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Lecture 8

Nov 12 Lecture 8 LIFESCI 2A03

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Department
Life Sciences
Course Code
LIFESCI 2A03
Professor
Joe Kim

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LECTURE 8 LIFESCI 2A03
1
Induced Pluripotent Stem Cells (iPSCs)
November 12, 2013
Reverting adult cell types back to pluripotent embryonic stem cell state two studies; Dr. John Gurdon and Dr. Shinya
Yamanaka
Dr. John Gurdon
Year 1962
Question Are adult stem cells terminally differentiated? Is there the possibility for a specialized adult cell to form any other
cell type? The DNA is there are there any other limitations? (Is the DNA terminally differentiated when the cell is terminally
differentiated, or is the DNA just turned off?)
Experiment removed the DNA (nucleus) from a frog egg and replaced it with the DNA (nucleus) of a mature cell taken from a
tadpole the egg developed into a healthy, cloned tadpole
o Mature cell (differentiated somatic cell) left intact nucleus from a different cell taken from an egg and put into
mature cell
Paper “The Developmental Capacity of Nuclei taken from Intestinal Epithelium Cells of Feeding Tadpoles
o These results show that a nucleus can promote the formation of a differentiated intestine cell and at the same time
contain the genetic information necessary for the formation of all other types of differentiated somatic cell in a normal
feeding tadpole
o It is concluded that the differentiation of a cell cannot be dependent upon the incapacity of its nucleus to give rise to
other types of differentiated cells
Nuclear Transfer
o Fixing the cell (holding it stationary) and fine needle injected into cell through membrane to remove nucleus
o Inject different nucleus into cell cell from one individual, nucleus from another
Assay for Pluripotency production of a living tadpole
o Two species being studied (different frogs)
o 78 total transplants (nucleus from intestinal epidermis transplanted into cell)
o Eggs uncleaved (not undergoing division)
o Aborted Cleavage
o Complete blastula (mid stage; cell divisions occurred but not fully developed)
o Neural folds (some differentiation occurring, migrating, changing shape)
o Normal swimming tadpoles 20/75 completed development from nucleus of intestinal epidermis
o Table the transplantation of nuclei from Hymenochirus and Xenopus into recipient eggs of Xenopus
Interviews
o Briggs and King believed that nucleus changes occur when cells specialize; believed
that when the cell differentiates and no longer needs certain genes, the genes lose
function
o Gordon results oppose this belief; people were skeptical (he was a graduate student
opposing credible scientists)
Genome remains intact grows fully functioning tadpoles from transplanted
nuclei; fundamental principle that set of genes is conserved throughout the
whole of cell differentiation led to his findings
Main Conclusion differentiated cells maintain all of the information in their genome to form all different cell types; they
maintain the ability to be pluripotent even if they are not acting as pluripotent cells
Dr. Shinya Yamanaka
Year 2006
Yamanaka looked at the experiments of John Gurdon
Question Can an adult cell be reprogrammed without using an egg and without transplanting the nucleus?
Experiment adding four genes to an adult cell can turn it into an embryonic-like state; showed this first in mice, then in
human cells a year later
Paper Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors
Morphological changes in cells
LECTURE 8 LIFESCI 2A03
2
o ES embryonic stem cell; pluripotent, have undifferentiated spherical shape
o MEF mouse embryonic fibroblast; differentiated cells, flattened appearance (adhering and interacting with surface
upon which they are growing)
o iPS induced pluripotent stem cell (from MEF); MED induced to become pluripotent; look more like cultured ES than
MEF
Ectopic expression of Yamanaka Factors (Oct ¾, Sox2, Klf4, and c-Myc)
o Looked at collection of 24 factors (proteins; growth fators involved in stem cell identity)
o A add all factors, except for 1 to MEF to observe whether cells are dividing and proliferation
Controls (far right columns)
All Factors see cell division
Mock no factors added see no division (control to observe whether artifacts will occur)
Result of removing factor
Little Cell Division (eg/ factor 1) factor important for inducing cell division
Large amount of cell division (eg/ factor 9; more division than control) factor may inhibit cell
division because more cell division is occurring in its absence
o B narrowed down to 10 factors
o C narrowed down to 4 factors; add all 4 factors except 1
Results
Column 1 (4 factors) adding all 4 factors allows for induction of cell proliferation
Column 2-5 (3 factors) observe effects of removing one factor (cell proliferation is affected)
o Klif4 or Oct3/4 missing minimal cell division
o C-Myc and Sox2 missing reduced cell division
Column 6-10 (2 factors) no cell proliferation; need at least 3 factors for any cell division
Column 11 (10 factors) all 10 factors result in cell proliferation; maximal threshold (control)
Column 12 (mock) no factors result in no cell proliferation (control)
Main Conclusion restore the ability to differentiated cells to divide and proliferate in culture; like stem cells
Assay for Pluripotency teratoma formation; 3 germ layers produced
o A Various tissues present in teratomas derived from iPS-MEF4-7 cells

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Description
LECTURE 8 LIFESCI 2A03 Induced Pluripotent Stem Cells (iPSCs) November 12, 2013  Reverting adult cell types back to pluripotent embryonic stem cell state – two studies; Dr. John Gurdon and Dr. Shinya Yamanaka Dr. John Gurdon  Year – 1962  Question – Are adult stem cells terminally differentiated? Is there the possibility for a specialized adult cell to form any other cell type? The DNA is there – are there any other limitations? (Is the DNA terminally differentiated when the cell is terminally differentiated, or is the DNA just turned off?)  Experiment – removed the DNA (nucleus) from a frog egg and replaced it with the DNA (nucleus) of a mature cell taken from a tadpole – the egg developed into a healthy, cloned tadpole o Mature cell (differentiated somatic cell) left intact – nucleus from a different cell taken from an egg and put into mature cell  Paper – “The Developmental Capacity of Nuclei taken from Intestinal Epithelium Cells of Feeding Tadpoles o These results show that a nucleus can promote the formation of a differentiated intestine cell and at the same time contain the genetic information necessary for the formation of all other types of differentiated somatic cell in a normal feeding tadpole o It is concluded that the differentiation of a cell cannot be dependent upon the incapacity of its nucleus to give rise to other types of differentiated cells  Nuclear Transfer o Fixing the cell (holding it stationary) and fine needle injected into cell through membrane to remove nucleus o Inject different nucleus into cell  cell from one individual, nucleus from another  Assay for Pluripotency – production of a living tadpole o Two species being studied (different frogs) o 78 total transplants (nucleus from intestinal epidermis transplanted into cell) o Eggs uncleaved (not undergoing division) o Aborted Cleavage o Complete blastula (mid stage; cell divisions occurred but not fully developed) o Neural folds (some differentiation occurring, migrating, changing shape) o Normal swimming tadpoles – 20/75 completed development from nucleus of intestinal epidermis o Table – the transplantation of nuclei from Hymenochirus and Xenopus into recipient eggs of Xenopus  Interviews o Briggs and King – believed that nucleus changes occur when cells specialize; believed that when the cell differentiates and no longer needs certain genes, the genes lose function o Gordon – results oppose this belief; people were skeptical (he was a graduate student opposing credible scientists)  Genome remains intact – grows fully functioning tadpoles from transplanted nuclei; fundamental principle that set of genes is conserved throughout the whole of cell differentiation led to his findings  Main Conclusion – differentiated cells maintain all of the information in their genome to form all different cell types; they maintain the ability to be pluripotent even if they are not acting as pluripotent cells Dr. Shinya Yamanaka  Year – 2006  Yamanaka looked at the experiments of John Gurdon  Question –Can an adult cell be reprogrammed without using an egg and without transplanting the nucleus?  Experiment – adding four genes to an adult cell can turn it into an embryonic-like state; showed this first in mice, then in human cells a year later  Paper – Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors  Morphological changes in cells 1 LECTURE 8 LIFESCI 2A03 o ES – embryonic stem cell; pluripotent, have undifferentiated spherical shape o MEF – mouse embryonic fibroblast; differentiated cells, flattened appearance (adhering and interacting with surface upon which they are growing) o iPS – induced pluripotent stem cell (from MEF); MED induced to become pluripotent; look more like cultured ES than MEF  Ectopic expression of Yamanaka Factors (Oct ¾, Sox2, Klf4, and c-Myc) o Looked at collection of 24 factors (proteins; growth fators involved in stem cell identity)
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