Lecture 3 - Pluripotent stem cells

Question 1

What are pluripotent stem cells (PSCs)?

Answer 1

Cells that have the ability to give rise to all cells of an embryo and adult, e.g., embryonic stem cells

Pluripotent stem cells include both embryonic stem cells and induced pluripotent stem cells.

Question 2

What is the capacity a stem cell must have to be functionally defined as a stem cell?

Answer 2

Capacity to:
* self-renew
* be clonal
* produce progeny with more restricted potential

Self-renewal refers to generating a daughter cell identical to the mother cell.
Clonal - single cell can generate more stem cells and differentiated progeny

Question 3

List the locations where stem cells can be found.

Answer 3

• Fertilised egg
• Early embryos
• Umbilical cord
• Adult tissues and organs
• Cancers

Question 4

What does totipotent mean?

Answer 4

Can form all tissues required for reproduction, e.g., fertilized egg

Question 5

Define multipotent stem cells.

Answer 5

Can form a limited number of cell types, e.g., most adult stem cells, HSC, NSC

Question 6

What are unipotent stem cells?

Answer 6

Can only form one type of cell, e.g., committed progenitor cells

Question 7

What is the significance of the inner cell mass in embryonic stem cells?

Answer 7

It is pluripotent and derived from the blastocyst stage of the embryo.

Cultured and colonies grown.

Question 8

What role does LIF (leukaemia inhibitory factor) play in mouse embryonic stem cells?

Answer 8

It maintains mESC in an undifferentiated, pluripotent state without feeders.

LIF plus serum in undifferentiated state.

Question 9

How is pluripotency maintained during embryonic stem cell self-renewal?

Answer 9

Through promotion of proliferation and suppression of differentiation.

Suppression of differentiation: extrinsic signals (growth factors and cytokines), signalling pathways, intrinsic factors (transcription factors)

Question 10

What are the key transcription factors expressed in human embryonic stem cells?

Answer 10

• Oct-4
• Sox-2
• Naong

Question 11

What are the challenges in culturing human embryonic stem cells compared to mouse embryonic stem cells?

Answer 11

Human ESCs dislike being separated into single cells and grow more slowly.

Question 12

What is the purpose of using feeder cells in stem cell culture?

Answer 12

To provide the best growth conditions for embryonic stem cells.

Question 13

What is the main difference between naive and primed pluripotent stem cells?

Answer 13

Naive stem cells are in a stable, undifferentiated state, while primed stem cells are closer to differentiation.

Question 14

What is the significance of the 2i conditions in mouse embryonic stem cell culture?

Answer 14

Dual inhibition of MEK and GSK-3 allows mESCs to enter a stable naive pluripotent state.

Mouse ESCs cultured in 2i conditions enter ‘ground state’ which is the naive pluripotent state.

mESCs cultured in 2i and LIF have uniform expression of key pluripotent transcription factors.

Question 15

Who was the first to derive human embryonic stem cells?

Answer 15

James Thomson in 1998.

Question 16

What are induced pluripotent stem cells (iPSCs)?

Answer 16

Stem cells generated from adult cells that have been reprogrammed to an embryonic stem cell-like state.

Question 17

What combination of genes is important in generating induced pluripotent stem cells?

Answer 17

• Oct-4
• Sox-2
• c-myc
• Klf-4

Question 18

What is the ethical concern regarding human embryonic stem cells?

Answer 18

They require the use of fertilized eggs, with strict regulations in place.

In UK, only IVF cells can be used.

Question 19

True or False: Human embryonic stem cells can be maintained in LIF.

Answer 19

False

Serum is required.

Question 20

What do BMP2 and BMP4 do in relation to mouse embryonic stem cells?

Answer 20

They work together with LIF to maintain pluripotency.

Question 21

Fill in the blank: The most famous sheep in the world, known for its role in induced pluripotent stem cells, is named _______.

Answer 21

Dolly

Question 22

What are iPSCs?

Answer 22

Induced pluripotent stem cells

iPSCs share many features with embryonic stem cells.

Chromatin structure differences between cell types

Question 23

Who were the first to induce pluripotent stem cells from mouse fibroblasts?

Answer 23

Takahashi, K. & Yamanaka, S.

Their study was published in Cell in 2006.

Question 24

List the four transcription factors used to reprogram mouse fibroblasts to pluripotency.

Answer 24

• Oct-4
• Sox-2
• c-myc
• Klf-4

c-myc is an oncogene.

Subsequent reprogramming achieved with fewer factors, Oct4, Sox2, Klf4.

Question 25

What is the efficiency of reprogramming fibroblasts into ES-like cells?

Answer 25

Approx 1 in 5000 cells reprogrammed (0.0002%) ## Footnote Based on a study by Wernig, M. et al. in 2007. Need to seed at low density, otherwise non-ES colonies predominate.

Question 26

How many days does the reprogramming process typically take?

Answer 26

30 days

Question 27

What is a potential application of iPSCs in medicine?

Answer 27

Cell replacement therapies ## Footnote This is part of regenerative medicine.

Question 28

What does 'Disease in a dish' models refer to?

Answer 28

Patient and disease specific iPSCs

Question 29

What percentage of interventional trials are using ESC-derived cells?

Answer 29

73%

Question 30

What region accounts for 60% of interventional trials targeting pluripotent stem cells?

Answer 30

Southeast Asia

Question 31

True or False: 77% of the classified clinical trials involving PSCs are observational.

Answer 31

True

Question 32

What is the significance of the study by Deinsberger, J. et al. (2020)?

Answer 32

It analyzed global trends in clinical trials involving pluripotent stem cells.

Question 33

Fill in the blank: iPSCs can be used in _______ to test drug efficacy.

Answer 33

drug discovery

Question 34

What publication discussed pluripotent stem cells and clinical trials in 2008?

Answer 34

Cell Stem Cell

Question 35

What is the main focus of the paper by Lin, X.; Tang, J.; Lou, Y.-R. (2021)?

Answer 35

Human Pluripotent Stem-Cell-Derived Models in Drug Discovery

Question 36

Do stem cells typically stay undifferentiated?

Answer 36

No, stem cells are hard to keep undifferentiated. Each differentiation stage losses cells (cell death).

Question 37

What is a blastocyst?

Answer 37

A hollow ball of 40-80 cells, consisting of an outer trophectoderm layer and inner cell mass.

Question 38

What is the blastocoel?

Answer 38

A fluid filled cavity within the blastula.

Question 39

What may transplanted embryonic stem cells cause?

Answer 39

Transplanted ES cells can form tumours called teratomas.

Question 40

What are MEFs?

Answer 40

Mouse embryonic fibroblasts.

Question 41

What does mitomycin C do?

Answer 41

Inhibits DNA synthesis and cell proliferation.

Question 42

Function of MEFs in cell culture

Answer 42

ICM grown on MEFs in conditioned cell culture and colonies form. ## Footnote ICM from diapuased blastocyst (E3.5) 1950s experiment - updated procedure now with BMP2/4 instead of conditioned media to reduce teratocarinoma.

Question 43

LIF signaling pathway | Diagram

Answer 43

Question 44

What is the issue with serum?

Answer 44

Differs between batches, instead take components and use to proliferate ES cells (BMP2/4)

Question 45

How does BMP4 phosphorylate Smads to inhibit differentiation? | Diagram

Answer 45

Question 46

Promotion of ES cell differentiation diagram | Diagram ## Footnote Oct4, FaF4. FGFr, etc...

Answer 46

Question 47

Which is main factor produced by feeders?

Answer 47

LIF (aka Differentiation inhibitory activity, Dia)

Question 48

What happens if signalling pathways activate LIF?

Answer 48

Contibute to maintenance of self-renewal.

Question 49

STAT3 as a transcription factor

Answer 49

Activated STAT3 maintains pluripotency without LIF. Dominant negative forms of STAT3 reduce self-renewal.

Question 50

What does BMP 2/4 bind to cause self-renwal?

Answer 50

TGFbR2, activating Smad4, which activates Id causing self-renewal. ## Footnote BMP = bone morphogenic protein Smad = transcription factor Id = inhibitor of differentiation, transcription factor

Question 51

Wnt signalling pathway | Diagram

Answer 51

## Footnote * In the presence of Wnt signalling, glycogen synthase kinase 3 is inhibited * This allows stabilisation of -catenin which accumulates in the nucleus * b-catenin relieves TCF-3 repression of transcription and reinforces self- renewal * Small molecule inhibitors of GSK-3 maintain pluripotency of mESCs * ESCs lacking b-catenin still self- renew * Wnt3a doesn’t support self-renewal of mESCs

Question 52

What else is required for maintaing pluripotent state? Other than LIF

Answer 52

LIF, inhibition of FGF receptor and MEK replaces requirment for BMP/serum

Question 54

Features of hESCs

Answer 54

* More difficult to culture than mESCs - don't like being separated into single cells * Need layer of 'feeder' cells * Cannot be maintained in LIF, required FGF-2 activin A * Express Oct4, Sox2 and Nanog

Question 55

Overview of iPSC derivation | Could be diagram

Answer 55

1. Choice of factors 2. Methods of factor delivery 3. Choice of cell type 4. Parameters of factor expression 5. Derviation conditions 6. Identification of iPSC colonies 7. Expansion and characterisation