Human Embryonic Stem Cells

EMBRYONIC STEM CELLS derived from preimplan—tation embryos are undifferentiated cells capable of differentiation into derivatives of all three embryonic germ layers. These cells are also capable of self—renewal and have an almost unlimited developmental potential.

Embryonic stem cells are derived from the inner cell mass of a preimplantation blastocyst about five days postfertilization.

During the last decade, there has been ongoing research into the isolation of inner cell mass (ICM), as it is useful in establishing embryonic stem cell lines, which in turn have the ability to develop into most of the specialized cells in the human body, including blood, skin, muscle, and nerve cells. They also have the capacity to divide and proliferate almost indefinitely in culture.

The method for establishing a human embryonic stem cell line comprises the following steps: [Page 246]isolate cells of an ICM from an isolated blastocyst stage embryo by creating an aperture in the blastocyst stage embryo by laser ablation, and remove cells of the ICM from the blastocyst stage embryo through the aperture; culture the cells of the ICM in the presence of an embryonic stem cell medium and an inactivated feeder layer to produce ICM—derived masses; and culture the ICM—derived masses to produce an isolated human embryonic stem cell line.

Human ES cell lines are derived from the embryos produced by in vitro fertilization, a process in which oocytes and sperm are placed together to allow fertilization to take place in a culture dish. ES cells are derived from the ICM of the preimplantation blastocyst approximately five days postfertilization. After a human oocyte is fertilized in vitro by a sperm, it forms a zygote. The zygote undergoes a series of cleavage, and by day five the cavity of the blastocyst is completed. The ICM begins to separate from the outer cells, which become the trophectoderm that surrounds the blastocyst. This represents the first observable sign of differentiation in the embryo.

The ICMs have the potential to generate any cell type of the body, but only before implantation. If the ICM is removed from its normal embryonic environment and cultured under appropriate conditions, the ICM—derived cells can continue to proliferate and replicate themselves indefinitely and still maintain the developmental potential to form any cell type of the body. Day five blastocysts are, therefore, used to derive ES cell cultures. They consist of 200–250 cells, and the trophectoderm must be removed from these cells to derive ES cell cultures. This is done either by microsurgery or immunosurgery. Immunosurgery is a process in which antibodies attacking the trophectoderm help break it down, thus freeing the ICM. These pluripotent ICM—derived cells are ES cells.

The ability to isolate ES cells from blastocysts and grow them in culture seems to depend in large part on the integrity and condition of the blastocyst from which the cells are derived. In short, the blastocyst that is large and has distinct ICMs tends to yield ES cells most efficiently. Several methods have been used for isolation of ICM for the establishment of embryonic stem cell lines.