The overall goal of the following experiment is to generate oligodendrocyte precursors from embryonic stem cells via a series of culturing steps over 30 days. This is achieved by first generating embryo bodies from mouse ES cells to initiate cell differentiation. As a second step, the embryo bodies are treated with the small molecules, retinoic acid and pure morphine, which induce the directed differentiation of the cells into a liga den.
Cyte precursors. As a third step embryo bodies are disaggregated into single cells after an additional 22 days. In culture, approximately 80%of the cells are OPCs.
Next, the MESC derived OPCs are transduced with bao viruses carrying NAV 1.2 subunit in order to generate spiking OPCs results are obtained that show the expression of OPC marker and spiking properties of these MESC derived OBCs based on immunofluorescence microscopy and electrophysiological recordings. Hi, I'm Pong Chiang from the laboratory of Dr.Ing in the Department of Cell Biology and Human Anatomy at University of California Davis. We are also affiliated with the Shriners Hospital for Children of Northern California.
Hi, I am vial Raj, also from the Deng lab. Today we'll show you a procedure for differentiation of mouse emron stem cells into genocide precursor cells. We use this procedure in our laboratory to study the properties of embryonic stem cell derd oligodendrocyte precursors in order to generate cells with better therapeutic potential.
So let's get started. Today, oligodendrocyte precursor cells or OPCs will be generated from the OG two GFP mouse embryonic stem cell line. These embryonic stem cells need to be passaged every three days to keep them healthy at an optimum density and to prevent spontaneous differentiation.Passage.
The ESS cells when they're at 70%confluence under a laminar flow hood. Remove the medium over the culture. Rinse with PBS and trypsin eyes with two milliliters of prewarm.
One x trip LE express for five minutes at 37 degrees Celsius. Stop the reaction and resuspend the cells in four milliliters of MES cell medium. Transfer the cells to a 15 milliliter conical tube and pellet them at 1000 RP for five minutes.
Resuspend in MESL medium plate, one 10th of the cells over a confluent layer of irradiated meth feeders in MES cell medium. After three days ES cells are approximately 70%confluent and are ready to be passaged or suspended to form embryo bodies to induce embryo bodies and begin differentiation Trypsin mouse ES cell colonies as before and then resuspend the cells in knockout serum replacement or KSR medium. Count the cells on a hemo TER, dilute the suspension in KSR.
Medium to 15, 000 cells per milliliter plate three milliliters into each well of an ultralow attachment six Well plate culture. The suspended cells at 37 degrees Celsius for four days. Change the medium completely every two days.
Starting on day four, replace the medium daily on the fourth day Supplement the KSR with 0.2 micromolar retinoic acid on the fifth day supplement with both retinoic acid and one micromolar primor famine on the sixth and seventh days. Feed the embryo bodies within two mediums, supplemented with 0.2 micromolar retinoic acid and one micromolar primor famine. After eight days in culture, the majority of embryo bodies express GFP and are ready to be disaggregated trypsin eyes and count the cells as before.
Then resus suspend them at 3.5 times 10 to the fifth cells per milliliter. In OPC medium reflate three milliliters of the suspension in OPC medium. Using poly ornithine coated 60 millimeter dishes approximately every week, the cells will become confluent and will look like this passage to the cells as before with a three minute trypsin ization.
30 days after the original resus suspension. In KSR medium, the cells show characteristics of oligodendrocyte precursor cells to generate spiking OPCs. Use mouse ESL derived OPCs that are between 50 and 70%confluent.
Exchange the medium for one XPBS to rinse the cells. To introduce voltage gated sodium. Channel 1.2 alpha subunit.
We use the back MAM sodium channel kit. Mix one milliliter of back MAM component A with four milliliters of one XPBS. Remove the PBS rinse and add the diluted component.
A incubate at 37 degrees Celsius for two hours. Replace the back memory agent with OPC medium enhanced with components B and C as described in the kit. Instructions incubate for an additional two hours at 37 degree Celsius.
After the incubation, replace the enhanced medium with fresh OPC medium. After another 24 hours of culture, the ESL derived OPCs can be assayed for spiking at this time point. The EBS do not show any GFP fluorescence.
The EBS show strong GFP expression and are ready for disaggregation culturing for about 30 days after original resus suspension. In KSR medium, the majority of cells are immuno positive for NG two staining, which is consistent with GFP expression. However, unlike brain OPCs, these MES cell derived OPCs fail to fire action potentials even when the cells were depolarized to zero millivolts.
After introducing NAV 1.2 subunit by viral transduction, these MES cell derived OPCs get excited and fire action potentials. We have just shown you how to differentiate mouse embryonic stem cells into OPCs and generate spiking OPCs by viral transduction. When doing this procedure, it's important to remember to store the retinoid acid per morphine and FGF two properly and always use fresh aliquots, discard any extra t retinoid acid after use.
So that's it. Thank you for watching and good luck with your experiments.
