Derivation of Human Embryonic Stem Cells by Immunosurgery

Hi, my name is Alice Chen. I'm a postdoc in the melting lab and today I'll be demonstrating to you how we derive embryonic stem cells from human embryos. We receive embryos at all different stages of development and we culture them in vitro until they reach the proper expanded blasts stage.

These are examples of how the embryo develops from the first, the one cell stage to the early blast assist, and then the expanded blast from which we derive human embryonic stem cells. High quality embryos chosen for derivation are then transferred through three drops of ACE roads where you'll leave it and watch for dissolution of the Z of lucita. The neurosurgery process is a non-specific process where the rabbit anti-human antibody will bind to the outer layer of cells.

In this case, it's the trifecta derm. Upon application of Guinea pig serum complement, all of these cells with antibody attached will be lysed. To begin, we want to prepare three plates.

The first plate is for ACE tyros, which we use to remove the zita. The second and third plates are for immunos surgery. I'll first begin by generating the plate where we will place the acid tyros drops and human embryonic stem cell derivation.

Media drops. Prepare three drops of acid tyros on the left side of the plate, and on the right side of the plate you'll prepare three drops of human ES culture media. The second plate consists of the primary antibody, which is a rabbit anti-human RBC.

Again, the left side of the plate will be dedicated to the primary antibody, three drops per embryo, and the right side of the plate will be dedicated to the human ES culture. Media drops, again, three drops per embryo for the complement plate. Again, we'll place three drops of complement on the left side of the plate, and then three drops of human ES culture media on the right side of the plate.

Again, each row is dedicated for an embryo. All three plates are covered with mineral oil to prevent evaporation and placed into the incubator for equilibration. This is a dissection scope inside of a picking good where there's laminar flow, just like for a tissue culture, and this is where we do the majority of our human ana manipulation.

The first thing that you want to prepare before you get started with your embryos is mouth pipettes. So this is the standard mouth pipette tubing, except that we've inserted a 0.2 micron filter between your mouth and where the pipette would go. And this is just to ensure sterility.

Let me demonstrate how to pull mouth pipettes. I like to pull a variety of sizes. You can get all of these from VWR and I like to pull 10 microliter, 50 microliter, and 100 microliter diameters.

Now am using a diamond pin to cut the mount pipe. For this first step, you want a rather large diameter because you're handling the Entire embryo. High quality embryos chosen for Derivation are then transferred through three drops.

Ofci roads just transiting from the first drop to the second drop to the third drop where you'll leave it and watch for dissolution of the zap palita. This process takes usually less than 30 seconds, and you can Actually see the zop palita dissolving away. When the zap palita has completely dissolved.

The embryos Then transferred through the three wash drops of human Es culture media transiting Through the first, second, and third drops of wash. Then the embryo is transferred into The primary antibody, drops again, transiting from the first drop to the second drop to the third drop where you'll leave it for half an hour of incubation in the tissue culture incubator lysis of the trifecta, derm becomes apparent when you see bubbling of the cells on the outside of the embryo. This is the beginning of the trifecta dermal cells beginning to ly.

When the lysis is apparent throughout the entire outer edge of the embryo, then the embryo is ready to be washed through the three drops of humanus culture Media. After immunos surgery, the Embryos are mechanically triturated using a smaller mouth pipette. This mouth pipette should be of a smaller diameter than the first pipette so that you can make the process of tation more efficient for shaving off the lys trifecta.

Dermal cells. At the end of mechanical ation, you're left with a cluster of cells in the center. There is the inner cell mass, which is intact.

Surrounding this, there is, there are many lys trifecta dermal cells, as well as some intact trifecta dermal cells. This inner cell mass isolate is then placed in a well that has been prepared with feeder layer cells of mouse embryonic. The plate of feeder layer cells were prepared the night before, and this morning I switched the media from mouse embryonic fibroblasts media to human es derivation media.

The inner cell mass isolate is then transferred from the wash drops after complement into this well containing the feeder layer cells. This dish is then returned to the incubator and left untouched for a couple of days to allow for attachment of the inner cell mass isolate. This is an example of how the outgrowth develops.

When the outgrowth becomes big enough. Then we can begin the first passage by dividing this outgrowth in half. Now, it's been 14 days since the original derivation, and here is an example of an outgrowth from one of our derivations.

This is an example of one such outgrowth where today I'm going to pick off half of the outgrowth. This outgrowth will be divided in half and then cut into square pieces, and each of these pieces will be transferred to a new well containing feeder cells. It's a good idea to leave half of the outgrowth untouched so that you have a continuing source of cells to go back to in order to amplify your cells and to back up any passages that you perform.

For the first two to three passages. It's a good idea to mechanically passage in this fashion and eventually adapt the cells to enzymatic dissociation for More efficient packaging. Our lab Consistently Derives human embryonic stem cell lines for use and understanding development and differentiation.

These lines are also distributed worldwide to stem cell researchers. I think there are four main factors that affect the success of the derivation. The first one has to do with the quality of the embryo, which also depends on the genetic background, how well the embryos were frozen and thawed, and also on the quality of in vitro culture up to the derivation stage at the blast assist.

The second factor is probably the quality integrity of the inner cell mass post isolation, and this can depend on the technique used for isolating the inner cell mass and can also depend on experience and skill. The third factor is how well the isolated inner cell mass attaches to the feeder layers, and the fourth factor is outgrowth of this ICM isolate. I don't think that it's technically a difficult process.

It just takes a bit of practice. In fact, each step is fairly straightforward, so as long as you can follow the protocol With a little bit of practice, I think anybody can do this. What we found in the lab is that every line is a Little bit different for reasons that we're not entirely sure we understand.

For example, if you were to test the differentiation potential of 10 lines, perhaps two or three of those lines may be more likely to develop into the type of cell that you're interested in. I suspect that these differences have to do with the stage in which the cells were, the embryonic stem cell lines were derived the stage of the embryo that is as well as in the manual handling of these cells. I think that their property sometimes changes the heterogeneity of the cell lines and their potential is something that every stem cell researcher needs to keep in mind.

Again, I don't know what causes this, but certainly as an example for my research, our goal ultimately is to make insulin producing beta cells of the pancreas. The first step towards making this type of cell line is to generate endoderm from embryonic stem cells, and in a comparison across 10 to 20 lines, what we found was that one or two of these lines can efficiently generate endoderm while half of them maybe can generate endoderm less efficiently, and some of them can't generate endoderm very well at all. So through this initial screening process, we select for cells that have a higher tendency to form the cell type of interest, and we pursue the remaining Differentiation using these cell types.

The first derivation of human embryonic Stem cells was reported by Thompson Etal in 1998, and in 2004, our lab reported derivation of 17 new human embryonic stem cell lines. By modifying these published protocols, and these modifications were really just to make the derivation process more streamlined. They consist of changes in the media composition, the way that the cells are passaged and the way that they're freeze thawed.

Since then, in every derivation, we look for new ways to improve the protocol and the rate of derivation. For example, our lab is exploring alternative ways to isolate the inner cell math, not using xeno reagents like the rabbit antibody or the Guinea pig sero complement, but more mechanically dissecting out the inner cell mass. Hopefully, these methods will lead to at least a comparable, if not better way of isolating the inner cell mass.