iPS & iN Technology in Disease Progression & Management Flashcards
What does iPS stand for?
Induced Pluripotent Stem cells
What is of great interest in relation to iPS?
- iPS generate unlimited stock, can differentiate into any cell type of interest
- iPS derived neurons hold the genetic properties in autologous form (i.e from patient’s own cells) to harbour disease- patient mutations are kept in IPSCs
- iPS can be generated with SM to avoid endogenous potential oncogenes
- Historic, well-validated iPS protocols now taken into clinical trials
→Lots we still don’t know
What are iPS generally used for?
For the modeling of disease in a patient OR to replace lost cells OR for leads to a novel drug gene target
To recreate a patient cell…
- Skin fibroblast
- Reprogramme it into any cell type via
→ IPSC modelling: forced expression of trans genes via viral vectors:- First need to undifferentiate them, then differentiate them into neurons by placing them in a neural culture situation
OR
→ BY Direct induced neuronal (iN): forced trans genes of viral vectors as well:- Directly redifferentiates, no undifferentiated middle stage
What were thought to be the only cells with pluripotent potential?
Embryonic stem cells
Characteristics of iPS cells
- Behave like embryonic stem (ES) cells
1. Pluripotent- can give rise to several different cell types
2. Reproduce indefinitely- grow and propagate in culture
[Unknown if this happens in the body- possible link to
tumorigenesis in vivo] - Retain an epigenetic memory
→ a pattern of chemical marks on their DNA that reflects their
original cell type
→ i.e., the ex-fibroblast will still have epigenetic patterns of the
fibroblast
Who worked for years and won a Nobel prize to showcase to pluripotent potential of iPS cells?
Shinya Yamanaka, in Japan 2006.
This opened up promise for regenerative and personalised medicine
the 4 necessary transcription factors for turning a somatic cells into a pluripotent state
- Oct4
- Sox2
- Klf4
- c-Myc
What limits the use of the transcription factors in cell replacement?
Overexpression of these TFs has potential to be carcinogenic
Why are skin fibroblast most commonly used in iPS?
- Easy to access (punch biopsy to top of arm or top of thigh)
- Stable in culture
- Easy to passage- take and then grow in culture
- Fast growing- prolific, taken from hypodermis
- Low reprogramming efficiency- may not reliably make iPSCs
2 main and most effective Transduction factors in iPS
Transcription factors via viral vectors, such as the Lentivirus
→ However, is mutagenic and can activate oncogenes - not desirable in clinical use. Alright for use in disease modelling
Use of small molecules: better for clinical trials and sue - efficacy and safety NB!
Uses in modelling disease?
The studying of the disease system in a dish is accurate, as it is prepared with the patients own cells and genome. Accounts for all possible cell variability within an individual patient
the use of iPSc in ALS / Lou Gehrig’s Disease
Sporadic condition, with some genetic components. However it still lacks a good disease model…
TDP-43 pathology → constitutes aggregation of motor neurons
=> iPS derived cells showcased this aggregation of motor neurons, however showcased clonal variations in how badly patients are affected
Isogenic cell lines needed to compare controls
What are isogenic cell lines?
use of CRISPR-cas9 to introduce disease-associated mutations into iPS cells then compare the two patient lines
the use of iPSc modeling in Alzheimer’s disease
Loss of neurons and synapses in the cerebral cortex and certain subcortical regions- global degeneration of grey and white matter
Misfolding of two proteins: amyloid (α) and tau
→ Comparing 2 familial AD patients to 2 sporadic AD patients to 2 healthy controls
Significantly higher RAB (trafficking protein) in sporadic AD and familial AD vs control
Confirmed a connection between APP (amyloid precursor protein) processing and Tau in human cells
Confirmed that toxic oligomers form inside of neurons
Cell replacement Therapy in Parkinson’s Disease & why it was problematic
Parkinson’s Disease characterised by loss of dopaminergic neurons in the striatum, Cell replacement attractive prospect
Fetal DA neurons were used - But these were problematic: Logistics, ethical implications of embryonic cells, standardising methods, quality control
the use of iPScs instead for Cell replacement in PD
Efficacy of using DA neuron cell replacement in PD (with rigorous tissue prep, patient selection, patient immunosuppression and delivery)
That could avoid graft-induced dyskinesias, which had been issues before
A gold standard protocol for stem cell studies
- Kyoto study in 2018- three PD patients donated DA neurons grown from the same donor
- US study 2020- patients received DA neurons grown from their own skin cells
Graft survived with no sign of tumour outgrowth nor troublesome dyskinesias (yet)
Issues with the reprogramming of iPSc lines into somatic cells
iPS cells seem to wind back time - they seem to become rejuvenated, in that they seem younger as common hallmarks of pathologies and age disappear. This is NB as as age is a major risk factor in many conditions like Parkinson’s this accounts for degeneration in cells causing a variability of cells.
such as DNA damage, inflammation and telomere shortening.
=> This only adds tot he cost and labour of generating these lines
What seems to be preferred nowadays to iPSc lines in models
Isogenic cell lines
iPS Vs iN
iN goes down direct reprogramming group from one somatic cell type to another, it compliments iPS routes.
iPS is very good at modelling monogenetic disorder, however there is a problem that cells are often rejuvenated back to a younger like state where they lose the characteristics of aging which are often risk factors associated with the condition in question
what does iN stand for?
induced Neuron
Directly changing a cell from one somatic cell type to another
What is transdiffrentiation?
a powerful tool for generating functional cell phenotypes without the need for iPSCs or embryonic stem cells
→ A wide array of cells has been successfully generated and their ability to mimic physiological cells shows great promise, especially with the advent of transdifferentiating cells in situ.
NB in personalized regenerative medicine and tissue engineering in the future
Describe the direct cell type conversionof fibroblasts to iN
- Circumvents rejuvenation and preserves hallmarks of cellular aging
- Rapid differentiation and maturation
- Retain their age- good for modelling neurodegenerative diseases
Limitations of iN use
- The age of the human donor has been negatively correlated with the percentage of iNs obtained
- Fibroblasts from adult human donors are resistant to Ascl1/Brn2‐based conversion
- Fetal fibroblasts are highly amenable (Drouin‐Ouellet J et al., 2017)
- Converting cells from one germ layer to another requires substantial chromatin remodeling to make target-cell-type-specific genes accessible to TFs
ie MET transition from mesoderm (fibroblasts) to ectoderm (neurons) - Starting material is finite, and no expandable stem cell stages are involved in the process (Unlike iPSCs)
What are the BAM factors?
Transcription factors Ascl1, Brn2 and Myt1l
What do BAM factors do to iN cells?
Induce them
