Exam 3 - Induced Pluripotent Stem Cells Flashcards
Who discovered induced pluripotent stem cells? How are they made?
Shinya Yamanaka, Nobel Prize in 2012 for the discovery that mature cells can be reprogrammed to become pluripotent
human adult fibroblasts + Yamanaka factors (Oct4, Sox2, Klf4, c-Myc) yields iPS
How is iPS different from SCNT for nuclear reprogramming?
rather than putting a somatic cell into an oocyte and creating an embryo that you can take stem cells from, the somatic cells are directly reprogrammed into iPS that resemble embryonic stem cells
Does iPS have the same mitochondrial concerns as therapeutic cloning?
No, because no oocyte is used
Describe the first mouse cloned from iPS
2009 in China, 37 iPS cell lines were made, 3 of them produced 27 mice
Tiny the mouse was one of them, went on to impregnate a female and produce young of its own
How was Tiny the mouse produced (scientifically)?
- isolate mouse skin cells
- infect the skin cells with the Yamanaka factors
- reprogrammed cells (the iPS) behave like embryonic stem cells
- injected into a 4n blastocyst, which serves only for the functional trophectoderm and has a nonfunctional inner cell mass
- developed embryo produces a fertile adult
How are iPS used for cloning (what do you do with the iPS to develop them into an adult)?
tetraploid complementation - inject them into a 4n blastocyst that has a functional trophectoderm but a non-functional or absent inner cell mass
What are the concerns of iPS?
- process (Yamanaka factor c-Myc) inhibits tumor-suppressing pathways and activates oncogenic ones
- disrupts control of gene activation or de-activation in unpredictable ways
- may introduce unintended mutations in the genome
How has the original Yamanaka process/factors been changed?
original factors were introduced by infection in retroviruses, permanently integrated the viral vector into the genome
recent modifications eliminated c-Myc (the oncogenic factor) and use vector free methods such as CRISPR Cas9
What is the newest route for nuclear reprogramming?
direct conversion from one type of somatic cell to another
How are cells directly converted from fibroblasts to neurons? How did scientists know they were functional?
three genes converted fibroblasts into induced neuronal (iN) cells
- expressed neuronal markers
- capable of firing action potentials
- received both excitatory and inhibitory synaptic connections when co-cultured with mouse neurons
- formed functional synapses with each other
What are arguments for and against continuing embryonic stem cell research?
continue:
- not clear if iPS and other reprogrammed cells are safe for human use (and the goal is therapeutic use)
- rate and efficiency of conversion is low for iPS
- reprogrammed stem cells appear to carry epigenetic memory as evidenced by DNA methylation patterns
end:
- it destroys embryos
- eggs are hard to obtain in large numbers
What are phenotypic and molecular similarities?
phenotypic similarity - morphology, growth, and ability to differentiate
molecular similarity - epigenetic modifications and gene transcription patterns
What are the relative molecular and phenotypic similarities of authentic ESCs, SCNT ESCs, iPSCs, and fibroblasts?
authentic ESCs are the best because they grow/differentiate the best and have no memory
SCNT ESCs are next best, then iPSCs
iPS cells retain evidence of DNA methylation patterns from their tissue of origin
ESC phenotype (ability to differentiate and grow) is achieved faster by iPSCs than their ESC molecularity (epigenetic modifications and patterns)
