Stem cell biology

Question 1

How can we experimentally demonstrate self-renewal of stem cells?

Answer 1

serial transplantation:

1. remove lacZ labelled sperm stem cells from a mouse donor
2. inject cells into recipient mouse into endogenous non-lacZ recipient
3. visualize that spermatogonia are repopulated into blue colonies from stem cells from the donor

Question 2

experimental demonstration of stem cell differentiation?

Answer 2

ability to form colonies in soft agar (in vitro) assay

Question 3

Which types of cells can form teratomas?

Answer 3

pluripotent ES cells

Question 4

what factors promote plurpotency of stem cells?

Answer 4

oct4, nanog, sox2

Question 5

HSCs have what type of potency?

Answer 5

multipotent, because they can make all lineages of cells within an organ system

Question 6

what do transit-amplifying cells do?

Answer 6

go-between for stem cells and differentiated cells

amplifies pool of progenitor cells to replace organ system faster

Question 7

How is potency determined experimentally?

Answer 7

lineage tracing: process of identifying all progeny of a single cell (in vivo)

• visualize cell lneage by reporter gene expression in stem cells that is passed onto progeny
• label starting stem cell with the reporter and see which cells end up containing the marker

Question 8

What is the effect of destroying the niche on a stem cell population?

Answer 8

Because the niche helps cells maintain stemness, it will cause them to differentiate.

Question 9

describe drosophila testis stem cell niche.

Answer 9

signaling from hub cells is proximity-based

• hub cell binds to germ SC and secreted “unpaired” protein
• unpaired miantains committed state of stem cells, so differentiated gonialblast cells divide away from the hub cell

Question 10

Describe the drosophila ovarian stem cell niche.

Answer 10

stemness maintained by proximity effect to cap cells

• cryoblasts divide and differentiate horizontally, so there is no TGF-beta from cap cells that reaches them to maintain them as stem cells
• in stem cells, TGFB signaling from cap cells activates BMP signal transduction
• BMP activates MAD which turns off expression of bam, which is required for differentiation into an oocye.

Question 11

Describe the adult intestinal stem cell niche.

Answer 11

• base of crypt houses ISCs. Here, Wnt2b is high, and Bmp4 is low.
• traveling up the crypt are the transit-amplifying (progenitor) cells, followed by more differentiated cells
• at the tip of the villi, cells die by anoikis (loss of contact)

Question 12

Describe the ISC types.

Answer 12

two types of ISCs:

1. crypt base columnar cells (CBCC): Lgr5+, divide every 9-12 hours, maintained for life (possible because they are next to paneth cells)
2. +4 cells: fourth stem cell in from the base, Bmi1+, quiescent

(stromal cells also contribute to stem cell maintenance via Bmp4 and Wnt2b by signaling)

Question 13

Describe the clonogenic nature of the ISC niche.

Answer 13

An entire crypt and villus can be populated from a single ISC at the very base of the crypt. Know this thanks to lineage tracing (single cell marked with lacZ).

Question 14

Describe the neutral competition model in the context of ISC niche.

Answer 14

If many CBCCs are placed in the base of a crpyt, one will win out as the ancestor to an entire crypt and villus.

Question 15

Describe the hematopoietic stem cell niche.

Answer 15

1. endosteal niche HSCs are quiescent
2. perivascular niche HSCs are rapidly dividing

Question 16

Which stem cells can function before they have a niche?

Answer 16

hair follicle

Question 17

How can we recreate the SC niche in vitro?

Answer 17

organoids

-a single SC will grow in soft agar (with signaling from niche cells, with ECM) and will generate the microenvironment consistent with its normal tissue type

Question 18

How can we study stem cells in vivo?

Answer 18

1. can study how long they live via label retention of DNA – will maintain in tissues for a long time because the cells are not dividing too much (stem cells often become quiescent so they don’t divide too much so they are protected from DNA damage and remain viable). Works by pulsing the cells with a nucleotide analog during S phase

Question 19

How is DNA damage repaired in quiescent stem cells?

Answer 19

Via NHEJ. Though this is error-prone, it is still better than rapidly dividing cells that accumulate much more DNA damage.

Question 20

Describe in vivo lineage tracing.

Answer 20

Can track progeny over time using a specific marker/reporter:

-Can use cre/lox to recombine a visual marker such as lacZ or GFP into a stem cell specific promotor

Question 21

Describe this model for lineage tracing.

Answer 21

in vivo, small intestine

Question 22

Define dedifferentiation and transdifferentiation.

Answer 22

dedifferentiation: terminally differentiated cells revert back to dedifferentiated cell within the same lineage (allows for regeneration of limbs in some organisms!)
transdifferentiation: one differentiated cell gives rise to another differentiated cell in an entirely different lineage (mammalian pancreas, ductal cell induces beta-cell)

Question 23

Describe regeneration in the mammalian intestine.

Answer 23

Loss of Lgr5+ CRCC stem cells can be replaced through the plasticity of their enterocyte-lineage Alpi+ daughter cells. These move down to the crypt, dedifferentiate, and are now multipotent and able to self-renew.

Question 24

Application and utility of IPS cells?

Answer 24

• disease modeling
• gene correction, differentiation, and transplantation

Question 25

Pros of IPS therapy?

Answer 25

Cells are autologous, no immune rejection

Question 26

What causes cancer cell relapse after drug treatment?

Answer 26

survival of cancer stem cells which are resistant to treatment due to their quiescence

Question 27

How can cancer stem cells be targeted?

Answer 27

niche therapy – disrupting niche cells will cause stemness of CSCs to be lost