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Flashcards in lecture 15 Deck (15)

What are stem cells?

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)


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

- 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)


How can the cells in our body be broadly categorised?

- sex cells and somatic cells


What is potency?

- 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


What is transdifferentiation (plasticity)?

- 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


What is special about oogonia?

- 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


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

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


What is the zona pellucida?

- protein coat that protects the blastocyst
- hatches out of this coat when ready to implant into the uterine wall


What is cell culture?

- 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


What have we learnt about mouse embryonic stem cell colonies?

- 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


How do we test for pluripotent stem cells?

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


What is the method to make tetraploid embryos?

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


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

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


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

- Nanog
- Oct 4
- Sox 2


What did Boyer et al show?

- 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?