Stem Cell Biology Flashcards
1892, stem cells are in between..
fertilised egg and committed germ cells
Stem cells have the potential to..
generate specialised tissue
copy themselves
Stem cells can be classified by: (3)
Age
Tissue of origin
Potency
Totipotent cells can..
produce all cell types of the body including throphoblast (placenta)
Multipotent cells can..
produce cell types specific to an organ or tissue
Pluripotent cells can..
produce cell types derived from the 3 germ layers
stem cells have a … cell cycle until…
Stem cells have a slow cell cycle until they become multipotent/restricted progenitors
External signals cause stem cells to..
speed up their cell cycle and differentiate into transit amplifying cells.
Stem cells expanded from a donor are called..
allogeneic stem cells
Autologous stem cells are..
from the same patient
As well as being used in therapies themselves, stem cells can also be used to..
Model new drugs
Teratomas are tumours which..
are non-invasive and contain multiple tissue types
Teratomas are produced from..
a single germ cell - from adult testes/ovaries
A single cell from a teratoma produced…when transplanted into another animal. This shows…
another teratoma. Shows that the cells are multipotent. They were termed embryonic carcinoma cells. They resemble pluripotent cells of blastocysts
ES cells have features of: (7)
Derivation from inner cell mass. Non-transformed. Indefinite proliferation/pluripotency. Stable diploid karyotype. Clonogenic Can be incorporated into chimeras Can be genetically manipulated
Functional assay for pluripotency
Transplant/inject cells into adult and teratomas form
In … the first ES cells isolated was from..
1981 from pre-implantation mice blastocysts. They formed embryos in vivo.
Stem cells grow with a layer of … which produce..
Feeder cells (carpet of fibroblasts) producing LIF (leukaemia inhibitory factor).
When LIF binds to its receptor, it causes transduction of..
Activation of G protein 130.
Activation of JAK.
Production of STAT3.
Causes pluripotency and self renewal.
Without LIF, what pathways are used
Absence of LIF activated the ERK pathway through SH2. This inhibits STAT3 so causes proliferation and differentiation.
For LIF to cause pluripotency or differentiation, it needs..
to be in serum.
LIF alone is insufficient for..
maintaining self renewal and blocking of differentiation
Factors in serum that LIF needs are:
BMPs
BMPs cause..
Smad and Id production, activating mesoderm and endoderm differentiation and blocking neuroectoderm differentiation.
STAT3 inhibits..
meso/endoderm fates and Smad
LIF and BMP work together by..
inhibiting meso/endo and neuroectoderm to sustain renewal
Mice ES colonies are … and … in appearance
Small and dome shaped
Human ES cells look
flat and defined
What is the human equivalence of LIFr-gp130
FGF2 and Activin-Nodal signalling
Mice and human ES cells have different: (3)
cell cycle and death rates.
LIFr and FGF2.
Surface antigens
Human ESC surface antigens are..
SSEA3, SSEA4 and TRA160
The only surface antigen mice have is..
SSEA1
Mice ESC renewal is dependent on LIF and..
ERK1 inhibitor
GSK3b inhibitor
Human epiblasts develop … before …
quickly before implantation of the embryo
Mice epiblasts are what shape.. When do they develop?
tubular. They develop later than human epiblasts, after implantation
The 2 states of ES cell are:
Naive
Primed
Naive ES cell features are..(4)
collected pre-implantation.
mouse ES like.
inner cell mass-like.
dome-shaped colonies.
Naive ES cells use Oct4..
distal enhancer
Primed ES cell features are..
collected post-implantation.
mouse epiblastic-like.
flat colonies.
Primed ES cells use OCt4..
proximal enhancer
Naive ES cells are better for..
understanding pluripotency and genetic modifications
Pluripotency stages are defined by..
a set of transcription factors expressed together in a careful balance
Transcription factors of pluripotency are:
Nanog
Oct4
Sox2
Transcription factors of pluripotency are all …. amino acids long and contain different …
300-350 amino acids long. They contain homeodomains
Sox2 has … for interactions with proteins
high motility groups (hmg)
Oct4 is the founding member of what family
POU transcription factor family
Oct4 is essential for … Upregulation causes … Normal levels cause … and low levels cause…
pluripotency.
Extra-embryonic endoderm and mesoderm.
Production of more ESCs.
Production of trophoectoderm.
hES were successfully obtained from an..
IVF generated human blastocyst. Induced teratomas in immunodeficient mice
Naive human ES cells cannot be captured as..
the time window is very short. Instead, primed ES cells have been undifferentiated
Human primed ES cells have been undifferentiated by inducing expression of Oct4 by what factors.. consisting of..
NHSM - LIF, TGFb1, FGF2, ERK1, GSK3, p38i, JNKi
NHSM factors cause primed ES cells to express..
pluripotency markers
Oct4 is expressed by different promoters in …and… ES cells.
naive and primed
Mouse epiblast SCs are characterised by low expression of pluripotency related genes:
Nanog, Rex2 and Klf4
naive human cells are more capable of growing as..
single cell clones with a shorter doubling time - good for scaling up production
Oct3/4 works in a …. with Cdx2 to..
reciprocal loop. represses trophoblast differentiation
Sox2 is needed in the..
early epiblast
without sox2, embryos fails to..
generate an epiblast, the embryo collapses
Both oct4 and sox2 are required together to..
form epiblast
Nanog is essential for…and was identified in 2 different experiments:
self renewal.
Seen by in silico screen and functional screen.
Self renewal with Nanog was observed in the presence and absence of LIF in ….. screens
In silico and functional
Nanog knockouts in ESCs show..
loss of Oct 4-pluripotency and differentiation into extra embryonic endoderm (completely).
When Nanog is overexpressed, what is not required?
LIF and BMPs
Pluripotent SCs show … of gene expression in populations
heterogeneity
Heterogeneity of pluripotent SCs was seen by tagging … with GFP, FACS and culturing cells. What was seen?
Nanog enhancers. After cells are cultured for 6 days, GFP+ and GFP- lines change.
Cells express certain amounts of .. and .. during different levels of commitment (for differentiation)
SSEA3 and TRA160
ESCs are triggered to differentiate in vitro (plastic without charge) by..
removing extrinsic conditions of self renewal
When ESCs are triggered to differentiate, how do they behave?
cells clump together and form aggregates called embryoid bodies
Embryoid bodies resemble..and have..(2)
gastrulation and early embryonic development. They show self organisation and axis formation
In vivo and in vitro, … activity is seen in embryoid bodies
Wnt activity.
Embryoid bodies are good since… however, there are difficulties controlling..
they are cheap to produce and generate 3 germ layers.
their aggregation, shape and size in a reproducible way
To change the shape of embryoid bodies, methods such as …. are used (2)
Hanging drop Controlled aggregation (plates with special geometry)
Embryoid bodies spontaneously form 3 different formations:
Cystic Bright cavity (cavitated) Dark cavity (dense)
cystic embryoid body structures are best at producing..
endoderm
bright cavity embryoid body structures show the best..and is the closest formation of..
organisation of the 3 germ layers, formation of a real embryo
dark cavity embryoid body structures show..
good production of the 3 germ layers
Oct3/4 causes expression of … and repression of…
activated expression of transcription factors such as Rox1.
Suppresses expression of Zfp42
Nanog is thought to suppress … to stop differentiation
GATA4/6 - induces extra embryonic endoderm
Nanog expression in the blastocyst is confined to the… Its expression is downregulation during…
inner cell mass.
implantation stage
Oct4, Sox2 and Nanog are lineage specifiers and suppress..
mutually exclusive fates.
Morphogen gradients influence …. to form specific cell types in embryoid bodies, eg…
cell lineage decisions.
Retinoic acid
Shh
Important aspects of growth factors to control during differentiating EBs:
Concentration
Time of addition
Substrates cells are grown on (laminin, fibronectin, collagen)
EBs are easier to manipulate during differentiation by…
plating cells as monolayers
EBs in cultures end up as a mixture of cell types after differentiation. They are sorted by..
their specific cell markers with FACS - (uses density gradients)
Polarised Wnt signalling is essential for…
primitive streak, antero-posterior polarity and EMT
In EBs, polarised Wnt signalling causes..
organisation into different germ layers
Aggregation kinetics of EBs causes different differentiation. Fast aggregations mediates…. production. Slow mediates…
Ectoderm production
Endoderm and mesoderm production
Pancreatic islet cells have been grown from ES cells and transplanted into type 1 diabetes patients. Pancreatic patterning involves..(4)
Foxa2, Sox17, Pdx1 and Ngn3
Pancreatic cells from ESCs were first poor at producing insulin and were more like foetal cells. What improved this?
A different staging was invented and transplanted into mice to test functionality and if hormones were produced. Successful but 15% mice developed tumours.
Methods of controlling EB production: (5)
removing self renewal factors. use non-adherent plates. growth factors - such as TGFb, bFGF, BMP4, NGF and EGF. adding stromal cells. changing shape for consistency.
Hanging drop method cannot be used for..(2)
hESCs.
Large scale prod of EBs
For large scale EB production, what techniques are used?
spinning flasks.
rotary cell culture systems.
They promote aggregation - greater EB formation.
Mouse ESCs were differented into neurons via EB formation. Sox2 was used with…
lacZ/neomycin so cells could be sorted by antibiotic selection
Mouse ESCs differentiated into neurons by EBs were determined by..
expression of neuronal markers - neurofilament chains MAP2, TAU and B tubulin
Producing pancreatic cells from ESCs required:
RA, inhibited Hh and Pdx1.
FGF10
Inhibition of Notch, Ngn3, neural TFs
Reprogramming SCs was experimented in frogs. An epithelial intestinal cell from albino frog was transplanted into an enucleated egg from green frog. What happened?
Eggs were incubated and complete embryos which were formed grew into albino adults.
After the green/albino frog experiment, reprogramming was taken to mammals..(what?)
Dolly the sheep
When reprogramming SCs, the …… allows genes to be de-repressed and undifferentiated
environment of the egg
The next step after Dolly the sheep is was…
Using patient-specific stem cell therapy (aka therapeutic cloning)
Reproductive cloning (growing human cells in utero) is..
widely banned
Yamanaka discovered..
Nanog and inducing pluripotent stem cells.
Green/albino frog experiment concluded: (2)
Genetic material is not lost, it is repressed/silenced.
Silencing can be reversed.
Yamanaka looked for genes controlling pluripotency. He knocked in..why?
Bgalactosidase and neomycin resistant genes. To select ESCs with fbx15 knocked out
Yamanaka introduced 24 candidate genes into mouse embryonic fibroblasts with Fbx15 KO by retroviral transduction. He eventually found:
Oct3/4, Sox2, Klf4 and cMyc
Thomsen’s factors were developed after Yamanaka factors:
Oct4, Sox2, Nanog and LIN28 (myc is an oncogene, LIN is an activator of myc)
iPSCs have been used to treat sickle cell anaemia in irradiated mice. iPSCs were..
differentiated into RBCs without mutation by homologous recombination
In iPSC sickle cell anaemia treatment, first … were harvested from mice
Tail tip fibroblasts
Sickle cell anaemia treatment with iPSCs showed what results in mice?
100% mutated haemoglobin to 70% normal haemoglobin over 4 weeks
Issues with hESCs: (6)
Genomic instability.
Continual supply of high quality embryos needed.
Can form tumours.
Unknown whether they differentiate into full grown adult cell types.
Rejected by immune system.
Ethical issues.
iPSCs can still cause immune response by…
reprogramming changing the antigen profile of cells.
Cells make many decisions on their path of differentiation. Attractors in the landscape are attractive because..
there is stability of a particular cell state
In an attractor of the landscape, cells can settle on different points on the valley slope. This allows..
heterogeneity - cells can transition into different positions
epigenetic landscape has been captured by… It produced..
3D phase map with differential expression of GATA and PU1. 3 different cell types were produced: high GATA, low GATA, combination of GATA and PU1
substates of hESCs were found when … were compared
normal hESCs and hESCs which were adapted to being in cell culture. These showed more proliferation.
hESC substates are defined by expression of………
SSEA3 - SSEA3+ showed more self renewal. SSEA3- still produced colonies
(in adapted h7)
SSEA3 expression is turned off when..
a cell differentiates
In the H7 study, normal hESCs showed:
adapted hESCs showed:
Normal showed SSEA3+ cells formed colonies and SSEA3- cells differentiated.
Adapted hESCs showed both SSEA3+/- formed colonies
Adapted h7 cells are different to normal h7 cells by:
extra chromosome 1.
lack of X chromosome inactivation (sign of naive ESCs).
As SCs progress towards the commitment barrier, they lose expression of …. then …
SSEA3, then TRA160
In culture, RA induces differentiation of ECs, turning off…
SSEA3 expression
Differentiated ECs by RA produce colonies after 12 days. Each colony had a varied…
% of neurons - ranging from 0 - 100
When exposure to RA was postponed in ECs, this allowed cells to divide before differentiation. Cells produced… This suggests…
more mixed colonies (0-30% neurons). this suggests that SCs have interconvertible neural and non-neural subsets during differentiation
Normal h7 SSEA3+ cells expressed..
genes associated with undifferentiated state: Nanog, POU, Sox2.
Adapted h7 SSEA3- cells expressed..
69% of these cells expressed genes of undifferentiated state
SSEA3 cells have heterogeneity of..
GATA6 expression
SSEA3+/low GATA6 formed…
SSEA3-/high GATA6 show…
stem cell colonies. 3-/6H did not form colonies
When 6H and 6L cells were separated to produce their own colonies….
They both regenerated each other’s population.
GATA6 expressing subsets showed … in EBs
increased expression of endoderm and mesoderm associated genes, and reduced expression of ectoderm genes.
Hearing loss affects more than ..% of the population who are over 60
50%
The 2 sensory cell types in the ear: …… are only produced during…
hair cells and auditory neurons are only produced during foetal stages
Sensorineural hearing loss accounts for … of cases. It is loss of…
90%. Loss of hair cells and neurons
Auditory stem cells were isolated from..
9-10 week old foetal cochlea (before terminal differentiation)
Cochlear SCs expressed typical TFs of cochlear progens:
Sox2, Pax2 and GATA3
With growth factors, cochlear SCs could express..
hair cell TFs: ATOH1 and BRN3C
Expansion of cochlear SCs occurred best in a serum-free medium containing…
bFGF, EGF and IGF (OSCFM)
OSCFM cells expressed otic progenitor markers: … and also pluripotent markers:
GATA3 and Sox2.
Oct4 and Nanog.
To differentiate OSCFM cells into neurons, genes expressed were…
Neurogenin1, POU4F1, Neurofilament200
In response to EGF and RA, OSCFM cells differentiated into..shown by..
hair cell phenotype.
ATOH1, MYO7A and POU4F3 expression
What is needed for otic placode formation? Why?
FGF3 and FGF10 produce high levels of Sox2 and Pax8
Double ko of FGF3 and FGF10 in mice showed..
no otic placode formation
Cochlear stem cells + FGF3 and FGF10 produce…which go onto produce…
Otic epithelial progenitors (hOEPs) - hair cells. Otic neuroprogenitors (hONPs) - spiral ganglions.
Proliferative capacity of cochlear SCs plateaus after..
20-30 weeks
Gerbils have been used to model death of spiral ganglia in deafness by use of chemical and where:
ouabain into the round window - induces apoptotic death
Otic neuroprogenitors were injected into gerbil cochleas lacking SGN. After 3-5 days, what formed? What did they do?
Ectopic ganglia formed and migrated. They formed projections to contact hair cells and the brainstem with functional synapses.
Functionality of ganglia formed by foetal cochlear SCs was tested by… What were the results?
ABR - auditory brainstem response. This allowed the auditory pathway to be seen in waves.
Thresholds increased by 50%, meaning gerbils could hear more.
Adult stem cells are also known as..
somatic
tissue-specific
multipotent
Research of adult stem cells started in..
Regeneration of limbs in salamander and Hydra
Mammals can regenerate what tissues?
Skin
Gut lining
Blood
RBCs are constantly replenished, with …. lost every day
10^11 cells
Haemopoietic SCs were the first adult SCs to be identified. Their location was shown by injecting…
bone marrow of healthy mice into irradiated mice (stopping production of blood cells). After injection mice survived.
Bone marrow injected into irradiated mice cause formation of..which showed..
colonies on spleens. This showed that injected cells could form colony-forming units - differentiation and self renewal properties.
Haemopoietic SCs form multipotent progenitors which form…
oligopotent progenitors -> lineage-restricted progenitors -> effector cells
MEPs are 1 of 2 myeloid progenitors. Without GATA1, MEPs will not differentiate into…
erythroid cell fates
GMP, a myeloid progenitor will differentiate into …. if exposed to GATA1
erythroid cell fates
GATA1 antagonises … fates and upregulated … fates
Downregulates myeloid fates and upregulates erythroid fates
GATA1 upregulates erythroid fate by…
removing c-Jun so that PU1 cannot be transcribed to cause myeloid fates
When PU1 is transcribed to upregulate myeloid fate, what action does it have on GATA1?
PU1 binds to GATA1 to prevent transcription of GATA1 target genes
MEPs and GMPs (myeloid progenitors) show …. to allow rapid production of cells if there is a wound.
multi-lineage priming
GATA1 is a … of erythroid lineage, meaning that it is essential.
master regulator
PU1 is a master regulator of…
myeloid lineage
Cytokine roles in haemopoiesis is either:
instructive - progenitors into specific cell type.
selective - act on progeny cells by stimulating their survival or death.
Human pluripotent SCs are currently being studied in the treatment of: (5)
Age-related macular degeneration. Parkinson's. Spinal cord injury. Diabetes. Myocardial infarction.
Age-related macular degeneration (AMD) is degeneration of..
retinal pigment epithelia
AMD is the leading cause of blindness in people aged over..
55
Degeneration of RPEs can be caused by:
AMD
Stargardt’s disease (genetic)
Retinitis Pigmentosa (genetic)
Treatment for AMD is very poor and limited, progression is only slowed down by..
vitamins and antioxidants.
Transplants of photoreceptor precursors have shown retinal repair in mice. Retinal progenitors which worked best were from… What are their features?
embryonic stages P3-P7 (post-fertilisation).
They express Nrl - post-mitotic progenitors
Transplantation of retinal progenitors into the retina from earlier stages
Nrl expression and survival but did not differentiate into photoreceptors.
P3-P7 retinal progens were transplanted into peripherin2 deficient mice retinas. Results showed…
Cell expressed new peripherin2 and were light-sensitive.
After transplant of retinal progens into mice retina, functionality was tested by…What were the results?
water maze. >50% showed functionality. Results also shown in vitro using 3D ESC cultures, integrating with adult retinas.
Foetal retina progens were tested in humans, how?
retinal sheets consisting of RPEs and neural retina were implanted into eyes.
What were the results and problems of implanting RPEs and neural retina in patients?
7/10 showed visual acuity.
Tissue source was limited
RPEs were differentiated from primate ESCs and transplanted into rats. What functional assay was used?
Rotating stripes to see if rats’ eyes were following the stripes.
The first clinical trial of hESCs -> RPEs was done in Stargardt’s and AMD. They were first observed in vitro and expressed:
MITF
Pax6
Bestrophin
hESCs - > RPEs were incubated at 37C with fluorescent photoreceptor bioparticles and injected into mice. This showed formation of…
A fluorescent layer of RPEs after 9 months with 2 types of RPE produced.
When hESCs-> RPEs were transplanted into human retina, what was observed?
Small improvement of visual acuity.
Takahashi used iPSC -> RPEs in 2 patients. What was seen?
Safety but no improvement seen.
Other approaches of delivering hESCs -> RPEs are being developed such as..
Patch of cells - provides good arrangement and easy removal. This was successfully delivered in 2 patients with visual acuity gain
Good manufacturing practices:
Pathogen free.
Reagents traced to be clear of any contact with other cells.
Source of cells.
Purification of cells.
Mesenchymal SCs can differentiate into..
bone (osteoblasts), cartilage (chondrocytes) and adipose (adipocytes) tissues
Transplants of bone marrow (mesenchymal SC) showed …. in vivo
bone production
Cell populations of bone marrow are highly..
heterogenous
MSCs have…..which defines them as not stem cells. After this discovery, they were called..
limited proliferative capacity.
limited ability to produce multiple lineages.
Mesenchymal stromal cells.
Mesenchymal stromal cells have… and express…
Plastic adherence.
Cell surface markers: CD105, CD73 and CD90.
MSCs (stromal) lack expression of …. which are expressed in endothelial and haemopoietic lineages.
CD45 and CD34, HLA-DR
MSCs produces bone except for…
around the head and neck. This is derived from neural crest mesenchymal cells
MSCs can also be isolated from: (6) but are limited so have to be purified
adipose tissue. placenta. umbilical cord. dental pulp. peripheral blood. endometrium.
MSC niche was found by screening foetal and adult tissues by immunofluorescence. Cells expressing BG2 and CD146 were found…
surrounding small blood vessels.
From MSC niche, CD146 were selected by FACS and transplanted into SCID mice. What happened?
Cells differentiated into muscle and bone. This assay identifies MSCs and plutipotency.
MSCs and multipotency is proven by what assay?
transplanting cells and observing muscle and bone induction.
MSCs are used in clinic for…(3)
replacement of bone, cartilage and cardiac muscle.
trophic and paracrine effects of factors which aid in healing and repair of acute injury.
anti-cancer tool - they are attracted to tumour microenvironments.
MSC attraction to tumour environment has been observed in…how?
gliomas in mice. hMSCs were injected into carotid arteries. Within 7 days, cells were located in the tumour. Also shown in vitro and in different tumour cell lines.
By transwell culture dishes with a porous membrane, MSC delivery was studied. Encoding … in MSCs by adenovirus causes…
Interferon beta causes apoptosis of cells
TRAIL-GFP activity (tumour necrosis factor) has been observed in in vitro co-cultures. What happens?
TRAIL-GFP is delivered by lentivirus and is activation dependent in the presence of doxycyline. Tumour cells are killed
Drosophila have a stem cell niche in the…
testes and ovaries
Hub cells are in contact with:
2 types of stem cells: germ line and somatic
Hub daughter cells of germ line/somatic cells either…
move out to become gonialblasts.
stay in contact with Hub cells.
Gonialblasts undergo rounds of mitosis and become..
spermatocytes.
Hub and Cyst (ovary) cells secrete…which activate..(2)
Dpp and Gbb which activate BMP downstream.
They also activate Upd and JAK signalling - maintains cyst cells.
In bones, endosteum divides bone tissue from… and is lined by..
bone marrow. Endosteum is lined by osteoblasts
Bone marrow is supplied with blood by arteries in..
cortical bone. Arteries branch into aterioles and venous sinusoids.
HSCs are distributed around…studied by …experiments
sinusoids - shown by ablation experiments
Vascular endothelial, perivascular stromal cells and megakaryocytes are essential components. They produce..
stem cell factor which binds to KIT receptor (on HSCs)
Organoids are made by aggregating pulripotent SCs with…this causes…
soluble cues and biomaterials cause cells to self-organise and form organoids
Organoids are physiologically relevant as they are experimentally tractable. They resemble..
Organs with multiple organ-specific cell types which are spatially organised and carry out specific organ function.
When organoids form, they recapitulate processes…
of self-organisation seen in development: cell sorting and restricted lineage commitment
