Lecture 2: Differentiation

Question 1

What is differentiation?

Answer 1

The process by which a less specialized cell becomes a more specialized cell.

Question 2

Pluripotent embryonic stem cells can be isolated from the inner cell mass of a ____

Answer 2

blastocyst

Question 3

Describe what the 3 germ layers help form/lead to?

Answer 3

Ectoderm = nervous system and skin
Mesoderm = hemopoietic organs
Endoderm = intestines, liver and lungs

Question 4

What if we only want to drive neural differentiation?

Answer 4

Scientists have used studies of development to guide them as they try to figure out how to generate a specific cell type from stem cells.
-One breakthrough In neural differentiation began in 1924 in tadpoles.

Question 5

Describe the tadpole experiment

Answer 5

In 1924, Dr. Spemann and Mangold transplanted the dorsal lip of a non-pigmented tadpole embryo into a pigmented host.
The resulting tadpole had 2 heads! The suggested that the dorsal lip must secrete an “organizer” protein that promotes neural tube formation.

Question 6

Describe the Spemann-Mangold discovery of the Organizer

Answer 6

Transplanted dorsal mesoderm produces a second notochord that induces host ectoderm to form neural tissue via secreted factors.

Question 7

What are the 2 main proteins produced by the dorsal lip that mediate the organizer?

Answer 7

Noggin and Chordin
Many studies have shown that Noggin and Chordin work by blocking BMP from binding to its receptors which normally induce Smad signals that repress neural genes.

Question 8

The TGFB pathway also blocks pro neural genes by ___ Smad signals

Answer 8

inducing

Question 9

What is SB431542?

Answer 9

This is a drug that can block TGFB signaling.

Question 10

How do you further drive ectoderm formation of EBs?

Answer 10

1) Treat pluripotent stem cells with noggin
2) Inhbit TGFB signaling with a drug
This leads to a bunch of neural genes turning on.

Question 11

How long do iPSC-derived human neurons take to mature?

Answer 11

several weeks.

Question 12

Describe how different NSCs give rise to different neuronal populations and glia.

Answer 12

-In development patterning along the rostro-caudal axes is mediated by gradients of FGF and retinoid acid.
- Patterning along the dorsal-ventral axes is mediated by Sonic Hedgehog and BMP signaling.

Question 13

What does one want to do after NSC differentiation?

Answer 13

You would want to further drive specific neuronal differentiation.

Question 14

How long does it take to make astrocytes from ES cells?

Answer 14

It can take 180 days to get astrocytes
and they come after neurons are formed.

Question 15

Neurons, astrocytes, and oligodendrocytes are derived from neural stem cells which are an ___ lineage

Answer 15

ectodermal

Question 16

True or False Microglia is NOT derived from ectoderm?

Answer 16

True!

Question 17

Describe the importance of microglia?

Answer 17

• Immune surveillance (similar to phagocytes, they are the trash man of the nervous system)
• Maintain neuronal homeostasis (provide G.F to neurons).
• Turnover of myelin (get rid of old myelin).
• Respond to brain injuries
  -Clear unwanted extracellular aggregates
• Regulate synaptic plasticity (ability of neurons to make connections and prune synapses, this can go away in AD).
  -Influence blood brain barrier function (provide nutrients to endothelial cells).

Question 18

Where do microglia arise from?

Answer 18

They arise from early hematopoietic cells (mesoderm) within the yolk sac that migrate into the developing brain.
Once micogrlia occupy the brain they self-renew to maintain their numbers.

Question 19

Why are microglia not stem cells?

Answer 19

This is because they can’t differentiate into other cells. They can however, make copies of themselves.

Question 20

iPS-microglia (iMG) protocols have been developed that aim to mimic ___ ____

Answer 20

developmental ontogeny

Question 21

What is an experiment that determined whether the resulting cells (from development) really represents naturally occurring versions of those cells?

Answer 21

One experiment found that >96% of iPS microglia co-express the microglial enriched proteins P2RY12 and TREM2

Question 22

IPS microglia secrete ___ and ___ in response to inflammatory stimuli

Answer 22

cytokines and chemokines

Question 23

IPS microglia can phagocytose relevant brain-derived substrates such as ____ and ___

Answer 23

beta-amyloid and synapses.

Question 24

What is the whole-transcriptome RNA sequence analysis of IPS microglia?

Answer 24

The closer the bands the more similar they are.
1 positive control was that there were 2 groups, Adult fetal MG and IPSC CD16 and CD14.

Question 25

___ approaches can be used to improve and guide the generation of specific cell types.

Answer 25

molecular

Question 26

What does a promoter do?

Answer 26

-It determines what cells will make mRNA from the gene and when.
-So every cell type has some promoters that are active and some that are suppressed.
- Different sets of promoters are active in pluripotent stem cells than neurons.

Question 27

What is green fluorescent protein?

Answer 27

It is a protein used to light up/color areas so we can study them.

Question 28

Describe how you can link an immature neuronal promoter (Doublecortin) to control the expression of GFP

Answer 28

You can modify the Doublecortin gene to code for a different mRNA sequence (GFP) but still have that new sequence driven by the Doublecortin power.

Question 29

What is the FACS machine?

Answer 29

It is a machine that uses fluorescent activated cell sorting of reporter lines and can improve purity.

Question 30

What are the conclusions of lecture 2?

Answer 30

-By understanding normal development we can design better differentiation protocols.
- To make most neuronal/glial cell types requires a long period of time with changing treatments to replicate the changing environment that occurs during development.
- Technological innovations such as small molecular compounds and reporter stem cell lines can further inform this process.