour on RHS
- gliomas induced by other lines (to show not a property of cell line used) and shown to be targeted by hMSCs
167
Was the tropism to gliomas unique to hMSCs?
- yes, when other cells transplanted, eg. fibroblasts, U87 inducing tumour, then did not locate w/ the original tumour
168
In the glioma experiment, how were the tumour cells attracting the hMSCs?
- explored in vitro using transwell culture dishes
* DIAG*
- measure migration of cells which move down t/ pores
- saw diff mols, eg. PDGF, were good at assoc, so could be potential factor involved
169
What are transwell cultures used to investigate?
- to see if soluble factor or direct interaction
170
How was it tested if hMSCs could be used to deliver an anti tumour agent?
- MSCs genetically mod w/ adenovirus to secrete IFN-β
- only IFN-β secreted by MSC delivered intracranially increased the survival of the animals = proof of concept that can use MSCs to deliver anti-cancer agents to tumour
- applied for other tumours
171
What is TRAIL, and what is its role?
- tumour necrosis factor-related apoptosis-inducing ligand
- type 2 tm death ligand that causes apoptosis of target cells t/ the extrinsic apoptosis pathway
- member of the tumour necrosis factor superfam, which inc tumour necrosis factor and Fas ligand
172
How was it shown that MSC delivery of TRAIL can eliminate metastatic cancer (esp lung)?
- hMSCs transduced w/ a TRAIL-GFP lentivirus that can be induced w/ doxycycline
- if give doxycycline too late then has little effect, but if give early on then vol v small and reduced tumour formation
- able to selectively induce apoptosis in transformed cells but not in most normal cells, making it a promising candidate for tumour therapy
- now in clinical trials
BMS6056 - Stem Cell Biology
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