The overall goal of the following experiment is to isolate human stem cell colonies in a quick and efficient manner. This is achieved by culturing human embryonic cells into a dense layer of mouse embryonic fibroblasts. Over time, expansion of the stem cell population increases the number of human stem cells, but also allows them to create island like colonies in the mes.
Next, the meth layer is removed by aspiration, leaving behind the individual colonies of human embryonic stem cells. Results of immunohistochemical staining for stem cell markers show that this is an effective isolation method for eliminating feeders and maintaining colony integrity. I first had the idea for this method when co culturing cells in our lab, I wanted to save the stem cell cultures from the over confluent culturing systems.
So the problem that most people will experience with having with this system is lack of human stem cell culture experience. We found this technique particularly useful when we need to isolate large numbers of human embryonic stem cells for experimentation Compared to other methods such as sorting or handpicking stem cell colonies. This method is faster and more efficient for isolating colonies from high density cultures.
The mouse embryonic fibroblast should be treated with mitomycin C or irradiated prior to co culturing with embryonic stem cells to keep them from going through division Two hours prior to meph seeding coat each 60 millimeter plasma treated culture dish with two milliliters of 0.05%Gelatin thaw a vial of treated mes spin down for five minutes at 1, 500 RPM and seed. A culture of approximately 20, 000 cells centimeter squared on the gelatin coated dish incubate the culture overnight for adherent cells, New cultures may be initiated from cryopreserved cells or packaging the current cultures onto meth coated dishes. When starting with a plate containing the large human embryonic stem cell colonies, wash once with three milliliters of warmed phosphate buffered saline.
Add three milliliters of one milligram per milliliter, collagenase four solution, and incubate for five to 10 minutes. At 37 degrees Celsius, The cells would not become distinct or ball up as you see with trypsin. Using the edge of a five milliliter pipette tip, create a grid pattern on the colonies and break them into small cell clumps.
Now holding the end of the pipette tip at a perpendicular angle to the culture plate, scrape the bottom of the culture plate and dislodge all of the cells from the surface. Transfer the cells into a falcon tube to harvest the cells centrifuge at 1000 RPM for five minutes and aspirate off the medium. Next, aspirate the medium from the mouse embryonic fibroblasts plated the day before.
Then plate the human embryonic stem cells at a one to three ratio from the original plate and feed with three milliliters of human embryonic stem cell medium. The human HECS are cultured at high density for 10 to 14 days. Place the tip of an aspirating pipette at the edge of the plate and use suction to gently pull up the edge of the meth sheet.
Remove the media from the dish. Allow the tip to catch the cell sheet and then slowly move the tip in an arced manner above the plate. If the sheet clogs the pipette manly, remove the sheet from the culture.
Tap the top of the culture dish with a aspirator to complete breaking up the cells and remove the cells through a complete aspiration. Replace the human embryonic stem cell medium immediately and return the culture to a cell culture incubator after seeding onto culture dishes. This co-culture system results in high density human embryonic stem cell colonies surrounded by mouse embryonic fibroblasts.
The aspiration technique removes the MES and produces small undisturbed human embryonic stem cell colonies. With very few mes surrounding the intact colony, the embryonic stem cell colonies are able to undergo significant expansion into very large colonies. For example, this human embryonic stem cell colony shown immediately after meth depletion expanded into a very large colony immunofluorescent identification of colonies up to 10 days after the meth.
Depletion shows that colonies maintain their expression of pluripotency markers and do not exhibit differentiation towards the mesodermal fate. These transmission light images show the morphology of immuno fluorescently stained colonies at different magnifications. DAPI stains the nuclei of the cells.
Importantly, the human embryonic stem cells express The human stem cell markers SS EEA four T three four and TRA 180 1. Note that the images depict a colony portraying the typical smooth borders. They do not express FLK one and early marker of mesoderm differentiation, nor did they contain any fibroblasts as shown by absence of DDR two staining.
After watching this video, you should have a good understanding on how to isolate human stem cell colonies using culturing techniques to manipulate natural environments. After mastering this, this can be done in seconds. Also, don't forget not to let your cultures dry out when removing the media and the layers of mess.
