STEM CELL TECHNOLOGY
Stem cells retain the capacity to self renew as well as to produce progeny with a restricted mitotic potential and restricted range of distinct types of differentiated cell they give rise to. The formation of blood cells also called haematopoiesis is the classical example of concept of stem cells. Indirect assay methods were developed to identify the haematopoietic stem cells. The process of haematopoeis is occurs in the spleen and bone marrow in mouse. In human beings about 100,000 haematopoietic stem cells produce one billion RBC, one billion platelets, one million T-cells, one million B cells per kg body weight per day.
Several methods have been developed to study haematopoiesis and stem cells:
a) Repopulation assay- Edmens Snell’s group created mice which were genetically identical by mating of sibling mice after 21 generations. Two groups of mice were lethally X- irradiated to destroy their blood cell forming capacity. One of this group was injected with marrow cells from the femur bone of a normal and healthy albino mice. It was observed that this group survived whereas the mice in the other group died. The spleen of mice which survived had the colonies of the bone marrow cells just like bacterial colonies on a Petri plate. This came to be known as colony forming units of spleen (CFU-S) and the technique is known as repopulation assay.
b) The in vitro clonal assay- In this assay, the stem cells proliferate to form colonies of differentiated cells on semi-solid media. This assay helps in identifying growth factors required for the formation of blood cells from the primitive stem cells. One of the first commercialized biotechnology product – erythropoietin was assayed by this procedure.
c) Long term marrow culture- In this method, the marrow cells from femur bone were grown under in vitro conditions on plastic surfaces. These techniques were helpful in bone marrow transplantation and treatment of blood cancer by releasing immature blood cells into the blood stream.
d) Embryonic stem cell culture- Embryonic stem cells are cell lines derived from the inner cell mass of fertilized mouse embryo without the use of immortalizing or transforming agents. The Inner cell mass (ICM) are the cells that are maintained in tissue culture in the presence of irradiated fibroblast cells. These cells are often used in creating chimeric mice. In 1998, J.A. Thomson developed the method to multiply the human embryonic stem cells. Human ICM can also be now derived either by IVF or from germ cell precursors and cultured on a Petri plate. The differentiation of these cells into lineage restricted (neuronal and glial) cells can be accomplished by altering the media in which the cells grow.
e) The ICM cells could be used to create chimeric mice. In chimeric mice it was possible to take ES cells from a black mouse and implant it into the embryo of an albino mouse (white). The progeny so developed had skin colour of black and white ( a chimera).
Following diagram shows the scheme of obtaining chimeras.
