lecture 17 Flashcards
What can we do with the stem cells in the nervous system?
1
Q
What are types of stem cells?
A
- 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
2
Q
What are induced pluripotent stem cells?
A
- 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
3
Q
How have iPS cells been received and used by the scientific community?
A
- 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
4
Q
What is the process when using iPS/hES cells?
A
- 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
5
Q
What is dual SMAD inhibition?
A
- 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)
6
Q
What is the default of neural induction?
A
- 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)
7
Q
What are layers of cortical neurons?
A
- 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)
8
Q
What do we do when we create iPS cells from ASD (autistic) patients?
A
- 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
9
Q
Why work with engineers? How do we use microelectrode arary technologies?
A
- 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
10
Q
What are vertical nanowire electrode arrays?
A
- 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
11
Q
How do we derive other cell types from other lineages?
A
- 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
12
Q
What is Friedreich’s Ataxia?
A
neurodegeneration
- 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
13
Q
What is the cause of Friedreich’s Ataxia?
A
- 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
14
Q
Why is Friedreich’s Ataxia a ‘low hanging fruit’?
A
- 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
15
Q
What has been slowing down the development of drugs to treat Friedreich’s ataxia?
A
- 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
