Identifying and isolating adult stem cells
Stem cell isolation and identification are critical procedures prior to cell culturing. However, there is no universal technique for isolating and identifying stem cells. In general, scientists use some laboratory tests to check for the following characteristics: undifferentiated marker and capability of self-renewal. Specific tests have been developed to identify embryonic and adult stem cells. The following are some general methods used:
To test for the presence of cell surface markers of undifferentiated cells
- Microscopic examination of chromosomes to assess any damage or change in chromosome. Undifferentiated cells should have no detection of genetic mutation in the cells.
To identify embryonic stem cells
- Clonogenic assay is an in vitro test to see whether a single cell can differentiate and self-renew.
- Examination by microscope to assess the health and status of the cells.
- Check for the presence of biomarker. Transcription factor, Nanog and Oct4, maintain the cell in an undifferentiated state and help to turn the genes on and off at the appropriate time. This is an important part of cell differentiation and embryonic development.
To check for pluripotency of the embryonic stem cell
- Allow the cells to differentiate spontaneously in cell culture and manipulate the medium to see whether a specific cell type is formed.
- Inject the cells into an immunosuppressed mouse to check for the formation of tetratoma (a benign tumor with a mixture of differentiated cells).
Isolating and identifying human adult stem cells is difficult, as it is rare in adult tissue, especially in the brain, the liver or the skin. There are limited resources and techniques to identify adult stem cells:
- Testing for bone marrow or haematopoietic stem cells (HSC) is carried out by transplanting one single cell to an individual without HSCs. If new blood and immune cells are produced, it demonstrates the potency, and this can minimize the harmful effect of the foreign cell.
- To culture the stem cells to see whether they can give rise to genetically identical cells. These cells after isolation can then repopulate or reform the tissue after being transplanted into an animal.
Generating specialized cells from stem cells
Before transplantation, the embryonic stem cells have to be stimulated and become specialized cells, as the stem cells may grow out of control and give rise to tumors. Therefore, many scientists have worked out the appropriate conditions for specialization and different stages of the whole process. The most important thing is to ensure no single embryonic stem cell is transplanted to the patients.
Integrating cells into the body
Once the specialized stem cells are obtained and cultured, the next obstacle is to transplant them into a human or an animal. The integration of tissue or organs into a new place allows them to function properly in the recipient's body. Nerve cells need to connect to the brain to work properly.
Overcoming rejection by the immune system
Stem cell therapies face the problem of rejection by the recipient's immune system. The immune system protects the body against disease development by recognizing foreign molecules, like microorganisms, other human cells and tissue. This results in organ failure after transplantation. Strong immunosuppressive medicines are required to minimize or prevent the rejection. However, this may also expose the recipients to other infections. Scientists have developed a technique, cell nuclear transfer — somatic cell nuclear transfer technique (SCNT) — to overcome this obstacle, by replacing the nucleus of donor stem cells to a recipient nucleus prior to any cell division. As the replaced embryo cell has been cloned, and is identical to the patient cells, they will not be rejected by the immune system after transplantation.
For more details on SCNT, watch the animation in Activity 5.
Now that you have a deeper understanding of how stem cells are identified and extracted for research, we will go on to see how stem cells are used for disease management.
Somatic cell nuclear transfer (SCNT) is one of the cloning methods. It is achieved by replacing the nucleus of a healthy egg with the nucleus of a donor egg. The resulting embryo can generate the embryonic stem cells with same genetic makeup of the donor.
Watch the following animation.
What cell has been used as the donor cell?
Activity 5 feedback
The skin cell has been used as the donor cell.