Chapter 5

Question 1

What is a stem cell?

Answer 1

A cell with high potential that can develop into any cell type. It maintains its own identity while producing different committed cells through aysmmetrical division.

Question 2

What is the lineage of adult stem cells?

Answer 2

Multipotent: Can self renew indefinitely. Can further divide. Could remain in body forever.
Committed: Fated to differentiate. Can self renew. Will further divide.
Progenitor (transit-amplifying): Will proliferate into differentiated cells, can self-renew.
Differentiated cells: self-explanatory. Specialized cells. Cannot self renew. Cannot divide further.

Question 3

Is a zygote a stem cell?

Answer 3

Yes. It can self renew and generate differentiated cells.

Question 4

What are the different potencies of stem cells?

Answer 4

Totipotent: can generate all the cell types of the embryo and extra-embryonic tissues
Pluripotent: Can generate all the cell types of the embryo
Multipotent: Can generate many cell types, tissue specific
Unipotent: Can generate one cell type, tissue specific

Embryonic or adult in origin!

Question 5

How were stem cells discovered?

Answer 5

Mice were irradiated lethally. Those untreated died. Those given bone marrow from non-irradiated mice recovered and lived healthy lives afterwards: thus something in the bone marrow must have saved them. Mice with this transplanted bone marrow showed self-renewing spleen colonies that healthy mice did not have after 8-12 days.

Question 6

What type of stem cells are hematopoietic stem cells?

Answer 6

Multipotent: give rise to a specific lineage of differentiated cells.

Question 7

How many progenitors do hematopoietic stem cells produce? How many progenitors do these have in turn? What are their roles in the immune system?

Answer 7

Lymphoid progenitor: T and B lymphocytes and NK cells. Adaptive immune.
Myeloid progenitor: Neutrophil, basophil, eosinophil, monocytes, platelets, RBCs. Innate immune.

Question 8

How are HSCs used in treating blood cancer?

Answer 8

1. Blood is taken from a healthy donor and 2. processed to remove the stem cells. 3. The patient undergoes chemotherapy to kill the cancer and weaken immune system to lessen likelihood of rejection.
2. Infused into patient.

Question 9

How are intestinal cells regenerated?

Answer 9

Via the Wnt pathway.

Question 10

What are the five key cell types of the small intestine epithelium and their roles?

Answer 10

1. Enterocytes: absorption of fluids/nutrients
2. Enteroendocrine cells: produce hormones for intestine function
3. Goblet cells: secrete mucus
4. Tuft cells: chemosensors/T-cell activators
5. Paneth cells: produce antimicrobial molecules, maintain SCs by activating Wnt pathway (provide Wnt3a)

Question 11

How was intestinal regeneration explored in vivo? In vitro?

Answer 11

LGR5+ in vivo was labelled with tdTomato+, the stem cells and their progeny was stained red.

LGR5+ in vitro was labelled with GFP+, the cells of the epithelium were isolated, each cell gave rise to organoids resembling crypts and villi.

Question 12

What are some tissues and organs with adult somatic stem cells?

Answer 12

Blood, intestine, lungs, brain, skin, muscle, mesenchymal stem cells (in marrow), adipose tissues, heart

Question 13

What is a model system for regeneration?

Answer 13

Planarian worms. Any part of the worm when cut will regenerate an entire worm. Only embryonic SCs can do this in humans.

Question 14

What defines a cell’s type?

Answer 14

Shape and function which are driven by gene expression.

Question 15

Give a basic overview of single-cell transcriptonomics and how it lets us find new cells.

Answer 15

Embryos have stem cells dissociated, a platform conducts genomics for many genes, similar cells cluster together which reveals different cell types, plotted by PCA or t-SNE (similar, just spreads the clumps for better visualization). Can label and identify these clusters and the degree of gene expression for each. Use markers too.

Question 16

Why can’t humans regenerate like planarians?

Answer 16

We don’t produce pluripotent adult SCs. Only embryonic.

Question 17

Where can you find pluripotent embryonic stem cells?

Answer 17

From blastomeres of the inner cell mass. Oct4 specifically is a marker of pluripotency required for it, KO results in death. The two major sources of these blastomeres are the inner cell mass and primordial germ cells.

Question 18

What happens if you put pluripotent stem cells in a media that promotes differentiation in vitro? In vivo?

Answer 18

Teratomas. They have all 3 germ layers. It’s characteristic of pluripotent stem cells.

Question 19

What aspects of stem cell pools are finely regulated?

Answer 19

Maintenance, rate of proliferation and self-renewal, differentiation of stem cell progeny.

Question 20

What are the main modes of stem cell regulation?

Answer 20

Extra-cellular:
Physical: Ex: Hippo mechanosensor
Chemical: Endocrine, paracrine (Wnt), juxtacrine (Notch)

Intracellular:
Cytoplasmic: Notch
* Asymmetric: Hippo
* Symmetric: B-catenin destruction complex
Transcriptional: Smads, B-catenin, YAP/Taz
Epigenetic: DNA accessibility b/w stem cells and progeny

Question 21

What is the role of the Hippo Pathway in Inner Cell Mass (ICM) formation?

Answer 21

It signals asymmetrical signalling from the basal surface of the pre-trophectoderm leading to the division of a cell which becomes the ICM.

This occurs because the Hippo pathway is linked to the expression of apical proteins in the trophectoderm called PAR (Partitioning defective) and aPKC (atypical protein kinase C) which associate with each other and angiomotin and inhibit it, allowing transcription in this cell.

In the ICM cell adhered through E-cadherin to the trophectoderm, this causes a hippo kinase cascade which signals degradation of LATS1/2 which inhibits Yap/Taz which inhibits the Wnt/B-catenin pathway and the TGF-Beta/BMP SMADS pathway which allows Oct 4 activity. This inhibition is necessary for the further propagation of the ICM to form primordial germ cells, a loss of Oct4 leads to epiblast formation.

Question 22

What are the three factors of Inner Cell Mass formation?

Answer 22

Apical base polarity, Asymmetrical Hippo signalling activation, Asymmetrical cell division

Question 23

What is the role of Notch signalling in adult stem cells?

Answer 23

From Intestinal stem cells, enteroblasts are formed. If Notch is activated, further differentiation of the EBs occurs to enterocytes, if it is inhibited, the EBs differentiate to enteroendocrine (secretory) cells.

If Notch is KO’d, asymmetrical cell division stops and it causes tumor-like conditions because of a lack of differentiation

Question 24

What is a stem cell niche?

Answer 24

The micro-environment that houses stem cells, protects their pool and controls their self-renewal. This can involve Notch and Wnt and Delta ligands in the intestine, for example, to maintain SC regeneration, otherwise they can lose their identity.

Question 25

Describe an experiment demonstrating how Stem Cell function in the intestine is controlled with expression and inhibition of the Wnt and Notch pathways.

Answer 25

Cultures of Lgr5+ ISCs were grown. Lyso marks the Paneth cells (which provide Wnt + delta ligands).

ENR (EGF, Noggin, R-spondin1 and ECM) was used in all medias. The medias were then exposed to either CHIR99021 which inhibits B-catenin destruction (like in Wnt), Valproic acid (active Notch w/out ligands), and Wnt3a. One media was exposed to C and V, this media had mainly intestinal SCs demonstrating how these pathways remaining ON constantly leads to regeneration of stem cells, while the progressive turning on and off at key points leads to differentiation into different cell types.

This was also explored with PSCs or ESCs being directed in different media and ligands to form different tissues.

Question 26

Define reprogramming as it pertains to stem cells.

Answer 26

Differentiated adult cells are converted back into a stem cell state by erasing cellular memory. These cells are called iPSCs (induced pluripotent stem cells).

Question 27

How do Dolly and other experiments show reprogramming?

Answer 27

By removing the nucleus from an egg cell and replacing it with the nucleus of a specialized cell from a donor, cloning was able to occur.

Question 28

What are the four transcription factors needed for iPSC formation?

Answer 28

Oct3/4, c-Myc, Sox2, Klf4

Question 29

How are iPSCs grown in vitro?

Answer 29

Differentiated mouse fibroblasts are cultured and infected to misexpress Yamanaka factors (regulatory transcription factors). They are also exposed to Oct3/4, c-Myx, Sox2, Klf4.
These cells are then selected for antibiotic resistance so only the infected cells grow, and the resulting cultures have iPSCs which can be directed to differentiate into many lineages.

Question 30

Can differentiated cells from adults be reprogrammed to pluripotency without an oocyte?

Answer 30

Yes with the 4 key TFs.

Question 31

What is one huge advantage of iPSCs?

Answer 31

They can be cultivated from a host’s own tissue, therefore there is no risk of rejection. It can also be used to study disease, such as sickle cell anemia (mouse model) or brain diseases (brain organoid cultivation).