The goal of this paper is to compare the utility of adult, embryonic and induced pluripotent stem cells (iPSCs) to treat Parkinson’s disease. As such several things will be assessed, dosage of stemcells, improvement in motor function, in combination with the presence of α-synuclein proteins and cell survival.
To give a short overview of the steps that will be taken to complete the study. Obtaining stem cells, whether adult, embryonic or induced, shall be done using healthy mouse models and after ethical approval has been gained. The process to derive them will be detailed below, however they are also purchasable commercially with the benefit of being well studied and accompanied by a detailed analysis of properties, however with a higher purchasing costs. All steps should be done using sterile techniques to avoid contamination, both to object and self. All (disposable) materials should be sterilized and (where possible) only for 1 time use, due to the sensitivity stem cells have to detergents. After verifying they are bonafide stem cells the next phase should be ready to commence.
Rats will be used as a disease model. All will be healthy, adult and a baseline PET scan and performance on a rotarod will be done. All but 1 set will then be subjected to a neurotoxin to mimic the diseased Parkinson state. Another PET scan and rotarod test will be done on all groups. The groups affected by the neurotoxin will then be subdivided into those receiving the different types of stem cells, one subgroup will receive no treatment and one group will receive medium (no cells). Next to this 2 different dosages will occur. Another PET scan and rotarod will be done to determine if any improvement occurs. Rats will then be sacrificed to determine viability of stem cells and if any have become infected by α-synuclein proteins.
Statistic tests will then be used to find out if there is a marked improvement in motor function in combination with stem cell type and dosage used. And analysis will one subtype shows greater vulnerability to α-synuclein proteins.
Culturing of mouse embryonic stem cells:
Many protocols have been utilized to culture mESC’s. Lin and Talbot have written a chapter on the culturing of both mouse and human embryonic stem cells. The culturing is done using 2 sets of cells, mouse embryonic fibroblasts (mEFs) to provide a feeder layer, and the culturing of the actual mouse embryonic stem cells (mESCs). Ensure reagents are at 37 degrees Celsius to prevent temperature shock to cells.
mEF medium contains the following: Dulbecco’s Modified Eagle’s Medium (DMEM), L-glutamine, penicillin/streptomycin and knockout SR-medium (preferable to FBS, since it can have changes in consistency between batches, also can promote differentiate embryonic stem cells).
Coat a T75-cm2 culture flasks with 0.2% gelatin to provide better adhesion surface for mEFs.
mEFs can either be purchased commercially or obtained in the following manner. Pregnant mice are...