The overall goal of the following experiment is to form oocyte like cells using somatic skin derived stem cells. This is achieved by utilizing clean undifferentiated stem cells derived from newborn mouse skin. As a second step, the stem cells are dissociated, which prepares them for differentiation.
Next, the stem cells are plated in defined differentiation medium. In order to induce germ cell formation, results are obtained that show the expression of oocyte specific markers based on semi-quantitative real-time PCR. This method can help answer key questions within the field of reproductive biology.
By providing a closed system in which to study germ cell, an eSight formation and development, All media required for the protocol should be prepared two to three hours in advance of use and placed with a loosened cap in the cell culture incubator where the experiment will be taking place. The two media used here are stem cell medium consisting of D-M-A-M-F 12, supplemented with B 27 BFGF, and EGF and germ cell differentiation. Medium consisting of M1 99 supplemented with FSH LH B-S-A-I-T-S, sodium pyruvate, tuin and EGF stem cells isolated from newborn mouse.
Pups are used for in vitro germ cell differentiation at passage two through four. Since the efficiency of cyte like cell or OLC formation is directly related to the quality of the starting stem cells, it is best to start the differentiation as soon as the population appears clean and healthy, which is generally around passage 2 48 hours prior to initiating differentiation subculture the stem cells using a serological pipette, remove all the suspended spherical cell aggregates and spent media from the culture dish and place in a 15 milliliter tube. It is important to only collect the suspended spherical aggregates of cells and not the cells attached to the bottom of the dish that are spontaneously differentiating.
Pellet the cells at 500 times G for five minutes, discard the supernatant and resuspend cells in 500 microliters of fresh stem cell media. Warm to 37 degrees Celsius. Gently pipette the aggregates to partially dissociate the cells.
Do not aggressively pipette the cells as this will reduce cell viability. Wash the partially dissociated cells in 9.5 milliliters of fresh prewarm stem cell medium on a low attachment 10 centimeter cell culture dish. Return the cells to a 37 degrees Celsius 5%CO2 cell culture incubator for 48 hours.
To begin the procedure for OLC differentiation, use a serological pipette to remove the stem cells from the culture dish and place in a 15 milliliter tube. Leave behind any attached cells. Pellet the cells at 500 times G for five minutes, discard the supernatant and resuspend cells in 500 microliters of sterile PBS vigorously pipette the cells using a wide bore 1000 microliter tip into clumps, not larger than 10 to 20 cells.
For the success of this protocol, it is critical to regularly check the level of dissociation in order to ensure that the correct level is obtained During dissociation. Periodically remove a small amount of cells and check under a white light microscope to confirm aggregated cells are in clumps of 10 to 20 individual cells. Do not over dissociate the cells as this will result in reduced cell viability.
Next, add 9.5 milliliters of PBS to the dissociated cells and transfer 15 microliters of this cell suspension to a hemo cytometer for cell counting. Pellet the cells at 500 times G for five minutes. After discarding the supernatant resuspend the cells in differentiation, medium to a concentration of 1.32 times 10 to the sixth cells per milliliter.
Plate the cells by adding 500 microliters per well into a flat bottom 24 well suspension dish. Place the 24 well dish into a cell culture incubator at 37 degrees Celsius in 5%CO2 for 48 hours After 48 hours. Remove 250 microliters of medium from each well and place in a corresponding labeled 1.5 milliliter tube.
Add 250 microliters of fresh differentiation medium to each well centrifuge the spent medium at 500 times G for five minutes, discard the supernatant and resuspend any pelleted cells in 50 microliters of fresh differentiation. Medium return cell suspension to the corresponding culture. Well in the differentiation plate, every 48 hours up to day 12 of differentiation change half the medium in the same manner.
During early stage differentiation, the cells will attach to the bottom of the culture dish and spread out. This representative image shows cells at 42 to 72 hours into differentiation cells positive for OCT four. A marker for undifferentiated stem cells are indicated in green nuclei are counterstain with hirst and indicated in blue.
As differentiation progresses, some cell aggregates will be seen with OCT four positive cells marked in green, surrounded by OCT four negative cells. The scale bar for this image is 100 microns. This figure shows a white light image of a follicle like structure following detachment from the culture surface, the scale bar represents 40 microns.
With continued culture, the LCS will grow to reach about 45 microns in diameter. The scale bar in this image represents 10 microns. Large OCT four positive lcs can be seen either surrounded by cells or singly within the culture.
These lcs can be collected with support cells or trypsin eyes to obtain LCS without other cells attached. In order to confirm that the LCS expressed similar transcripts as natural ol sites, the LCS were collected and tested for the expression of common OO site markers such as Z-P-C-S-C-P three CMOs, T four BMP 15, and vasa. This figure shows the transcript levels of these markers in 10 lcs relative to 10 newborn mouse cytes.
The results show that the LCS express less ZPC than cytes. Since ZPC is a component of the CYTE specific zop lucidum membrane structure, this may explain the fragile nature and thinner zita membrane of lcs. The expression of the meiosis SCP three is higher in the LCS when compared to natural cytes.
However, a different meiosis marker CMOs is expressed at a lower level. This discrepancy in expression levels may partially explain the current failure of LCS to complete myosis. Other markers such as BMP 15 and vasa are not significantly different between lcs and O sites.
When attempting this procedure, it is important to remember that the health of the starting stem cell population is critical to its success.
