iPS & iN Technology in Disease Progression & Management

Question 1

What does iPS stand for?

Answer 1

Induced Pluripotent Stem cells

Question 2

What is of great interest in relation to iPS?

Answer 2

• 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

Question 3

What are iPS generally used for?

Answer 3

For the modeling of disease in a patient
OR
to replace lost cells
OR
for leads to a  novel drug gene target

Question 4

To recreate a patient cell…

Answer 4

• 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

Question 5

What were thought to be the only cells with pluripotent potential?

Answer 5

Embryonic stem cells

Question 6

Characteristics of iPS cells

Answer 6

• 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

Question 7

Who worked for years and won a Nobel prize to showcase to pluripotent potential of iPS cells?

Answer 7

Shinya Yamanaka, in Japan 2006.

This opened up promise for regenerative and personalised medicine

Question 8

the 4 necessary transcription factors for turning a somatic cells into a pluripotent state

Answer 8

1. Oct4
2. Sox2
3. Klf4
4. c-Myc

Question 9

What limits the use of the transcription factors in cell replacement?

Answer 9

Overexpression of these TFs has potential to be carcinogenic

Question 10

Why are skin fibroblast most commonly used in iPS?

Answer 10

• 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

Question 11

2 main and most effective Transduction factors in iPS

Answer 11

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!

Question 12

Uses in modelling disease?

Answer 12

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

Question 13

the use of iPSc in ALS / Lou Gehrig’s Disease

Answer 13

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

Question 14

What are isogenic cell lines?

Answer 14

use of CRISPR-cas9 to introduce disease-associated mutations into iPS cells then compare the two patient lines

Question 15

the use of iPSc modeling in Alzheimer’s disease

Answer 15

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

Question 16

Cell replacement Therapy in Parkinson’s Disease & why it was problematic

Answer 16

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

Question 17

the use of iPScs instead for Cell replacement in PD

Answer 17

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)

Question 18

Issues with the reprogramming of iPSc lines into somatic cells

Answer 18

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

Question 19

What seems to be preferred nowadays to iPSc lines in models

Answer 19

Isogenic cell lines

Question 20

iPS Vs iN

Answer 20

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

Question 21

what does iN stand for?

Answer 21

induced Neuron

Directly changing a cell from one somatic cell type to another

Question 22

What is transdiffrentiation?

Answer 22

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

Question 23

Describe the direct cell type conversionof fibroblasts to iN

Answer 23

• Circumvents rejuvenation and preserves hallmarks of cellular aging
• Rapid differentiation and maturation
• Retain their age- good for modelling neurodegenerative diseases

Question 24

Limitations of iN use

Answer 24

• 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)

Question 25

What are the BAM factors?

Answer 25

Transcription factors Ascl1, Brn2 and Myt1l

Question 26

What do BAM factors do to iN cells?

Answer 26

Induce them