Stem cell therapy Flashcards
guiding principle of regenerative medicine?
the use of endogenouos or transplanted stem cells to restore normal function of damaged tissues
Promise of stem cell research?
from cultured pluripotent stem cells:
- identify drug targets and test therapeutics
- toxicity testing
- tissues/cells for transplantaion
- study cell differentiation
- understanding prevention and treatment of birth defects
In vitro fertilization to generate stem cells?
at five day point, take inner cell mass from blastocyst. Can freeze, implant, or culture cells Can no longer implant cells taken from a blastocyst.
Stem cell manipulation by somatic cell nuclear transfer?
method of therapeutic cloning:
- remove nucleus from egg, and add nucleus from somatic cell in the body
- stimulate cell division
- remove inner cell mass from blastocyst (restricted from implanting this in humans)
- culture pluripotent stem cells and do what you would like (correct genetic defect via CRISPR, differentiate, implant, etc)
Risks of embryo gene editing with CRISPR?
- unknown embryo genome sequence
- off-target effects
- mutating some WT embryos
- optimization takes 1000s of embryos
- abortion or high risk pregnancy
Reproductive cloning?
- nuclear transfer in domestic animals
- low success rate
- high rate of abnormalities (large size, immune deficiency, cellular aging based on donor nucleus, incorrect epigenetics in donor DNA)
Establishing human embryonic stem cell lines?
May take up to a year:
blastocyst –> primary colonies –> subcultures –> cell lines established (after 30-60 passages!) –> cell lines for distrubution (2 million cells needed per vial!)
What is an issue with establishing ES cell lines?
we want the cells to remain undifferentiated, so we cannot divide them forever (may cause DNA damage). they also take a very long time to establish. If animal feeder is used with culture, may also be contamination.
Issues with using ES cells for therapy?
- differentiation protocols are needed and may take a long time to develop. Also do not want contamination of any pluripotent cells remaining (may cause teratomas)
- immune rejection: must suppress immune system unless using autologous IPS cells, but this is very expensive
ES cell differentiation protocol for cell replacement in Parkinson’s disease?
generation of domaminergic neurons:
- pluripotent stem cells are given oct4
- rosette neural precursors are given pax6+, sox1+, nestin+, and NCAM+
- committed DA precursors are given pax2, pax5, lmx, and en1
- finally, DA neurons arise after map2, th, aadc, and vmat2
takes many months!
Challenges and progress with using IPS cells?
Challenges:
- low efficacy
- insertion of 4 txn factors into the genome is random
- concern of teratomas if pluripotent cells remain after differentiation
progress:
- don’t need as many txn factors to induce pluripotency
- don’t need to use oncogenes anymore
- can use plasmids and miRNA rather than viral vectors to get txn factors into cells
- can mimic txn factors with chemicals
Direct reprogramming of differentiated cell types?
Instead of reversing a somatic cell type to a pluripotent cell, can add factors to directly reprogram one somatic cell type to another directly.
How can we prove that certain cell types come from a single multipotent stem cell in adult tissues?
in vivo, ablate the system (hematopoeitic for example) in mice and transplant potentially multipotent stem cells back in. See if it is able to generate a fully viable organism
Can also do lineage tracing with cre/lox system using markers like GFP to follow a potential stem cell.
Lineages of stem cells in bone marrow?
- hematopoeitic stem cells (blood, immune system)
- stromal stem cells (bone, cartillage, fat)
Sites of neurogenesis in the brain?
regions associated with learning and memory
- rostral migratory stream
- subventricular zone
- hippocampus
Describe the starting steps of stem cell therapy in the CNS.
