Stem Cell Lectures Oct 1

Question 1

How is the pool of stem cells maintained when they divide into daugher cells?

Answer 1

Stem cell division displays self renewal because because division produces two daughter cells: one of which remains a stem cell.

Question 2

From the stem cell division, the daughter cell that enters the differentiation pathway because what type of cell first?

Answer 2

A committed transit amplifying cell (or a progenitor)

Question 3

What does a committed transit amplifying cell do?

How is comitted transit amplifying cell division different from stem cell division?

Answer 3

They are very mitotically active and will proliferate (amplify) as they begin to differentiate.

Unlike stem cells, progenitor cells can only divide a finite number of times.

Question 4

What did the early stem cell studies with irradiated mice show?

Answer 4

Irradiation halts blood cell production

Injecting bone marrow cells from healthy donors into irradiated mice brought back blood cell production

Taking those stem cells from the “fixed mouse” and injecting them into another irradiated mouse also brought back blood cell production in the second mouse

This showed that hematopoietic stem cells can be serially transplanted

Question 5

Where are adult stem cells usually found?

Answer 5

They are found in adult stem cell niches in tissues and organs that undergo continual renewal - like skin, intestine, breast, and blood

Question 6

How do stem niches keep stem cells from differentiating?

Answer 6

They produce paracrine factors that regulate stem cell proliferation and prevent differentiation.

Once the cells leave the niche, they will begin differentiating

Question 7

What signalling pathways are involved in the stem cell niche of the small intestinal crypts?

Answer 7

Wnt (maintains cell proliferation in the crypts)

Hedgehog

BMP (makes sure the stem cells niche remain in the crypts and don’t establish themselves elsewhere)

Notch (Differentiation of progenitor cells into the different cell types of the villus)

Question 8

Describe a stem cell’s path from the base of the crypt to the to top of the villi?

Answer 8

THe stem cells down in the crypt slowly divide.

As the now-progenitor cells are pushed up the wall of the crypt, they will rapidly divide and Notch signalling will push them in certain differentation directions

As they near the beginning of the villus (top of the crypt), the projenitor cells become terminally differentiated and stop dividing.

WHen they reach the very top of the villus, they slough off into the lumen.

This whole process takes about 3-5 days

Question 9

Where is the stem cell niche in the epidermis?

How do stem cells lead to regeneration of the epidermis?

Answer 9

At the tips of the dermal palillae

Stem cells at the tips will slowly divide into progenitor cells.

THe progenitor cells will rapidly divide and slide down along the sides of the papillae untilt hey reach the base.

They will then be pushed off the side of the papillae to move toward the surface of the skin and differentiate

Question 10

What cell interaction is necessary for the self-renewal of stem cells in the bone marrow (for hematopoiesis)?

Answer 10

The stem cells need to be in contact with stromal cells like osteoblasts.

To survive and self renew, a stem cell tyrosin kinase receptor called Kit msust remain bound and activated by Kit ligand in the stromal cells.

Question 11

What is the main epigenetic mechanism for determining cell fate?

Answer 11

Methylation silencing of gene activity, resulting in a restriction of developmental potential - these are heritable changes

Question 12

What does epigenetic reprogramming entail?

Answer 12

In order to regain developmental potency, epigenetic changes that induced restriction of developmental potential has to be wiped or “reset.”

Question 13

Sperm and Ooctyes have different patterns of methylation through imprinting, so they’re not totipotent. How can they give rise to a totipotent zygote them?

Answer 13

1. Shortly after fertilization (preimplantation reprogramming) - demethylation is incomplete with methylation at imprinted regions being maintained, which allows the zygote to attain totipotency while still allowing for paternally–an maternally–derived genes to be differentially expressed
2. Then during the PGC determination, demethylation IS complete in order to allow for a resetting or imprinted regions accoring to the sex of the embryo. Subsequently, specific genes are imprinted in sex specific patterns during spermatogenesis and oogenesis.

Question 14

How does demethylation of the paternal pronucleus differ from the demethylation of the maternal pronucleus?

Answer 14

In teh zygote the paternal genome is actively demethylated after fertilization, perior to S phase and the first cell cycle

Maternal genome is demethylated more passively through suppression of methylation during the early cleavage divisions

Question 15

What step marks the first genome-wide de novo methylation for the zygote?

Answer 15

the formation of trophoblast and inner cell mass (when the inner cell mass becomes pluripotent)

Question 16

How do the transcription factors Oct4 and Nanog result in pluripotency for cells?

Answer 16

Oct4 and Nanog promote the expression of microRNAs including miR302.

miR302 is highly effective at driving pluripotency because is suppresses the expression of epigenetic modulators like methyltransferases

This causes global demethylation and reactivation fo the genome including Oct4 and Nanog expression (so there’s a positive feedback loop that promotes pleuripotency)

Question 17

What two methods do stem cels use to greatly reduce the extent of genomic damage incurred by the stem cells over time?

Answer 17

1. DNA replication is not perfect, so the number of division that stem cells undergo is greatly reduced. Progenitor cells are the ones that will divide rapidly.
2. Stems cells have the ability to segregate their replicated chromosomes so that the daughter cellst hat remains a stem cell receives all of the parental chromosomes template strands. This is the “immortal strand hypothesis”. THis reduces the chance of the stem cell line being altered.

Question 18

What are the advantages and disadvantages to using either embryonic or adult stem cells for research?

Answer 18

Embryonic stem cells are perfect because they are pluripotent (can become all 3 germ layers), have a rapid division rate and the srouce is well defined - we know where they are. THe problem is the ethical concern of destroying an embryo to obtain them.

Using adult stems cells ammeliorates the ethical issue, but adult stem cells are multopotent at best, they’re difficult to ideanity/isolate/purify, and have a low rate of cell division

Question 19

WHen adult stem cells are used for treatment or research, which type is most commonly used? WHere are they found?

Answer 19

Mesenchymal Stem Cells (MSCs)

they are multipotent adult stem cells found in severl adult tissues, especially the bone marrow.

It can give rise to numberal mesenchymal cells types inclding bone, cartilage, muscle and fat

Question 20

Besides embryonic and adult stem cells, which cells can exhibit pluripotency in the embryo?

Answer 20

germ cells (after epigenetic reprogramming anyways)

Question 21

What is the result of an embryonic germ cell that has been transformed to a malignant type?

Answer 21

a malignant growth called a teratocarcinoma

Question 22

What are the two sources of teratomas?

WHere do they tend to be located?

Answer 22

Teratomas can arise from germ cell transformation - these will be located in the ovaries or tested

Teratomas can also arise from very early embryonic cells that become transformed prior to commitment or specification - these are usually found along the midline of the body at the sacrococcygeal area

Question 23

How can differentiated cells be induced back to pluripotency in the lab?

What are these cells called if successful>

Answer 23

Enforced expression of transcription factors like Oct4, Nanog, Sox2, and Tcf3 will induce a differentiated cell back to pluripotency.

these cells are called induced pluripotent stem cells (iPS)

In general, it’s imperfect and low probability of success

Question 24

Why were Klf4 and c-myc replaced by Sox2 and Tcf3 in the cocktail for producing iPS cells?

Answer 24

Klf4 and c-myc were found to promote tumorigenesis in mice who were injected with iPS.

Question 25

Describe somatic cell nuclear transfer.

Answer 25

SCNT involves taking the nucleus of an adult somatic cell and place it in the cytoplasm of an egg (replacing the egg’s nucleus).

The egg is then activated to divide and the resulting duaghter cell is genetically identical to the somatic donor cell, but is not re-activated to a totipotent state.

This can then be used to give rise to a complete individual that is genetically identical to the somatic cell donor

This is cloning!

Question 26

Why is cloning so much easier to do in lower animals than in mammals?

Answer 26

Development of totipotency by an egg involves removal of epigenetic modifications via demethylation of CpGs.

This epigenetic reprogramming is much harder in mammalian somatic cells than in lower animal cells.

Question 27

What type of cell was using in the cloning of Dolly?

Answer 27

A mammary cell - this is probably why they were successful when so many others had failed because mammary cells are already very dynamic by their nature

Question 28

In therapeutic cloning, what is the the embryo produced through SCNT used for?

Answer 28

It is dissociated and grown in a culture dish to provide pluripotent cells for regenerative medicine uses.

This will produce pluripotent cells that are genetically identical to the donor without destroying an embryo, so it a little less iffy on the ethics (although is does require an egg)

Question 29

What are the 4 sources of stem cells for regenerative medicine purposes?

Answer 29

adult stem cells

embryonic stem cells

iPS cells

Therapeutic cloning

Question 30

What are some of the risks of stem cell therapies?

Answer 30

tumor formation

unwanted immune repsonses

transmission of adventitious agents

engraftment in unwanted location

toxicity