1 Flashcards
1
Q
what is a stem cell
A
cells that have the potential to generate specialised tissues (differentiate) as well as copies of themselves (replication)
2
Q
what can totipotent stem cells differentiate into
A
all cell types of the body
3
Q
what can pluripotent stem cells differentiate into
A
derivatives of the 3 germ layers (ectoderm, mesoderm and endoderm)
4
Q
what can multipotent stem cells differentiate into
A
different cell types from a tissue or organ (tissue specific)
5
Q
what can unipotent stem cells differentiate into
A
only a single cell type
6
Q
what is an allogenic transplant
A
stem cells are derived from a different donor and are expanded in the lab – eg embryonic stem cells, cord blood cells
7
Q
what is an autologous transplant
A
stem cells transplanted are derived from same patient – expanded and treated outside the body and re-implanted into same patient eg auto transplant of bone barrow cells or producing iPSCs
8
Q
what are the pluripotency factors
A
SOX2, OCT4, MYC and KLF4
9
Q
what can stem cells provide models for?
A
- models to screen new drugs
- models to study genetic conditions
- models for oth
10
Q
what are embryonic carcinoma cells
A
Embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and the malignant counterparts of embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos
11
Q
EC and ESCs grow better on a feeder layer T/F
A
T
12
Q
list properties of ES cells
A
- derived from ICM of blastocysts
- indefinite proliferative potential
- clonogenic
- pluripotent
- germline transmission to chimeras
- permissive to genetic manipulation
13
Q
what is LIF (leukaemic inhibitory factor) important for?
A
self renewal state
14
Q
how does LIF work
A
interacts with LIF receptor which combines with gp130. Activates JAK pathway which upregulates STAT3 which is critical to maintain proliferation
also activates ERK1/2 kinase by binding SH2 which has an inhibitory effect on STAT3 and induces differentiation
15
Q
what determines the balance of self renewal and differentiation
A
balance of STAT3 and ERK1/2
16
Q
if concentration of LIF is low what happens to cells
A
differentiate
17
Q
if conc of LIF is high what happens to cells
A
self renew
18
Q
LIF alone is not sufficient to do what
A
maintain pluripotency
block neural differentiation
19
Q
what did ying 2003 show that you need BMP4 and LIF for
A
LIF blocks differentiation into mesoderm and endoderm
BMPs induce genes that block differentiation into neurons
overall lead to blocking of differentiation and maintenance of pluripotency
20
Q
what surface markers do both hES and mES show
A
ALP+, OCT4+ and Nanog +
21
Q
what surface markers do hES show not including ALP+, OCT4+ and Nanog +
A
SSEA3, SSEA4, TRA-1-60, GCTM2, Thy1, MHC
22
Q
what surface markers do hES show not including ALP+, OCT4+ and Nanog +
A
SSEA1
23
Q
what is mouse hES pluripotency maintained by
A
presence of LIF and inhibitors of ERK1/2 and GSK3b
24
Q
what is human hES pluripotency maintained by
A
FGF2 and activin
25
what do the mouse and human epiblasts differ in
the timing of their formation
26
what are human escs the equivalent to in mouse
mouse epiblast cells
27
what are the two stages of pluripotency and what cells represent them
naive - mES
| primed - mEpiESC or hES
28
what did gaffini do to try and capture a true naive hESC
exposed cells to different combinations of factors. Used Oct4-GFP to visualise pluripotency. Oct4 is controlled by different enhancers in naive and primed so can tell what state the cell is by deleting either of the enhancers. Found 8 factors (NHSM) which changed a primed cell into a more naive cell
29
why are naive HESC important
- give fundamental understanding of pluripotency
- easier to modify genetically
- potential application for humanised organs as evidenced by human-mouse chimerism (can't be done with primed)
30
what are the intrinsic factors that control pluripotency
SOX4
OCT4
NANOG
31
The Niwa 2000 paper found that relative levels of Oct3/4 influence ES cell fate. T/F
T
32
what does upregulation of Oct4 lead to
extraembryonic endoderm and mesoderm
33
what does downregulation of Oct4 give
trophoectoderm
34
without oct4 was does the embryo fail to acquire
the potential for the production of different lineages
35
the interaction between oct4 and what determines trophoectoderm differentiation (Niwa 200)
Cdx2
36
OCt4 and Cdx2 bind in a complex that inhibits their individual transcriptional activity. When theres low expression of Oct4 there is high expression of Cdx2. What has forced overexpression of cdx2 been found to lead to
trophoblast differentiation
37
embryos where Sox2 is deleted fail to generate what?
epiblast
38
nanog was identified in 2 independent screens by who
Mitsui (2003) and chambers (2003)
39
how did the mitsui in silico screen identify nanog
look for genes highly expressed in ES cell population
| found that nanog rescued LIF deficiency
40
how did the chambers functional screen identify nanog
transfected cells with ES cell genes
looked for cell displaying more pluripotent properties. Put nanog in cells that were deficient in LIF receptor and showed it could maintain pluripotency
41
what happens to nanog deficient cells
lose pluripotency and differentiate to form the extraembryonic endoderm
42
using FACs for Nanog GFP+ found what
nanog expression is heterogenous in the cell population. it may act as a rheostat to provide variable resistance to differentiation
43
what are embryoid bodies
heterogenous aggregates that resemble an organised embryo
44
what signalling mediates self organisation and axis formation in embryoid bodies
Wnt signalling
45
what are the pros of using embryoid bodies to produce differentiated cells
cheap to produce
| generate 3 germ layers
46
what are the cons of using embryoid bodies to produce differentiated cells
difficult to control aggregation in a reproducible way
number of days before differentiated cells can be collected
dont get enrichment of particular lineage
47
how can you isolate desired cell types
- FACs
- using density gradients
- inserting selectable markers (good for research)
48
summarise the generation of purified neural precursors from ES cell paper Li (1998)
used Sox2 to drive ectodermal differentiation
replaced one allele with casette that had LacZ reporter and neomycin resistance
enriched for cells expressing Sox2 (survive on neomycin)
obtained relatively pure population of neurons
49
summarise the D’Amour et al(2006) Productin of Pancreatic hormone-expressing
endocrine cells from human embryonic stem cells study
developed a differentiation protocol that mimicked in vivo formation of pancreatic cells
measured C peptide to measure levels of processing insulin
first derived fetal cells
50
how did Kroon 2008 follow D'amour 2006
adapted the protocol with hope to avoid fetal cells.
| tested these cells in vivo in mouse models of diabetes
51
how did they test if the pancreatic cells worked in vivo
implanted into mice.
measured each mouses response to glucose via the production of C peptide (made when insulin is processed)
measured in a mouse diabetes model
The transplanted cells were able to reduce blood glucode
52
what did kroon 2008 do to confirm that it was the transplanted cells that reduced the blood glucose
explanted the cells (removed them) and saw that the blood glucose increased rapidly
53
although the results from kroon 2008 were promising. What negative results were seen
15% of the mice developed cells suggesting that the transplanted cells were contaminated
54
how did kelly 2011 cause no animals to obtain tumours
enriched the cells for endocrine pancreatic progenitors using the cell surface marker CD142
none of the implanted animals formed tumours
55
what did the osafune paper show
that there are differences in the differentiation propensities between different cell lines
56
what is said to be the future of 3D differentiation
organoids - get the formation of an organ with the lineage that you want.
57
what did the expereiments by john gurdon find
found that mature cells could be reprogrammed to become pluripotent.. Found all cells had the same genes. When nucleus for an intestine cell was placed in an enucleated it was able to form progeny that had phenotypic characteristics of the frog that the intestine cell was taken from
58
what is therapeutic cloning
take cells from a patient and do SCNT to create a blastocyst. Stem cells can then be obtained from this blastocyst that can be differentiated into different tissues for therapeutic purposes
59
Hwang 2004/2005 paper about SCNT derived embryonic stem cells proved to be a fabrication why
issues with data
60
who reported in 2013 that somatic cell nuclear transfer could be used to produce human embryonic stem cells
tachibana
61
what are the main points of the takahashi and yamanaka (2006) paper - induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors
developed an assay to read pluripotency. Knocked in geomycin resistance gene at fbx15 loci
if cells are pluripotent they will be resistant to the antibiotic
24 genes collectively gave resistance
withdrew factors and found only 4 genes were needed (Oct3/4, Sox2, c-Myc and Klf4)
nanog was indispensible
injected iPSCs created this way in vivo and showed the formation of teratomas
injected into blastocysts and saw the formation of chimeras
62
what are the yamanaka factors to create iPSCs and why did people have problems with them
Oct3/4, Sox2, Klf4 and c-Myc
c-Myc is an oncogene so people didnt want to subject cells to it
63
what are the thomson factors to create iPSCs
Oct4, Sox2, Nanog, Lin28
lin28 represses inhibitor of Myc, indirectly activating myc
64
what are the preferred ways of generating pluripotent stem cells from somatic cells
exposure to transcription factors and small molecules
- but can be a slower process
65
what is the problem for generation of pluripotent cell from somatic cells by cell fusion
not good for therapeutic application as you generate a tetraploid cell
66
what are the 4 different ways of reprogramming a cell
1. somatic cell nuclear transfer
2. cell fusion with pluripotent stem cell
3. transcription factor expression
4. small molecule exposure
67
what are the current methods to make iPSCs by exposing them to factors
- integrating virus
- non integrating vector
- Excisable vector
- protein
- small molecule replacements
68
you dont want to use integrating virus' for the reprogramming of cells as this can interfere with the genetic makeup of the cell. What are the best alternatives
- proteins
- transposons
- RNA
- small molecules
69
what are the main points from the Hanna (2007) paper - Treatment of sickle cell anaemia mouse model with iPS cells geenrated from autologous skin
- used mouse models of thalassaemia and harvested tail tip fibroblasts
- reprogrammed them in vitro and corrected the mutation
- differentiated to haematopoietic progenitors and implanted back into animal
- showed its possible to take cells from diseased individuals, reprogram, correct the mutation, differentiate and put back in organism to show functional recovery
70
what are the issues with ES cells
- genomic instability
- need continual supply of high quality embryos
- potential for tumour formation
- questions regarding functional differentiation
- problem of immune rejection
- ethical controverst
71
what are the advantages of iPSCs
- no requirement for the administration of immunosupressive drugs
- opportunity to repair genetic defect by HR
- opportunity to repeatedly differentiate iPSCs into desired cell type for continued therapy
- less ethical issues
72
what are the main concerns surrounding iPSCs as evidenced by Zhao 2011
reprogramming event may trigger mutations
- cells derived from B6 EiPSCs can be immunogenic in B6 mice. abnormal overexpression of proteins directly contributes to this immunogenicity
73
what is direct differentiation
differentiation from one differentiated cell type to another
74
what are the main points fro the vierbuchen 2010 paper regarding direct differentiation
able to direct the differentiation of fibroblasts to functional neurons.
Screened factors responsible for this conversion and found: Brn2, Myt1l, Zic1, Olig2 and Ascl1
75
what do we need to do/know before using iPSCs as therapy
- are all iPSCs the same?
- need to develop robust and reliable differentiation protocols
- need to know relative efficiency of these different differentiation methods
- need to devise a way that iPSC can be screened for quality
76
who described mesenchymal cells as "adherent fibroblast-like colonies from monolayer cultures of bone marrow, thymus and spleen"
Friedenstein
77
the true identity of mesencymal stem cells (Caplan 1991) was determined as stem cells when what
transplants of clonal bone marrow MSCs produced bone in vivo
78
why are mesenchymal stem cells also referred to as mesenchymal stromal cells
mounting evidence indicates that cell population isolated from bone marrow are highly heterogenous and consist of several subpopulations all of which do not fulfil the criteria to be considered stem cells
79
what is the minimum criteria to define a MSC
- remain plastic adherent under standard culture conditions
- express CD105, CD73 and CD90
- lack expression of markers of endothelial and haematopoietic cells
- differentiate into osteoblasts, adipocytes and chondrocytes in vitro
80
evidence suggests that MSC may be present virtually in any vascularised tissue throughout the whole body
T
81
the Crisan (2008) paper - A Perivascular Origin for Mesenchymal Stem Cells in Multiple Human Organs - contributed to the understanding that MSC came from around blood vessels as a quick source for tissue repair. What were the main findings
- in all tissues detected NG2+ and CD146+ cells surrounding small blood vessels
- characterised cells expressing CD146 using flow cytometry - selected out ones that were negative for certain markers
- expanded in vitro - maintined expression of CD146
- transplanted into SCID mouse - differentiated into muscle and bone
- isolated cells that showed same markers as MSC confirming their identity
82
MSC are attractive for autologous transplants why
can be obtained from bone marrow or fat tissue (becoming more popular)
83
MSC have been used in several clinical trials for cell replacement. what have they particularly shown promise in
treating racehorse injuries
84
MSCs secrete factors such as TGF-b, IL-2, prostaglandin and PGE2 which attract what
immune cells such as macrophages
85
how do MSCs secrete factors to attract immune cells and what are current therapeutic strategies targetting this
vesicles. There are efforts to purify these vesicles so they can be directly administered
86
MSCs have an inherent tropism to tumours why?
tumour creates a microenvironment and produces MMPs to digest the ECM. MMP cleaves and activates PAR1
87
what are the main points from the nakamizo 2005 paper "Human bone marrow-Derived MSC in the treatment of gliomas"
hMSCs labelled in red were injected into carotid artery
within 7 days they located the tumour
when other cells were transplanted they did not locate the tumour - property is unique to hMSCs
88
how did nakamizo directly test that the migration of MSCs was directly due to the tumour
used transwell culture dished
hMSCs were placed in upper well and conditioned medium from U87 gliomas was placed in the lower wells. Exposure to conditioned media from U87 cell produced significant hMSC migration
89
what did nakamizo do as a proof of principle that MSCs were able to deliver antitumour agen
genetically modified hMSCs by transfecting with cDNA of IFN-B. These cells were cocultured with gliomal cell line and found to inhibit their growth. also proved in vivo that IFN-B secreted by MSC delivered intercranially increased the survival of mice
90
what are the key point of the paper - "mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer" Loebinger 2009
TRAIL is a member of the tumour necrosis factor superfamily. that is able to selectively induce apoptosis in transformed cells but not normal cells.
MSC were tranduced with TRAIL-GFP lentivirus that can be induced with doxycycline
in vivo - early administration of Dox reduces volume of the tumour
91
what was the evidence that stem cells existed in the bone marrow
transplantation of bone marrow into an irradiated mouse meant it was able to survive
92
how were haematopoietic stem cells discovered
noticed colonies on spleen of mice that had been transplanted with bone marrow. all colonies contained differentiated blood cells and new colony forming units. these colony forming units also lead to rescue of irradiated mice
93
why are HSC multipotent
they can only form 2 lineages - myeloid and lymphoid
94
what play a large role in stem cell linage fate
transcription factors
95
what are features of master regulators of stem cell fate
1. required for the development into a specific lineage
2. can change the fate of the cells that theyre introduced to
3. antagonise the opposite lineage prorams
96
what is the master regulator of the erythroid lineage
GATA1
97
what is the master regulator of the myeloid lineage
PU.1
98
describe GATA1 mediated antagonism of PU.1
- to regulate the expressio of genes, PU.1 has to bind DNA and bind the cofactor c-Jun
- GATA1 outcompetes c-Jun meaning PU.1 cant drive the expression of target genes
99
describe PU.1 mediated antagonism of GATA1
- GATA1 is a transcription factor that binds to GATA sequences in DNA
- PU.1 can bind to GATA1 and stop it binding sites in the DNA
100
when TF are equal the cell remains undifferentiated T/F
T
101
HSC are heterogenous with respoect to:
- Self renewal upon transplantation
- cell cycle properties
- differentiation
102
what causes HSC heterogeneity
- niche localisation
- replication errors
- differing methylation
- asymmetrical segregation
- stochasticity
103
what are the consequences of HSC heterogeneity
ageing
| haematological malignancies
104
what is a niche in stem cell biology
a local tissue microenvironment that hosts and influences the behaviours or characteristics of stem cells
105
who first proposed the idea of the stem cell niche
Schofield 1978 "The relationship between the spleen colony-forming cell and the haemopoietic stem cell"
106
what were the key points of a stem cell niche when it was first proposed by schofield
- has a defined anatomical location
- regulates self renewal of stem cell
- removal from niche results in stem cell differentiation
107
what are the niche producing cells in the drosophilia testes
the hub cells (present at the anterior end of the testes)
108
the hub cell (niche producing cell) of the drosophila tetes are in contact with which 2 type of stem cells
germline
| somatic (cyst)
109
when not in contact with hub cells what do stem cells differentiate into
gonialblast
110
what does division of germline stem cells in the drosophila testis lead to
the production of 2 cells. One stays in contact with the hub cell and therefore remains undifferentiated. Other moves posterior due to spatial restrictions and differentiates
111
gonialblast undergoes 4 mitotic divisions with incomplete cytokinesis to form ___ spermatogonia which mature into spermatocytes
16
112
the hub cell produces which ligand that activates JAK/STAT signalling in the germline and cyst stem cells
unpaired
113
JAK/STAT signalling is sufficient for the self renewal of the stem cells in the testis T/F
it is sufficient for the self renewal of cyst stem cells
| germline stem cells require this and the supression of the expression of Bam through activation of the BMP pathway
114
which ligand produced by the hub cell activates the BMP pathway and supresses the expression of Bam
Gbb/Dpp
115
what are the niche producing cells of the ovaries
Cap
116
what are the stem cells in the drosophila ovary
germline
117
what happens when the germline stem cells in the ovary divide
do so asymetrically leading to one losing contact with the cap cells and differentiation into cystoblast
118
what signalling is not required in the ovarian niche to maintain undifferentiated stem cells
JAK/STAT
119
what signals does the cap cell provide the germline stem cells with in the ovary
Gbb/Dpp activates BMP in the stem cells which activates mad and inhibits Bam leading to no differentiation
120
how were the niche cells of haematopoeitic cells identified
ablation experiments
121
what are key components that maintain HSC
Stem cell factor
| CXCL12 (activates CXCR4)
122
in a simplistic stem cell niche what 3 types of interaction are there
cell: cell interaction
cell: ECM interaction
cell: soluble signal interaction
123
what is AMD3100
a CXCR4 inhibitor. blocks the binding of CXCL12 so the HSC are mobilised - therapy
124
summarise the walkley 2007 paper
"A microenvironment induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency"
healthy bone marrow transplanted into a RAR null environment resulted in increased proliferation seen in myeloproliferative syndromes
125
why is the 2D in vitro culture of cells a simplistic model
- soluble gradients absent
- force apical basal polarity
- continuous layer of matrix
- high stiffness
- no third dimenstion
Cells in this form of culture lose some of their in vivo phenotype
126
what are examples of 3D stem cell cultures
organoids
127
what is an organoid
a stem cell culture that can recapitulate the complexity of a whole organ. They have to have multiple organ specific cell types, be capable of recapitulating some function and are spatially organised similar to the organ
128
what is the differential adhesion hypothesis of the formation of organoids
similar cells create more bonds with each other based on their differential adhesion leading to an organised architecture
129
where do the stem cells of the gut reside
in the crypts between lumen of the intestine
130
what differentiated cell types are present in the gut (intestine) epithelium
enterocytes
paneth
goblet
enteroendocrine
131
what cells secrete factors that are important for the maintenance of intestinal stem cells
paneth cells
132
intestinal stem cells express what
leucine rich repeat GPCR 5 (Lgr5)
133
what are the key points from the Sato 2009 paper: Single Lgr5 stem cells build crypt-villus structures in vitro without mesenchymal niche
1. Isolated mouse intestinal crypts – labelled LGR5 with GFP – can do FACs
2. Crypt preparations were suspended in Matrigel (protein mix derived from mouse sarcoma cells – rich in ECM)
3. Optimisation of culture medium – titration of key signalling molecules (EGF, R-Spondin, Noggin)
were also able to generate similar organoids from single lgr5+ cells following dissociation of crypts
134
what are particular applications of organoids
experimental tool
diagnostic tool
therapeutic tool
135
what virus have organoids been successful in increasing the understanding of
zika virus
136
organoids can allow personalised medicine T/F
T - first application for this was seen in cases of intestinal organoids for cystic fibrosis
137
what have organoids also been produced for
snake venom glands
138
what is aging an imbalance favouring
favouring differentiation
139
what is cancer an imbalance favouring
self renewal
140
what are the effects of ageing on the blood
decreased immunity
anaemia
increased incidence of bone marrow failure
141
what happens to populations of HSC upon ageing
see an increase in number but a reduction in reconstitution potential. The HSC also have a myeloid lineage bias
142
what are intrinsic factors that drive HSC ageing
- DNA damage
- ROS
- polarity shift
- altered proteostasis
- impaired autophagy
- epigenetic drift
143
how does aging increase DNA damage
HSC are quiescent (protected from replicative DNA damage)
| - upon ageing there is an increased proportion of cycling cells - increase DNA damage and production of ROS
