Lecture 7: Organoid Systems

Question 1

What do Cadherin-mediated stem cell-niche adhesions do?

Answer 1

Cadherin-mediated physical cell-cell adhesion helps anchor stem cells to their niche.

-catenin and -catenin, which associates with the intracellular domain of cadherins

help to cluster cadherin molecules and form adherens junctions (AJs).

Question 2

What do Integrin-mediated stem cell-niche adhesions do ?

Answer 2

Integrin-mediated cell-extracellular matrix (ECM) interactions help anchor stem cells to the niche,

Niche contains a number of ECM components.

The intracellular domains of integrins interact with the actin cytoskeleton network through talin proteins to cluster integrin molecules together.

Question 3

What do Cadherin/integrin-mediated stem cell-niche adhesions do ?

Answer 3

Cadherin-mediated cell interactions and integrin-mediated cell-ECM interactions can work together to anchor stem cells to the niche.

Question 4

Non-cadherin/integrin-mediated stem cell-niche adhesion

Answer 4

In addition to cadherins and integrins, other molecules, such as Delta/Notch, SCF/c-Kit, CD44/hyaluronic acid (HA) and gap junction components, are also involved in stem cell-niche adhesion.

Question 5

What does direct/ facilitating signalling for adhesion molecules do?

Answer 5

Cadherin and integrin complexes can signal directly downstream or facilitate receptor-mediated niche signalling to regulate stem cell self-renewal, proliferation and survival.

Question 6

What can asymmetric cell division and polarity do ?

Answer 6

Cadherin and integrin molecules are required to regulate asymmetric cell division and possibly to maintain cell polarity; the actin cytoskeleton network associated with cadherins or integrins helps anchor one centrosome to the apical side of the cell to ensure that its mitotic spindle is always orientated perpendicular to the niche surface.

Question 7

What does competition and quality control in adhesion molecules entail?

Answer 7

They send signals that regulate stem cell renewal, proliferation and survival.
Also asymmetric cell division and polarity.

Question 8

How does again affect stem cells ?

Answer 8

The expression levels or functions of adhesion molecules in stem cells and niche cells are affected by aging. The adhesion between stem cells and their niche is, therefore, also affected by aging.

Question 9

What is one of the best-defined adult stem cell niches?

Answer 9

The mammalian gut crypt (at the base of the villi). The crypt is a tube of cells with stem-like cells in a niche at the distal end and differentiated cells at the proximal end.

A portion of the basement membrane in the stem cell region may be specialised. Stem cell progeny known as transit amplifying (TA) cells move upwards and differentiate. Underlying mesenchymal cells send signals that help regulate stem cell activity.

Question 10

What is the role of Enterocytes ?

Answer 10

Uptkae ions, water, nutrinets, vitamins and absorption of unconjugated bile salts.

Question 11

What do Enteroendocrine cells secrete?

Answer 11

Secrete hormones, such as GLP-1 and GLP-2, PYY, CCK and seretonin.

Question 12

What do Goblet cells secrete ?

Answer 12

Mucin and create a protective mucus layer

Question 13

What do Tuft cells do ?

Answer 13

They monitor intestinal content using succinate and sweet/ bitter taste receptors.

Question 14

What do Paneth cells do?

Answer 14

They have highly specialised secretory functions to regulate the composition of the intestinal flora and cell populations. They produce EGF, Notch and Wnt and alpha-defensins.

Question 15

What do Wnt signals do in the crypt ?

Answer 15

Wnt signals are highest at the bottom of the crypt, where the Intestinal stem cell (ISC) population resides. These signals are critical for maintaining the stem cell population and promoting their self-renewal.

Question 16

What do BMP signals do in the crypt?

Answer 16

BMP signals are highest at the top of the crypt, where the differentiated cells are located. BMP signals promote differentiation of progenitor cells into specialized cell types.

Question 17

What do EGF signals do in the crypt ?

Answer 17

EGF signals are highest in the middle of the crypt. These signals promote the proliferation of both ISC and progenitor cells.

Question 18

Where are Spacial gradients formed ?

Answer 18

Spatial gradients of Wnt, BMP and EGF are formed along the crypt axis

Question 19

Why do Lgr5+ intestinal CBC cells adhere to Paneth cells ?

Answer 19

They do this to receive signals for stem cell maintenance

Question 20

What are EGF, Notch and Wnt essential for ?

Answer 20

They are essential for intestinal epithelial stemness whereas BMP negatively regulates stemness. For full Wnt activation in the intestinal epithelium, R-spondin-Lgr4/5 signal is required.

Question 21

How are Adult stem cell-derived Organoids made from gut stem cells?

Answer 21

1. Lgr5+ stem cell is isolated. It is found in adult small intestine, in the crypt of the villus and cryot is isolated to retrieve it.
2. It is put inside a Matrigel matrix
3. Development of organoids take place
4. This makes epithelial organoids (Enteroids) which then dissociate into stem cells
5. Creates an organoid derived-monolayer

Question 22

How are single stem cells sorted from the gut epithelium ?

Answer 22

Using FACS (Fluorescent activated cell sorting)

Question 23

What is the mechanism of the self-organising architecture of a mini-gut ?

Answer 23

An organoid derived from Axin2-LacZ knock-in mice. Axin2-LacZ (blue) expression recapitulates Wnt activation.

Wnt activation induces local proliferation and EphB expression. Local cell expansion and Eph-Ephrin repulsive force generate bud formation.

Question 24

How normal are the epithelial mini-guts?

Answer 24

In terms of normality, epithelial mini-guts closely mimic the cellular composition and architecture of the human intestine. They contain multiple types of intestinal cells, including absorptive enterocytes, mucus-secreting goblet cells, hormone-producing enteroendocrine cells, and stem cells. They also exhibit key features of the intestine, such as the formation of crypt-like structures and the presence of villi-like projections.

Question 25

What are epithelial mini-guts ?

Answer 25

Epithelial mini-guts, also known as organoids or intestinal organoids, are three-dimensional structures derived from stem cells that resemble the lining of the intestine. These mini-guts have become a valuable tool in biomedical research as they provide a model system to study the structure, function, and diseases of the human intestine.