lecture 15

Question 1

What are stem cells?

Answer 1

The defining properties of a stem cell:
1. it is not itself terminally differentiated
2. it can divide without limit (animal lifetime)
3. when it divides, each daughter has a choice:
a. it can either remain a stem cell
b. or it can embark on a course leading to terminal differentiation
(asymmetric division)

Question 2

What are the different degrees of potency that a stem cell can have? What are examples?

Answer 2

• Totipotent: whole organism, e.g. fertilised egg (up to 8 cell stage in humans, very specific)
• pluripotent: all cell types of embryo (the three germ layers) (embryonic stem cells), e.g. blastocyst
• multipotent: don’t fulfill the criteria for pluripotent stem cells e.g. umbilical cord blood stem cells, blood, muscle, bone, cartilage, (can bank cord blood) e.g. adult stem cells: brain; cornea, retina; dental pulp; bone marrow; gut; skin; liver; (somatic stem cells)

Question 3

How can the cells in our body be broadly categorised?

Answer 3

• sex cells and somatic cells

Question 4

What is potency?

Answer 4

• stem cells are categorised by potency, which denotes the potential of the cell to derive other cell types — how many and what cell types
• potency is the range of developmental options available to a cell
• totipotent: ability to form the entire organism. In a mammal only the zygote and the first cleavage blastomeres are totipotent. not demonstrated for any other mammallian mmalian stem cell type
• pluripotent: ability to form all lineages of the body. example: embryonic stem cells and embryonic germ (EG) cells
• multipotent: ability to form multiple cell types from one lineage. e.g. haematopoietic stem cells which form all the blood type cells
• unipotent: ability to form one cell type, e.g. spermatogonia which can only form sperm

Question 5

What is transdifferentiation (plasticity)?

Answer 5

• controversial notion that some stem cell types have broadened potency and can generate cells from other lineages
• examples from literature: haematopoietic stem cells that can be persuaded to form cartilage or muscle cells

Question 6

What is special about oogonia?

Answer 6

• when female mammals are born they no longer have oogonia - all our eggs form during in development - frozen at prophase I when we are born and they stay that way until every cycle a few decide to ‘complete’ meiosis
• technically meiosis is not completed in a female without fertilisation

Question 7

Where are embryonic stem cells found? How do we use them?

Answer 7

• blastocyst
• specifically inner cell mass
• isolate ICM culture in vitro
• grow in clumps
• immortal (unlimited numbers)
• self-renew
• pluripotent

Question 8

What is the zona pellucida?

Answer 8

• protein coat that protects the blastocyst

- hatches out of this coat when ready to implant into the uterine wall

Question 9

What is cell culture?

Answer 9

• we take plasticware e.g. culture disk, flask, culture wells
• you add media (liquid with nutrients e.g. amino acids, fetal calf serum) that allows the cells to grow
• cells grown in nutrient rich solution (media)
• house in incubator at 37 degrees with 5-20% and O2 and CO2
• cells grow, divide, can be induced to become specialised cells

Question 10

What have we learnt about mouse embryonic stem cell colonies?

Answer 10

• maintain normal karyotypes (chromosome numbers)
• express stem cell markers: e.g. alkaline phosphatase, Oct4, Nanog, SSEA1, Sox 2, etc
• need fibroblast feeder layer, LIF (leukaemia inhibitory factor) and Serum to stay pluripotent and to grow

Question 11

How do we test for pluripotent stem cells?

Answer 11

Test 1:

• differentiate spontaneously in vitro into derivatives of the three germ layers: ectoderm, mesoderm, endoderm (when LIF and serum are removed)
• happens randomly
• least stringent criterion:
• • the expression for differentiation markers is not a test for functionality;
• • marker expression can be due to cellular stress response

Test 2:

• take your cells (couple of hundred)
• put them under the skin, or under the kidney capsule of an immunodeficient mouse
• these cells form a tumour
• within that tumour you can see muscle cells, glandular cells, pancreatic cells, blood, liver, bone, etc
• stem cells able to create cell types from the three germ layers in vivo
• form teratomas (not metastatic) when injected into immunodeficient mice
• differentiate spontaneously in vivo into derivatives of the three germ layers: ectoderm, mesoderm, endoderm
• due to loss of pluripotency and exposure to signals in the new environment that induce differentiation
• does not test for the ability to promote normal development

Test 3:

• use coat colour to see if the cells added have contributed to the mouse
• take one blastocyst from a white coat-coloured mouse and one from a black coat-coloured mouse
• take ES cells from black mouse (at this stage can do all sorts of things e.g. genetically target/manipulate etc)
• inject ES cells from black mouse into blastocyst from white mouse (~10-12 cells)
• put this blastocyst into a pseudopregnant female
• chimera
• when injected into donor blastocyst, ES cells contribute to all tissues of the resulting offspring
• does not test for epigenetic defects that could interfere with development

Test 4:

• tetraploid complementation
• produced by injecting ES cells into a tetraploid (4n) (rather than 2n) blastocyst
• most stringent test for pluripotency
• because 4n host cells cannot contribute to somatic lineages, embryo is exclusively composed of test cells
• doesn’t allow you to test for the ability to form trophectoderm (placental) lineage

Question 12

What is the method to make tetraploid embryos?

Answer 12

1. inject a diploid nucleus to a zygote
2. induce fusion of 2 diploid blastomeres
3. duplicate genome without cell division

Question 13

How do stem cells divide to produce daughters with different fates?

Answer 13

environmental asymmetry

• environmental factors maintain ‘stemness’ of daughter cell
• or environmental factors change and alter fate of daughter cell

divisional asymmetry
- determinants found in stem cell are distributed asymmetrically between daughter cells

Question 14

What are some important transcription factors expressed in embryonic stem cells?

Answer 14

• Nanog
• Oct 4
• Sox 2

Question 15

What did Boyer et al show?

Answer 15

• looked at the three important transcription factors and looked at which genes were turned on when that particular transcription factor was present in a cell
• there were 353 genes that were incredibly important to all three
• this data suggests that Oct4, Sox2, and Nanog function together to regulate a significant proportion of their target genes in ES cells
• are these 353 genes the basis of pluripotency?