Exploring mental health using stem cells

Question 1

What are IPSCs?

Answer 1

Induced Pluripotent Stem Cells

• capacity to generate different cell types: foetal and adult bodies
• > nervous system, heart and circulatory systems, muscles

Question 2

What are pluripotent cells?

Answer 2

Cells with capacity to generate different cell types

• foetal and adult bodies
• > nervous system, heart and circulatory systems, muscles

Question 3

Which cells have an ephemeral (temporary) pluripotent property?

Answer 3

Inner cell mass cells, in the blastocyst

• these cells are incredibly ephemeral
• > their pluripotency only lasts a few days and never reappears during the entire life cycle
• > hard to study

Question 4

Which cells have a permanent pluripotent property?

Answer 4

Embryonic stem cells

• derived directly from inner cell mass
• permanent pluripotency
• they can give rise to all different cell types that make up the body

Question 5

Who initiated the discovery of the biological basis for pluripotency?
What was his/her experiment?

Answer 5

John Gordon
> Used enucleated egg (nucleus destroyed with radiation) containing pluripotent cells of Frog A
> Implanted nucleus of Frog B into egg -> egg with transplanted nucleus
> Egg develops into tadpole -> clone of Frog B
-> pluripotent constructed cell

• > Nucleus, even from fully differentiated cell, had all info. to generate an organism
• > There are factors in the enucleated egg that, once combined, tell the nucleus that it has to start behaving as a pluripotent cell

=> Existence of factors in the cytoplasm of pluripotent cells that dictate pluripotency

Question 6

Who found the biological basis for pluripotency which led to the discovery of iPS cells?
What are the Yamanaka factors?

Answer 6

Shinya Yamanaka
> Suggested the factors must be gene products = proteins
-> List of 24 potential pluripotency factors

Takahashi and Yamanaka (2006, 2007)
> Step 1: Have an assay for pluripotency: a way to recognise he’d produced pluripotency in cell that weren’t pluripotent
- pluripotent cells always seem to have the Fbx15 gene active -> a reporter for pluripotency (if cells turn blue they’ve become pluripotent)
- transfection of fibroblasts with the 24 pluripotency factors
-> within these 24 factors are the important ones leading to pluripotency

> Step 2:

• Repeated experiment several times, leaving a factor out each time
• factors needed to induce pluripotency vs. factors not needed

> Step 3:
- Repeated experiment with the 10 remaining factors, sequentially leaving one out at a time

=> Yamanaka factors: Oct3/4, Sox2, KIf4, c-Myc

• if he uses these 4 factors, he gets blue cells, believed to be pluripotent ; leaving out 2 of them, he doesn’t get blue colonies
• > all of the 4 remaining factors are needed to generate pluripotent cells
• > these 4 factors are sufficient on their own

> Step 4:

• generate embryoid bodies with 3 germ layers: ectoderm, mesoderm, endoderm
• it is still a hypothesis that the blue cells are pluripotent

> Step 5:

• take fibroblasts that had been transduced with the 4 factors, grow them into embryoid bodies and show each germ layer is represented in the embryoid bodies
• histological sections of mice confirmed the cells had contributed to creation of all different body tissues = iPSCs
• fibroblasts transduced with the 4 factors, injected in blastocyst, significantly contributed to the formation of germ line -> made it possible to produce mice that were entirely derived from these

=> Generate a whole line of mice derived from those transduced fibroblasts which were indeed pluripotent

Question 7

What happened after Takahashi and Yamanka’s 2006 experiment on mouse (skin) fibroblasts?

Answer 7

> 2007: Takahashi and colleagues: human (skin fibroblasts)
Onwards: Yamanaka’s procedure accepted worldwide
- changes to protocol

Question 8

How was the generation of IPSC lines used for the study of childhood disorders?

Answer 8

iPS cells can be derived from any cell type in the body:

• blood cell sample
• urine sample
• hair sample -> suitable for the study of childhood disorders (e.g. autism)

Hair keratinocytes

• reprogramming with Yakamana factors (Oct3/4, Sox2, KIf4, c-Myc)
• > iPS cell colonies

Question 9

How to make iPS cells to create neurons?

Answer 9

1. Collect sample: blood cells, urine, hair
2. Reprograming using Yakamana genes (Oct3/4, Sox2, KIf4, C-Myc)
3. You get iPS cell colonies
4. iPS cell line
   - used in the study of brain development disorders
5. Neural ‘rosettes’ - progenitor cells
6. Neurons

Question 10

Which genes are the Yakamana factors?

Answer 10

• Oct3/4
• Sox2
• KIf4
• C-Myc

Question 11

What is the cultured neuralisation timeline?

Answer 11

> Day 1: iPS cell lines

• SMAD signalling pathway inhibition to induce the iPS cell lines to produce neuroepithelium
• > add SMAD inhibitors to the culture of these induced pluripotent cells

> Day 21: neural ‘rosettes’
- progenitor cells that can do tissue histogenesis (compared to NSCs found in other contexts)
- proper polarised neuroepithelium -> cells are trying to make a two-dimensional neural tube
= two-dimensional cell culture (in vitro)

> Day 28 to 35: young neurons

> Day 35 to 53: mature neurons

Question 12

What is reassuring about the cultured neuralisation and iPSCs technology?

Answer 12

It is a slow process (53 days to get mature neurons)
- reassuring because it reproduces the timing and the differentiation processes that seem to underly human neural development

=> iPSCs allow us to look at human neural development in a cultural dish

Question 13

What is histogenesis?

Answer 13

Processes that begin with the generation of neuroblasts, to the morphological and biochemical differentiation of mature neurons in the cortical plate

Question 14

What is specific about the histogenesis of iPSCs?

Answer 14

• If you allow cell development, the iPS cells try and undergo proper histogenesis
• iPS cells have this capacity for histogenesis remarkably larger than any other neural developing systems in vitro

Question 15

Why does the cultured in vitro differentiation process and neural development ceases after cerebral tissue is developed?

Answer 15

Lack of blood supply which does not exist in the culture dish

Question 16

What is a cerebral organoid culture?

Answer 16

Cortical structure developed entirely from iPS cells growing in a culture dish

Question 17

What are the possible studies using iPSCs to address questions around the aetiology of neurodevelopment disorders?

Answer 17

> Compare lines from patients and controls

> Induce mutations in iPSCs
- inducing genetic variation (genome editing) into cells in vitro: CRISPR-cas9 system ; ZNFs ; TALENs

> Study environmental risk factors

• e.g. autism risk increases if the mother suffers from influenza during her first trimester
• pro-inflammatory cytokines -> altered neurodevelopment

Question 18

What kind of assays / phenotypes are we going to run on iPSCs to detect differences?

Answer 18

> Gene expression

• patients or controls
• genetic mutations or exposure to environmental risk factors
• study differences in expression during developement

> Physiological

• iPS cells eventually become electrophysically active
• they develop the channels and receptors commonly present in human neurons

> Morphogenetic
- compare histogenesis of iPSCs derived from patient cells vs. control cells

Question 19

How did Pasca and colleagues (2011) use iPSCs to study disease pathophysiology?

Answer 19

Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy Syndrome
(caused by point mutation in CACNA1C and encodes a sub-unit of a calcium channel)
> Patients vs. controls in terms of neurons and behaviour of the calcium channel
> iPS cells showed a predicted phenotype: carry mutation we know is associated with the disease
> They tried to generate cortical neurons
> In the differentiated iPS cells, authors used different markers to know if they got the normal distribution of upper-layer vs. infragranular cortical neurons

• > Neurons derived from Timothy syndrome iPS cells had a greater propensity to make upper-layer neurons, and reduced propensity to make lower-layer neurons
• > Smaller proportion of the lower-layer cells showed expression of SATB2
• these lower-layer cells take either one of 2 fates:
  1. Subcortically projecting neurons: project to subcortical brain regions
  2. Callosal projecting neurons (with SATB2-positive cells): project across the corpus callous to the cerebral cortex on the other hemisphere

=> Neurons from Timothy syndrome patients have a lower proportion of the SATB2-positive cells
- lower proportion of callosal projecting neurons

> Transgenic mouse engineered to carry the mutation found in Timothy syndrome

• > same histogenic phenotype in the cortical structure of the mouse as seen in cells carrying the calcium channel mutation
• > lower number of SATB2 positive cells in lower layer of cortex of the mouse compared to controls

Question 20

What are the advantages of cellular models of neurodevelopmental disorders?

Answer 20

> True human cells used
> Good construct validity
> Good controls
> Tractable system
> High through-put screening
> Able to be manipulated genetically and phenotypically

Genome editing makes it possible to remove particular mutations from cells
e.g. with CRISPR-cas9

Question 21

What are the disadvantages of cellular models of neurodevelopment disorders?

Answer 21

> Variability: genetic and epigenetic differences between individuals
- higher variability in human cells when compared to cells derived from mice, which can be genome controlled to become genetically identical

> System properties inaccessible
- disorders don’t concern the property of a single cell population, but of properties of the brain as a whole

> Slow development
- long wait required for the development of iPS cells is a disadvantage in terms of practicability and logistics

> No behaviour

• iPS cells lack the behaviour phenotypes present in several neurodevelopment disorders (e.g. autism)
• > to what extent the cellular and molecular phenotypes observed in culture actually relate to human behaviour

=> To observe behavioural changes, animal models are still needed