Embryonic SCs and cloning Flashcards
How can human ESCs be retrieved?
The ESCs can be retrieved from the inner cell mass (ICM) of the human blastocyte, which is the zygote 5-6 days after fertilization. The blastocytes can be created with in vitro fertilization.
How would you test for pluripotency of embryonic cells?
In vitro:
- Expression of molecular markers specific for endoderm, mesoderm, and ectoderm.
In vivo:
- The ESCs can be injected into mice where they develop into teratomas. The teratoma can be checked for the presence of the 3 germ layers.
- The ESCs can be marked (e.g. GFP) and implanted into mouse blastocyte where they will develop into chimera. With breeding an entirely ESC derived mouse can be obtained.
- Implant 2n ESCs into a 4n blastocyte which cannot contribute to the developing embryo. The embryo will be entirely ESC derived.
How can you create pluripotent stem cells that are not derived from the embryo?
Four different methods:
- Somatic cell nuclear transfer. The nucleus of a somatic cell can be transferred to a denucleated donor oocyte. The epiblast can later be isolated from this cloned blastocyte to retrieve the PSCs.
- Cell fusion. Fuse a somatic cell with an ESC. The reprogramming factors from the ESC can make the somatic nucleus transition into PSC inside a heterokaryon.
- Parthenogenesis. Make an oocyte remain diploid (2n) by a chemical or electrical shock so that it can develop without fertilization and PSCs can be harvested.
- Induced PSCs. Make a somatic cell pluripotent again by expressing certain TFs or adding chemical compounds.
Explain two ways to reprogram cell fate.
Indirect reprogramming: take a somatic cell and overexpress a cocktail of TFs to convert the cell to a iPSC. Direct programming: expressing certain combinations of TFs can differentiate somatic cells in a different cell type.
