Stem Cell Technology
The cells which are capable to divide and renew and also to produce progeny are called stem cells. However, their mitotic potential is restricted. The stem cells can differentiate into a variety of different cell types.
For example, tissues (such as skin, blood and intestinal epithelium, bone marrow, etc.) continuously renew themselves throughout the life. They also maintain sufficient number which in turn retains proliferation potential.
The concept of formation of blood cells (hematopoiesis) in spleen and bone marrow (in mouse) was developed about a century ago.
Hematopoietic stem cells are already used to restore hematopoietic cells. The other stem cell type also may soon routinely be employed for replacement of other cells and tissues.
The stem cells have two properties which increase their importance: (i) they have potential to form more differentiated cells, and (ii) they are self-renewing, because each division of a stem cell creates at least one stem cell.
1. The Morphological Approach :
Earlier workers attempted to identify the morphology studied and indirect assay methods developed for identification of hematopoietic cells. For this study, genetically identical population of mice is required.
Edmond Snell and co-workers developed such population by sibiling mating of mice. He obtained 21 generations. Snell was awarded Nobel Prize for this pains taking work.
You would have observed identical albino mice. Reproducible and comparable experiments could be performed by using two identical albino mice, one as experimental and the other as control. In this you can have two group of populations.
In one of the experiments, the blood cell forming ability of the two groups of mice was destroyed, when they were lethally irradiated by X-rays.
Bone marrow cells from femur of normal mice were taken out and injected into one group of mice. It was noticed that the irradiated mice which were injected with bone marrow cells survived and were healthy.
While the other irradiated mice, uninjected with bone marrow cells of normal albino died. Colony forming units (CFUs) of spleen of surviving mice were similar to bacterial colonies growing on Petri plates. This method is called repopulation assay.
When the spleen cells were injected into other irradiated mice, they also survived and were healthy. These findings supported the basis strengthening the concept of hematopoietic and stem cells associated with this phenomenon.
2. In vitro Clonal Assay:
In clonal assay system in vitro stem cells proliferate to form colonies on semi-solid media or clones of differentiated cells. This character resembles with the bacteria that multiply and form colonies on nutrient media. This feature helps the scientists to perform many assays and procure differentiated and semi-differentiated cells.
Through this assays one can find out the growth factors which are needed to differentiate blood cells from primitive stem cells. You must keep in mind that erythropoietin was the first commercialised biotech product which was assayed following this method.
3. Long-term Marrow Culture :
In recent years artificial marrow can be created on plastic surface. The idea of such work helped the scientists to use marrow cells from femur bone and study hematopoiesis [i.e. erythrocyte (blood cell) formation] under in vitro conditions. A typical process of erythropoiesis has been shown in Fig. 10.12. It occurs in the following four stages.
1. Stem cells retain the potential to proliferate and self-renew.
2. Progenitor cells have greater differentiation potential and limited self-renewal capacity.
3. Precursor cells differentiate and lack self-renewal capacity.
4. The mature cells are fully differentiated. They have no capacity of differentiation and self-renewal.
Several types of human blood cells arise from a single haematopoietic stem cell.
By using bone marrow culture technique, blood cell formation and growth factors affecting blood formation could be understood. For the last 20 years these techniques also supported bone marrow transplantation when the purified hematopoietic stem cells are used.
Furthermore, immature blood cells are incorporated into blood stream of patients suffering from blood cancer i.e. leukemia, so that mature erythrocytes could work and save the life of the sufferers.
4. Embryonic Stem Cell Culture :
Embryonic stem (ES) cells are the pluripotent cells isolated from inner cell mass of early embryos. ES cells are isolated without injection of immortalising (transforming) agent in mice.
For many years it has been possible to grow mouse ES cells as cell lines in laboratory. These ES cells can be induced to generate many different types of cells. Mouse ES cells has been shown to give rise the muscle cells, nerve cells, liver cells, pancreatic cells as well as hematopoietic cells.
The ES cells can be used for cloning in two ways: (i) creation of chimera, and (ii) nuclear transplantation.
In the presence of irradiated fibroblast cells, the ICM should be maintained in tissue culture.
It was interesting to note that these cells (i) retained the characteristics of the embryo founder cells even after prolonged culture, (ii) fully irradiated into embryogenesis when returned to early embryo, (iii) could be used to produce chimeric mouse, (iv) maintained a stable euploid karyotype, and (v) self-renewal without differentiation into cell types.
The ES cell lines have a great value in the area of production of transgenic animals. Since the cells are plentiful and maintained in culture, they can be transformed very easily. The resultant transformants can be fully characterised in vitro. Their genome can be modified. This may be the area of greatest impact on live stock.
Using in vitro fertilisation (IVF) technique you can fertilise a mouse egg artificially i.e. outside the animal body and grow in tissue culture. You can observe that the embryo will undergo several steps of cleavage. The dividing cells accumulate at one corner of embryo. These accumulated cells are called the inner cell mass (ICM).
(a) Production of Chimeric Mouse:
Different steps for production of chimeric mouse are given in Fig. 10.14. An early embryo is isolated from the fertilised mouse of black colour. The ES cells of trophoblast stage of embryo are grown on culture medium.
A small number of ES cells can be injected into blastocoels space of an embryo of a white (albino) mouse through microinjection technique. The ES cells of black mouse intermingle with that of albino.
The microinjected embryo is transplanted into the uterus of a surrogate mother (another mouse of which ova are not used). The progeny born have black and white skin colour. Such mouse was called chimera or chimerical mouse.
Ideally, all tissues of the mature chimeric animals were the mixture of the two cell genotype. That is why patches of different coloured fur were present on chimeric mouse.
If the germ cells are also chimeric, a proportion of progeny will result from ES cells. Crossing male and female chimeras allow the selection of a homozygous strain of mice derived solely from the ES cells.
(b) Knockout Mice:
Transgenic mice that carry a knockout gene (i.e. gene of interest replaced by a non-functional gene) is called knockout mice. Now it is possible to select and knockout (remove) a gene and make genetic modifications in the ES cells and mouse.
Different types of model mouse can be developed to understand the function of various genes e.g. disease development.
For example, knockout mice have helped the immunologists to understand the effect of knockout gene on immune system in animals. Various knockout mice are being used in immunological research. Production of knockout mice (gene targeted) is accomplished in the following steps.
1. Isolation and culture of ES cells from inner cell mass of a mouse embryo.
2. Induction of a mutant or disrupted gene into the cultured ES cells and selection of homologous recombinant cells in which genes of interest have been knocked out.
3. Injection of homologous recombinant ES cells into a recipient mouse embryo and transfer of manipulated embryo into uterus of surrogate mother mice.
4. Mating of chimeric offspring heterologous for disrupted gene to produce homozygous knockout mice.
(c) Human ES Cell Culture:
J.A Thompson (1998) announced that the human ES cells are able to multiply and grow on medium. Human inner cell mass of blastomere can be obtained either by IVF technique or from human germ cell precursors (before initiating meiosis).
Then the ICM is cultured on growth medium in a Petri plate. These cells can differentiate in culture to form more restricted stem cells for neural, blood or muscle lineage.
The differentiation of ES cells into lineage-restricted (neuronal and glial) cells can be accomplished by altering the media in which the ES cells grow. Specific cells were produced in the presence of specific growth factors e.g. fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF).
This endeavour has opened an area for gene manipulation and cell engineering in ES cells of human too. A variety of PDGF are released by the activated plate lets on the damaged expithelial tissue.
5. Cell and Tissue Engineering :
Animal biotechnologists can grow blood cells, cardiac cells, skin cells, etc. by using human ES cells.
The objective of cell and tissue engineering is very clear. Using this technique such type of body parts can be prepared that can be used to repair damaged tissues and organs with causing an immune response, infection or multilating other body parts with ease, stability and safety.
In the wide range of emerging techno-logies and therapeutic applications, these primary cell types (e.g. kiratinocytes, endothelial cells and epithelial hepatocytes) have potential commercial value.
The other important applications of cell and tissue engineering are gene therapy, pseudo-organ and model cell systems. The therapeutic approaches have been developed to combat with dangerous human diseases like genetic disease. A brief account of gene therapy has been given in preceding section.
6. Nuclear Transplantation Technique :
Using nuclear transfer technique, the world’s first mammalian clone -Dolly, was born in February 1996. In 1995, Ian Wilmut and his research group (Scotland) took out udder from a six year old sheep a called clone mother, and put in a special solution.
Nucleus of udder cell was taken out and put in a solution. At the same time an unfertilized egg was taken out from another sheep B called egg mother.
Nucleus of the egg was removed and enucleated egg was put in a culture medium. The nucleus of udder cell and enucleated egg cell were put together followed by mild electric shock.
Consequently nucleus was taken up by the enucleated cell. This cell was incubated onto growth medium then transferred into a surrogate mother. A little lamb Dolly was born in February, 1996.