Embryonic Stem Cells

Embryonic stem cells (ES cells) are pluripotent stem cells that originate from the inner cell mass (ICM) of a blastocyst, which is an early-stage preimplantation embryo formed after the fourth day of development.

Human embryonic stem cell lines can be isolated from three main sources.

  • Established embryonic cell lines that already exist.

Some organizations and people oppose the use of human embryonic stem cells (hESCs) under any circumstances. However, the usage of already established cell lines is one of the most widely accepted sources of stem cells. This acceptance is based on the argument that what’s done is done. Even if harvesting the embryos in the first place could be considered morally wrong, nothing can now be done to save the original embryos from which the lines were created.
hESCs hold great potential in the treatment of various debilitating diseases in human beings. The technology is moving towards being more and more cost-effective as time goes on. But sourcing of these cells is limited by moral and ethical dilemmas as well the fact that there are only a certain number of cell lines suitable for human usage.

  • Spare embryos that have been leftover from fertility treatments.

Usually, in assisted reproductive treatments extra embryos are created and cryopreserved in case of future attempts at achieving pregnancy should the first time fail. With consent from eh parties involved the stem cells from the extra embryos may be used for the purposes of research and treatment. Research has shown that a large percentage of couples consent the usage of leftover embryos since they would be discarded over time regardless.

  • Custom-made embryos that have been created by the process of somatic cell nuclear transfer (SCNT), This particular technique was used to create Dolly the Sheep - the first cloned animal.

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Therapeutic Cloning

Therapeutic cloning is a technique used to create stem cells from an individual. It is performed by using a technique known as Somatic Cell Nuclear Transfer (SCNT). SCNT is a laboratory technique that is used to create a viable embryo from a somatic (body) cell usually a skin cell and an egg cell that is both obtained from one person or animal.
In the technique, an enucleated oocyte (egg cell whose nucleus is removed) is taken and implanted with a donor nucleus from a somatic (body) cell. The egg then begins dividing as it contains a complete complement of genetic material and the cells are all clones of a single individual. A regular egg would have had only half the amount of genetic material and would only divide once it is inseminated and would contain genetic material from two individuals hence it is not a clone. These stem cells can then be used for treating disorders present in the individual. 

Furthermore, implanting the embryo in a surrogate and allowing it to divide to form a living organism is called reproductive cloning. Dolly the Sheep, famously known for being the first mammal that was successfully cloned, was created using this exact process using the egg and somatic cell of an adult sheep.

SCNT has been the solution to many issues concerning embryonic stem cells (ESC) and the destruction of viable embryos in research and is readily being utilized in stem cell research, with a particular focus on "therapeutic cloning", which is also termed as regenerative medicine.

Embryonic Stem Cell Cultivation

Human embryonic stem cells (hESCs) can be created by the transfer of cells from a preimplantation-stage embryo into a plastic cell culture dish containing culture medium that contains the nutrients necessary for the growth. The cells then divide and spread over the surface of the dish.

A feeder layer of cells may be used as a co-culturing mechanism along with the stem cell. They are treated so that they don't divide themselves. They coat the bottom of the dish in order to provide a better surface to adhere to and to release more nutrients into the medium. Previously mouse embryonic fibroblasts (MEFs) were used but were replaced by the use of human embryonic fibroblasts to prevent of risk of contamination.

If the cultured cells divide and survive positively they can be subcultured in order to avoid overcrowding. Each cycle of creating fresh plates of cells from existing cultures is known as passaging.
As long as the ESC’s in culture are grown under the appropriate conditions, they will remain undifferentiated. But if the cells are allowed to clump together leading to the formation of embryoid bodies, they will begin to differentiate spontaneously. Even though spontaneous differentiation is a good indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce cultures of specific cell types. So, to generate cultures of specific cell types scientists will control the differentiation of the cells by altering the chemical composition of the culture medium, altering the surface of the culture dish, or modifying the cells by the insertion of specific genes.

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Source: https://stemcells.nih.gov/

Once the required cell line has been maintained and grown in the same conditions for over six months, the cells can be considered pluripotent in nature and termed as an established embryonic stem cell line. The cells can then be cryopreserved for future use or utilized for further experimentation.

 

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