Embryonic stem cell lines are made from four-five-day-old human embryos. At this stage of development, the embryo is a sphere of cells with two distinct cell populations; an outside layer of flat trophoblast cells and an inner clump of round inner cell mass (ICM) cells. The embryo consists of ~100 cells four days after fertilization, and ~150 cells five days after fertilization.
Embryonic stem cell (ESC) derivation involves the removal of the trophoblast cells (which are collectively called the trophectoderm) and the isolation of the ICM cells. There are several ways to remove the trophectoderm, but the most commonly-used technique is “immunosurgery,” which uses antibodies that bind to proteins on the surfaces of the trophectoderm, and serum to initiate destruction of the trophoblast cells. The isolated ICM cells are then cultured, and if they grow, they may produce an embryonic stem cell line.
Immunosurgery was first perfected by Davor Solter and Barbara B. Knowles on mouse embryos. They used an antiserum that was raised in rabbits when the rabbits were immunized against mouse spleen tissue. When mouse embryos were incubated with this antiserum plus serum from mice, all the cells of the mouse embryo died. However, if they used the rabbit antiserum and serum from guinea pig, then only the trophoblast cells were destroyed. For human embryonic stem cell derivation, the rabbits are immunized again human red blood cells, and this rabbit antiserum is used with guinea pig serum. The serum contains proteins called “complement,” which bind to cells that have antibodies attached to them and bore holes in those cells, thus destroying them.
When ICM cells are cultured, they are placed on a layer of mouse cells that have been treated with a chemical called mitomycin C to prevent them from dividing. These non-dividing cells act as “feeder cells” that keep the ICM cells from differentiating. Because ICM cells are grown on animal cells, they cannot be used for clinical purposes, since they will possess animal proteins can carbohydrates on their surfaces, which would be attacked by the patient’s immune system. However, several ESC lines have been derived without animal products, and it is possible to make ESC lines that would be safe or human use.
ESC derivation results in the destruction of human embryos. There is not two ways about it. Even though there are potential ways around this problem, the majority of ESC lines were made literally over the dead bodies of very young human beings. All the rationalization in the world (the embryo is too young, too small, too inchoate, too unwanted, going to die anyway, in the wrong place at the wrong time) do not undo the fact that the embryo is a very young human being, and making an ESC line from it ends his/her life.
Getting the ESC line to differentiate in what you want it to be is another problem. If any undifferentiated cells remain after differentiation, they can cause tumors. Therefore, there is a need to ensure that differentiation is efficient and complete. To this end, Doug Melton’s lab at Harvard University has published a remarkable paper in the journal Nature that uses mesenchymal stem cells from particular organs to direct the differentiation of ESC lines.
Melton’s lab, in particular Julie M. Sneddon and Malgorzata Borowiak (say that fast five times), established 16 lines of tissue-specific mesenchymal stem cells (MSCs) from embryonic, neonatal and adult mouse intestine, liver, spleen, and pancreas and human pancreas too. Then they cultured mouse ESCs on these MSC lines to determine if they could drive the ESCs to differentiate into pancreas cells. In the embryo, pancreatic precursors express several genes in a nested, hierarchical fashion. First, they express Sox17, which is a common endodermal marker, and then pancreatic progenitors all express Pdx1. Of these pancreatic progenitors, some express Ngn3 and these will become endocrine rather than exocrine cells, and othe the Ngn3-expressing cells, a few will become beta cells that make insulin.
Melton and his co-workers tried to determine if any of these genes was up-regulated in their ESC lines if that were co-cultured with their established MSC lines. They discovered that four lines – MSC1, 2, 3, & 4, all affected gene expression when co-cultured with ESCs. MSC 1 and 2 induced and increase in Sox17 expression and MSC 3 and 4 increased the expression of Ngn3 in ESCs.
These changes in gene expression were due to increased cell proliferation of cells actually expressing these genes and not due to differential survival. Also, no combination of growth factors could achieve the same results as the accompanying MSC lines. Thus there is more going on here than the MSCs just secreting the right growth factors. The MSCs must be making contact with the ESCs and inducing them to differentiate into a particular cell type.
Next Melton and his colleagues determined if this interaction with MSCs caused the ESCs to lose their ability to self-renew. The answer was a clear “no.” Even though these ESC lines were expressing genes characteristic of endodermal or pancreatic tissue, they did not lose their ability to differentiate into pancreatic tissue when appropriately induced to do so, and they also id not lose their ability to self-renew and grow competently in culture.
In a more stringent test, these ESCs that had been grown on tissue-specific MSCs were implanted into mice. As Melton points out in the paper, the “most efficient published protocols for in vitro differentiation of pluripotent cells to beta-cells yield only a small percentage (typically 0-15%) of insulin-positive cells, and these do not secrete insulin in a glucose-responsive manner.” Could the MSC-conditioned ESCs do any better?
Before implantation, the ESCs were differentiated into endodermal progenitors (Sox17-expressing cells), and co-cultured with MSCs for at least 3-7 passages. Then they were differentiated into beta cells and transplanted into mice. There were a few important controls that were used; Just saline, implantations of MSCs alone, and ESCs that had been differentiated into beta cells, but had never been passaged on MSCs. Finally, human pancreatic islets were used as a positive control.
The results were interesting to say the least. The saline and MSC alone implantations showed no insulin production with or without glucose. Likewise the human pancreatic islets made insulin in a glucose-dependent manner (no surprise there). The ESC-derived beta cells that had never been passaged on MSCs made insulin, and even showed some ability to respond to glucose and make more insulin after glucose ingestion. However, the beta cells derived from ESCs that had been passaged on MSCs made insulin in a glucose-dependent manner. The experiment produced a wide range of variability since the number of transplanted cells differed between each trial, but the implanted beta cells derived from ESCs passaged on tissue-specific MSCs definitely performed the best, and even did as well or better than the implanted human beta cells in some cases.
Melton notes at the end of his paper that this technique worked rather well for coaxing ESCs to form pancreatic derivatives, but it could very well be applicable to other systems as well. Also, it could probably work with induced pluripotent stem cells, which have many (though not all) of the characteristics of ESCs and can be made without killing human embryos. Thus another technique for increasing ESC differentiation seems to be on the table.