By Dr Ananya Mandal, MD
In 1961, scientists found that adult bone marrow could yield stem cells. This bone marrow could be coaxed to form all the different types of blood cells. It was not until 1988 that these cells could be isolated in their pure form. The time lag can be explained by the fact that adult stem cells are inconspicuous in shape, size, and function and are present in very small amounts in the tissues. This makes their identification and isolation into pure cell lines difficult.
Identification of stem cells
There is a standard battery of tests that can help identify stem cells. For embryonic stem cells, serial subculturing for many months without differentiation is the first test that determines capability of long-term self-renewal. The cells are examined under the microscope to see if they are healthy and remain undifferentiated.
Another important test is to look for presence of a protein called Oct-4 that is typically made by undifferentiated cells. Oct-4 is a transcription factor that helps turn genetic switches on or off. Chromosomes of the cells are also examined to look for damage if any.
For many adult stem cells molecular markers, use of molecules that recognize and attach to specific surface proteins and can fluoresce under certain wavelengths of light, can help in identification.
Culturing cell lines and stimulating differentiation
Obtained stem cells from the inner cell mass of a blastocyst or from adult stem cells are more often than not present in very small amounts. These are then cultured in the laboratory to make them proliferate and multiply. This helps in actual use of the stem cells in research and in therapeutics.
Cell culture refers to the growth and maintenance of cells in a controlled laboratory environment outside the organism. For a successful stem cell culture the cells should be healthy, dividing, and unspecialized for initial and series of subcultures, re-plating and transplantation onto other fresh culture media. This series of subcultures may go on for months to yield the desired number of cells.
The stem cell culture is the first step in establishing a stem cell line. A cell line is a propagating collection of genetically identical cells. Cell lines are important because they provide a long-term supply of multiplying identical cells that can be used by more than one scientist in different laboratories for research and development of new therapeutic methods.
According to the National Academies report “Stem Cells and the Future of Regenerative Medicine (2001)”, maintaining a healthy stem cell line is the most challenging part of stem cell research. The report says that “Over time, all cell lines…change, typically accumulating harmful genetic mutations. There is no reason to expect stem cell lines to behave differently.”
After a stable cell line is established, the cells are then coaxed to form specialized cells. This is brought about spontaneously for adult stem cells and by changing chemical environments or genetic triggers for embryonic stem cells. Embryonic stem cells cannot be used before full differentiation as this can lead to a type of tumor called a teratoma.
Reviewed by April Cashin-Garbutt, BA Hons (Cantab)