Lecture 1 - intro Flashcards
What is the main aim of regenerative medicine?
To replace, repair or regenerate cells, tissues and organs in order to restore health
What are the different types of stem cells?
Totipotent - can form all embryonic and extra embryonic tissues, are very rare
Pluripotent - can form all embryonic tissues
Multipotent - all lineages of a tissue/organ
Oligopotent - several but not all lineages of a tissue/organ
Unipotent - single lineage of a tissue/organ
What are autologous and allogenic cells?
autologous cells come from the patient and will not be rejected, whereas allogenic cells come from a donor and need to be matched with cell surface antigens to prevent rejection
How can gene therapy be used (overall process)?
take a functional gene, place in vector and inject into the patient. The vector finds the tissue, delivers the cargo into the cell and the functional gene produces a functional protein
How can gene editing be used (overall process)?
elimination or insertion of a mutation, replacing the faulty gene. Can design complementary RNA to deliver cas9 to a specific part of the genome and edit the sequence.
How can tissue engineering be used (overall process)?
isolate cells, expand their number, seed them on a suitable scaffold, culture under certain conditions to generate a mature tissue, and implant tissue in patient.
How can xenotransplantation be used (process)?
using animals to generate organs that can be harvested and used in humans
How can detection and use of stem cell markers be used? How can expression be determined and which techniques can enrich cell populations?
Stem cells are identified by the expression or absence of a number of TFs and cell surface markers. These markers can be used to enrich stem cell populations for tissue engineering
Western blotting, PCR, immunocytochemistry and flow cytometry can be used to determine expression
Cross-linking and centrifugation, MACs and flow cytometry can be used to enrich cell populations
How does density centrifugation work?
Can be separated into layers using centrifugation
anti-cell antibodies bind and recognise unwanted cells
the other end binds to glycophorin A, which is found in RBCs
unwanted cells are cross linked with RBCs and end up in the pellet, so we can collect cells of interest
How does MACS work?
magnetic-activated cell sorting uses antibodies that are expressed on the cell of interest, but not on the cells we want to get rid of
pass them through a column with a magnet - unwanted cells do not have magnetic nanoparticles and are able to pass through the column (positive selection)
untouched isolation - depletion of contaminant cells
sequential sorting - depletion followed by positive selection
How does flow cytometry worK?
A method for detecting specific molecules on and within cells
cells are passed through a laminal flow through a laser, which scatters light and excites different fluorophores on the cell surface
the light either passes straight through or bounces off cells
when the cells pass through the laser beam, the light is dimmed - the time it is dimmed for is proportional to the size of the cell
How does FACS work?
Fluorescent-activated cell sorting
A droplet can be given a charge depending on the antigen. Various electrodes have different charges and can be used to separate the cells. Can look at combinations of markers.
