Lecture 11 - Pluripotent stem cells Flashcards
What are stem cells?
Relatively primative cells capable of self renewal and differentiation. Can build embryos and tissues (development) and repair tissues (regeneration)
Pluripotent stem cells
mESC and human naive ESC
hESC and mouse EpiSC
What are the origins of human ESCs?
Teratocarcinoma, ICM of embryo and iPSCs
What are the properties of stem cells?
Self renewal and differentation
What are the applications of stem cells?
Regenerative medicine, drug discovery, toxicology and disease models
How are ES cells derived?
- Grow in IVF medium to blastocyst
- Anti-trophectoderm antibody and complement to induce complement-induced killing
- ICM cells replated onto feeder cells (originally MEF)
How do we characterise HPSCs?
Flow cytometry/in situ staining
Genetic/epigenetic markers
Gene expression: microarrrays/RNA seq/qPCR
Functional - clonogenic assays and differentiation
How do you assess if a cell is a stem cell?
Test it’s clonogenic capacity
What markers can be used to look at stem cells?
Oct4, Sox2, Nanog, LeftyA, SSEA3/4
What are the functional tests for stem cells?
Chimerisation and teratoma formation are gold standard
What assays are used to check if a cell has differentiated properly?
Functional assays
What do the mutations in stem cells tell us?
Selective pressure - tend to occur in genes that prevent cell death
How does methylation change in iPSC?
Needs to be similar to ESC not the cell it was derived from
How can we test for pluripotency?
Embryoid, spin EB, spin EB and gf, monolayer and gf, teratoma formation
What is induced pluripotency?
Application of facotrs to a differentiated cells
