Cloning and regenerative medicine Flashcards
Nuclear transfer shows that “terminal differentiation” can be reversed
Inter-species transfer allows proteins to be easily analysed, e.g. human myosin can be distinguished from mouse myosin
Experiment: fuse nucleus of human liver cell with mouse muscle cells and saw that activation of human muscle gene expression, mouse and human muscle proteins are produced, shows human nucleus instantly starts coding for muscle cells instead liver cells, means something (probably in cytoplasm) telling nucleus it is now muscle cell
Damage can also induce reprogramming of cells
Iris cells transdifferentiate into lens cells to regenerate missing tissue
Experiments done with newts as are knows to be able to regrow lost limbs
They removed the lens and iris regrew a new lens
Can terminal differentiation be reversed?
Gene expression in nuclei from terminally differentiated cells can be reprogrammed only under special circumstances
Points towards idea that gene expression can be controlled by cytoplasmic factors
Genes not los when cells differentiate
tissue loss can sensed in some animals resulting in regeneration, implies some form of extra cellular signal
Doesn’t show that terminally differentiated cell can be made totipotent, just very specific cell types that they go on to become
What do we mean when cells are clonal?
A clone is group identical cells that share common ancestry (derived from same cell), implies genetically identical
In vivo not very useful except when talking about WBC which can undergo chromosomal rearrangement to generate antibodies
When culturing cells in vitro, is important because often generate transgenes that make cells different
In organisms it indicates that individual are genetically identical (twins)
Frogs first animal cloned in lab
First done with tadpole gut cells in 1962
Then adult skin cells in 1975 because are though to be more terminally differentiated
Nuclei removed from cells and placed into new ones can form tadpoles but at a low rate
Soma (body) i.e. not the germline of the nucleus
This method sometimes called somatic cell nuclear transfer
Blastula nuclei far more successful suggesting that younger cells more stem-like, if you remove several nuclei from blastula and then implant then in enucleated eggs they then go on to form genetically identical frogs
Both John B. Gurdon and Shinya Yamanaka shared Nobel prize in medicine in 2012 for their separate research on these frogs
Cloning sheep - Wilmut et al., 1997
Take mammary epithelial cells from an adult sheep and culture the in low serum - cells exit the mitotic cycle (into G0)
At the same time you remove an unfertilised egg from another sheep and then remove the chromosomes
You then induce the fusion of mammary cells with unfertilised lacking chromosomes egg by electric current
This gets nucleus of mammary cell inside the egg
Embryo culture begins to form and is transferred to foster mother
Dolly was first mammal to be cloned
Stem cells in regenerative medicine
Scaffolds are fabricated out of biodegradable materials to support 3 dimensional growth of cells, transplanted into the area to aid growth
Stem cells should be from patient to avoid tissue rejection
In vitro methods:
- Add stem cells that then change into the types of cells needed for repair these will then attach to the scaffolding of straight to the damaged tissue
In Vivo method:
- Add drugs or transgenes near the affected area, these then diffuse into normal tissue and then into the damaged tissue
Three different strategies to create insulin producing cells
Take skin fibroblast and produce iPS (way 1 - induced pluripotency) cells or ESC (way 2 - somatic cell nuclear transfer) then put the cells through differentiation protocol with growth factors and small molecules become Pdx-1 expressing cells and then Insulin-producing glucose-responsive cells
(Way 3 - transdifferentiation in vivo) Take adult exocrine pancreas liver from either Xenopus or mouse, then transfection of organs with Pdx1 and three other transfection factors or with activated Pdx1 and then you get insulin-producing glucose-responsive cells
Insulin lacking in diabetes patients, normally produced by pancreas
Pdx1 is regulator pancreas differentiation (marker cell for insulin producing cells)
iPS cells come from patient so less likely to be rejected
In vivo production insulin in Xenopus and mice
Stem cell therapies based upon iPS cells
Good for:
- Correcting genetic defects, like junctional epidermolysis bullosa
- Replacing simple tissues or single cell types (bladder, retinal cells)
- Testing how your cells will respond to different pharmaceuticals (personalised medicine)
Limitations:
- Culturing cells introduces genetic changes
- Transplantation
- Organogenesis very complex - tissue engineering scaffolds have had limited success
Harnessing innate power of cells to self-organise (by using organoids)
Experiments done by Henry Van peters Wilson in 1907
Dissociated sponges by putting them through fine sieve then watched as cells reorganised into intact sponges
Adhesion molecules and cell signalling play roles in the process
Organoids formed by stem cells when cultured in special media
Hope is that complex tissues or organs could be grown for study and regenerative medicine
First example of these are intestinal organoids (look at diagram)
