Lesson 19 Flashcards
how can we test of cells are pluripotent?
differentiate the cells in vitro and see if you derive any adult cell type or to transplant the iPCS cells subcutaneously under the skin of an immunodeficient mouse that cannot reject the cells
what is the first thing to do when reprogramming fibroblasts?
assess if they express pluripotent markers
what is a crucial hallmark to verify human iPCS generation?
the formation of teratomas
what is the method of choice to generate iPSCs?
Sendai Virus Vector (SeV) → virus of a single strand of RNA
how does SeV work?
it expresses the 4 Yamanaka factors th RNA which enter to the cells will just produce the 4 transcription factors to initiate the reprogramming process
why is SeV better than other previous methods?
does not leave any trace into the genome of the cells and since the level of viral RNA in high the level of expression of the factor is high and the process of reprogramming is very efficient → traceless efficient and technically very simple method
what is an advantage of generating human iPS cells?
they can be differentiated into a specific cell type for a specific diseases in order to create an “in vitro human model” for study
what did the Sendai kit make possible in the field of research?
it is possible to generate iPSC from peripheral blood mononuclear cells → easy method to have patient specific iPSC that we can use to differentiate into the cells of interest
it is possible to correct genetic mutations in the iPSCs with gene editing, generating what?
isogenic control iPSCs
how are isogenic control iPCs used?
the “corrected” isogenic control cells can be compared to the persons cells in order to test different therapies
what type of neurons die in Parkinsons disease?
dopaminergic neurons
how were isogenic controls used to study Parkinsons?
they created a panel of iPSCs lines all isogenic between each other which were different only for the number of copies of a-synuclein → these iPSCs were then differentiated into neural progenitors and then mature neurons, using a specific protocol which allows to direct the differentiation of pluripotent stem cells into neuronal stem cells
what mutation can cause Parkinsons?
a mutation in OPA1
how did scientist recreate in vitro neuronal circuits, in particular the dopaminergic striata connection?
a microfluidic chip - has very small chambers where you can grow different populations of neurons
what can we see with microfluidic chips?
we can see single axons in the microchannel and study the synapses in this other channel
how were micofluidic chips used to study Parkinson’s?
The aim was to analyse the axons of the DA neurons with the mutation in OPA-1
what did they discover with the microfluidic chips?
if these are mitochondria among the axons of the control neurons and these are the mitochondria along the axons of OPA-1 mutant → indicate that there is a strong impairment of fragmented mitochondria to travel along the axons and to reach the synapses
if synapses lose mitochondria, what occurs?
they degenerate
why can you not transplant an individuals specific iPSCs derived from neurons back into the substantia ganglia?
this connection is built during embryogenesis → when the brain is mature there are a lot of lipids which block the growth of axons so you cannot re-create an axon bundle
what idea was tested instead of transplanting neurons into the substantia ganglia?
you transplant the neurons directly where they have to release dopamine → in the basal ganglia
what are organoids?
organs or tissues differentiated in vitro to different iPSC derived from different cell types
describe a brain organoid:
is nothing like a brain, but it recapitulates some morphological features of embryonic brain structure → some people refer to them as neuron aggregates instead
what are brain orgainoids induced to form?
aggregates which float freely in the calcium medium, so these aggregates can grow and then can be induced into neuronal progenitors
what do the neural aggregates form, and what can be formed from further differentiation?
large bulges; if we cut these bulges in sections and let them differentiate each will start to form a laminar neuronal organization
why do neuronal aggregates form layered structures?
during the generation of organoids each of this layer start to differentiate these cortical neurons in a layer
organization really resemble what happens in brain development
what is a challenge regarding neuronal aggregates?
the maturation timing is a challenge in order to make sure it its mature enough to form a well-organized structure
what did the first study with brain organoids focus on?
microencephaly → they made organoids from healthy donors and patient with microencephaly, and they could recapitulate the defects of microcephaly in the organoids
what is an assembloid?
when two organoids of different neuronal cell types are fused together, when they fuse the axons from the cerebral cortex penetrate inside the striatal organoids and form contacts within neurons → can generate axonal connections between cortical neurons and striatal neurons in a 3D structure
what is a technique to activate neurons and record their activity?
optogenetics
how does the retina and lens form?
From the neuronal tube there is an evagination which will the contact the external epidermis and through a
double interaction this part of the neuronal tube will give rise to neuronal retina, and the overlaying epidermis will form the lens
What happens when we get an aggregate of iPSC, and we provide the right signals in form of culture medium molecules?
You have an aggregate of undifferentiated cells which get committed to a retinal faith and this retinal faith which forms the neuronal retina
what current research is being done to cure blindness?
The idea is to make photoreceptor transplantation to restore the visual activity into patient in which
there is the genetic loss of these cells
what germline layer is the liver generated from?
interior endoderm
what is the primordio?
induced by the endoderm and during the embryo development, this primordio grows always connected
to the endoderm which generate the gut tube where is going to be connected liver, pancreas and gut
tube
how can we generate liver organoids?
Using human iPSC-derived
hepatoblast and combining
them with mesenchymal stem
cells and endothelial cells. if
we combined these 3 cell types
together in the right calcium
medium and signals, these
cells form an aggregate of
cells
