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 sacrificed between 12,5 and 13,5 days after mating. Embryos are removed from uterus and placed in sterile PBS. Head and internal organs are removed from embryo. In fresh PBS 1mm sections are cut then transferred into trypsin/EDTA. Stir cells for 40 minutes, add DNase if it looks viscous and clumpy. Proceed by adding mEF medium, then strain the solution through a 100 m cell strainer, repeat twice, then spin down 270 g for 4 minutes. Discard supernatant. Resuspend pellet using fresh mEF medium. Remove gelatin from T75-flask and replace by mEF medium. Add cells to T75. Check cells under microscope and incubate at 37 degrees Celsius. Change medium after 24h, then every other day until 90-95% confluency is reached. They are then ready for passaging or cryostorage.
To create the feeder layer, use a T25 flask coated in gelatin. Replace by mEF medium. Add passage 2 or 3 mEF cells (so the end passage is 3 or 4 when using in combination with embryonic cells) and allow to grow to 90-95% confluency. To stop cell division of mEFs before adding stem cells, use mitomycin C or irradiation. Before use with the embryos, add mEF-medium containing Leukemia Inhibitory Factor (LIF) several hours before adding embryos and leave in incubator.
To obtain embryonic stem cells remove uterus 3,5 days after conception from mouse and place in petry dish containing FHM-medium. Cut uterus in half and flush out embryos with some medium while using dissecting microscope. Collect all embryos in a collection plate containing FHM-medium, repeat with other uteri, all embryos can be collected together. Wash embryos by moving them into another 3 wells consecutively, one embryo at a time. Then place them on feeder layer plate, one embryo per well. Continue process until 25-40 embryos are obtained (may take up to 2 breeding cycles). Leave undisturbed for 6 days.
Before breaking up the embryos make more feeder plates. However, use mEF medium with FBS instead of SR-medium (and for every passage after, SR is used for refreshing medium) before adding cell clumps this time. To disaggregate the embryo, prepare a well containing trypsin covered by sterile filtered mineral oil. Then wash embryo with sterile PBS 2 times. Leave last PBS wash on to prevent drying while dissociating embryo and surrounding feeder layer. Then place embryo in trypsin and leave in incubator for 5 minutes. Remove a small amount of medium from the destined feeder well and add it to embryo, then suck up the embryo up and down to break it down into clumps. Place each clump in the feeder well and spread them out so they form a single layer. Leave for a day in incubator, then replace medium with mEF containing SR-medium. Four days later cells mESCs should be present. Wash plate with PBS, add trypsin and incubate again. Then plate the cells on a feeder plate with mEF medium containing FBS. Replace medium the day after, only now containing SR-medium. This should provide an established mESC line. After 1 or 2 passages, test to see if mESC will survive independent of feeder layer, however the plate has been coated with gelatin.
iPSCs can be obtained from several different cell types. Although fibroblast were used initially, it was found that adipose cells have a much higher success rate in becoming iPSCs. They also do not require a feeder layer, cutting out a lot of time and money from the process and reducing the risk of xenobiotic-material being introduced. Since adipose cells are a viable alternative to fibroblasts with high accessibility in PD patients they are of interest and will be used in this particular study.
To obtain adipose cells remove fat pads from 8-10 week year old mice and place in Hanks Balanced Salt Solution (HBSS). Then move to collagenase solution. Cut up tissue into small pieces, briefly spin at 100 rpm and place in 37 degree Celsius shaking hot waterbath for 30-60 minutes, while checking progress every 10 minutes. Filter through 250μm nylon filter. Then, filter through a 100μm cell strainer. Centrifuge at 400g for 5 min at room temperature. Discard any floating cells and remove supernatant. Resuspend pellet in HBBS and repeat centrifuge at 400g and re-suspension another 3 times. (If there is any red in the pellet use erythrocyte lysis buffer). Resuspend pellet in DMEM with FGF and incubate at 37 degrees Celsius for 1 hour. Transfer non-adherent cells to another dish and allow to cells to grow to 80% confluency while refreshing medium after 24 hours and every 3 days. Remove medium and use Dubelco-PBS to wash 3 times, then add trypsin-EDTA and incubate for 5 minutes, tap to dislodge cells, add DMEM containing FGF to inactivate trypsin and centrifuge at 400 g for 5 minutes. Discard supernatant and re-suspend at cell density of 5,000 cells/cm2 in DMEM with FGF. Repeat Passaging to ensure majority of cells are adipose stem cells.
To produce the retrovirus use 293T cells at ~1.2 × 105 cells per ml, then culture until 90% confluent. Then remove medium wash with PBS, use trypsin/EDTA to dissociate cells, add complete DMEM, centrifuge at 400g for 2 minutes, and discard supernatant. Resuspend in complete DMEM and reseed at 1.2 × 106 cells. Prepare the Lipofectamine 2000 and DNA complex then mix pMX, gag-pol and VSV-G–expressing plasmids for co-transfection. Make a separate dish for Oct4, Sox2, Klf4 and c-Myc in pMX vectors, use pMX-GFP as a control and add each lipofectamine/dna complex to the 293 T cells, still with the pMX vectors in separated plates. Incubate overnight at 37 degrees Celsius. Replace medium with complete DMEM and check if transfection occurred. Leave for another 24 hours, and replace again with DMEM, but collect the viral supernatant for each plasmid this time, repeat after another 24 hours. Filter supernatant with 0.45μm syringe filter then add polybrene.
Creating the iPSC is done by using the mASC after passaging and plating at 40,000 cells per well (for 12 well dish only use 10, leaving one for cell count and another for checking transduction efficiency) and incubate at 37 degree Celsius overnight. Once 7-% confluency has been reached, add the oct4, sox2, klf4 and c-myc viral supernatants together to the wells, add the GFP virus supernatant to the empty one and then incubate overnight again 32-37 degrees Celsius. Then remove supernatant, wash 2 times with sterile PBS and add DMEM with FGF. After a day, passage cells, count the untreated well and plate out in mEF medium containing FBS on gelatin coated wells. Then change medium after 2 days to mEF using SR-medium. Re-fresh medium every other day for a week. Change medium every day then and continue to select desired colonies of iPSCs.
Culturing Adult (Neuronal) Stem Cells:
Newborn mice will be used to provide brain tissue, because these tend to provide the most neurospheres. They are euthanized with CO2 and then decapitated. Pups should be hairless but use Betadine soaked gauze to sterilize and then repeat with 95% ethanol. Make a midline incision with scalpel along skull and pull back skin to expose skull. Cut towards sides to remove skin beyond ears. Cut skull, then use forceps to break off skull without cutting brain. Slide forceps underneath brain to cut of spinal cord, nerves and mayor blood vessels. Then remove brain and place into dissection solution. Strip all meninges off, (can be a potentially source of contaminated cell types) then rinse carefully, then place brain on another dish containing dissection solution.
Although the ventricles provide the most neurospheres, the subventricular zone (SVZ) is of particular importance since these have seen to still contain NSC’s in aged and Parkinson’s affected individuals. Therefore only this region will be used to source the mNSC’s, since it may represent a source in affected individuals. Placed in another dish containing dissection solution the SVZ is cut up into small sections 1.0 mm3 large. Pieces are the centrifuged at 250g for 1 minute, supernatant is removed and trypsin, Type 1-S Hyaluronidase and Kynurenic Acid c. Mixture is placed in 37 degree Celsius shaking water bath for 1,5 hours, while dissociation takes place, ensure it is triturated every 20 minutes. Centrifuge at 250g for 5 minutes and remove supernatant. Add trypsin inhibitor, break up pellet and place in water bath for another 10 minutes. Centrifuge at 500g for 5 minutes, remove supernatant, then add SFM-medium and re-suspend mixture into single cells. Calculate viable cell density, then plate at 10-15 cells /μl using SFM-medium. Incubate at 37 degrees Celsius, 6% CO2 until at least 25 neurospheres have been formed before cryopreservation or passaging.
Typing Stem Cells:
Before any of the stem cells can be used, verification needs to be obtained that they are bonafide stem cells, especially for neurospheres the results will be a mixture of cell types at slightly different stages of differentiation. Therefore certain markers need to be tested for in order to determine the state of the cell.
There are several mRNA transcripts can be used to determine cell differentiation status, and how far differentiated a mixture of cells may be. Nanog, Sox2, Oct3/4 and Esg1 are the most important for the ESC and iPSC’s (Takahashi et al., 2007). Next to this, teratoma formation containing ectodermal, mesodermal and endodermal cell in an immunosuppressed mouse would be another way to assess lineage status (Lerou, P.H. et al 2008).
To determine if neurosphere cell contains stem cells, see if astrocytes, oligodendrocytes and neurons are formed after repeated cultures of single neurospheres.
Animal disease model:
To start the assessment precise and comparable parameters will allow for a fairer comparison among the different sets of experiments. The direct area to be affected needs to be easily replicated among the different subjects. In the study of Parkinson’s, different animal models are used for this purpose; neurotoxin rodent models have proven to be predictive in outcome. The use of mice or rats affected by 6-Hydroxy-Dopamine (6-OHDA) to study Parkinson’s has been well established. It causes oxidative stress to DAergic neurons, causing them to die quickly. By increasing the dosage it is possible to replicate disease severity. Although they do not display all of the Parkinson traits, 6-OHDA causes motor behavior that becomes asymmetrical if it is injected in only one half of the brain in the substantia nigra directly. This allows for quantifiable motor behavior when animals are assed on automated rotameter bowls, when given amphetamine, to see if turns towards one side is favored over the other, in this case on the same side as the lesion. The ratio of left to right turn becomes more skewed when DAergic neurons have been lost. Next to this, software is available that keeps track of movement, enabling easy access to quantifiable data and its analysis, with less chance of human error occurring when observing, next to being able to asses several subjects at the same time.
Administering Stem Cells:
Prior to administering the Stem Cells, rats will be injected with immunosuppressors to prevent rejection. This is started a day before the operation and continues throughout the experiment.
It was suggested that a lower dose of embryonic stem cells 1,000-2,000 cells / μl might be more beneficial due to more direct contact with the neuronal environment (Bjorklund et al., 2002. Therefore next to this, there will be another dosage used at 9,000-10,000 cells / μl to compare the effect for all 3 types of stem cells. There will also be a group that only receives medium, containing no stem cells to measure) the effect of the surgery procedure. To ensure the same location is used as the 6-OHDA injections, the atlas of Franklin and Paxinos was used to provide the exact location for reference again.
Rotameter bowl. In order to asses the effects of the stem cells, several moments of rotational behavior assessment will need to take place across several different groups. All rats will be assed before the use of 6-OHDA. After 6_OHDA administration they will be assed again. To approximate the amount of damage, it was found that 97% decrease in DAergic neurons if turns exceeded 500. They would be assed again every 2 weeks until there was no more improvement occurring after receiving stem cells.
Pet Scan. PET scan was done under anesthesia. To asses the dopamine transporter during PET the specific DAT ligand arbon-11-labeled 2b-carbomethoxy-3b-(4-fluorophenyl) tropane [11C]CFT was used after every assessment moment used for the rotameter bowl. This to represent the stem cells differentiating into DAergic neurons.
Immunohistochemistry. To determine the survival of the stem cells and proceed with immunohistochemistry rats needed to be euthanized and the brains fixed.
For those cells involved in dopamine production, the enzyme Tyrosinehydroxylase is used as a marker, to be used in conjunction with NeuN to find the neuronal cells. To determine whether the origin was of the stem cells or residential (rat) cells, this also needs to be labelled with a mouse specific antibody.
To determine if any α-synuclein proteins had infected the injected stem cells, antibodies were utilised with them, these would then be compared with the presence of mouse antibodies to determine the origin of the affected cell.
Sample size should ideally be as large as possible. Realistically however this would not be practical or economically viable. Mead’s resource equation may therefore be used to give an estimate on group size (Mead, 1988). E=N-B-T (with E= degrees of freedom, N=number of rats -1, B= blocking component and T= treatment groups, minus control). E should be between 10-20. Therefore if 20=N-0-6 ⇒ N= 14.
This would indicate 15 rats should be spread between the 7 groups (there are 3 different types of stem cells, given at 2 different dosages, and one control group. Which would work out at 2-3 rats per group. To allow for mortality this number should be slightly higher however. At an approximate of 3-4, getting a total between 21 and 28. Therefore 28 rats should be used for the experiment.
The statistical test to track behavior would be a two way-ANOVA to analyse the rotarod performance (ie score of last last improvement) among the different stem cell types in combination with the dosage administered. Therefore
H0 = there is no statistical difference between type of stem cell and dosage.
H1 = there is a statistical difference between type of stem cell and dosage.
Effect of stem cells: F(3,28) = x, P < 0.0001
Effect of dosage of stem cells: F(1,28) = x, P < 0.0001
Interaction genotype x treatment =
F(3,28) = X, P < 0.0001
If found significant, a further analysis should be done using a Post hoc Analysis, Tukey’s HSD test would therefore give the result that most benefitted by the admission of stem cells.
The use of different types of stem cells are being investigated in animal models in order to find a cure for Parkinson’s Disease. This study attempts to determine if origin and dosage has an influence in the success of restoring motor function in the rat after a 6-OHDA lesion of the substantia nigra in a single hemisphere and if the injected cells would become infected by α-synuclein proteins.
Bjorklund LM, Sánchez-Pernaute R, Chung S, Andersson T, Chen IY, McNaught KS, Brownell AL, Jenkins BG, Wahlestedt C, Kim KS, et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA. 2002;99:2344–2349.
Lerou, P.H. et al. Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos. Nat. Protoc. 3, 923– 933 (2008).
Mead R. 1988. The design of experiments. Cambridge, New York: Cambridge University Press. 620 p.
Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).