Introduction to Stem Cells and Ethical Issues: Part 1 Lecture Notes

Key words and terms

Embryonic stem cells, adult stem cells, self-renewal, differentiation, transit-amplifying cells, blastocyst, totipotent, multipotent, unipotent, ethics, somatic cell nuclear transfer, induced pluripotent stem cells, regenerative medicine.

Lecture notes

What are stem cells?

There are two basic types of stem cells: embryonic stem (ES) cells, and adult stem cells. They share the ability to endlessly self-renew – they can divide continuously to generate daughter stem cells of the same type. However, they can also divide to produce transit amplifying (TA) cells which proliferate rapidly to quickly increase in number before differentiating into specialized cell types. Normally, a stem cell divides to produce daughter cells with different fates – one daughter remains as a stem cell, while the other becomes a TA cell.

Embryonic stem cells are totipotent and can be cultured in vitro.

ES cells are totipotent because they give rise to all of the tissues in an animal. ES cells make up the ‘Inner Cell Mass’ of early embryos. At the embryonic stage called the blastocyst, the inner cell mass is surrounded by a layer of cells called the trophectoderm. The trophoblast cells in the trophectoderm provide support to the ES cells in the inner cell mass as they develop into the fetus.

Scientists can remove ES cells from blastocysts and culture them in vitro. Once these ES cells have been isolated, they can be maintained in culture indefinitely, because, as stem cells, they can divide and self-renew endlessly. However, the growth conditions can be manipulated to induce the ES cells to reform blastocysts. A single ES cell can be induced to form a blastocyst (including the trophectoderm layer) which can then be implanted back into a ‘pseudopregnant’ mouse, where it will develop normally and be born three weeks later. Thus, a single ES cell has the potential to give rise to an entire animal.

Cultured ES cells can also be induced to differentiate in vitro into many different tissues by treating them with different cocktails of growth factors. For example, treating ES cells with Nerve Growth Factor cause them to develop into neurons. Many other cell types can be formed in this way too – the only limitation is knowing the precise culture conditions to use. This is why ES cells hold such great promise for regenerative medicine (see below).

Adult stem cells are either multipotent or unipotent.

During development, stem cells become increasingly restricted in what they can make. Adult stem cells can’t give rise to all the tissues of the body. Instead, they are restricted to particular tissues. They allow these tissues to undergo homeostasis (natural turnover throughout the tissue’s lifetime) and to be repaired following wounding or other types of injury. Adult stem cells may be multipotent (can develop into several different cell types and lineages) or unipotent (can only differentiate into one specific cell type).

Examples of multipotent adult stem cells include:

Hematopoietic stem cells (give rise to all the cells of the blood, including both red blood cells and immune cells)

Hair follicle stem cells (give rise to hair follicles, sebaceous glands and the epidermis)

Examples of unipotent adult stem cells include:

Epidermal stem cells (only give rise to the epidermis, which is constantly turning over and so needs to be continuously replenished with new cells)

Liver stem cells (only give rise to hepatocytes of the liver).

Adult tissues may contain large or small numbers of stem cells, depending on the tissue’s requirement for new cells in maintaining homeostasis (for example, the pancreas and the central nervous system has few adult stem cells).

Stem cells and regenerative medicine

Because stem cells (particularly ES cells) can be manipulated in vitro to form a variety of different cell types, they hold great potential for regenerative medicine. ES/totipotent cells are of most interest as they have the most developmental potential, including the ability to differentiate into tissues in which adult stem cells are only present with very low abundance.

Stem cells could be differentiated into cells able to replace defective or damaged cells in many different diseases and conditions. Examples include:

Nerve cells to treat patients with Parkinson’s disease, Alzheimer’s disease or spinal cord injuries.

Pancreatic islet cells for diabetics.

Skeletal muscle for patients suffering from muscular dystrophy.

Heart muscle to replace damaged or diseased heart tissue.

Immune cells for patients with immunodeficiencies.

Skin and hair cells for patients with skin disease, baldness or with burned or otherwise damaged skin.

A greater understanding of stem cells may also help us better understand cancer and provide new treatments. The ‘cancer stem cell’ hypothesis holds that stem cells within tumors produce, by self-renewal and differentiation, the cancerous tissue. These stem cells may persist after normal anti-cancer treatments, thereby causing relapses and metastases.

Ethical issues surrounding human ES cell research

The major ethical dilemma associated with human embryonic stem cell research is the concern that the derivation of human ES cells from fertilized embryos often entails the destruction of that embryo and thus, in some people’s view, the destruction of a human life.

In vitro fertilization (IVF) technologies allow fertilized embryos and blastocysts to be created in the laboratory. The techniques often results in excess embryos that either go unused or are discarded. Some believe that using these embryos poses fewer ethical problems, since the embryos were not created in the mother’s womb, and were not destined to develop into a fetus anyway. However, this does not satisfy everyone: some believe that IVF embryos still constitute human life, and that they should only be used for implantation into mothers and not for research.

It is possible to isolate a single ES cell from an 8 cell stage IVF embryo and for the remaining 7 cell embryo to remain viable (although not without risk to that embryo). The single isolated ES cell can be cultured and expanded to provide material for stem cell research. This technique is, in fact, already in use for genetic screening of embryos derived from couples at high risk of passing on a serious genetic disease to their unborn child. Nevertheless, some people believe that the danger posed to the embryo in extracting one of its cells is not justified by the potential it offers for stem cell research.

Alternative methods for producing totipotent stem cells

Ultimately, generating stem cells free of any ethical issues may require using alternatives to the isolation of ES cells from human embryos. Note that finding a way to produce totipotent stem cells holds the most promise for regenerative medicine as they have the capacity to differentiate into every possible tissue of the body. The lecture discusses one alternative method – nuclear transfer. Since this lecture was recorded, a second potential alternative has received much attention, namely induced pluripotent stem (iPS) cells. Both are discussed below.

Nuclear transfer

Nuclear transfer was the technique used to clone Dolly the sheep in 1996. The same approach was originally used by John Gurdon to clone frogs in 1962, and many animals, including mice, have been cloned in similar fashion since Dolly.

The technique involves removing the nucleus from an unfertilized oocyte and replacing it with the nucleus of an adult somatic cell. This produces a hybrid cell in which the oocyte cytoplasm somehow ‘resets’ the developmental history of the somatic nucleus, restoring its totipotent potential. Single hybrid cells can be cultured, induced to form blastocysts, and then implanted into mothers where they develop normally. The nuclei of mouse skin cells, for example, can be injected into enucleated oocytes and used to generate ES cell-like totipotent hybrid cells. This is actually a relatively efficient process, although subsequent attempts to generate cloned mice by implanting cloned blastocysts are much less efficient.

iPS cells

Induced pluripotent stem (iPS) cells are a relatively new approach to generating ES-like cells without running into so many ethical concerns. The technique was first demonstrated in mouse cells in 2006 in Shinya Yamanaka’s laboratory, and with human cells in 2007 by Yamanaka and by James Thomson’s group.

The technique involves reintroducing activated versions of a select few genes to adult somatic cells which then revert to an ES cell-like state. In Yamanaka’s original work, just 4 transcription factors – Oct-3/4, SOX2, c-Myc, and Klf4 – could induce this reversion when transduced into adult mouse fibroblasts using retroviruses. However, mice derived from these iPS cells have a high incidence of tumor formation. Many researchers around the world are working to improve the efficiency and safety of iPS cell technology, using slightly different combinations of genes and different ways of introducing them into various types of adult cells. Importantly, this technology has been adapted to human skin keratinocytes, raising the possibility of generating ES-like cells tailor made to human patients simply from a small skin biopsy.

Potential uses of nuclear transfer and iPS cell technologies

It should be stressed that scientists are not interested in using either of these technologies to clone humans. Instead, totipotent cells produced by these alternatives to ES cells have the potential to be differentiated in vitro to generate cells for research and regenerative therapy.

In terms of therapies, both of these techniques offer the intriguing advantage that stem cells could be derived from a patients own tissue. This would avoid issues of immune rejection, as implanted cells would be a genetic match to the patient, preventing their immune system from recognizing the cells as foreign and eliminating them. On the other hand, if a patient’s own cells are to be used as the source of totipotent cells, any genetic defect in the patient’s cells will need to be corrected.

In terms of research, differentiated cells produced in vitro from either iPS cells or nuclear transfer hybrid cells will be useful tools for understanding disease processes and for drug development. For example, the skin cells of an Alzheimer’s disease patient can be converted into iPS cells, which can then be differentiated into neurons. These genuine “Alzheimer’s neurons” are invaluable, as we cannot take a brain biopsy from a living Alzheimer’s patient, and we can presently only study the endpoint of the disease rather than study its onset.

Potential dangers of stem cell therapies

However they are attained – whether isolated from embryos, produced by nuclear transfer or by iPS techniques – totipotent cells have a danger, as well as benefits, in their developmental potential. These cells have the potential to form teratomas – tumors which derive from germ cells and contain multiple different tissue types. These tumors can form if ES/ES-like cells are implanted into animals due to uncontrolled differentiation. Future regenerative therapies must ensure that any ES cells derived by these technologies have been fully and irreversibly differentiated into the desired cell type prior to implantation into a patient. How long it will take before this research becomes clinically applicable is currently uncertain. However, the research holds promise for the future.

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