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What are types of stem cells?

- totipotent: fertilised egg
- pluripotent: embryonic stem cells, iPScs
- multipotent: fetal stem cells, adult stem cells, cord blood stem cells
- unipotent: fetal stem cells, adult stem cells


What are induced pluripotent stem cells?

- take adult cells and introduce four/five transcription factors that can induce pluripotency
- initially used viral techniques now a lot of non-viral
- once made into iPS you differentiate into the lineage of choice: this is a massive challenge in the field itself
- at the moment, these differentiated cells are mostly used for in vitro screening of drug candidates on healthy and diseased cells
- one day hopefully suitable for transplantation
- if you isolate them from a cell with a particular genetic mutation you can change that --> add, change, delete genes etc


How have iPS cells been received and used by the scientific community?

- excellent: now we have a new way to complement existing animal models with having human cells, especially diseased cells
- didn't care how they were going to analyse them, they just said let's just make them and we will worry about that later
- many groups working on neurodegenerative diseases, especially one's that carry inherent mutations, ran off and made stem cells from them
- many of these papers don't show a particular phenotype of this disease
- lots of reasons for this that demonstrate the challenges associated with stem cell research
- some make them from highly complex diseases e.g. schizophrenia, spectrum disorder like autism in children


What is the process when using iPS/hES cells?

- pluripotent stem cell: how do we make the differentiated cell, i.e. neuron, in the first place
- neural differentiation system (used by most groups around the world)
- you can break up any differentiation protocol into stages of development --> need to understand developmental process to get from stem cell to desired cell type
- can divide into stages of neural induction
- going from pluripotent into a neural epithelial cell type e.g. neural plate
- takes about two weeks, different methods
- once they become committed to the neural lineage we can isolate these cells (mechanically, under the microscope)
- cultured in suspension, aggregate together and form neurosphere
- neurosphere = aggregates of neural stem cells but within the sphere itself all cells like to differentiate - never completely homogenous: neural stem cells, neural progenitors, neurons, glials etc
- can make neural spheres even from feotal neural stem cells
- can keep them in culture for several weeks: can passage, expand
- can plate them down onto different substrates to differentiate them into more mature cell types of specific lineages


What is dual SMAD inhibition?

- directed induction of human pluripotent stem cells
- noggin (inhibits BMP or SMAD1 signalling - trophectoderm)
- SB431542 (inhibits Activin/Nodal or SMAD 2/3 - mesoendoderm)

- usually one of the very first lineages that pluripotent stem cells want to differentiate into is endoderm: extraembryonic endoderm or difinitive endoderm, and in particular with extraembryonic endoderm there is a cell type called trophectoderm
- these are the cell types that are involved in implantation into the uterus
- so it makes sense that a pluripotent stem cell, like an embryo, initially wants to make cell types that support the growth of the embryo
- also seen in a culture dish: the first lineage made is usually endoderm
- we know that the proteins that drive this differentiation are usually BMPs and for mesoendoderm, Activin/Nodal pathway
- downstream signalling pathway of BMP is SMAD1, and for Activin/Nodal pathway SMAD2/3
- if we inhibit these pathways they default to the next differentiation pathway
- next default pathway in early devolpment is neural ectoderm/ectoderm
- block it by adding BMP inhibitor (noggin) or use small molecule compounds that mimic these inhibitors

- add these inhibitors to a culture for two weeks – find that these pluripotent stem cells start expressing markers of early neural stem cells
- earliest marker of neural stem cells is the transcription factor PAX6 (human) and SOX2
- SOX2 also expressed in pluripotent stem cells but persists in early neural stem cells (switched off in other lineages)


What is the default of neural induction?

- early most anterior forebrain regions
- in development of the nervous system you have different sections of the brain and spinal cord: forebrain, midbrain, hind brain
- forebrain gives rise to the cortex --> most anterior part that then forms a carpert of cells that covers the whole brain
- this is the first lineage that develops from neural stem cells
- interesting because this is the most highly evolved part of the brain (however takes longest to develop)
- very large so development of sulci (more sulci = greater intelligence)


What are layers of cortical neurons?

- if you take a column of cortical neurons you see there are layers and many cell types
- at least 6 layers
- what type of cortical neurons do you want?
- have found that most of the neurons at the early stages are forming the deep layer cortical neurons (4,5)


What do we do when we create iPS cells from ASD (autistic) patients?

- differentiate into neurons
- develops into relevant cortical neurons
- how do we model autism?
- representative of newborn baby in someways - unmyenlinated, not connected to anything/the right cell type
- want to test neuronal sub-populations, synaptogenesis connectivity, functionality


Why work with engineers? How do we use microelectrode arary technologies?

- try to develop ways that we can culture neurons in a dish that model the way they work in a brain
- microelectrode array technologies for measuring functional connectivity between cultured human stem cell-derived neurons
- to show functionality in a neuron a lot of people use electrophysiology/patch clamping: individual neuron, go in with glass electrode and measure the electrical activity of individual neurons
- add inhibitors etc and monitor impact on firing

- 60 electrodes on array:
- culture neurons
- can measure simulatenously all the firing patterns
- use it to teach neurons to start talking to each other
- once they start talking to each other can measure it
- this is only a 2D system

- also work with engineers to create 3D matrices of neurons
- a better model
- silk fibroins
-- bioresorbable
-- optically transparent
-- mechanically robust
-- biologically and chemically functionalisable
-- non-immunogenic
-- integration with electronics


What are vertical nanowire electrode arrays?

- targeted/localised delivery of different agents (plasmid DNA, proteins, peptides, and low molecular weight compounds)
- ultra sensitive sensors for electrical, real time detection of chemical and biological species --> fast and accurate medical diagnosis analyser
- poke into cell to measure surface electrical and intracellular chemicals
- trying to make hollow so possible to inject things into cell


How do we derive other cell types from other lineages?

- e.g. Freidreich Ataxia, Hirschsprung's disease, hearing loss from neural crest/PNS neurons and glia
- e.g. multiple sclerosis from neuroepithelial --> CNS neurons and glia
- e.g. parkinson's disease from floor plate --> mesencephalic dopamine neurons

- have to mimic conditions seen in development - requires a very good understanding


What is Friedreich's Ataxia?

- loss of balance and coordination
- muscle weakness
- vision and hearing impairments

heart disorders
- tachycardia
- hypertrophic cardiomyopathy
- atrial fibrillation

- very rare: 1:30,000
- most common out of inherited ataxias
- symptoms start in childgood
- ataxia = loss of balance
- within a matter of years in a wheelchair
- heart disorders tend to be lethal

- not every neural cell type is affected:
-- cerebellar neurons
-- sensory neurons (pain, feeling, pressure)
-- pressure/feeling generally lost
-- imagine trying to walk etc without feeling the ground


What is the cause of Friedreich's Ataxia?

- inherited mutations in Frataxin gene resulting in the body producing insufficient levels of Frataxin protein
- function in the mitochondria
- maintenance of iron metabolism
- iron build up in the mitochondria - neurotoxicity
- even though frataxin is expressed in every cell in the body, for some reason neurons and cardiomyocytes are more sensitive to these mutations

- mutations occur in the intron regions of the gene
- between exon 1 and 2 --> GAA repeats (20 in normal state, 500+ in freidreich ataxia)
- extra repeats causes chromatin modelling, refolding etc that affect transcription
- hard for transcription to occur in this region because of extra expansion
- 10% or less of frataxin protein
- recessive --> need expansion on both alleles


Why is Friedreich's Ataxia a 'low hanging fruit'?

- we know how to cure the disease: correct the mutations within the frataxin gene, increase Frataxin protein levels in the body
- treatments: rescue and/or prevent further tissue degeneration, antioxidants

- for this reason a lot of money has gone into, a lot of research, drugs etc, some decrease iron toxicity, increase transcription, frataxin-protein replacements


What has been slowing down the development of drugs to treat Friedreich's ataxia?

- human Friedreich ataxia cells are needed to develop treatments
- to study degenerative mechanisms of having low Frataxin levels
- human cellular models for drug screening
- lymphoblast and fibroblast cell lines from patients are available, but not representative of all cell types affected


How did iPS cell lines contribute to research about Friedreich's Ataxia?

- got patient fibroblast samples
- characterised them and made iPS cells
- two cell lines: FA3 and FA4
- low levels of frataxin transcription compared to controls
- showed GAA repeat expansions
- you do see some genetic mutation when turning a cell into a stem cell: especially in unstable sections of DNA e.g. these very large expansions, sometimes you see further expansion in the stem cell, sometimes you see shrinkage
- however always in the pathological range (>50)
- initially differentiated the cells into the neurons without worrying about the exact neuron type --> just make into cortical neurons --> neurons and glial cells
- looked at the phenotype in these cells --> cell death over a long time not significantly different compared to control, looked at electrophysiology
- importantly looked at mitochondrial activity, (lots of stuff) --> saw almost nothing


Why might there be a lack of disease phenotype observed in Friedreich's Ataxia iPS cells?

- frataxin protein levels still too high ??
-- even though FA3 had 39% and FA4 had 28% perhaps this is not low enough
- need to analyse specific neuronal cell type?
-- create the sensory neurons that are normally affected by the disease as opposed to studying the cortical neurons
- need to analyse correct stage of maturation, at the functional stage?
- cells need to be stressed?
- microenvironment plays a role?

important to recognise that iPS cells are never 100% reflective of the original patient
transplanted into rat cerebellum - a lot differentiated into neurons and followed exogenous tracts (no extra cell death, unsurprising)

field moving rapidly


Can iPS cells be used in therapies?

no, not yet
trying to find ways to make them more stable
iPS ≠ ES


How have stem cells been used in therapies?

- iPS cells currently still haven't been used because too dangerous
- human ES cells the safest - used in a privately funded clinical trial where they turned hES cells into oligodendrocytes and inserted them into patients with recent spinal injury --> prevented scar formation and allowed for the regrowth of axons (usually prevented by scar tissue)
- unfortunately the cost of the trial far outweighed the number of people who benefitted so had to be stopped

- now a clinical trial underway for using hES cells to treat Age-Related Macular Degeneration
- often happens with the endothelial cells around the eye
- cell type of the nervous system, but external, so doesn't have to cross blood brain barrier
- don't have to worry about using immunosuppressants
- fantastic results --> really good reversal

in the cardiomyocyte field:
- really good success in differentiating hEScells into cardiomyocytes
- shown proof of principle that if they inject these hESCs into animals that have had heart attacks they do see recovery and regeneration
- just last year approved a clinical trial for using hESC derived cardiomyocytes in patients that have had very severe injuries (End Stage Heart Failure)


What can we do with stem cells?

Do we actually need stem cells?
- reprogramming and transdifferentiation
- can we make anything from anything?
- if you stimulate the right genes you can even bypass using stem cells
- already shown that you can get dopaminergic neurons etc


Why are hES stem cells important?

- without them we never would have discovered how to make iPS cells
- allow us to give back
- help us understand a lot about reproductive biology