Lecture 22: Direct Reprogramming

Question 1

What did Takahashi and Yamanaka work on?

Answer 1

They worked on the induction of pluripotent stem cells from adult human fibiroblasts by defined factors.

Question 2

___ cells can be made from virtually any ___ cell type

Answer 2

1) iPSC
2) proflierative

Question 3

Is there perhaps a simpler way to make neurons from skin cells?

Answer 3

Yes, this method is called direct reprogramming.

Question 4

What is the process of direct reprogramming?

Answer 4

The researchers used fibroblasts taken from a mouse that was genetically modified so that all neurons will express green fluorescent protein. They then tested a pool of 19 candidate transcription factors.

Question 5

Reprogramming of fibroblasts to induced neurons (iNs) takes only ___

Answer 5

12 days

Question 6

How many transcription factors can make iNS?

Answer 6

They found 5 transcription factors that could make iNs and then they tested different combinations.

Question 7

What did they ultimately find when combining the various transcription factors?

Answer 7

Ultimately they found that one TF was critical (Ascl1) but when it was further combined with Brn2 and Myt1l the neurons were more complex and exhibited a more typical neuronal electrophysiological pattern.

Question 8

What does direct reprogramming sound like?

Answer 8

It is similar to the process of making induced pluripotent stem cells. The Fbx15 promoter is only turned on and only makes Fbx15 protein in mouse pluripotent embryonic stem cells. It is not expressed in any differentiated cells.

Question 9

Why might we want to use direct reprogramming instead of making iPSCs and then neurons?

Answer 9

Saves time and money
Quicker (12 days vs. more than 2 months).
Retention of age-related epigenetic changes.
Epigenetic changes occur either directly to DNA or to the Histones around which DNA is wrapped.
These changes influence the ability for a gene to be transcribed or not.
Transplant neurons without teratomas.

Question 10

Describe age-related epigenetic changes.

Answer 10

As we age our DNA undergoes characteristic age-related epigenetic changes.
We can now even use measurements of these epigenetic changes to accurately predict someone’s age.

Question 11

Direct reprogrammed neurons ___ many of these ___ epigenetic marks

Answer 11

a) retain
b) age-related

Question 12

What happens to epigenetic marks and age-related changes in gene expression during the iPSC reprogramming process?

Answer 12

Epigenetic marks and age-related changes in gene expression are erased during the iPSC reprogramming process.

Question 13

Describe the differences between fibroblast aging genes vs. iPSC aging genes

Answer 13

The red dots are genes that significantly differ between young (<40) and older (>40) patient fibroblasts. In contrast, only one age-related gene is retained after iPS.

Question 14

What technique works well for treatment vs. modeling of aging?

Answer 14

You would want baby/younger cells (iPSC ) for treatment.
You would want older cells to model aging (fibroblasts.
IPSC treatments extend telomeres which helps with preventing aging.
No extension of telomeres with direct reprogramming.

Question 15

The researchers then made iNS from the same ___ fibroblasts that were used to make the iPSCs

Answer 15

18

Question 16

There is ___ overlap between age-related genes found in fibroblasts and those found in iNS? Why is that?

Answer 16

a) little
b) When cell differentiates epigenetic changes occur that are related to the cell’s identity.
Age-related changes also affect the cell broadly.
Cell types are defined by differing epigenetic that either activate or repress sets of genes that are needed for that cell’s differentiation, identity and function.

Question 17

So in each cell-type a subset of specific active genes may also be further altered by ___epigenetic changes

Answer 17

age-related

Question 18

What do altered genes in the iNs point towards?

Answer 18

They point towards impairments in nuclear cytoplasmic compartmentalization (NCC).
So they designed a system to quantify NCC deficits.
GFP with a nuclear export signal.
RFP with a nuclear import signal.
If NCC is disrupted then more RFP will stay in the cytosol and more GFP will stay in the nucleus.

Question 19

What happens to fibroblasts from older patients?

Answer 19

Older patients exhibit worsening nuclear cytoplasmic compartmentalization (NCC).

Question 20

What happens to iNS made from older patients?

Answer 20

They also exhibit worsening nuclear cytoplasmic compartmentalization (NCC).

Question 21

True or False: NCC is not altered in iPSCs from aged patients.

Answer 21

True

Question 22

Are there any drawbacks to iNs vs. neurons that are differentiated from iPSCs?

Answer 22

Fibroblasts have a limited capacity to divide and so the number of iNs one can make is also quite limited. In contrast one can make an endless supply of neurons from iPSCs.
Direct reprogramming exhibits variable efficiency and so one often doesn’t have a pure population of neurons to study.

Question 23

What are some ways to induce age-related phenotypes in iPS cells?

Answer 23

Modify epigenome
Change telomeres
Damage DNA repair enzyme
This work is still in its early days.

Question 24

Direct reprogramming exhibits ___ efficiency and purity

Answer 24

variable

Question 25

What can be used to sort and purify immature neurons before they become too complex?

Answer 25

FACS sorting

Question 26

Analysis of mRNA expression shows ____ neuronal genes and neuronal ontology after sorting

Answer 26

increased

Question 27

Can you also make other cell types from fibroblasts?

Answer 27

Yes, you can make them into other things such as functional astrocytes.
But you need a cell that is proliferative to directly reprogram it.

Question 28

Can you make induced neurons from other cell types?

Answer 28

Yes you can.
You can reprogram human astroglial cells into functional neurons using small molecules to activate downstream gene expression (transcription factors).
In one study they identified drugs that could activate key neuronal transcription factors so they didn’t need to add in those genes.

Question 29

Can we reprogram cells in vivo?

Answer 29

There have been attempts to do this such as reprogramming astrocytes to neuroblasts in the adult brain. They used astrocyte promoter paired with NSC gene and then use a virus to turn on a gene causing it to change into young neurons

Question 30

What is the challenge in reprogramming cells in vivo?

Answer 30

Getting transcription factors into the brain is hard to do efficiently because it’s difficult to cross the blood brain barrier.

Question 31

Reprogramming of astrocytes to neurons is more efficient in ____ mice.

Answer 31

younger mice
This is because there is less convergence in older animals and more coverage in younger mice.
Neurons can also increase doublecortin expression when damaged. Aka “upset neuron”.

Question 32

In what disease could in vivo reprogramming offer therapeutic potential?

Answer 32

Parkinsons’s disease!
One study attempts to reverse a model of PD with in situ mitral neurons. .
Here they lesioned one side of the brain and looked at the dopaminergic neurons.
Then they saw if the direct reprogramming led to more dopaminergic neurons.

Question 33

What is the controversy about some in vivo reprogramming studies?

Answer 33

After lineage tracing (the gold standard to claim direct reprogramming in vivo) was used. It was found that the study where astrocytes were converted to neurons could not be replicated.