Cockburn Lectures

Question 1

What are the core properties of stem cells?

Answer 1

1) Self-renewal: Immortal, or at least capable of many cycles of division.

2) Multi-potency: ability to differentiate into different types of cells.

Question 2

The HayFlick Limit

Answer 2

• Normal cells stop replicating after a certain number of divisions.
• Mixed young and old cells and saw that the old cells did not regain the ability to divide, proving the limit was internal. Telomere shortening was found to be the cause, explaining why cells stop dividing.

Question 3

How would multi-potency discovered?

Answer 3

The discovery of multipotency began with observations that mice exposed to radiation developed anemia and died. However, when these mice received bone marrow from another mouse along with radiation, they survived. In a follow-up experiment, mice were given radiation and a small amount of bone marrow from another mouse, after which their spleens were harvested. Each nodule on the spleen contained a mixture of erythropoietic cells (red blood cells), megakaryocytes (platelets), and immune cells, such as neutrophils and macrophages. The cells within each nodule shared the same karyotypic abnormality, indicating that each nodule originated from a single hematopoietic stem cell. This experiment demonstrated the existence of multipotent stem cells, which are capable of differentiating into multiple types of cells.

Question 4

Embryonic Stem Cell (ESCs)

Answer 4

• Derived from the inner cell mass of the early mammalian blastocyst (structure formed a few days after fertilization). The blastocyst has two main parts: inner cell mass (ICM - source of ESCs) and the trophoblast (forms the placenta).
• ICM cells can become any cell type in the body, marking them as pluripotent. These cells eventually develop into all tissues and organs of the body.
• Capable of indefinite self-renewal and maintaining an undifferentiated state in lab conditions.
• Gastrulation generates the three embryonic germ layers (the cells of ICM go into each).
  1) Ectoderm: forms skin, brain, nervous system.
  2) Mesoderm: muscles, bones, blood
  3) Endoderm: internal organs like lungs and digestive tract.
• ESCs are extracted from the ICM and cultured in lab to keep them in pluripotent state, their ability to differentiate into any cell type from all three germ layers makes them very valuable for research & regenerative medicine.

Question 5

How was Pluripotency demonstrated in Embryonic Stem Cells?

Answer 5

1) when injected into immunocompromised mice, ESCs form teratomas (tumours containing cells from all 3 germ layers), this confirms that ESCs can differentiate into a wide range of cell types.

2) ESCs can spontaneously aggregate in culture to form embryoid bodies; 3D structures contain cell types from all three germ layers.

3) Directed differentiation of ESCs into specific cell types by mimicking developmental signals through precise combinations of growth factors & conditions.

Question 6

Chimerism & Germline Transmission

Answer 6

• ESCs can be injected into a developing embryo, contribution to tissues in the host, resulting in chimera (an organism with cells from different genetic background)

Question 7

What can ESC-derived cells integrate into?

Answer 7

• Epiblast: become the fetus.
• Primitive endoderm: becomes extra-embryonic tissues.
• Trophectoderm: forms the placenta and supporting structures.

Question 8

How is Chimerism a test for pluripotency and totipotency?

Answer 8

• Creating a tetraploid embryo by fusing cells (making it non-viable in forming a complete organism) and combining it with ESCs can lead to a chimera where only ESC-derived cells contribute to the embryo proper.
• Gold standard: successfully forming a full organism from ESCs alone in a chimera demonstrates their complete pluripotent capability.

Question 9

What are the reprogramming factors “Yamanaka Factors” and what are their purpose?

Answer 9

• Set of transcription factors essential for inducing and maintaining pluripotency in adult cells.
• Oct4, Sox2, Klf4 and c-Myc.

Question 10

Purpose of Reporter Assays

Answer 10

• Reporter for “Gene X”: produces an easily detected gene or selectable marker in cells where Gene X is normally expressed.

-Example 1: Easily detected gene (GFP, Luciferase, Galactosidase)
—> Oct4 promoter -> GFP coding sequence, it is green if it expressed OCT4 and if it doesn’t it is not.
-Example 2: Selectable marker (eg. Puromycin resistance gene).
—> If it expresses OCT4 then it also produced puromycin.

• Reporters can be at an: endogenous locus (hard to make, can affect the expression of the gene)

Question 11

How do ESCs participate in nuclear reprogramming?

Answer 11

• ESCs contain factors that can reprogram a T cell nucleus to an embryonic-like state.

Question 12

What are Induced Pluripotent Stem Cells (iPSCs)?

Answer 12

• They are created by reprogramming adult cells, like T cells, back to a pluripotent state.
• Introduces genetic resistance to G418, a marker for successful programming.
• Programmed cells generate a blue colour when X-gal is added, indicating successful reprogramming.
• These cells show pluripotent characteristics, confirmed by their ability to form teratomas and embryoid bodies. BUT they do not support germline transmission or tetraploid complementation, making them less capable than ESCs in creating an entire organism.

Question 13

Applications of iPSCs

Answer 13

• Disease Modelling and Drug Discovery: iPSCs can be generated from patient cells to study diseases in lab and screen potential treatments.
• Patient-specific cell therapy.

Question 14

Where is the most active stem cell population?

Answer 14

• Most adult tissue contain resident stem cells.
• Tissue with the highest turnover have the most active stem cell population.

Question 15

In terms of tissue resident stem cells, define the following terms: Niche, Committed Progenitor Cells, Differentiated cells.

Answer 15

Niche:
- specialized microenvironment.
- provides stimuli that influence stem cell behaviour.

Committed Progenitor Cells:
- cells that have begun differentiating but can still divide.
- Can make a smaller number of cell types.

Differentiated cells:
- Specialized cell types with diverse functions (secretion, absorption, phagocytosis, barrier formation)

Question 16

What are characteristics of Tissue-Resident Stem Cells?

Answer 16

• Located in the intestine at the base of crypts.
• Crypt structure: the top contains differentiated cells, below are committed progenitor cells, the niche is on the bottom left and the stem cells are on the bottom right.
• Stem cells divide frequently, especially in high-turnover tissues like the intestine.
• Stem cells express specific markers (TF, signalling pathways).

Question 17

Describe a positive feedback loop associated with Stem Cells?

Answer 17

• There is high Wnt activity in stem cells which leads to high levels of Lgr5, which is a marker for stem cells in multiple tissues.

Question 18

What are the challenges associated with stem cell markers?

Answer 18

• There is no universal stem cell marker. Often, markers must be used in combination for accurate identification.
• Marker expression can vary based on cell state (e.g., during homeostasis or injury)

Question 19

Stem cells can give rise to various differentiated cell types within the tissue, how can the lineage be traced?

Answer 19

• Cre Recombinase: enzyme that targets specific DNA sequences, and it can be controlled by a specific promoter.
• By fusing Cre to the estrogen receptor (ER), activation is controlled by adding estrogen or tamoxifen, allowing for permanent labelling of the cell and its progeny.
  –> example: cells expressing marker B are stem cells, but differentiated daughter cells do not express marker B.

-> Differentiated cells are labeled with LacZ = goblet cells, paneth cells, enteroendocrine cells.

Question 20

Function of Paneth Cells

Answer 20

• Play a critical role in the stem cell niche.
• Closely associated with stem cells, secreting Wnt ligands and aditional cues (EGF and Notch ligands)
• Mesenchymal cells and other neighbouring cells also contribute important signals for stem cell maintenance and function (other niche components).

Question 21

What are Organoids and what are their applications?

Answer 21

• Organoids: mini versions of organs that can be grown in culture.

1) It is not always easy to study stem cells in an intact tissue (and impossible to study them in human tissues)

2) Patient organoids can be used for drug screening or personalized medicine.

3) Potential source of transplantable tissues for regenerative medicine.

Question 22

What are 3 characteristics that define organoids?

Answer 22

o Self-organization: cells arrange themselves into a 3D structure reminiscent of the organ.

o Multicellularity: the structure consists of multiple cell types found in that organ.

o Functionality: the cells carry out at least some of the functions they carry out in vivo.

they can be derived from tissue resident stem cells or pluripotent stem cells

Question 23

What is a multipotent stem cell?

Answer 23

• can only differentiate into a limited range of cell types within a specific tissue or organ, and moderate potential, example is hematopoietic stem cells (produce blood cells).
• limited to a specific tissue-related cell type.

Question 24

What is a pluripotent stem cell?

Answer 24

• can differentiate into any cell type in the body, high potential, can give rise to cells from all three embryonic germ layers, example is embryonic stem cells (ESCs) and Induced Pluripotent stem cells (iPSCs).
• can form organoids with diverse cell types.

Question 25

What are strategies to generate organoids? (culture conditions)

Answer 25

• Goal is to mimic the 3D environment and structure that cells would encounter in a real organ.
• First need to grow the cells (expand), then the symmetry starts to break (cells start to organize into a more complex structure), and the morphogenesis will take place (cells form shapes resembling tissue structures).
• cell sources:
• single cells: initiates organoid from individual cells.
• multiple cells: uses aggregates for organoid formation.
• pure cell population vs. mixed cell types: some organoids may need a mixture of cell types for complexity.
• Intestinal Organoids: Created from single intestinal stem cells (GFP+).
  Process: Expansion → Symmetry Breaking → Morphogenesis → 3D organoid with differentiated cells.

*Optic Cup Organoids:
- Derived from aggregates of embryonic stem cells, forming complex eye-like structures.

Question 26

Applications of Organoids

Answer 26

• Drug Screening for Cystic Fibrosis
• Cystric fibrosis is caused by mutations in the CFTR gene, which encoded chlorine channel affecting ion and water transport.
• Mutations lead to thick mucus buildup in lungs and gut.
• There are many CFTR mutations, making it hard to predict drug responses.
• Organoid use: allows testing drug efficacy for patients with specific CFTR mutations, helping personalize treatment, especially for rare mutations.

Question 27

What could explain why male cells stop dividing in the following?

Answer 27

• Male cells stop dividing because they have reached their replicative limit, known as replicative senescence, due to telomere shortening over successive divisions. Since replicative lifespan is cell-autonomous, each cell line retains its own division history, meaning the late-passage male cells cease dividing independently of the early-passage female cells, even when mixed together.
• Hayflick Limit.

Question 28

There are four common experiments used to demonstrate pluripotency:
Teratoma assay, Creation of embryoid bodies, directed differentiation and chimerism & germline transmission. Explain how pluripotency would be observed in the results of each of these experiments?

Answer 28

1) Teratoma Assay: Pluripotent stem cells are injected into an immunocompromised mouse, forming a teratoma, a benign tumor containing tissues from all three germ layers (e.g., neural tissue from ectoderm, cartilage from mesoderm, and gut-like cells from endoderm). The presence of these varied tissues confirms pluripotency.

2) Creation of Embryoid Bodies: When pluripotent cells are cultured in suspension, they form embryoid bodies (3D clusters of cells) that spontaneously differentiate into diverse cell types representing all three germ layers. Examining the embryoid bodies for markers of these layers confirms the cells’ pluripotency.

3) Directed Differentiation: Pluripotent cells are exposed to specific conditions or signaling molecules that encourage differentiation into particular cell types. Successfully generating differentiated cell types from all three germ layers (e.g., neurons, muscle cells, and liver cells) indicates the cells’ pluripotent capacity.

4) Chimerism & Germline Transmission: Pluripotent cells are injected into a developing embryo, leading to chimeric animals in which the cells contribute to tissues throughout the body. If these cells contribute to the germline, producing offspring that carry the introduced cells’ genetic material, it confirms pluripotency and full differentiation potential across all germ layers.

Question 29

Explain the concept of reporter assay and how it shows gene activation. How might this be useful in the study of stem cells?

Answer 29

A reporter assay is an experimental technique used to study gene activation by linking a gene of interest to a reporter gene — a gene that produces an easily measurable product, such as luciferase, GFP (green fluorescent protein), or β-galactosidase. When the target gene is activated, the reporter gene is also expressed, producing a detectable signal (e.g., light emission or fluorescence). This signal directly indicates the activation status of the target gene.

In stem cell research, reporter assays are valuable for monitoring specific gene expression patterns associated with different stages of cell differentiation or pluripotency. By attaching reporter genes to markers of pluripotency or differentiation, researchers can observe when and where these genes are activated, providing insight into how stem cells transition between different cell states. This helps in understanding the mechanisms of gene regulation in stem cells and optimizing conditions for their differentiation into desired cell types.