STEM CELLS
Describe the ethical and practical challenges involved in investigating the potential of totipotent/pluripotent stem cells.
Technical difficulty
Legislative barriers such as laws prohibiting reproductive cloning in humans, use of embryonic stem cells (except for leftovers from IVF)
Can prove, it's just not legal
Brown and white pure-breeding mouse - cannot replicate this in humans
Mt disease (Meaghan)
Rigorous regulatory environment
Licensing bodies
Institutional approval
In vitro
In vivo
FACS
Lgr5+ Gut crypt stem cells
Axin2 + alveolar epithelial cells
CD34+ HSCs
CD73+, CD90+, CD105+ MSC, negative for myeloid and lymphoid markers
Surrogate clonogenic assays
Will a single stem cell differentiate into multiple cell types or a homogeneous population? Characterising potential
Different colony characteristics (e.g. BFU, CFU) correlated with certain potentials
Animal model
Congenic mouse model
Non-competitive re-population assay
Competitive repopulation assay
Competitor is a standard, or radioprotector
K/O or pertubation model
Repopulate with stem cells to see how well stem cells can repair
Surrogate = modelling in vivo
Discuss how the cellular microenvironment/niche influences stem cell behaviour. What are ways of/challenges of replicating this in tissue engineering?
Discuss how stem cells may be used as disease models and the various strengths and challenges of each approach.
Intrinsic regulation = transcription factors expressed, euchromatin vs heterochromtain
Extrinsic regulation
Surrounding cells
Stimulatory (permissive) or inhibitory (restrictive) signals
Example: Paneth cells in the intestinal crypts to regulate Lgr5+ stem cells
Example: MSCs regulating HSC differentiation and proliferation
Example: Liver stem cells --> Cholangiocytes (differentiation promoted by TGF-beta) and Hepatocytes (differentiation promoted by Notch signalling and ECM proteins)
Growth factors
Anti-inflammatory cytokines
Anti-fibrosis
Tissue specific niches
Cannot generalise about characteristics
MSC mess
Inconsistency
Tissue of origin
Genetic background
Differentiation protocols
Nomenclature
Transcriptional noise, inter-clonal variation
Characterisation/definition
Effective extrinsic signals
Some researchers simply use plastic adherence as marker for MSC
Current guidelines: 3 requirements
Plastic adherence
Differentiation into adipocytes, chondrocytes and osteoblasts
Markers: CD105+, CD90+, CD73+
Must replicate 3 aspects - tissue mimetic
Biological
Mechanical
Biochemical
Bioreactors that e.g. facilitating stretching for myocytes
Stiffness
Porosity for delivery of soluble factors
Layer by layer technique regulates release of heparin and bFGF
Sandwiching protects from degradation of soluble factors
Inkjetting technology and thermally induced phase separation
Modulates rate of delivery/release kinetics
Spheres made at varying levels of porosity and cross-linker levels as well as capping layers
bFGF vs alpha MSH
bFGF is bigger than alpha MSH
Crosslinking influences bFGF
No of capping layers influences aMSH
NOT HAVING A GOOD NICHE - FBR
Adsorbs layer of denatured proteins
Nts and macrophages aggregates around denatured proteins
Forms multinucleated giant cells
Release of cytokines recruits fibroblasts
Collagenous fibrosis, formation of acellular collagenous bag
Vasculature
In vivo bioreactors: angiogenesis by planting a blood vessel in bioreactor surgically
Signalling molecules, e.g. VEGF - consider release kinetics
Tools
Gene editing
Applications
Knockouts
Insert disease polymorphism into healthy individual
Insert reporter gene
Lineage tracing
Lineage tracing!
Working out contribution of gene to disease
Example: Parkinson's Disease
K/O of LRRK2 and SCNA to work out contribution of these polymorphism to disease
Insert LRRK2 and SCNA into normal background to observe contribution to disease and whether there are other precipitating factors
Insert LRRK2 OR SCNA separately - are these polymorphisms alone enough to produce diseased phenotype
Emphasis now on using well defined singular genetic background to study individual genes as opposed to inconsistency
Characterisation of differentiation protocols e.g. dopaminergic neuron development - k/o of different genes and observing effect on development of neurons in specialised areas of the brain
Reporters
Examples
Why are reporters used?
Guide in vitro control of stem cell differentiation
Observing drug efficacy
Lineage tracing
Allows straightforward readout of complex models
Luciferase (bio luminescent)
Fluorescent proteins (GFP, mCherry)
Pros:High through put screening of small molecular drugs
beta-lactamase
Pros: no substrate required
Cons: can be weakly or unpredictably expressed
Con: needs a substrate
Pros: good for live cell imaging
Disease modelling
Examples
Drug efficacy testing
Limitations
Why are they used?
Animal models are less predictive
Grown efficiently with robust differentiation protocols
Consistency
Ease of genetic manipulation
Personalised medicine
Specific to individual as opposed to generalised disease model
Polygenic diseases
Low penetrance and late onset
Alzheimers
Parkinsons
PSEN1, PSEN2, APP - highly penetrant/causative but <5% have this gene
APOE4 and TREM2 - intermediately penetrant/medium risk
BIN1 - low penetrance but high frequency in population
Used GWAS to identify these but can use k/o studies to investigate contribution further
Poorly replicated microenvironment
Inconsistent genetic backgrounds
Epigenetics disregarded
Schizophrenic patient iPSCs
Model Schizophrenia
Test drug efficacy in improving neuronal connectivity - done using rabies model looking at synaptic integrity
Rhett's syndrome
Alzheimer's
Major issue in drugs is cardiotoxicity
due to amine groups
Dog models have poor predictive capacity (50%) whereas iPSC models have predictive capacity that is 95% accurate
Current approach is reductionist
Using biologically irrelevant immortalised cells (transfected with receptor or interest that they're trying to target) - not reflective of normal biology
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over expression of target can lead to signalling artefacts (noise)
using enzymatic reporters to quantify gene expression
e.g. inducing Lmx1a expression through use of different kinase inhibitors using luciferase (sensitive plate based assay)
Organoids vs intermediate systems (combining cells) vs individual cells
Organoids
Cons: Only epithelial cells, cannot be replicated well --> therefore not suitable for drug testing (can't test multiple drugs on one organoid), not suitable for high throughput screening
Pros: 3D structure, multiple cell types
Intermediate systems
Lack of 3D structure
Poorly replicates micro environment
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iPSCs or ESCs allow for better lead selection and phenotypic screening