Broccoli: Lecture XIX

Question 1

What does iPSC stand for?

Answer 1

induced pluriportent stem cells

Question 2

What are iPSC?

Answer 2

they are embryonic-like stem cells that have been reprogramed from adult mice cell using the 4 Tfs:

Oct3/4
Sox2
c-Myc
KLF4

Question 3

When was the 1st successful attempt of human pluripotency stem cells?

Answer 3

2007, and it was done without using a reporter gene unlike the mouse

Question 4

What are some drawbacks to the human fibroblast reprogramming?

Answer 4

it is not as efficient as it takes 6 weeks where mice take 2/3 weeks

Question 5

How can we test if human cells are pluripotent?

Answer 5

differentiating them in vitro

transplanting the iPSCs subcutaneously under the skin of an immunodeficient mouse, and see if they proliferate and form teratomas, which shows differentiation

Question 6

What did the harvard study find?

Answer 6

iPSCs are very similar to human ESCs derived from human blastocysts

Question 7

Since retroviruses pose the danger of integrating into the genome and causing genotoxicity, what methods can be used to ensure iPSCs leave no trace of reprogramming?

Answer 7

using “Floxed viral vectors”

using “Episomal vectors”

using “adenoviral vectors”

using Sendai Virus Vector

Question 8

What are floxed viral vectors?

Answer 8

retroviruses with 2 loxP sites that use a Cre recombinase to excise the viral gene and in the end, only 1 remains

*doesn’t work properly or efficiently

Question 9

What are episomal vectors?

Answer 9

plasmids that transiently express 4 TFs, so the iPSCs proliferate a lot

*they lose the vector so this method is not efficient

Question 10

What are adenoviral vectors?

Answer 10

vectors that are not efficient like episomal vectors or floxed viral vectors

Question 11

What is the Sendai Virus Vector?

Answer 11

virus of a ss RNA that expresses the 4 TFs when recombinant, and it then enters the cells and the viral protein only expresses the 4 Yamanaka factors

*no trace is left and reprogramming is efficient

**in 6 weeks, the 1st primary iPSCs are obtained

Question 12

What is an advantage to generating human iPS cells?

Answer 12

we can generate an “in vitro human model” to study the basis of human disorder

Question 13

What is it possible to do with the Sendai kit?

Answer 13

generate iPSCs from peripheral blood mononuclear cells (PBMC)

Question 14

What is the design of the study using human iPSC?

Answer 14

generation of the iPSC from patients and from healthy individuals that are comparable for age and sex

differentiation of the iPSC into the specific cell type

comparison from the healthy cells to the disease cells

Question 15

What are isogenic control iPSCs?

Answer 15

patient’s cells in which we have the corrected disease mutations and serve as a control to compare the patient’s cells with the mutation

Question 16

What is a disease that has used iPSC modeling in vitro?

Answer 16

parkinson disease

Question 17

Lewy bodies = ?

Answer 17

aggregates = fibrils of 𝛼-synuclein protein

Question 18

What are the neurons that die in parkinson’s?

Answer 18

dopaminergic neurons

Question 19

Why are the aggregates formed in Parkinson’s toxic?

Answer 19

they block endocytosis, ER trafficking to golgi, block trafficking to synapses, etc.)

Question 20

Neurodegenerative diseases are sporadic and have a very small genetic contribution. What is an exception?

Answer 20

Parkinson’s as 50% of the cases are genetically inherited and caused by mutations in single genes

Question 21

How many genes are known to cause familiar genetic forms of Parkinson disease, which are generally more severe and arise much earlier?

Answer 21

15 genes

Question 22

What is another way a person can develop parkinson’s?

Answer 22

mutation (1 or 2 copies) in the OPA1 gene

Question 23

What was found in OPA1-Parkinson’s neurons?

Answer 23

the neurons had fragmented mitochondria

• OPA1 is responsible for the fusion of the mitochondria

Question 24

What was a hypothesis that was found using the microfluid chip (very small chambers where you can grow different populations of neurons)?

Answer 24

PD patients with an OPA1 mutation have mitochondria that cannot move along an axon and reach their synapse, so the synapse will degenerate and can lead to the degeneration of dopaminergic neurons

Question 25

Why can’t iPSC derived from dopaminergic neurons be transplanted into the substantia nigria?

Answer 25

in the adult brain, these neurons will have a hard time regenerating the connectivity to send to their axons and connect to the basal ganglia as this connection is established during embryogenesis

*mature brain has lots of lipids, due to oligiodendrites, which block the growth of axons

Question 26

What is an alternative proposition since DA neurons cannot be transplanted into the substantia nigra?

Answer 26

transplant the DA neurons into the striato (basal ganglia) so that the neurons can differentiate, release dopamine, and rescue the neuropathy

Question 27

What is currently in clinical trial in regards to Parkinson’s?

Answer 27

transplanting DA neurons into the striato (basal ganglia)

Question 28

Describe how 3D models are being used?

Answer 28

there are many 3D protocols that have been written to differentiate iPSC into intestinal, kidney, brain, spinal cord, or retinal organoids

Question 29

Scientists created an assembloid to study the brain and how it makes neural connections. What is an assembloid?

Answer 29

2 organoids from different neuronal cell types that are fused together

Question 30

Describe the retinal organoid:

Answer 30

it is an organoid that was developed from the neuronal tube that fully formed photoreceptors

*in the future, the idea is to restore the visual activity of patients where there is a genetic loss of these cells

Question 31

What generates the liver?

Answer 31

interior endoderm (on end of the germinal layers)

Question 32

What did the liver organoid demonstrate?

Answer 32

the human and mouse vessel were able to fuse and the human hepatocytes were differentiated and released human albumin that was detectable in the blood of mice

*indicates the transplant was becoming a functional piece of human liver which release liver enzymes into circulation