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Lecture 8

Nov 12 Lecture 8 LIFESCI 2A03

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Life Sciences
Joe Kim

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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