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.

Stem Cell Research

Stem cells are the raw material from which all of the body’s mature, differentiated cells are made.  Stem cells give rise to brain cells, nerve cells, heart cells, pancreatic cells, etc. They have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses.
They can replicate themselves over and over for a very long time.
Understanding how stem cells develop into healthy and diseased cells will assist the search for cures.

There are two types of Stem Cells

Embryonic (also called “pluripotent”) stem cells

Embryonic stem cells are capable of developing into all the cell types of the body. There are two Sources of Embryonic Stem Cells-

1) Excess fertilized eggs from IVF (in-vitro fertilization) clinics can be used as a source of embryonic stem cells. Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment. These surplus embryos can be used to produce stem cells. Regenerative medical research is finding modern methodology to develop these cells into new, healthy tissue to treat severe and often fatal illnesses.

2) Therapeutic Cloning (Somatic Cell Nuclear Transfer)

In the Somatic Cell Nuclear Transfer, the nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example). No sperm is involved in this process, and the embryo are not created to be implanted in a woman’s uterus. The resulting stem cells can be induced to develop into specialized cells that are useful to treat dangerous illnesses.


Adult stem cells

Adult stem cells are less versatile and more difficult to identify, isolate, and purify.

The stem cells are extracted from a 5-7 days old blastocyst. Stem cells can divide in culture to form more of their own kind,
thereby creating a stem cell line. Later these are induced to generate healthy tissue needed by patients.

The Importance of Stem Cell Therapy

Stem cells allow us to study how organisms grow and develop over time. Stem cells can replace diseased or damaged cells that cannot heal or renew themselves. To develop and research and find new drugs and medicines, stem cells can be used to test these chemical and drugs. Stem cells can helps us to understand what is called the “genetic machinery”. All ready tremendous efforts are going on to treat diseases like Parkinson’s Disease, Leukemia (Bone Marrow Transplants) diabetes, multiple sclerosis, etc using stem cell therapy. Another area of importance is to regenerate tissues to be used as skin grafts to treat patients suffering severe burns.

The Controversy regarding Stem cell Therapy

The supporters of Stem cell therapy argue that embryonic stem cell research (ESCR) fulfills the ethical obligation to alleviate human suffering. The end justifies the means. If the research is directed towards making the human species disease and pain free, any kind of research including Stem cell research should be allowed and pursued. They further argue that as excess IVF embryos will be discarded anyway, they should better be used in research. As far as Stem cell nuclear transfer SCNT (Therapeutic Cloning) is concerned, it produces cells in a petri dish, not out of pregnancy.

Those who oppose the stem cell therapy accuse the researchers involved in this as murders. Their argument is that extracting the stem cells from a human blastocyst leads to the destruction or killing of the embryo, which amounts to murdering, or killing a potential human life. Further there is risk of commercial exploitation of the couples who willingly or unknowingly become participants in ESCR. Some opponents also predict that in the future this will lead to reproductive cloning.

It is not easy to decide or resolve the serious ethical issues surrounding this area of research. Often question is asked as to at what stage of embryonic development the blastocyst should be regarded as a “person with life”. Often the blastocyst used in the stem cell research is microscopically so small with no nervous system, that the supporters of the stem cell therapy do not consider it as live. There is also a conflict between embryonic stem cell research science and religion, as most of the major world religions do not support using embryos for research. There have been major hurdles in creating and formatting a human public policy on this issue due to sensitive nature of the problem.

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.

Genetic Engineering of animal cells and their applications

The mammalian cells are genetically modified by introducing the genes needed for specific purposes such as production of specific proteins or to improve the characteristics of a cell line. The methods used to introduce the foreign genes/DNA into mammalian cells are: Electroporation, Lipofection, Microinjection and/or fusion of mammalian cells with bacteria or viruses.

After the integration of the foreign DNA into the mammalian cells, the transfected/transformed cells are selected by using suitable markers. Some of such markers in use are: Viral thymidine kinase, Bacterial dihydrofolate reductase, Bacterial neomycin phosphotransferase. It has been possible to overproduce several proteins in mammalian cells through genetic manipulations e.g. tissue plasminogen activator, erythropoietin, interleukin-2, interferon- beta, clotting factors VIII and IX, tumor necrosis factors. The recombinant mammalian cells are also conveniently used for the production of monoclonal antibodies.

Manipulation of Gene Expression in Eukaryotes

The eukaryotic organisms have the capability to bring about the post-translational modifications such as glycosylation, phosphorylation, proteolytic cleavage etc which ultimately helps in the production of stable and biologically active proteins. Due to these reasons the use of eukaryotic expression system is preferred however it is difficult to conduct experiments with eukaryotic cells. The introduction of a foreign DNA into animal cells is called transfection. The insert DNA in the eukaryotic cells may be associated with vector or integrated into the host chromosomal DNA. Among the various hosts used for the expression of cloned genes, the common yeast Saccharomyces cerevisiae is the most extensively used. Besides this, the cultured insect cells are in use for expressing cloned DNAs. Baculoviruses exclusively infect insect cells. The DNA of these viruses encode for several products and their productivity in cells is very high to the extent of more than 10,000 times compared to mammalian cells. The baculoviruses not only carry a large number of foreign genes but can also express and process the products formed. By using baculovirus as an expression vector system, a good number of mammalian and viral proteins have been synthesized. The most commonly used baculovirus is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). It grows on the insect cell lines and produce high levels of polyhedrin or a recombinant protein.

The mammalian cell expression vectors are used for the production of specific recombinant proteins and to study the function and regulation of mammalian genes. However, large-scale production of recombinant proteins with engineered mammalian cells is costly. The mammalian vector contains a eukaryotic origin of replication from an animal virus such as Simian virus 40 (SV 40) and a prokaryotic origin of replication. It has a multiple cloning site and a selectable marker gene, both of which remain under the control of eukaryotic promoter and polyadenylation sequences. These sequences are obtained from either animal viruses (SV40, herpes simplex virus) or mammalian genes (growth hormone, metallothionein). The promoter sequences facilitate the transcription of cloned genes (at the multiple cloning site) and the selectable marker genes. On the other hand, the polyadenylation sequences terminate the transcription.

Collection and purification process of Recombinant proteins

As the recombinant proteins start accumulating in the host cells, it becomes important to collect and purify them. This is a tricky process since many times the recombinant protein is a foreign body for the host cells and the enzyme machinery of the host cell becomes activated to degrade the outside protein. One of the strategies adopted is the use of bacterial strains deficient in proteases or alternatively, the recombinant proteins are fused with the native host proteins. The fusion proteins are resistant to protease activity. Sometimes, the foreign proteins accumulate as aggregates in the host organism which minimizes the protease degradation. The best way out is to quickly export and secrete out the recombinant proteins in to the surrounding medium. The recovery and the purification of foreign proteins is easier from the exported proteins. The efforts have been made to develop methods to increase the export of recombinant proteins.

Some of the species of the bacterium, Bacillus subtilis normally secrete large quantities of extracellular proteins. A short DNA sequence called signal sequence from such species is introduced into other B. subtilis. These bacteria produce recombinant DNA tagged with signal peptide, which promotes export and secretion. This signal peptide is removed after the purification of foreign protein. The techniques used for the purification of recombinant proteins from the mixture of secreted proteins are affinity tagging, immunoaffinity purification etc.

Organ culture and Histotypic cultures

The cell-cell interaction leads to a multistep events in in vivo situations. For example, hormone stimulation of fibroblasts is responsible for the release of surfactant by the lung alveolar cells. Androgen binding to stomal cells stimulates the prostrate epithelium. In other words, hormones, nutritional factors and xenobiotics exert stimulating effects on the cells to function in a coordinated manner. Xenobiotics broadly refers to the unnatural, foreign, and synthetic chemicals such as pesticides, herbicides, refrigents, solvents and other organic compounds. It is impossible to study these cellular interactions that occur in the in vivo system with isolated cells or cells in culture. This has lead to the attempts to develop organ and histotypic culture with the aim of creating in vitro models comparable to the in vivo system. The three types of such cultures are:

a) Organ culture- In this type of culture, the whole organs or small fragments of the organs with their special and intrinsic properties intact are used in culture.
b) Histotypic culture- The cell lines grown in three dimensional matrix to high density represent histotypic cultures.
c) Organotypic cultures- A component of an organ is created by using cells from different lineages in proper ratio and spatial relationship under laboratory conditions.

Organ culture

In the organ culture, the cells are integrated as a single unit which helps to retain the cell to cell interactions found in the native tissues or organs. Due to the preservation of structural integrity of the original tissue, the associated cells continue to exchange signals through cell adhesion or communications. Due to the lack of a vascular system in the organ culture, the nutrient supply and gas exchange of the cells become limited. In order to overcome this problem, the organ cultures are placed at the interface between the liquid and gaseous phases. Sometimes, the cells are exposed to high O2 concentration which may also lead to oxygen induced toxicity. Due to the inadequate supply of the nutrients and oxygen, some degree of necrosis at the central part of the organ may occur. In general, the organ cultures donot grow except some amount of proliferation that may occur on the outer cell layers.

Techniques and Procedure for organ culture

In order to optimize the nutrient and gas exchanges, the tissues are kept at gas limited interface using the support material which ranges from semisolid gel of agar, clotted plasma, micropore filter, lens paper, or strips of Perspex or plexiglass. The organ cultures can also be grown on top of a stainless steel grid. Another popular choice for growing organ cultures is the filter-well inserts. Filter-well inserts with different materials like ceramic, collagen, nitrocellulose are now commercially available. Filter well inserts have been successfully used to develop functionally integrated thyroid epithelium, stratified epidermis, intestinal epithelium, and renal epithelium.

The procedure for organ cultures has the following steps:
a) The organ tissue is collected after the dissection.
b) The size of the tissue is reduced to less than 1mm in thickness.
c) The tissue is placed on a gas medium interface support.
d) Incubation in a CO2 incubator.
e) M199 or CMRL 1066 medium is used and changed frequently.
f) The techniques of histology, autoradiography, and immunochemistry are used to study the organ cultures.

The advantages of organ culture

The organ cultures can be used to study the behavior of an integrated tissue in the laboratory. It provides an opportunity to understand the biochemical and molecular functions of an organ/tissue.

Limitations of organ culture

It is a difficult and expensive technique. The variations are high with low reproducibility. For each experiment, a new or fresh organ is needed as organ cultures are not propagated.

Histotypic cultures

Using histotypic culture, it is possible to use dispersed monolayers to regenerate tissue like structures. It the growth and propagation of cell lines in three-dimensional matrix to high cell density that contributes to this. The techniques used in histotypic cultures are:

a) Gel and sponge technique- In this method, the gel (collagen) or sponges (gelatin) are used which provides the matrix for the morphogenesis and cell growth. The cells penetrate these gels and sponges while growing.

b) Hollow fibers technique- In this method, hollow fibers are used which helps in more efficient nutrient and gas exchange. In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed to be used for histotypic type of cultures. The cells get attached to capillary fibers and increase in cell density to form tissue like structures.

c) Spheroids – The re-association of dissociated cultured cells leads to the formation of cluster of cells called spheroids. It is similar to the reassembling of embryonic cells into specialized structures. The principle followed in spheroid cultures is that the cells in heterotypic or homotypic aggregates have the ability to sort themselves out and form groups which form tissue like architecture. However, there is a limitation of diffusion of nutrients and gases in these cultures.

d) Multicellular tumour spheroids- These are used as an in vitro proliferating models for studies on tumour cells. The multicellular tumour spheroids have a three dimensional structure which helps in performing experimental studies related to drug therapy, penetration of drugs besides using them for studying regulation of cell proliferation, immune response, cell death, and invasion and gene therapy. A size bigger than 500 mm leads to the development of necrosis at the centre of the MCTS. The monolayer of cells or aggregated tumour is treated with trypsin to obtain a single cell suspension. The cell suspension is inoculated into the medium in magnetic stirrer flasks or roller tubes. After 3-5 days, aggregates of cells representing spheroids are formed. Spheroid growth is quantified by measuring their diameters regularly. The spheroids are used for many purposes. They are used as models for a vascular tumour growth. They are used to study gene expression in a three-dimensional configuration of cells. They are also used to study the effect of cytotoxic drugs, antibodies, radionucleotides, and the spread of certain diseases like rheumatoid arthritis.

Organotypic cultures

These cultures are used to develop certain tissues or tissue models for example skin equivalents have been created by culturing dermis, epidermis and intervening layer of collagen simultaneously. Similarly models have been developed for prostrate, breast etc. Organotypic culture involves the combination of cells in a specific ratio to create a component of an organ.

   
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