BIOLOGY 1A Lecture Notes - Lecture 25: Mario Capecchi, Inner Cell Mass, Embryonic Stem Cell
Stem Cell Biology:
Stem Cell:
Cells that have the capacity to self-renew and generate differentiated
cells. They can generate daughter cells identical to their mother as well as
produce progeny with more restricted potential (differentiated cells)
○
Working definition: A clonal, self renewing entity that is multipotent and
can generate several differentiated cell types
○
•
Classifications:
Embryonic vs. Adult Stem Cells
Embryo development
§
Blood turns over about every 7 weeks, replenishment through
blood stem cells
§
○
Totipotent, Pluripotent or Multipotent
Totipotent: able to give rise to ALL cell lineages ('extraembryonic '
tissue)
§
Pluripotent: able to give rise to cells of all embryonic germ layers
endoderm, mesoderm, ectoderm
□
WE CAN ONLY MAKE PLURIPOTENT
□
§
Multipotent: able to give rise to multiple lineages
Can't give rise to all germ layers
□
Only certain types of neurons
□
§
○
•
Waddington's epigenetic landscape
Stem cells starts out of totipotent and can roll down to all the valleys
○
•
Stem Cells of the Early Embryo
Inner cells have not defined who they are- ability to differentiate
○
Have not committed to certain parts
○
Pluripotent at LEAST
○
How do we know they make different lineages?
Transcription factors regulate at different gene sets
Accumulate to make totipotent state
□
Regulate gene sets that are essential for making stem cell
population
□
§
○
•
Discovery of stem cells: Mario Capecchi, Martin Evans, Oliver Smithies
Inner cell mass in blastocyst stage can make every cell type
○
Took mouse embryos, took out inner cell mass, cultured it and separated
it into individual cells
○
Each individual cell would make clonal population to see if it could make
ectoderm, endoderm and mesoderm
Cells could keep self renewing and differentiating
§
○
•
Mouse genetics:
Can these cells that are isolated make skin cells, heart cells, etc?
Follow coat color of mice
See if cells can mix in with other cells in embryo to make coat
color of mouse
□
§
Take out stem cells and genetically alter it to tag it
Put them into naïve embryo that is developing and we should
see all the tissues with host cells and injected cells
□
Could get heterogeneity of skin color□
§
○
Start with "agouti" mouse (light brown)- take stem cells which are all light
brown
Inject the cells into blastocyst from black mouse
§
Black and brown mixture
§
○
•
Experiment: inner cell masses were taken out and genetically modified for Z
gene (beta galactosidase)
Has embryonic stem cells so embryo is blue from Xgal
○
Can genetically define for specific genes (reparation, etc.)
○
•
The potential of human embryonic stem cells:
Can we take the stem cells and turn them into other cell types (neurons,
muscle, blood) and put it back into people w defects?
○
James Thomson: Human Embryonic Stem cells
○
•
Derivation of pluripotent stem cells from somatic cells: John Gurdon
John Gurdon showed us there was ability to reprogram
Took a developing frog oocyte and removed nucleus. Embryo would
die without genetic material.
§
Took a skin cell from another frog (tadpole) and took its nucleus and
put it back into embryo without nucleus. He took it from a tadpole
not an embryo. Wanted to see if this embryo was viable
§
It was! Developed into fully functional frog
§
Skin cell--> embryo---> reprogramming
§
That nucleus had two dots (nucleolus) that was unique to that
particular frog species. Could see the developed one had two
nucleoli so he could mark it.
§
○
Somatic Cell Nuclear Transfer: Dolly was born with lost nucleus. Cell from
another sheep was used. Clone animal is completely normal. Can change
fate of nuclei
○
•
Capecci: did the cloning in mice, sheep, frogs•
Induced pluripotent Stem Cells: shiya yamanaka
Show us that there is a program that can be changed ; defined factors
determine whether we can convert skin cell into pluripotent cell
○
Take cells from mouse (skin) and convert into embryonic cell
○
Or take frozen material and grow up the cells and convert back to
embryonic
○
Transferred genes into cells taken from skins which were reprogrammed
into stem cells that could develop into all cell types
○
How does it do it?
Experiment: have different markers that show they are embryonic
stem cells
§
He knew there was a bank of transcription factors expressed during
embryonic development. Gordon showed us that there must some
reworked transcription factors. Yamanaka characterized every
factor turned on. 24 different factors were turned on. Ectopically
added it back to skin cells and asked which will drive it back into
pluripotent?
Narrowed down which cocktail of factors did it. □
4 factors that are ESSENTIAL to convert skin cell to embryonic
stem cell
C-MYC, Klf4, Sox2, Oct3/4
®
Normal transcription factors for development-
combination converted differentiated cell into stem cell
®
□
§
○
When we add 4 factors, how do we know it is a stem cell growing up?
When he took one cell out, he could see it convert into muscle cell
or nerve cell or skin cell
§
Cells had potential to make all cells in body
§
How do we know a cell is pluripotent?:
For human and mouse cells:
Proof of marker genes, teratoma formation (see all
three germ layers when put into mouse) , cell
differentiation
®
□
For mouse cells only:
Proof of chimera formation, tetraployid
complementation (karyotyping, no rearrangements,
chromosomes are normal)
®
□
§
○
•
Adult stem cells: undifferentiated cell, found among differentiated cells in a
tissue or organ. The adult stem cell can renew itself and can differentiate to
yield some or all of the major specialized cell types of the tissue or organ.
Some in brain, blood, and eye.
○
MULTIPOTENT- can only make certain lineages
○
•
Blood stem cells erythrocytes (blood cells), T and B cells (lympcytes),
macrophages and mast cells for infections
All cells made from a HEMATOPOIETIC STEM CELL
○
•
McCulloch and Till: studied radiation in the Cold War
Deplete the bone marrow and tissue to make blood during radiation-
animal gets anemia and can't make blood
○
Get rid of bone marrow, add missing material to repopulate tissues to
generate blood
Mouse survives and blood system is reconstituted from donor bone
marrow
§
Need to transport at least 10,000 cells to repopulate blood system;
within 10,000 you would have 100 or so stem cells to reconstitute
the system
§
Radiate it again. Unique chromosomal abnormalities would show us
where the stem cells originated. By doing second radiation, the new
chromosomal abnormalities were from donor cells.
Showed us the origin of stem cell population
®
§
○
•
Mouse--> irradiate---> stem cells transduced with lentiviral vectors expressing
red, green or blue fluorescent proteins
All constituents in lineage would be a certain color
○
Take out spleen--> see all the different colors that we marked stem cells
with. Will find different types of cells w all the colors.
○
We marked donor, put into recipient and it gave rise to all stem cells
○
•
Adult stem cell: undifferentiated cell, found among differentiated cells in a
tissue or organ. The adult stem cell can renew itself and can differentiate to
yield some or all of the major specialized tissue types of tissue or organ.
MULTIPOTENT
○
•
Lecture 25 3/21
Sunday, April 8, 2018
2:03 PM
Stem Cell Biology:
Stem Cell:
Cells that have the capacity to self-renew and generate differentiated
cells. They can generate daughter cells identical to their mother as well as
produce progeny with more restricted potential (differentiated cells)
○
Working definition: A clonal, self renewing entity that is multipotent and
can generate several differentiated cell types
○
•
Classifications:
Embryonic vs. Adult Stem Cells
Embryo development
§
Blood turns over about every 7 weeks, replenishment through
blood stem cells
§
○
Totipotent, Pluripotent or Multipotent
Totipotent: able to give rise to ALL cell lineages ('extraembryonic '
tissue)
§
Pluripotent: able to give rise to cells of all embryonic germ layers
endoderm, mesoderm, ectoderm□
WE CAN ONLY MAKE PLURIPOTENT□
§
Multipotent: able to give rise to multiple lineages
Can't give rise to all germ layers□
Only certain types of neurons□
§
○
•
Waddington's epigenetic landscape
Stem cells starts out of totipotent and can roll down to all the valleys
○
•
Stem Cells of the Early Embryo
Inner cells have not defined who they are- ability to differentiate
○
Have not committed to certain parts
○
Pluripotent at LEAST
○
How do we know they make different lineages?
Transcription factors regulate at different gene sets
Accumulate to make totipotent state□
Regulate gene sets that are essential for making stem cell
population
□
§
○
•
Discovery of stem cells: Mario Capecchi, Martin Evans, Oliver Smithies
Inner cell mass in blastocyst stage can make every cell type
○
Took mouse embryos, took out inner cell mass, cultured it and separated
it into individual cells
○
Each individual cell would make clonal population to see if it could make
ectoderm, endoderm and mesoderm
Cells could keep self renewing and differentiating
§
○
•
Mouse genetics:
Can these cells that are isolated make skin cells, heart cells, etc?
Follow coat color of mice
See if cells can mix in with other cells in embryo to make coat
color of mouse
□
§
Take out stem cells and genetically alter it to tag it
Put them into naïve embryo that is developing and we should
see all the tissues with host cells and injected cells
□
Could get heterogeneity of skin color
□
§
○
Start with "agouti" mouse (light brown)- take stem cells which are all light
brown
Inject the cells into blastocyst from black mouse
§
Black and brown mixture
§
○
•
Experiment: inner cell masses were taken out and genetically modified for Z
gene (beta galactosidase)
Has embryonic stem cells so embryo is blue from Xgal
○
Can genetically define for specific genes (reparation, etc.)
○
•
The potential of human embryonic stem cells:
Can we take the stem cells and turn them into other cell types (neurons,
muscle, blood) and put it back into people w defects?
○
James Thomson: Human Embryonic Stem cells
○
•
Derivation of pluripotent stem cells from somatic cells: John Gurdon
John Gurdon showed us there was ability to reprogram
Took a developing frog oocyte and removed nucleus. Embryo would
die without genetic material.
§
Took a skin cell from another frog (tadpole) and took its nucleus and
put it back into embryo without nucleus. He took it from a tadpole
not an embryo. Wanted to see if this embryo was viable
§
It was! Developed into fully functional frog
§
Skin cell--> embryo---> reprogramming
§
That nucleus had two dots (nucleolus) that was unique to that
particular frog species. Could see the developed one had two
nucleoli so he could mark it.
§
○
Somatic Cell Nuclear Transfer: Dolly was born with lost nucleus. Cell from
another sheep was used. Clone animal is completely normal. Can change
fate of nuclei
○
•
Capecci: did the cloning in mice, sheep, frogs•
Induced pluripotent Stem Cells: shiya yamanaka
Show us that there is a program that can be changed ; defined factors
determine whether we can convert skin cell into pluripotent cell
○
Take cells from mouse (skin) and convert into embryonic cell
○
Or take frozen material and grow up the cells and convert back to
embryonic
○
Transferred genes into cells taken from skins which were reprogrammed
into stem cells that could develop into all cell types
○
How does it do it?
Experiment: have different markers that show they are embryonic
stem cells
§
He knew there was a bank of transcription factors expressed during
embryonic development. Gordon showed us that there must some
reworked transcription factors. Yamanaka characterized every
factor turned on. 24 different factors were turned on. Ectopically
added it back to skin cells and asked which will drive it back into
pluripotent?
Narrowed down which cocktail of factors did it. □
4 factors that are ESSENTIAL to convert skin cell to embryonic
stem cell
C-MYC, Klf4, Sox2, Oct3/4
®
Normal transcription factors for development-
combination converted differentiated cell into stem cell
®
□
§
○
When we add 4 factors, how do we know it is a stem cell growing up?
When he took one cell out, he could see it convert into muscle cell
or nerve cell or skin cell
§
Cells had potential to make all cells in body
§
How do we know a cell is pluripotent?:
For human and mouse cells:
Proof of marker genes, teratoma formation (see all
three germ layers when put into mouse) , cell
differentiation
®
□
For mouse cells only:
Proof of chimera formation, tetraployid
complementation (karyotyping, no rearrangements,
chromosomes are normal)
®
□
§
○
•
Adult stem cells: undifferentiated cell, found among differentiated cells in a
tissue or organ. The adult stem cell can renew itself and can differentiate to
yield some or all of the major specialized cell types of the tissue or organ.
Some in brain, blood, and eye.
○
MULTIPOTENT- can only make certain lineages
○
•
Blood stem cells erythrocytes (blood cells), T and B cells (lympcytes),
macrophages and mast cells for infections
All cells made from a HEMATOPOIETIC STEM CELL
○
•
McCulloch and Till: studied radiation in the Cold War
Deplete the bone marrow and tissue to make blood during radiation-
animal gets anemia and can't make blood
○
Get rid of bone marrow, add missing material to repopulate tissues to
generate blood
Mouse survives and blood system is reconstituted from donor bone
marrow
§
Need to transport at least 10,000 cells to repopulate blood system;
within 10,000 you would have 100 or so stem cells to reconstitute
the system
§
Radiate it again. Unique chromosomal abnormalities would show us
where the stem cells originated. By doing second radiation, the new
chromosomal abnormalities were from donor cells.
Showed us the origin of stem cell population
®
§
○
•
Mouse--> irradiate---> stem cells transduced with lentiviral vectors expressing
red, green or blue fluorescent proteins
All constituents in lineage would be a certain color
○
Take out spleen--> see all the different colors that we marked stem cells
with. Will find different types of cells w all the colors.
○
We marked donor, put into recipient and it gave rise to all stem cells
○
•
Adult stem cell: undifferentiated cell, found among differentiated cells in a
tissue or organ. The adult stem cell can renew itself and can differentiate to
yield some or all of the major specialized tissue types of tissue or organ.
MULTIPOTENT
○
•
Lecture 25 3/21
Sunday, April 8, 2018 2:03 PM
Document Summary
Cells that have the capacity to self-renew and generate differentiated cells. They can generate daughter cells identical to their mother as well as produce progeny with more restricted potential (differentiated cells) Working definition: a clonal, self renewing entity that is multipotent and can generate several differentiated cell types. Blood turns over about every 7 weeks, replenishment through blood stem cells. Totipotent: able to give rise to all cell lineages ("extraembryonic " tissue) Pluripotent: able to give rise to cells of all embryonic germ layers endoderm, mesoderm, ectoderm. Multipotent: able to give rise to multiple lineages. Stem cells starts out of totipotent and can roll down to all the valleys. Inner cells have not defined who they are- ability to differentiate. Regulate gene sets that are essential for making stem cell population population. Discovery of stem cells: mario capecchi, martin evans, oliver smithies. Inner cell mass in blastocyst stage can make every cell type.
