What is parkinson's?
-Progressive degeneration of midbrain dopamine neurons
What is the target with stem cell therapies for Parkinson's?
-getting the right type of dopamine neuron in, not just a generic one, should be the motor one
What are the current Parkinson's treatments?
-pharmacological treatments: L-DOPA
-L Dopa is the main one (pre cursor to dopamine, used as it can cross the blood brain barrier)
-number of problems= patients very responsive in the first 5 to 10 years and then efficacy decreases, the graph shows the motor responses of a parkinson's disease
-the extremes= too much dopamine= too much movement, little dopamine and little movement
-excessive movement when high dopamine
-this depends on the dopamien neurons to change L Dopa into dopamine and once they die the dopa doesn’t work
-no disease modification! through this
What are the main issues with the current treatment of Parkinson's?
-No disease modification
What are the types of stem cells and how do they differ?
3 stem cell classes
- totipotent= any cell in the embryo and placenta
-pluripotent= from inner cell mass of blastocyst (can become anything in the embryo) (now can make these)
-multipotent= more restricted, reflective of the tissue they come from, like neural stem cells can become neurons or glia
What are the main sources of neural stem cells?
-we want neural stem cells here so they don't grow into something else
- either developing embryo or from an adult particularly in the hippocampus, and the lateral ventricle (SVZ) = for olfactory bulbs
What ways are being explored in how to make the dopaminergic neurons regenerate?
-fetal and adult
-this is looking at cell replacement therapy, or can do a chaperone cell or trophic cell (cell that will secrete trophic
-induced pluripotent stem cells from patients= maybe to use
-final option= activate quiscent population of stem cells in the brain SVZ and hippocampus (but really far away!)
What is special about the salamander's ability to regenerate its neural cells?
-salamander= extreme regenreation capabilities -can they grow midbrain dopaminergic neurons
-kill the neurons and see what happens
-cut coronal section and look
-so test in salamander= inject amphetamine (causes release of dopamine from terminal and inhibits degradation)
-at 3 days move only a bit, at 30 days move rapidly reflective of hyper dopamine state
-amphetamine promotes DA release
-correlate restoration of motor function with number of cells in the midbrain
-in the lesion animals at 3 days only 10% motor function, as more cells in midbrain then get more motor function
What is most of the stem cell research going into?
-research is mostly into stem cell replacement therapy
-the issue here is that you are injecting into the forebrain instead of the midbrain to avoid the need to have the dopamine pathway
What was the human trial with injecting the dopamine neurons into the forebrain?
-here transplanting new cell into the forebrain so don’t have to send long projections to the forebrain
-lot of new cells in the brain and restore motor function to mice
-human trials: -in some cases very successful, patient 3 years later complete motor function
-200% increase in dopamine levels in the hemisphere where the transplant was in, and 45% reduction in dopamine levels in the hemisphere the transplant was not in.
-after 3 years!
-the only success is using aborted fetal tissue
-unfortunately it didn't quite work
-only 30% of people improved some had very bad side effects!
-lot of variability in patients, only 30% robust improvement, 30% no effect, and 20% adverse debilitating effects
-the issue with it was the variability with the donor tissue, storage, age of tissue etc. (make it more like the environment in which the neurons normally develop)
What are the ways in which we can improve the treatment of Parkinson's where tissue is injected?
1. Standardization of donor tissue (& availability)
- Isolation of appropriate DA neurons
- Selective expansion of donor tissue
- Standardization of pluripotent stem cells
2. Environments conducive to graft integration
- Exposure to trophic cues
- Promoting plasticity
- Scaffolding support
How can you standardize the donor tissue?
-want to control which type of DA neurons is coming in (motor-Substantia nigra not the reward-limbic)
-can do this via brith dating analysis
How can you do the birth dating analysis to get the donor tissue standardized?
-Current transplants of mouse embryonic day 12.5 (E12.5) VM with no knowledge DA neuron composition
- Dopamine neurons are born from E10.5 to E14.5
- Inject pregnant mouse at E10.5, E12.5 or E14.5 with BrdU (to label cells undergoing division)
-Aim: Assess when dopamine neurons are born in the SNpc (motor) and VTZ (limbic/reward)
- at E10.5 2-fold more GIRK/TH (motor) dopamine neurons
-so target the E10.5 neurons!
What effect does the age of tissue have on the graft?
-Younger donor tissue results in larger graft -as the motor DA neurons are on day E10.5 instead of the now used 12.5
-younger tissue enriches for motor-like DA neurons
-Younger donor tissue enhances striatal innervation, dorso-lateral fiber density & DA levels
Does younger tissue eliminate the issues with serotonin in the treatment?
-the side effects in the treatment= due to mishandling of dopamine by serotonin neurons, serotonin grows later so younger tissue doesn’t have it= get younger= better effects
What is a mitogen?
-An agent that encourages a cell to commence cell division
- epidermal growth factor
- fibroblast growth factor
What is a morphogen?
-A protein (gradient) governing the position of various specialized cell types within a tissue.
- Sonic hedgehog (Shh)
- Fibroblast growth factors (FGF)
What is the second approach with developing stem cell treatment?
-making the environment more like where the neurons develop normally
-introducing the appropriate morphogens, mitogens and even semi-hypoxia
What is the role of semi-hypoxia in stem cell treatment, what is the rationale behind using it?
second approach to mitigating the issues:
-low O2 is more like the developing neurons
-adopting knowledge from these studies and take younger tissue and use morphogens to enhance proliferation= tenfold increase in tissue so almost one to one (fetus to patient ratio, before it was 8:1)
What effect does adding Wnt5 have on the graft?
-these proteins work and release dopamine
What was the test with mice and transplanted tissue?
-only one side of the brain has no dopamine
-so have control and the animal will turn in circle and can use that as a measurement of parkinsons
-can see how this behaviour decreases and from that determine how motor function is establishing
-improved markedly with growth factors! Wnts and VMN
Are there human trials about to begin with PD?
-Selective expansion of human neural stem cells
-Multi center trials about to commence
- Still a limited tissue source
- Ethical considerations
-Potential for pluripotent stem cells
What is the third approach to improving PD treatment?
-looking at the use of pluripotent stem cells
-3. Regulating pluripotent stem cell differentiation
Why would getting the pluripotent cell treatment be great and what are the challenges?
1. Unlimited cell source
- Risk of neural overgrowth and tumors
2. Ability to generate all cell types
- How to control DA differentiation
• Identification of novel regulators in DA development
• Refinement of DA differentiation from PSCs
What are some of the advantages of using the pluripotent stem cells?
-can get them anytime
What is the 4th thing to do in treatment?
-Cell sorting of pluripotent-derived neurons
-the ideal scenario
-in reality get a mix of NS cells and mDA progenitors to the brain, and sometime the ES cells and this causes teratoma growth! to remove this option, have ways of removing the cells: next slide, the progenitors express green fluorescent protein so can tell them apart
How can you sort out which pluripotent stem cell you have, which neurons it made?
-attach reporter line
-then can sort them via fluorescence
What is the 5th approach to the treatment?
-Restoring DA connectivity following transplantation
-the other approach: improving the host environment, improving the integration of teh graft
-since most of the transplants are ectopic (forebrain) and don’t have the circuitry
-Exploitation of DA axon guidance cues following transplantation (Identify dopamine axon growth and guidance cues in development)
-here try to inject into the correct spot and guide them to the connection
-Exploiting developmental trophins and guidance cues to promote graft integration (GDNF for example)
-VM+GDNF =up to a seven fold increase in the axons, improvement in grafts!
What is the 6th approach to the treatment?
6. Alternative approaches for promoting graft plasticity
-another way of improving the graft integration
-if can promote plasticity of transplanted neurons
-so have group with stimulating environment and group without
-enrichment of the environment
-the enriched group do much better!
What is the 7th approach to the treatment?
￼7. Engineering scaffolds to support stem cell grafts in neural repair
-another way of approaching it: improving the physical environment
-provide surface for axons to grow on, use three types -aslo can functionalise the
-nanofibrous scaffold, hydrogels and some mix it and make composite scaffolds
-Modified scaffolds promote NSC proliferation & differntiation
-Nanofibrous scaffold including GDNF immobilization promotes neural stem cell engraftment
What is the prognosis to date?
• Proof of principle for cell transplantation in PD
• requires tissue standardization
• requires alternative cell sources (availability/ethics)
Pluripotent stem cells (PSC) present viable options, especially iPSC for transplantation, drug discovery and disease modeling HOWEVER
• PSCs require improved differentiation (>30% efficiency)
• PSCs require guaranteed safety from persistent dividing cells and residual oncogenes (iPSCs)
This is rapidly progressing field but could still take many years to achieve the end point goal...
What is the ideal for the future in terms of PD treatment?