L4 - In Vitro Stem Cell Models Flashcards
Why is the mouse a goof model to test the effect of ESCs?
Can be genetically modified
Short gestation period
Where are early embryonic cells found?
In inner cell mass
They are pluripotent
What two things show that a cell is pluripotent?
Expression of pluripotency factors (only expressed in inner cell mass)
- Nanog
- Oct4
- Sox2
Teratocarcinoma formation
- Pluripotent cells grafted onto kidney of host mouse - give rise to teratocarcinomas
- They are tumours containing all cell types
What evidence is there that stem cell potency diminishes as the embryo develops?
At E4 we find pluripotent cells
At E9 we find neural stem cells (bipotent)
What does the ectoderm form?
Skin
CNS
What does the mesoderm form?
Blood
Muscle
What does the endoderm form?
Gut
Lung
Why are stem cells in vivo difficult to study
Small cell numbers
In utero development
Ethics
What is the importance of capturing cell in vitro in petri dishes?
In vitro modelling of embryonic development can lead to production of clinically relevant cell populations
- Drug screening
- Cell replacement therapies
How are embryonic stem cells captured in vitro?
- Signals to maintain cells in self-renewing, undifferentiated state and can replace feeders
a. Mouse - Leukaemia Inhibitory Factor, BMP
b. Human - FGF2, TGFβ - Plate single E4 cells on layer of feeder cells - irradiated stromal cells derived from later embryos which support ES cell growth
- Once embryonic cells have divided a few times, disaggregate and re-plate
- ES cells express a transgene encoding GFP
How do you reprogram adult somatic cells to a pluripotent cell fate?
- Get skin or biopsy from an adult with a disease or normal
- Ectopically overexpress reprogramming factors
- Reverses cells back into embryonic stem cell state
More ethical than using embryos
What do embryonic stem cells express?
Express the pluripotency factors Oct4, Nanog, Sox2
Resemble the cells present in the inner cell mass of early embryo
When transplanted into permissive environments what do ESCs form?
Teratocarcinomas
What happens if mouse ES cells are reintroduced to normal mouse embryos?
They contribute to normal development
- Mice derived from donor embryonic cells
- Shown using GFP
How do you capture neural stem cells in vitro?
- Dissociate cells
- Plate on laminin in the presence of the cytokines FGF2 and EGF
- NS cells express undifferentiated markers - RC2
- No expression of genes indicative of differentiation
a. Glia - GFAP
b. Neuron - TUJ1 - A single cell can generate identical daughter cells - stem cell
- Can differentiate into glia and neurons after removing FGF2, EGF
What are the two main approaches to in vitro differentiation?
3D
2D/adherent
What is the 3D approach to in vitro differentiation?
Remove signals that keep cells in an undifferentiated state
- Mouse – BMP and LIF
- Human - FGF2, TGFβ
Grow in aggregates (embryoid bodies/organoids) in presence or absence of signals
What are the advantages of the 3D approach?
Recapitulates more accurately embryonic environment
What are the disadvantages of the 3D approach?
Difficult to observe role of individual signals
What is the 2D approach to in vitro differentiation?
Plate a defined number of cells on the right substrate or extracellular matrix
Remove signals that keep cells in an undifferentiated state
- Mouse – BMP and LIF
- Human - FGF2, TGFβ
Grow in defined medium with appropriate amounts of signals - FGF, WNT
What are the advantages of the 2D approach?
More tractable system – live imaging
- E.g. GFP expression reflects T(Bra) (early mesodermal marker) expression during ES cell Easier to test the role of specific signals
What are the disadvantages of the 2D approach?
Loss of cell interactions that may occur in vivo
What is microcephaly
Neurodevelopmental disorder
Infants are born with an abnormally small brain
Neurological defects and seizures
What is the cause of microcephaly?
Due to various autosomal recessive mutations
Mouse mutant fail to recapitulate the condition
What is the possible treatment for microcephaly?
Taken skin biopsy’s from infected patient and a normal sibling
Generated induced pluripotent stem cells by expressing pluripotency factors
Generated mini brains in petri dishes
- Smaller number of neurons and get smaller number of cerebral organoids
What is the potential application for microcephaly on the development of mini brains?
Small molecule screens using mutant differentiated cells can help identify suitable drugs
What is familial dysautonomia?
Genetic disorder that affects
Development and survival of neurons that control involuntary actions - digestion, breathing, tears, regulation of blood pressure and body temperature
Sensory nervous system - taste and the perception of pain, heat, and cold
Familial dysautonomia facts
Problems related to this disorder first appear during infancy
No treatment - 50% of the affected individuals by age 40
Usually caused by mutation in IKBKAP gene
What is the possible treatment for familial dysautonomia?
Took skin biopsy from FD patient and healthy individual
Produced induced pluripotent stem cells
Differentiated these into neurones we know are affected by FD
What is the role of Kinetin in familial dysautonomia?
Candidate drug testing to discover molecules that reverse the phenotype
Kinetin treatment induces an increase in number of in vitro derived neurons
What are the symptoms of Parkinson’s?
Tremor Slowness of movement Rigidity Dementia Progressive loss of dopaminergic neurons
What is the potential treatment for Parkinson’s?
Generate dopaminergic neurones in petri dish using hES cells
Tyrosine hydroxylase - enzyme involved in dopamine synthesis
Neurones transplanted into mice which showed evidence of improvement in motor function
How can you treat macular degeneration?
Development of retinal pigment epithelial cells from hPSCs
