L7 Stem Cell Niches I

Question 1

What must a stem cell niche have?

Answer 1

Both anatomic and functional dimensions i.e. a local tissue microenvironment that hosts and influences the behaviour of characteristics of stem cells.

Question 2

Where did the concept of a niche come from?

Answer 2

In 1978, Schofield noticed that haematopoietic stem cells needed to reside in the bone marrow in order to maintain their infinite potential. His first proposal of the niche hypothesis included:

• Must have a defined anatomical location.
• Regulates self-renewal.
• Removal from the niche results in stem cell differentiation.

Question 3

Why is it useful to comprehend the function of stem cell niches?

Answer 3

This will enable the design of improved treatments and better research techniques as we cannot maintain adult stem cells for prolonged periods of time in vitro).

Question 4

Describe the Drosophila testis niche.

Answer 4

The Drosophila testis niche is a tube-like structure which is closed at one end (apex) and at the other has non-mitotic cells called hub cells. These are in constant contact with germ-line stem cells and somatic stem cells (these surround the germ-line stem cells).

The hub cells produce a signal that keeps germline cells in a self-renewing state, but they will divide asymmetrically to produce one germline stem cell and another that will differentiate and form a gonialblast. This will eventually form sperm.

Question 5

How are germline and somatic/cyst stem cells attached to the hub cells?

Answer 5

Through adhering junctions.

Question 6

Describe the interacting signals between the Hub, Somatic (Cyst) and germline stem cells.

Answer 6

• Hub cells produce a signal called unpaired ligand (Upd) which leads to JAK/STAT activation in both germline and somatic/cyst cells.

For the somatic stem cells, this is enough to keep them in a self-renewing state.
For germline stem cells, this signal is important but not sufficient.

• Gbb (glass bottom boat) and Dpp are BMP ligands that are produced by both hub and somatic cells and lead to BMP activation.

The BMPs then act through Mad to repress transcription of the gene Bam. This stops transcription of differentiated genes.

Question 7

Describe the Drosophila ovary niche.

Answer 7

At one end there are terminal filament cells followed by cap cells (only 4-8 of them). These are similar to hub cells in male Drosophila. The cap cells are in contact with germline stem cells via altering cell junctions and lie close to the escort stem cells.

Question 8

Explain BMP activation in the Drosophila ovary niche.

Answer 8

BMP activation is both required and sufficient.

• Gbb and Dpp in cap cells causes BMP activation in germline stem cells.
• BMPs then act through Mad to suppress transcription of Bam.
• NURF also inhibits Bam in germline stem cells.

Question 9

What is asymmetrical division and why is it important in the Drosophila niche?

Answer 9

This is where one daughter cell remains in contact with the niche and is an exact replica of the original cell, and the other moves away from the niche and, as it doesn’t receive signals anymore, differentiates.

This is important because it allows only the stem cells to be in contact with the main signals from the niche and means the stem cells are not exhausted.

Question 10

List the stem cell niches in the human body - which have a constant turnover?

Answer 10

• Bone marrow.
• Intestine.
• Basal layer of the epidermis.
• Hair follicle.
• Subventricular zone and subgranular zone in the brain.
• Skeletal muscle.

The top 4 have a constant turnover.

Question 11

What are the two types of HSCs?

Answer 11

1) Quiescent (not cycling). These cells are not dividing and are necessary for prevention of cell exhaustion.
2) Cycling HSCs.

Question 12

How can you determine between cycling and quiescent HSCs?

Answer 12

You can use pulse-chase experiments to determine between quiescent and cycling HSCs.

You feed cells a media with a label (such as BRDV) that is incorporated into the DNA when cells are replicated. Cells are kept in this medium for long enough for all of the cells to be replicated and carry the label.

Over time, the proliferating cells will dilute the label, and the quiescent cells will retain it.

You can also use time-lapse microscopy to visualise the cells as they divide.

Question 13

What are the two main stem cell niches in the bone marrow?

Answer 13

1) Endosteal is between the bone and marrow and is where the quiescent cells are found.
2) Perivascular is closer to the vasculature and is where the cycling cells are found.

Question 14

What are the three main interactions in HSC niches and why are they important?

Answer 14

1) Cell-cell interactions. The niche contains stem cells as well as stromal support cells. They interact with each other via cell-surface receptors, gap junctions and soluble factors.
2) Cell-ECM interactions. These interactions provide an anchor for the cell to the niche. They also provide signals and are a platform for signalling.
3) Cell-soluble signal interactions. These can be secreted by the stem cells themselves or other cells (these would be paracrine factors). Studies have shown that some of these factors are required to maintain stem cell identity.

Question 15

What is the role of macrophages in HSCs?

Answer 15

Macrophages are thought to regulate nestin-positive mesenchymal stem cells.

If macrophages are ablated from the bone marrow, the nestin mesenchymal stem cells are still present, but are no longer able to give signals to the stem cells.

Question 16

What do mammalian stem cell niches provide and do?

Answer 16

They provide physical support, soluble factors and cell-mediated interactions to maintain and regulate the function of stem cells.

Question 17

What question did Calvi et al (2003) attempt to answer and how?

Answer 17

They asked the question ‘do osteoblasts contribute to the unique micro-environment in the bone marrow?’

We know that osteoblasts are activated by parathyroid hormone (PTH) through the receptor PPR. Therefore, Calvi et al (2003) used an experimental model consisting of a transgenic mouse with a constitutively active PPR receptor.

Question 18

What did the results of Calvi et al (2003)’s experiments show?

Answer 18

The transgenic mice had generated a lot more bone than the WT. The HSC numbers were also increased. However, this could have been due to the direct effect of PPR on HSCs, or the indirect effect of stromal cells (osteoblasts) on HSCs.

In order to test how the increased numbers of HSCs came about, they tested the ability of stromal cells to support HCSs in vitro. The cells from the transgenic mice were better performing indicating that it was indeed the stromal cells that were increasing the amount of HSCs.

They then replaced a constitutively active PPR receptor with the exposure of WT stromal cells to PTH. Not only did the osteoblast numbers increase, but it led to a survival advantage after bone marrow transplants.