Cell biology

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When you place side-by-side human and animal embryonic cells, or human and animal fetal precursor cells of the same type you find out that they look alike, and even most of the available cell-surface markers are the same. 

The only way to tell the cells of one species from another is by their karyotype, the number and shape of chromosomes that are temporary structures created from the genetic material of each cell during one short phase of the mitosis. 

The embryonic cells have unique capability to renew themselves, i.e. proliferate, and are pluripotent, that means that they have the potential to differentiate into any and all specialized cells of the body, with a characteristic shape, and function. 

They remain in an undifferentiated state, uncommitted, until they get a signal to develop into one of specialized cells of the body.

A puzzling problem is that embryonic cells apparently do not exist for any prolonged period of time in real life, i.e. in a living embryo, only in the laboratory dish.

The current optimism about embryonic stem cells is based on theoretical expectation of:

- their enormous ability to proliferate that makes them suitable for a factory level manufacturing of cells for therapeutic use,

- their capability to be manipulated so that they differentiate into any desired cell type to be used for cell transplantation treatment of patients.

The unlimited potential of embryonic cells to proliferate sounds wonderful, but only until one does not realize that in cancer growth likewise one kind of cell stopped responding to the commands of the patient’s body, became independent, and decided to become a ‘cell factory’.

A manipulation of embryonic cells into differentiation, whereby precursors / progenitors of any and all specialized cells of the body are created in a laboratory dish, is an exciting feat, and a formidable task, but currently without a solution.

But since fetal precursor cells cannot create in a laboratory conditions a three-dimensional body, or an organ, or even a tissue, the question arises whether these cells grown in a laboratory dish are indeed the same fetal precursor cells that can be obtained from a fetus, where they have developed in a natural way, and where they created three-dimensional tissues and organs. 

The kind of environment in which fetal precursor cells are growing, i.e. either in tissue culture or in live human and animal body, makes a lot of difference for the direction of cell differentiation. 

Let's Now Focus On Fetal Precursor Cells

When the fetal precursor cells are taken from fetus, that already reached the stage of organogenesis, such cells are no longer pluripotential but are committed to follow a predetermined path of differentiation along one lineage only, in other words such fetal precursor cells are directed to produce cells specific for the kind of tissue where these cells normally reside, and to follow the body commands. At the same time they retain the ability to proliferate without pre-determined differentiation for a long time, i.e. capability of long term self-renewal. (Those fetal precursor cells obtained by manipulation of embryonic cells in a laboratory dish remain pluripotential to some degree.) This is because in fetal body the undifferentiated stem cells live in a milieu of various specialized (differentiated) cells, and there is a lot of interaction between them, and that is not the case when undifferentiated stem cells grow in tissue culture.

It appears more physiological to take fetal precursor cells for transplantation from their natural environment in the fetal body,  that means taking them along with other cells of the same ‘family’, in a cell-to-cell contact with cells of the same ‘family’, together with cells of various generations of the same ‘family’ and then grow them in a primary tissue culture in order to have sufficient time for observation and safety tests, as well as to minimize their immunogenicity so that they can be implanted without immunosuppression.

Following this principle, the described method of preparation of fetal mprecursor cell transplants is based on the primary tissue culture of tissue fragments, or cell clusters, and not on the primary cell culture of dispersed cells. Besides that, it has been proven beyond any doubt that cells in the tissue fragments communicate via contact, via soluble factors, and also via their electromagnetic fields. All of these factors are missing in the cell culture of dispersed cells.

Besides that the process of disintegration of an explant into individual cells requires the use of chemicals and proteolytic enzymes that damage cell membranes, sometimes even cell nuclei, so that the cell culture begins with decreased quantity of intact healthy cells. In our method of tissue culture a much more physiological mechanical disintegration is utilized, so that the quality of cultured cells is superior.

The use of cell lines for preparation of fetal precursor cell transplants is another questionable area. Cell transplants prepared from cell lines have never been used for the patient treatment in the past. It has been a ‘res ipsa loquitur’ for practicing cell transplantologists in Europe, because there has been a strong opinion that cell transplants prepared from cell lines are not clinically effective due to the following facts.

Development of cells in an organ or tissue culture is influenced by a variety of interactions between cells, and due to a lack of such interactions the growth of dispersed cells in cell culture is difficult to impossible. Culturing of dispersed embryonic cells growing outside their natural environment is possible only on the cell matrices, currently known as ‘feeder layer’ of cells. The only other way is by culturing fetal precursor cells in their natural environment, inside of a living organism. Bone marrow, for example, is a perfectly structured micro-environment for ‘production’ of stem cells. Hematopoetic stem cells are capable of growth and development into structurally and functionally competent cells only under influence of unique stimuli of all types of cells in the given micro-environment.

In cell lines of one type of cells only, as a result of living in an artificial conditions of cell culture, the cells are almost always heteroploid, and due to the influence of selection they are so changed, that they often cannot be recognized as derived from their tissue (or organ) of origin. In cell lines sex chromatin disappears, the mitoses run without any controls, there is a decreased production of acids released into the culture medium, cell membranes of daughter cells are incomplete, there is a lack of histotypical differentiation. Cell lines have lowered resistance against viral infections.

On the other side, the primary tissue cultures, as used in the described method of preparation of BCRO fetal precursor cell transplants, have a limited lifespan, determined by the lifespan of the tissue source of the culture, or of the donor. In primary tissue cultures the cells maintain diploid set of chromosomes, typical for the normal somatic cells of the animal source of the tissue culture. They do not differ from the cells of the original organ or tissue planted on the tissue culture neither structurally, nor biochemically. These cells grow in practically the same functional environment as when they were a part of an organism from which they were taken.

Adult (stem) cells have a low therapeutic potential, despite their apparent ability of long-term self-renewal, differentiation along a predetermined cell lineage, and ability to give rise to just one cell type for the purpose of maintenance of homeostasis.

Stem cells obtained from the fetus are much more numerous than rare adult (stem) cells, and they possess certain unique properties, such as:

- high level of readiness to differentiate and undergo changes in response to environmental stimuli, or in accordance with their own genetic make-up;

- easy adaptability, due to the plasticity of tissues (including growth, migration, mobility, ability to create cell-to-cell contacts), that in the course of normal fetal development gradually decreases, and finally disappears at the completion of development;

- much more frequent and faster cell division, and proliferation, as compared with adult stem cells, depending upon the type of tissue and stage of fetal development;

- production of large amounts of various biological substances, i.e. growth factors, etc., which facilitate the survival and growth of fetal precursor cells after implantation, and stimulate damaged cells, tissues and organs of the host;

- lowered immunogenicity, with a consequently much weaker immune response of the host, as compared with an implantation of adult cells or tissues;

- ability to survive on energy supplied by glycolysis alone, and thus on lesser amount of oxygen, that is important during the preparation of fetal precursor cell transplants, and during the first hours after implantation;

- lack of cell extensions easily damaged during processing of cell transplants.


‘Homing’ means that the respective fetal precursor cells do not have to be implanted into a damaged organ, i.e. liver cells into liver, they can be implanted into more accessible superficial tissues, as for example under the aponeurosis of the rectus abdominis muscle, because they find their way into the damaged organ from there, as if  ‘attracted’ by it.

Lymphocyte spends most of its life within solid tissues, entering the circulation only periodically to migrate from one resting place to another. At any moment only 1% of the total lymphocytes are found in blood: this part of lymphocytes is in motion, shuttling through organs, surveying body for foci of infection or foreign antigens. Lymphocytes in the blood are attracted back into solid tissues by way of specialized blood vessels known as high endothelial venules, modified form of postcapillary venules found in all lymphoid organs. Some lymphocytes passing through these venules tend to bind tightly to the specialized endothelium and then infiltrate between endothelial cells and penetrate into the tissue.

Binding occurs because proteins known as ‘homing receptors’ on the lymphocyte surface have a strong affinity for determinants, called vascular addressins, on the high endothelial venules. High endothelial cells in different target organs express addressins that are characteristic of that organ, and are recognized by a specific type of homing receptor. When a circulating lymphocyte expresses a particular homing receptor, it tends to bind to and infiltrate only a tissue or organ with the corresponding addressin.

High endothelial venules are a constant feature of lymphoid organs but may also appear transiently at any site in the body where an immune response is occurring, i.e. in a diseased organ or tissue. They arise by differentiation of pre-existing capillaries in response to IFN-? produced locally by activated CD4 T cells. These cells serve as a portal for circulating lymphocytes to enter the site and join in an immune response. By such means the lymphocytes and other reactive cells can accumulate wherever they are needed in the body. VI.BIBLIOGRAPHY [108, 180, 165]

A cascade of adhesive molecules, of which L-selectin is expressed on naive T lymphocytes, interacts with vascular addressins, with the resulting homing of naive T cells to lymphoid organs. In order for naive T cells to cross endothelial barrier into the lymphoid tissue two other types of adhesion molecules are necessary: integrins, and the immunoglobulin superfamily. VI.BIBLIOGRAPHY [165]

This homing principle has been recognized to be of key importance for a succesful long-term engrafment of stem cells in bone marrow transplantation. VI.BIBLIOGRAPHY [20] After intravenous injection hematopoietic stem cells circulate through organs, such as the liver, lungs, kidneys, spleen, where they undergo few mitoses and form small colonies. But, they seed only bone marrow, and establish sustained hematopoiesis there. Homing represents a cascade of adhesive interactions between hematopoietic cells and bone marrow extracellular matrix. VI.BIBLIOGRAPHY [21, 179] Interestingly, only 75% of labeled cells could be accounted for in these experiments. VI.BIBLIOGRAPHY [19]

Intrathymic transplantation of syngeneic islets into mice significantly reduced the severity of insulinitis, and development of diabetes mellitus, but not of sialitis, which proved that it induced a tissue specific protection. Subsequently there was a marked decline of thymic insulin content within 48 hours post-implantation, which indicated a rapid loss of implanted islet cells from thymus. Within a week the thymic insulin content was <1% of the initial content. Histology confirmed the rapid elimination of the islet syngrafts, and by the day 7 only an occasional granulated ß-cell, as well as non-ß endocrine cells, were seen, and 20 weeks post-implantation no surviving islet cells were detectable in the thymus. Thymus histology and thymic insulin content revealed a rapid loss of implanted Beta-cells, and despite that there was tolerance induction by adoptive transfer of splenic leukocytes to another breed of mice. VI.BIBLIOGRAPHY [10]

Let’s compare the modern scientific facts of the last two paragraphs with the description of findings of the school of cell therapy of a few decades ago. F. Schmid states that the implanted cells disappear from the implantation site, with different speed, usually within 48 hours, and that the removal is effected by macrophages, and the vitally stained cells are mostly incorporated in the respective organ or tissue, following the rules of ‘organospecificity’. He also refers to the ‘Halsted principle’ according to which implanted cells migrate to the place of need, the fact that is difficult to prove or refute by way of experiment. VI.BIBLIOGRAPHY [95]