The overall goal of this procedure is to differentiate mouse embryonic stem cells into motor neurons. First culture mouse, embryonic stem cells on primary mouse embryonic fibroblasts, and supplement with leukemia inhibitory factor. Then enhance the normalization process by adding noggin BFG F and FGF eight induce motor neuron specification by incubation with retinoic acid and smoothened agonist, which is followed by axon elongation and motor neuron maturation.
Finally, use immunofluorescence microscopy to examine differentiated MES cells that express strong GFP fluorescence Generating large quantities of motor neuron facilitated research in motor neuron diseases compared to current existing protocols. This approach of differentiating mouse embryonic stem cells into motor neurons is more efficient. This method can provide insight into spinal muscular atrophy.
It can also be applied to other motor neuron diseases such as amyotrophic lateral sclerosis To plate primary embryonic mouse fibroblasts coat four, 100 millimeter tissue culture dishes with eight milliliters of 0.1%gelatin solution. After 30 minutes at room temperature, discard the excess liquid. Now dilute one vial of mitomycin C in activated PMEF into 40 milliliters of PMEF media plate onto the four plates and culture for up to one week.
Next, defrost one vial of MES cells in a 37 degree water bath. Wash the cells with five milliliters of E ES media in a 15 milliliter tube. After centrifuging at 800 RPM for five minutes.
Aspirate the supernatant and resuspend MES cells in 20 milliliters of ES media with freshly added leukemia inhibitory factor. Proceed to remove 10 milliliters media from the PMEF cultures and replace with 10 milliliters of the HBG three ES cell suspension. Monitor the progression of MES cells over time from small colonies at 24 hours to colonies of approximately 100 micrometers in diameter at 72 hours after plating.
Replace media daily to maintain proliferation of cells for induction of MES cells. Differentiation into motor neurons. The mouse embryonic stem cells must first be separated from the PMEF feeder cells and then cultivated in a suspension environment on day of induction.
For each 100 millimeter ES culture, prepare a T one 50 flask coated with 0.1%gelatin After 30 minutes at room temperature, remove the excess gelatin and wash three times with PBS. Continue to carefully aspirate the media from the ES culture, making sure not to dislodge loosely attached colonies. Then wash once with 10 milliliters PBS.
Now to separate MES cells from PMEF, add three milliliters of 0.25%trypsin EDTA and incubate for three to five minutes. At 37 degrees Celsius, verify that the stem cells and the PMEF have detached from the plate. Then add 15 milliliters of fresh ES media and disrupt colonies by pipetting.
Transfer the cell suspension into the prepared gelatin coated T one 50 flask incubate for 30 minutes at 37 degrees Celsius for PFS to attach to the gelatinized surface. Now harvest the floating MES cells in a 50 milliliter tube and pellet by centrification. Resuspend the cells in 10 milliliters of neural differentiation.
Medium enumerate using a hemo cytometer and then plate onto a 100 millimeter suspension culture dish on differentiation. Day one, verify by microscopy that the MES cells have formed small floating aggregates called embryonic bodies. Since any carryover PMEF attached to the dish, transfer the ES suspension cells and media into a 15 milliliter tube, then centrifuge at 500 RPM for three minutes.
To harvest the embryonic bodies aspirate off media carefully add 10 milliliters of fresh neural differentiation, medium to the pelleted ebs, and then plate cells in a new bacterial dish culture for 24 hours on differentiation. Day two, swirl the culture dish and transfer the media and EBS into a 15 milliliter tube. Let EBS settle by gravity, then aspirate off the medium carefully and resus suspend EBS and 10 milliliters of motor neuron differentiation.
Medium culture EBS in a new bacterial dish for five days on differentiation. Day seven, verify that EBS are approximately 150 to 200 micrometers in diameter and express strong GFP signals on differentiation. Day five, two days before dissociating EBS, place 12 millimeter round cover slips on the bottom of a 24 well plate.
Then add 0.5 milliliters of 100 micrograms per milliliter, poly DL ornithine, and incubate at four degrees Celsius overnight. Aspirate the poly DL ornithine air dry plates and cover slips in a tissue culture hood. Rinse the plate wells with double distilled water three times.
Let air dry for an to coat the cover slips. Make a fresh dilution of two micrograms per milliliter mouse laminin in five milliliters of iced cold one XPBS. Add 0.5 milliliters per well and incubate at four degrees Celsius overnight.
Wash twice with PBS on differentiation. Day seven, collect the EBS into a 15 milliliter tube and allow EBS to settle by gravity. After four washes with PBS, add one milliliter of ACU max to the EBS gently mix and incubate for five minutes at room temperature, then aspirate excess ACU max covering the ebs.
Now add three milliliters of MND medium and dissociate EBS by pipetting approximately 20 times using a one milliliter einor micro pipette, then incubate for two minutes at room temperature, pass the cell suspension through a cell trainer cap into a 12 by 75 millimeter tube. Repeat this dissociation step with remaining clumps and cells retained by the filter so as to enrich the yield of single cells in a final single cell suspension of six milliliters. Gently mix the single cell suspension and count cells using a hemo cytometer dilute cells into complete MND medium to a density of two times 10 to the fifth cells per milliliter plate.
0.5 milliliters cell suspension per well of the prepared 24 well plates containing poly DL ornithine laminin coated cover slips. The differentiated cells extend long neurites after two days. In culture, HBG three is an ES cell line derived from a transgenic mouse expressing EGFP under the control of a motor neuron specific promoter HB nine with a defined differentiation protocol.
MES cells can be used to derive motor neurons. The undifferentiated MES cells form round colonies on the top of a fiberblast feeder layer. They have weak GFP expression.
These MES cells were separated from feeder layers and cultured on low attachment dishes to form embryonic bodies. Differentiated MES cells expressed strong GFP fluorescence after day seven. Differentiation EBS were dissociated and plated on poly DL ornithine laminin coated plates.
The differentiated cells extend long neurites from the cell bodies of the plated cells after two days in culture, the MES cell derived motor neurons can be characterized by immunofluorescence staining. These GFP positive cells express the pan neuronal marker neurofilament and the two motor neuron specific markers. Eyelid one and choline acetyl transferase DPI stains the nuclei taken together.
Our two-step differentiation protocol provides an efficient way to differentiate MES cells into spinal motor neurons. After watching this video, you should have an understanding of how to efficiently differentiate mouse embryonic stem cells into motor neurons. In conclusion, remember a high qualities of mouse embryonic stem cells is the most critical parameter for efficiently generating motor neurons.
