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BMS6056 - Stem Cell Biology > Rivolta > Flashcards

Rivolta Flashcards

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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
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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
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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*
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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
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5
Q

How is age of development used to classify SCs?

A
  • embryonic or adult
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6
Q

How is tissue of origin used to classify SCs?

A
  • neural SCs, hematopoietic (blood), umbilical cord etc.
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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)
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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
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9
Q

What diff strategies are there for use of SCs as therapies?

A
  • allogenic
  • autologous
  • recruitment of endogenous SCs from the same tissue
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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
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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
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12
Q

What factors must be induced to gen iPS cells?

A
  • SOX2, OCT4, MYC, KLF4
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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

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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
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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
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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
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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*
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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
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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
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20
Q

When do EC cells grow better?

A
  • if have a layer of feeder cells
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21
Q

What tissues in the body can ESCs form?

A
  • all the tissues in the body

- ie. ectoderm, mesoderm, endoderm and germ cells

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22
Q

From where are ESCs derived?

A
  • ICM of blastocysts
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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
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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
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25
Q

How were transgenic animals created?

A
  • 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
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26
Q

What happens when ESCs are injected into a competent adult isogenic host?

A
  • form teratomas

- diff structures inc gut-like, neural epithelium, bone, cartilage, striated muscle and glomeruli like

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27
Q

What extrinsic factors are there for self renewal?

A
  • LIF (leukaemia inhibitory factor) or feeder layers
  • once withdrawn prolif continues but differentiation induced
  • initially LIF comes from feeder layers
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28
Q

What is the LIF pathway?

A
  • 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
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29
Q

What is the consequence on cell fate due to LIF also acting on SHP-2?

A
  • 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
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30
Q

What is the result when LIF is exp in serum free conditions in mESCs?

A
  • LIF alone is insufficient to maintain pluripotency and block neural differentiation
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31
Q

What did Ying et al (2003) show?

A
  • 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
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32
Q

How do mESC and hESC colonies differ?

A
  • mESCs grew forming small colonies that tend to project out

- hESCs usually flat w/ well defined edges

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33
Q

How do markers exp differ between mESCs and hESCs?

A
  • SSEA-1 marker only in mESCs

→ SSEA-4 only in hESCs

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34
Q

What pathways for self renewal are conserved between mESCs and hESCs?

A
  • Stat3 signalling
  • Nanog
  • Oct-Sox
  • FGF signalling
  • TGFβ signalling
  • BMPR1α
  • microRNAs
  • methylation, eg. X-inactivation
  • cell cycle (eg. Rb)
  • Igf2-H19
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35
Q

What are some known diffs between self renewal in mice and humans?

A
  • 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)
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36
Q

How do ES cell phenotypes differ between humans and mice?

A
  • hESCs / mESCs
  • SSEA1- / SSEA1+
  • SSEA3+ / SSEA3-
  • SSEA4+ / SSEA4-
  • TRA-1-60+ / TRA-1-60-
  • GCTM2+ / GCTM2-
  • Thy1+ / Thy1-
  • MHC+ / MHC-
  • ALP+ / ALP+
  • Nanog+ / Nanog-
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37
Q

What are human ESCs more equivalent to in mice, how was this found?

A
  • 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
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38
Q

Are mESCs and hESCs primed or naive?

A
  • hESCs primed
  • mESCs naive as less dev
  • EpiSCs are primed
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39
Q

How does the differentiative potency change t/ dev?

A

DIAG

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40
Q

How was the ground state of human naive pluripotency captured?

A
  • 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
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41
Q

Why was it challenging to capture the ground state of human naive pluripotency?

A
  • as dev into primed state so quickly
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42
Q

How was the role of enhancers shown to be important in hESCs?

A
  • 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
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43
Q

How was robust gen of cross species chimeric humanised mice achieved?

A
  • microinjection of naive human iPSCs into mouse morulas
  • prod chimeras
  • were incorp into mature tissues
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44
Q

What are the 2 stages of pluripotency?

A

1) A naive or ground state (ICM like)

2) A primed state (epiblastic like)

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45
Q

How does doubling time differ between naive and primed cells?

A
  • reduced in naive

- increased in primed

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46
Q

How do X chroms differ between naive and primed cells?

A
  • active in naive

- inactive in primed

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47
Q

How does ability for single cell cloning differ between naive and primed cells?

A
  • poss in naive

- poor in primed

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48
Q

How does enhancer use differ between naive and primed cells?

A
  • naive use Oct4 distal enhancer

- primed use Oct4 proximal enhancer

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49
Q

How does ICM integration differ between naive and primed cells?

A
  • poss in naive

- low in primed

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50
Q

How does factors dependent on differ between naive and primed cells?

A
  • naive are LIF dep

- primed are activin/FGF dep

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51
Q

How does interaction between naive and primed cells occur in mice?

A

DIAG

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52
Q

How does interaction between naive and primed cells occur in humans?

A

DIAG

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53
Q

Why are naive human pluripotent ESCs important?

A
  • 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?)
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54
Q

How was comparability of the many hESC lines investigated?

A
  • 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
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55
Q

Why do mESC lines tend to be reasonably similar?

A
  • inbred strain, so little variability
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56
Q

How does capacity to differentiate differ between hESC lines?

A
  • differing efficiencies and efficacy
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57
Q

What does the stage of pluripotency imply?

A
  • the ability of a cell to self renew and gen lineages from the 3 germ layers
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58
Q

What confers the ability of a cell to self renew?

A
  • by a set of TFs, whose exp is carefully balanced to achieve the right balance
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59
Q

How does the mouse epiblast dev?

A
  • 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)
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60
Q

What is the battlefield model of pluripotency?

A
  • group of factors conflicting between pluripotency TFs that seek to to direct ESC differentiation to opposing lineages
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61
Q

What do Nanog, Oct4 and Sox2 define?

A
  • Nanog important for endoderm
  • Oct4 for mesoderm
  • Sox2 for ectoderm
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62
Q

What prot doms are in Nanog, Oct4 and Sox2?

A
  • DIAG*
  • green are DNA BDs
  • Nanog is typical homeodomain TF
  • Oct4 has POU dom
  • Sox2 can interact w/ Oct4 via TAD dom
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63
Q

How do relative levels of Oct4 influence ESC fate?

A
  • 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*
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64
Q

What is Oct3/4 and how is exp critical?

A
  • 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
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65
Q

Why is continuous Oct4 function necessary to maintain pluripotency in ESCs?

A
  • otherwise results in trophoblast
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66
Q

How do Oct3/4 and Cdx2 interact, and what is the importance of this interaction?

A
  • 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
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67
Q

What is the importance of Sox2, and how was this shown?

A
  • 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
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68
Q

How was Nanog identified as a pluripotency factor by an in silico screen?

A
  • 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
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69
Q

How was Nanog identified as a pluripotency factor by a functional screen?

A
  • 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
Q

How was it shown that Nanog is essential for self renewal?

A
  • 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
Q

How do diff pluripotency factors endow ESCs w/ multilineage potential?

A
  • DIAG*
  • shows primary roles of each pluripotency factors
  • interaction between 4 keeps cell pluripotent
72
Q

How was transcription levels of Oct4 and Nanog monitored, and what were the findings?

A
  • 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
Q

How was the functional implication of Nanog levels differing in a pop studied?

A
  • 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
Q

How does FACS work?

A
  • 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
Q

What does the model that Nanog may act as a rheostat providing variable resistance to differentiation mean?

A
  • initially can switch between +ve and -ve (ie. transient)

- when get to point of commitment, -ve cells become very -ve and differentiate

76
Q

What is the Waddington landscape?

A
  • 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
Q

How is heterogeneity useful in a Waddington landscape?

A
  • 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
Q

How can cell surface markers be used to look at Waddington landscapes?

A
  • 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
Q

Why is heterogeneity important?

A
  • cells from diff states may preferentially dev into particular lineages
80
Q

What options do cells have in terms of their fate?

A
  • can either self renew, differentiate or die
81
Q

What can factors preventing differentiation or death impact on?

A
  • could have direct effect on self renewal
82
Q

What is the consequence of impacting on self renewal (genetic stability)?

A
  • 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
Q

How can differentiation of ESCs be triggered, and how can this happen?

A
  • 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
Q

What are the properties of EBs?

A
  • highly heterogeneous

- initially obtained from embryonic carcinoma cells

85
Q

How does organised gastrulation in EBs occur?

A
  • 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
Q

How does presence of Wnt or DKK1 in media influence EB dev?

A
  • if Wnt in media then early dev of EBs

- if DKK1 in media then late dev in Wnt responsive areas

87
Q

What are the advs of EBs?

A
  • cheap to prod

- gen 3 germ layers

88
Q

What are the disadvs of EBs?

A
  • difficult to control aggregation in a reproducible way (shape, size)
  • no. of days before they are collected
89
Q

What diff EBs prod what parts in hESCs?

A
  • 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
Q

How can EBs be prod in a more controlled manner?

A

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
Q

How can directed differentiation be induced?

A
  • 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
Q

Why is it necessary to isolate the desired cell type from a culture?

A
  • 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
Q

How can the desired cell type be isolated from a culture?

A
  • 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
Q

What happens if use activin or Wnt to block ectoderm formation?

A
  • Wnt early in dev, so get prod of mesoderm

- activin exp later, so get prod of endoderm

95
Q

As well as being a pluripotency factor, what is an important role of Sox2?

A
  • drives endodermal differentiation
96
Q

How were purified neural progenitor cells purified from ESCs by lineage selection?

A
  • 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
Q

How were ESCs used to cure a man on TID?

A
  • transplant of pancreatic islets allowed patient to become insulin independent
98
Q

How does dev of islet cells and pancreas occur in mice?

A
  • 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
Q

What protocol did D’Amour et al create to study GFs, what were the findings?

A
  • 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
Q

How is insulin prod in the body?

A
  • prod as proinsulin, then C-peptide released outside cell, can measure this to get idea of rest of insulin
101
Q

How did Kroon et al improve the protocol of D’Amour et al?

A
  • 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
Q

What were the relevance of the results of Kroon et al?

A
  • 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
Q

How did Kelly et al study cell surface markers to isolate pancreatic cell types derived from hESCs?

A
  • 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
Q

What is ViaCyte researching?

A
  • testing cell therapy in the clinic
105
Q

What are organoides (ie. the future of 3D differentiation)

A
  • conceptually diff from EBs
  • targeting more complex, late differentiation into organs
  • achieve good tissue architecture and good differentiation
106
Q

What have organoides been used to study?

A
  • studying Zika virus and how infection works
107
Q

What are the diff fates of cells in Waddingtons epigenetic landscape model?

A
  • 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
Q

How was direct conversion of SCs found?

A
  • from neoplastic lesions, go t/ transdifferentiation, to prod diff lineages
109
Q

What were the 2 possibilities for what could happen to genes during differentiation (Gurdon 60/70s)?

A
  • as gene exp changes and many downreg, is genetic material lost OR are genes repressed/silenced (therefore reversible)
110
Q

What early experiments did Gurdon carry out in the 60/70s?

A
  • 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
Q

What did early experiments by Gurdon in the 60/70s show?

A
  • that genetic material present in mature nucleus was intact and can be reset
112
Q

How was the 1st cloning of a mammal achieved?

A
  • 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
Q

How can patient specific SC therapy be carried out?

A
  • 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
Q

How could reproductive cloning be carried out in humans (in theory)?

A
  • take cells from patient (biopsy), SCNT, develop to blastocyst stage
  • blastocyst implanted into uterus of surrogate mother
115
Q

Has SCNT been carried out in humans?

A
  • 2 papers by Hwang claimed to (2004/5)

- but both retracted, as fabrication of data and ethical issues

116
Q

What did Tachibana et al (2013) study?

A
  • hESCs derived by SCNT
  • in vitro
  • but this technology was overtaken by other advances in the field (ie. Takahashi & Yamanaka)
117
Q

What did Takahashi & Yamanaka (2006) aim to do, and why was this idea rejected?

A
  • understand factors which needed to be present in cells to confer pluripotency
  • but too many factors so rejected
118
Q

How did Takahashi & Yamanaka (2006) start develop an assay to read pluripotency (ie. what gene was used)?

A
  • 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
Q

How many candidate genes did Takahashi & Yamanaka (2006) select as having a pivotal role in maintaining ESC identity and how was this narrowed down?

A
  • 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
Q

Which of the candidate genes were found to be req for induction of pluripotent SCs (Takahashi & Yamanaka, 2006)

A
  • 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
Q

What did Takahashi & Yamanaka (2006) do after identifying OSKM factors?

A
  • 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
Q

What did Takahashi & Yamanaka (2006) find loss of Sox2 resulted in?

A
  • only undifferentiated cells (which could not differentiate)
123
Q

How did Takahashi & Yamanaka (2006) repeat their experiment for further validation?

A
  • 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
Q

How have others built on Takahashi & Yamanaka’s (2006) experiment?

A
  • slightly alt protocol
  • obtained germline transmission
  • and also derived human iPSCs
125
Q

How do the Yamanaka and Thomson factors compare?

A
  • Yamanaka: OSKM

- Thomson: Oct4, Sox2, Nanog, Lin28

126
Q

What was a problem people had w/ Yamanaka’s factors?

A
  • c-Myc is an oncogene
127
Q

What diff way of reprogramming a cell are there?

A
  • 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
Q

What happens to cells when OSKM factors are introduced?

A
  • 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
Q

What current methods are there to reprogramme factor deliver to make iPSCs?

A
  • integrating virus
  • non-integrating vector
  • excisable vector
  • protein
  • small mol replacements
130
Q

Which of these methods are best for making iPSCs?

A
  • non integrating or protein
131
Q

Why is sickle cell anaemia such a prevalent disease?

A
  • heterozygotes have greater malaria resistance
132
Q

How did Hanna et al (2007) use iPSCs to treat a sickle cell anaemia mouse model?

A
  • 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
Q

What did Hanna et al’s (2007) experiment show?

A
  • showed could take iPSCs from organism w/ genetic disease, correct them and transplant them back in to correct phenotype
134
Q

What is an app of producing human iPSCs to study human disease?

A
  • to prod cells w/ disease to use for testing drugs
135
Q

What are some of the issues w/ hESCs?

A
  • 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
Q

Are ESCs and iPSCs completely equivalent?

A
  • DIAG*
  • big overlap but also diffs
  • can have bad quality pluripotent SCs
137
Q

What are the advantages of human iPSCs?

A
  • 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
Q

What is the main issue w/ using human iPSCs for patient specific therapy?

A
  • 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
Q

How could the process of reprogramming trigger changes in the antigen profile of cells?

A
  • 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
Q

How direct differentiation be obtained from 1 phenotype to another?

A
  • 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
Q

What needs to be studied before human iPSCs can be used therapeutically?

A
  • 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
Q

What are the advantages of human iPSCs?

A
  • 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
Q

What is the main issue w/ using human iPSCs for patient specific therapy?

A
  • 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
Q

How could the process of reprogramming trigger changes in the antigen profile of cells?

A
  • 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
Q

How direct differentiation be obtained from 1 phenotype to another?

A
  • 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
Q

What needs to be studied before human iPSCs can be used therapeutically?

A
  • 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
Q

What is the history of mesenchymal SCs?

A
  • 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
Q

How was the true identity of mesenchymal SCs demonstrated?

A
  • when transplants of clonal bone marrow MSCs prod bone in vivo
149
Q

Are all MSCs SCs, why?

A
  • 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
Q

What is the criteria a cell must fulfil to be considered a SC?

A
  • unlimited proliferative capacity and ability to prod multiple lineages
151
Q

What does MSC stand for?

A
  • 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
Q

What are the minimum criteria to define MSCs?

A
  • 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
Q

What is the lineage potential of MSCs?

A
  • can prod osteocytes, adipocytes, chondrocytes and myocytes
154
Q

Do the origins of MSCs vary?

A
  • yes

- tissues in head/neck come from neural crest –> migrates out and forms these tissues

155
Q

Where can MSCs be iso from

A
  • 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
Q

In what tissues are MSCs present, and what does this suggest?

A
  • 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
Q

How was a perivascular origin for MSCs in multiple human organs uncovered?

A
  • 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
Q

What are SCID mice?

A
  • immunosuppressed, ie. don’t recognise transplants as foreign
159
Q

What diff clinical uses for MSCs are there?

A
  • for cell replacement
  • trophic, paracrine effect
  • immunomodulation
  • anti-cancer tools
160
Q

How are MSCs being used for cell replacement?

A
  • 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
Q

Where are MSCs typically derived from for clinical use?

A
  • traditionally obtained from bone marrow, but adipose tissue increasingly becoming source material
162
Q

How can MSCs be utilised clinically for their trophic, paracrine effect?

A
  • MSCs secrete factors that help healing and repair

- when culture MSC will prod mols such as IL-2 and TGFβ/PGE-2

163
Q

How can MSCs be used for immunomodulation?

A
  • can affect IS t/ secreted factors and contact mediated interaction
  • role in chronic inflammation, so their app can cause problems
164
Q

Why do MSCs have potential as anti-cancer tools?

A
  • 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
Q

What are gliomas?

A
  • highly aggressive tumours, prod by glia cells, particularly in brain
166
Q

How were human bone marrow derived MSCs used for the treatment of gliomas?

A
  • 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
Q

Was the tropism to gliomas unique to hMSCs?

A
  • yes, when other cells transplanted, eg. fibroblasts, U87 inducing tumour, then did not locate w/ the original tumour
168
Q

In the glioma experiment, how were the tumour cells attracting the hMSCs?

A
  • 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
Q

What are transwell cultures used to investigate?

A
  • to see if soluble factor or direct interaction
170
Q

How was it tested if hMSCs could be used to deliver an anti tumour agent?

A
  • 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
Q

What is TRAIL, and what is its role?

A
  • 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
Q

How was it shown that MSC delivery of TRAIL can eliminate metastatic cancer (esp lung)?

A
  • 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 (3 decks)

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