36 Stem Cells

Dr. M. N. Gupta

1. Objectives

•  To learn about types of division of stem cells.

•  To understand the difference between totipotent, pluripotent and unipotent stem cells.

•  To learn about sources of stem cells

•  To learn what we know about progression from totipotentcy to unipotency.

2. Concept Map

3. Description

The promises of stem cells go even beyond organ/tissue transplant. If we can use stem cells to repair/reconstruct what has become damaged, the stem cells will differentiate appropriately and the new tissue/organ will be in place. Obviously, the problem of immune rejection is here as well. There are, along with greater promise, greater challenges. Let us understand how cells differentiate from stem cells onwards.

We have actually introduced the concept of stem cells twice before without discussing it in detail. Firstly we talked of pluripotent haematopoetic cell which gives rise to various cells of the blood very early in this paper.

More recently, while talking of cancer, we mentioned that a terminally differentiated cell (as opposed to a stem cell) starts proliferating to form the neoplasm.

Let us first discuss the basics of cell differentiation. It all starts from a stem cell.

A stem cell can divide without limit during the lifetime of the animal.

Upon division, each daughter has two optional routes. It either remains a stem cell itself and follows the route of its parent cell all over again. That is what the first property means. It can, alternatively, follow the path of a terminally differentiated cell.

A stem cell which can give rise to several cell types is called pluripotent. As we mentioned earlier, a pluripotent haematopoietic cell can give rise to several types of blood cells.

It should be noted that in the above example, the pluripotent cell, (while not terminally differentiated), has committed or determined its fate: it is going to be differentiated into a blood cell (and not a liver cell or heart cell !).

Unipotent cells are stem cells which can give rise to only one type of cells. An epidermal stem cell is going to be differentiated into a keratinized epidermal cell. The spermatogonium is the stem cell for spermatozoa.

At some point a cell decides its future: it will become a specialized cell. This decision may be partial such as to become a pluripotent haemotopoitic stem cell or more complete: to become a skin cell of a particular organ. In both cases, we say a cell has “determined”.

At this point, the developmental potential to carry out the specific biological function or to assume a particular morphology, is in place.

Cell differentiation is a process by which selective gene expression takes place which results in the specific cell type.

A terminal differentiation in many ways, leads a cell to lose its ability to proliferate. A RBC has no nuclei and does not divide. The nucleus in the outermost layers of skin is digested away. In the striated muscle cells, myofibrils hinder mitosis and cytokinesis.

Thus some cells in mammals e.g. nerve cells, heart muscle cells, receptor cells in eyes and ears and eye lens fibres neither undergo cell division nor are replaced, there is no cell renewal.

However, in some cells, cellular components are renewed. In adult eye lens, lens proteins are formed at embryonic stage. Within eye, while photoreceptor cells do not divide photosensitive protein molecules have a turn over. So, biosynthetic activities continues and within some cells, some renewal is possible.

Majority of cells in a vertebrate are undergoing apototosis and replacement on a continued basis. The new differentiated cell is either formed from by cell duplication or formed by differentiation of a stem cell.

Two important examples of cells which renew by simple duplication are liver cells and endothelial cells.

Liver is an example of an organ where its cells hepatocytes- renew at a slow rate. However, even there a homeostatic mechanism ensures that cell division and cell death balance each other and organ size remain same.

In case of intentional (in animal experiments) or incidental case where large cells have died, cell renewal accelerates in response signals from blood in the form of hepatocyte growth factor.

Under normal circumstances for all tissue components regeneration is coordinated. Liver has, besides hepatocytes, kupffer cells (which we had mentioned earlier as liver macrophages) and few fibroblasts supporting framework of connective tissue.

Excess alcohol intake frequently repeated, does not allow adequate rate of renewal of hepatocytes. Fibroblasts continues to grow and end up clogging the tissue irreversibly with connective tissue. This is the well known liver cirrhosis.

The endothelial cells lining blood vessels follow more flexible renewal rate which is adjusted to local requirement. In all parts of the vascular system, endothelial cells divide by duplication. These cells also can move. Some damage at a site, such as insertion of tubing by surgeon, new endothelial cells move and cover the surface.

The cells lining the gut are good example of cells which are renewed via stem cells. Here, epithelium is formed by a monolayer of cell. This covers both vilii (projections in the gut) and crypts (underlying connective tissues). The stem cells are in the crypts. Upon differentiation, these reach vilii tips ultimately. Here, apoptosis occurs.

Epidermis, epithelium which covers the skin is multilayered. Here also, renewal by stem cells involves, outer surface cells being replaced by differentiation of stem cells in the lower layer.

In all such cases, stem cells fulfil the need to replace existing population of differentiated cells which cannot divide. As we have already discussed, in several tissues, differentiated cells cannot divide further for multiple reasons.

Let us look at the stem cell differentiation more closely as an example. Only basal cells on the basal lamina undergo mitosis. Over the top surface are dead cells which do not have any distinguishing organelles and form squames (flat scales) rich in protein keratin.

About one out of 10-12 basal cells is a stem cell. As a cell line, these remain throughout the life time of the animal.

Hence each patch of epidermis has at least one such immortal stem cell. Upon division, an immortal cell live can divide to give one stem cell +one cell which can differentiate, to ultimately become a part of squamus.

Apparently, not all basal cells are alike. Basal cells which have larger expression of extracellular matrix receptors of integrin family bind better to basal lamina. These are able to proliferate, differentiate and leave the basal layer. These similar to -10% constitute subset of basal cells and are immortal stem cells.

This influence of basal lamina is not constant. The tissue culture (in vitro), studies show that proliferation of differentiated cells can accelerate if the need arises. Thus, in case of a wound, renewal of skin cells at the top require that this proliferation is accelerated. Stem cells upon differentiation shed away the receptor which were binding them to the basal lamina.

Once the skin at the top (squamus cell layers) are restored to its original thickness and coverage area, division of the basal cells drops back. A skin disease psoriasis caused by proliferation of basal cells is high but cells from top layer fall of prematurely before complete keratinisation has taken place.

What we talked about stem cells, in fact, has been a discussion on what are called adult stem cells. As we have seen, these are a small % of cells present among the population of differentiated cells in a tissue or organ. The primary role is to maintain/repair/renew the tissue.

The adult stem cell is yet to differentiate into a cell type to have a specialized structure and function in the tissue/organ. Their differentiation happens all the time during the life time of an individual. The frequency of the differentiation/proliferation depends upon many factors and vary from organ to organ. Some scientists prefer the term somatic stem cell to adult stem cell.

Initially, it was not known that such cells occur within the organ/tissue. The discovery of their occurrence in many tissues raised the hope of their use in tissue engineering.

It was in 1960s that two types of cells haematopoitic stem cells and bone marrow stromal cells were discovered in bone marrow.

While former can differentiate into all types of blood cells, the later can be differentiated into bone, cartilage, fat cells and fibrous connective tissues.

Around the same time, adult brain was found to contain stem cells which can transform into two major non-neuronal cells (astrocytes and oligodendrocytes) and neurons (nerve cells).

By now adult stem cells have been discovered in brain, bone marrow, peripheral blood vessels, skeletal muscles, skin and liver. In some cases, adult stem cells may remain as stem cells and start dividing in case of an injury or a pathological situation.

Adult stem cells are identified in the following ways:

•  Label the tissue cells with molecular markers and track their progeny whether it forms specialized cells.

•  Take cells from a living animal, grow them in a culture, label and transplant the labelled cells to the animal and check if the transplanted cells survive.

•  Grow isolated cells and manipulate by addition of factors/introduction of new gene etc. to see whether a differentiated cell type different from the original cell emerges.

The basic criterion is that a stem cell should be able to form a clone of identical cell types.

Hence, what we have discussed so far are the cases in which adult stem cells were identified and their differentiation route in some tissues tracked. However it is not always easy to identify an adult stem cell unequivocally.

The pluripotency, the ability to differentiate to form different cell types, is also called plasticity or transdifferentiation.

We still do not understand the mechanism of somatic stem cell transdifferntiation. Insight into these help us in divising protocols for converting stem cells from a healthy tissue and use it for tissue engineering (repair/repopulate) an unhealthy/dead tissue part.

Some challenges which remain at least in part are:

•  In what all tissues, adult stem cells occur and of how many types. As we have discussed, increasingly we are learning about this in more and more organs/tissues.

•  How do somatic stem cells originate in different organs/tissues?

•  Given identical environments, why some are stem cells and other are not, like in epithelia, why ~10% cells in the basal layer are stem cells whereas rest lack these properties ?

•  What factors induce stem cells to change their differentiation/proliferation rate and why only in some cases relocation of the stem cells/differentiated cells is more facile?

•  Finally adult stem cells are pluripotent but they are not totipotent. What are totipotent cells and when do they lose totipotency?

•  How can we control the rejection of implanted cell mass during tissue engineering by the immune system?

Sources of stem cells

At present there are different ways of obtaining stem cells. Adult stem cells can be obtained from many tissues now. Foetal stem cells are obtained from aborted foetuses or umbilical cord blood.

Embryonic stem cells can be obtained from:

•  Either discarded samples of in vitro fertilizations.

•  Or by a technique called somatic cell nuclear transfer (or simply SCNT or nuclear transfer)

Briefly, SCNT consists of placing a normal cell into a fertilized egg from which nucleus has been removed. Fertilized egg provides the necessary microenvironments for reprogramming the cell to become a precursor primordial form.

There are obvious ethical barriers in working with embryonic stem cells. One argument is that adult stem cells are available and are a promising system for stem cell therapy. The two kinds of stem cells have however many differences.

We still do not completely understand the nature of adult stem cells in tissues.

Purely in terms of gaining insights of fundamental nature, working with both types of cells:

adult and embryonic helps.

To remove common misconception is necessary. Embryonic stem cells are not obtained from a woman’s reproductive organs. These are, as stated above, obtained from in vitro fertilization clinics.

Human embryonic stem cells are derived from embryos which are about 4-5 days old. At this stage embryos are hollow spheres of cells and are called blastocyst.

A blastocyst has three structural components: Trophoblast (the surrounding layer cells), blastocoel (hollow cavity) and inner cell mass (- 30 cells) present at one end of blastocoels.

For growing in the laboratory, the following steps are followed:

•  Inner cell mass is transformed to a dish containing culture medium. Inner surface of the dish has a coating (feeder layer) of undividing mouse skin stem cells. The feeder layer provides an adhering surface to the dividing cells from the inner cell mass.

•  Mouse cells also release necessary substances into growth medium. As these are potential source of viruses and other biological structure which may get transferred to the growing human embryonic cells, lately methods have been developed which do not require this feader layer.

•  After sufficient growth, cells are removed and distributed to different dishes again for subculturing. Each subculturing cycle is called a passage.

•  After about -6 months, -30 cells yield million of stem cells. If these remain undifferentiated, these are embryonic stem cells line.

•  An established cell line facilitates collaborative research which can be validated among different groups.

Tests to identify whether cells obtained are embryonic stem cells

• One obvious way is keep these subculturing and examine the cells microscopically to confirm that no differentiation has occurred.
• Surface markers which are characteristics of undifferentiated cells can be tested. Undifferentiated cells also produce a transcription factor oct-4 whose presence can be checked.
• To check for the number of and damage (if any) to chromosomes
• Allow the cells to differentiate into a specific cell types to check its pluripotency.
• Inject the cells into an immunosuppressed mouse and see whether a benign tumour called tetroma is formed. A tetroma contains both differentiated and partly differentiated cells and its formation indicates pluripotency.

How to differentiate embryonic stem cells

Embryonic stem cells (EST) are totipotent. Hence, they offer enormous opportunities in regeneration medicine. The applications however require that we learn to make them differentiate into a specific cell type:

The EST upon clumping form embryoid bodies which begin to differentiate spontaneously.

However, this does not help in obtaining specific cell type.

Protocols for differentiating EST into specific cell type involve nature of nutrient broth, surface of growth and genetic manipulation of cells.

Understanding progression from totipotency to unipotency

Animal systems have contributed a lot to our understanding of cell differentiation from embryonic stem cells onwards.

A growing embryo is surrounded by membranes. To start with cells which form extraembryonic tissues later on cover the pre embryonic cells.

The thick lining, zona pelluda is part of the egg cell cover. Cell divisions form first morula and than a blastocyst with blastocoel. So, these stages are similar to what has been observed with stem cells.

There is a proper embryo with an epithelial lining called trophactoderm. At this point blastocyste emerges from zona pellucida and trophactoderm becomes extraembryonic cells.

Figure 10

After additional divisions egg moves from ovary to uterus and attaches to endometrium. The blastocyst is engulfed by endometrial cells.

Cells of trophectoderm (trophoblasts) differentiate into cytotrophoblasts (encircling the inner cell mass) and syncytotrophoblast, a multinuclear cell which attaches blastocyst to uterine wall.

In all animals, gastrulation creates 3 layers of cells which are of different tissue types. These are called primordial germ layers.

Different organs form from different germ layers. The central cavity of the somites-clumps of mesodermal cells differentiate to become chest and abdominal cavities, muscles and skeletal system cells.

Some mesodermal cells, called mesenchymal cells interact with endodermal cells to become different cell types to form organ

Mesenchymal cells are highly proliferative, pluripotent stem cells and their sources include bone marrow adipose tissues and umbilical cord blood.

Umbilical and mesenchymal cells do not express MHC class II (HLA-DR) antigens-neverthless, these are not rejected 4 months after transplantation even without immunesuppresive. These stem cells can also differentiate into neurons and thus potentially restore neurogenesis, angiogenesis and synaptic plasticity.

Recent studies suggest that adult stem cells from a tissue can differentiate into cell types of other tissues. A term multipotent adult progenitor cells (MAPCs) has been suggested.

As an example, mesenchymal stem cells were found to differentiate into cell types of visceral mesoderm, neuroectoderm, and endoderm. When injected into an early blastocyst, MAPCs could differentiate into many somatic cell types: hematopoitic, epithelium of liver, gut and lung. It was found that MAPCs require culture parameters similar to embryonic stem cell and in fact express some markers of ESC (Oct-4, Rex-1 and SSEA-1) Similarly, bone marrow stromal cells could undergo “unorthodox” differentiation to possibly form neural cells.

This returns us to the question of when cells lose totipotency. It is believed that loss of totipotency taken place at different developmental stages in different organism.

In mammals, a blastocyst can give rise to a normal animal when implanted in a host uterus but the host mother has to be treated with appropriate hormones.

Many studies indicate that it is group of cels and not a single cell which is progenitor of developing structures.

The loss of totipotency is adult (stem) cells may occur because of irreversible rearrangement/changes in the genes during differentiation.

There is enough evidences that during differentiation of cells in developing tissue and organs, inter-cellular interactions play important roles.

Endoderm-mesoderm interaction dictates the internal organ formation in some cases. In other cases, the other germ layers take part in inducing the differentiation.

Tissues like skin, blood and intestinal epithelium have short life of few days. Stem cells, thus are necessary to regenerate cells to maintain the adult organ.

In some cases, differentiation to a new cell during stem cell division may be delayed by initial formation of a precursor to differentiated cells. Thus, the population of differentiated cells in a tissue is controlled.

While we have limited information that cell determination occurs at some stages. In what ways, (at the molecular level), this commitment to follow a particular differentiation path differs from the events which occur during differentiation is not much understood.

We do know that cell-cell interactions are important but cell-cell signalling is not completely understood.

During cell determination, cell proliferation occurs at high rate. After differentiation, cell proliferation slows down. Finally, as mentioned before terminally differentiated cells like in skin and RBC do not divide. We do not yet completely understand controlling mechanisms for this cell growth vs. differentiation paradigm In many cases, use of adult stem cell is yielding exciting results in stem cell therapy.

Greater understanding of cell determination and cell differentiation will enable further progress.

The challenges to avoid immune rejection in stem cell therapy are more or less similar to what we discussed during organ transplantation.

Stem cell therapy and regenerative medicine is a frontier area at the interfaces of cell biology, immunology and medical sciences. What is discussed above is just a quick overview of basic background and a glimpse of what is possible. In many cases, as happens in any fast developing area, claims and counterclaims are frequent.

Hence, we have refrained from discussing details of individual cases of tissue engineering/stem cell therapy.

Summary

•  Embryonic stem cells and adult stem cells.

•  Sources of stem cells

•  Totipotency, pluripotency and unipotency

•  Cell determination and cell differentiaton