Stem cells, master cells for all seasons: a primer

1998 saw the publication of two papers describing the growth in vitro of human embryonic stem (ES) cells derived either from the inner cell mass (ICM) of the early blastocyst or the primitive gonadal regions of early aborted foetuses. Work on murine embryonic stem cells over many years had already established the amazing flexibility of ES cells, essentially able to differentiate into almost all cells that arise from the three germ layers. The breakthrough in 1998 was to keep human ES cells in a state of prolonged undifferentiated proliferation by the use of a blocking factor (leukaemia inhibitory factor, LIF) that, when removed allowed the dividing cells to go down specific, directed differentiation pathways. The realisation of such pluripotentiality (see below) has, of course, resulted in the field of stem cell research going into overdrive with the establishment of many new biotechnology companies (http://www.stemcellresearch news.com/catalog1677.html), stem cell banks and Regulatory bodies to oversee their use, with a genuine belief that stem cell research will deliver a revolution in terms of how we treat cardiovascular disease, neurodegenerative disease, cancer, diabetes and the like. However, many people believe that early human embryos should be accorded the same status as any sentient being and thus their ‘harvesting’ for stem cells is morally unjustifiable (1,2). With this in mind, other sources of malleable stem cells have been sought. In the adult, organ formation and regeneration was thought to occur through the action of organ- or tissue-restricted stem cells (i.e. haematopoietic stem cells making blood, gut stem cells making gut). However, we now believe that stem cells from one organ system, for example the haematopoietic compartment can develop into the differentiated cells within another organ system, such as the liver, brain or kidney. Thus, certain adult stem cells may turn out to be as malleable as ES cells and so also be useful in regenerative medicine. In this overview we summarize the important attributes of stem cells from a variety of sources, clarify the terms used and try and get beyond the hype that so often accompanies apparent new ‘breakthroughs’ in medical research. We also emphasise the importance of stem cell biology to the development and treatment of cancer.

Morbidity and mortality as a result of malfunctions in vital organs plague even the most technologically advanced societies. Because of a dearth in transplantable organs there is a growing hope that stem cells may be the answer to mankind’s prayer to be able to replace tissues worn out by old age and ravaged by disease. Indeed, it is impossible to open a newspaper today without seeing yet another apparent ‘breakthrough’ in stem cell research; the more optimistic hoping for an elixir of life – the promise of immortality. More realistically, regenerative medicine is already delivering results, for example, biotechnology companies like Osiris Therapeutics, Inc are making “off-the-shelf” products from human mesenchymal stem cells for bone (OsteoCel^TM) and joint (Chondrogen^TM) repairs. This type of tissue repair uses the body’s own three-dimensional matrix and growth factor milieu; more difficult will be the realisation of the holy grail of tissue engineering – the creation of whole complex internal organs such as the liver and kidney outside the body.

ES cell lines are invariably derived from the ICM of 5-day-old embryos (blastocysts) or foetal gonadal tissue (Figure 1). Blastocysts are usually from in vitro fertilization (IVF) programmes that would have otherwise been discarded, though some have been deliberately created. These blastocysts are composed of about 100 cells, of which 30-40 make up the ICM. A misconception is that ES cells are ‘totipotent’, i.e. they have the potential to form an entire human being. This is incorrect because they have been separated from the supporting trophectoderm, a tissue that protects the ICM and is what implants in the uterus. Legislation regarding the use of ES cells varies around the globe. In countries like the UK and Australia (3) new cell lines can be created from spare embryos with the uncompensated permission of the donors, but in the USA at the moment in a compromise between proponents and critics of ES cell research, federal funds (taxpayers’ money) can only be used on ES cell lines created before the 9^th August 2001 (~ 70 existing cell lines); the rationale being that such cells while exhibiting pluripotency have not the ability to develop into a whole human being, thus the sanctity of human life is not compromised by their use. However, many of these ‘approved’ cell lines are now not available, and most of the others were grown in the presence of mouse feeder cells (to supply essential growth factors), exposing human cells to potentially pathogenic murine viruses and proteins. With such considerations in mind, along with the realisation that up to 3,000 Americans die every day of diseases that could be combated with ES cells, not forgetting that the nation’s best scientists may move abroad to more supportive environments, intense political lobbying is now hoping to reverse this decision. Further impetus has been given to the ES cell lobbyists in the US by the realisation that other countries, as well as privately funded entities are forging ahead perhaps with irresponsible research, while NIH funded research would be (hopefully) carefully regulated and have safety as a major priority. In the UK, the Human Fertilization and Embryology Authority (HFEA: http://hfea.gov.uk/Home) licences and monitors all human embryo research, including using embryos for stem cell extraction. Moreover, on May 19th 2004 the world’s first stem cell bank opened in the UK, jointly overseen by the MRC and BBSRC (http://www.ukstemcellbank.org.uk/), acting as a repository and supplier of all types of human stem cells, not just embryonic but also those derived from foetal and adult tissues and discarded cord blood.

Of course, for ES cell research to have a major impact on regenerative medicine, cloned human blastocysts will be needed because replacement cell therapy, like whole organ transplants, must overcome the obstacles posed by immune system incompatibility (graft rejection). Somatic cell nuclear transfer (also called ‘therapeutic cloning’) offers the possibility of using the patient’s own genome to generate ES cells and so overcome this problem. Therapeutic cloning involves taking a cell from the patient and inserting it into an enucleated egg from an anonymous female donor (not as easy as it sounds), nurturing it to the blastocyst stage and then harvesting the ES cells from the ICM. Each cell would be almost identical in genetic terms to the cells of the patient who would be treated with them, and the first successful demonstration of human ES cells derived in this manner has now been published (4). Many argue that cloning embryos for regenerative medicine is not exactly therapeutic for the embryo (true!), and really what is happening is placing society on the slippery slope to reproductive cloning (see below). Nevertheless, in August 2004 the HFEA awarded a licence to the University of Newcastle to carry out the first cloning of a human embryo.

Nuclear transfer technology has in fact been with us for over 50 years, and scientists such as John Gurdon were able to clone frogs by transplanting nuclei into enucleated frogs’ eggs. Unsurprisingly cloning was more successful when relatively primitive cells like blastula cells were used, rather than adult skin or gut epithelial cells. However, it was the birth of Dolly the sheep in 1996 that attracted so much media attention, being the first mammal to be cloned from a cell (a mammary cell) extracted from an adult. Scientists and lawmakers in particular make an important distinction between ‘therapeutic cloning’ and ‘reproductive’ cloning, the latter described as implanting a cloned embryo into a woman’s womb – a practice that is strictly illegal in most countries, including the UK. Nevertheless, maverick fertility expert Severino Antinori has recently claimed to have several women pregnant with cloned embryos under his care, though few have taken these claims seriously. Apart from the moral boundary between therapeutic and reproductive cloning, the most vociferous criticisms of human reproductive cloning come from scientists themselves finding that almost all cloned animals develop one or more abnormalities (5,6). So while no one really doubts that ES cells are likely to be the most flexible of all stem cells, the ethical issues surrounding their use have prompted the search for alternative adult sources.

1) A hierarchy of potential: As we have already seen, the appeal of ES cells is the fact that they are pluripotent, able to differentiate into almost all cells that arise from the three germ layers, but not the embryo because they are unable to give rise to the placenta and supporting tissues. On the other hand, most adult tissues have multipotential stem cells, cells capable of producing a limited range of differentiated cell lineages appropriate to their location, e.g. small intestinal stem cells can produce all four indigenous lineages (Paneth, goblet, absorptive columnar and enteroendocrine), CNS stem cells have trilineage potential generating neurons, oligodendrocytes and astrocytes (7), while the recently discovered stem cells of the heart can give rise to cardiomyocytes, endothelial cells and smooth muscle (8). However, describing tissue-based stem cells as ‘multipotential’ may be incorrect if, as it appears, that some adult stem cells, when removed from their usual location can transdifferentiate into cells that arise from any of the three germ layers (so-called plasticity). The least versatile are unipotential stem cells, cells capable of generating one specific cell type. Into this category we could place epidermal stem cells in the basal layer of the skin that produce only keratinized squames and certain adult hepatocytes that have long-term repopulating ability (9). Some would argue that there is no such thing as a unipotential stem cell, and really these cells should be called committed progenitors. While there is no doubt that in some tissues, e.g. the gastrointestinal tract and haematopoietic renewal systems, there are committed stem cells (progenitors) with more limited division potential than their multipotential stem cells, in the epidermis these unipotent cells do have a large clonogenic capacity capable of producing large sheets of cells for the treatment of burns patients.

3) Proliferation, clonogenicity and genomic integrity: Stem cells are slowly cycling but highly clonogenic. Teleologically it would seem prudent to restrict stem cell division because DNA synthesis can be error-prone. Thus, in many tissues stem cells divide less frequently than transit amplifying cells. In the intestine, stem cells cycle less frequently than the more luminally located transit amplifying cells, and in the human epidermis the integrin-bright cells have a lower level of proliferation than the other basal cells. In hair follicles, the hair shaft and its surrounding sheaths are produced by the hair matrix that is itself replenished by the bulge stem cells. As befits true stem cells, the bulge cells divide less frequently but are more clonogenic than the transit amplifying cells of the hair matrix. Combined to an infrequently dividing nature, stem cells would also appear to have devised a strategy for maintaining genome integrity. Termed the ‘immortal strand’ hypothesis or Cairns hypothesis, stem cells can apparently designate one of the two strands of DNA in each chromosome as a template strand, such that in each round of DNA synthesis while both strands of DNA are copied, only the template strand and its copy is allocated to the daughter cell that remains a stem cell (13). Thus, any errors in replication are readily transferred (within one generation) to transit amplifying cells that are soon lost from the population. Such a mechanism probably accounts for the ability of stem cells to be ‘label retaining cells’ after injection of DNA labels when stem cells are being formed (14).

4) Adult stem cell identity: In many tissues and organs the identity of the stem cells has remained either elusive or at least equivocal. However, in the bone marrow the recognition of cells with the properties of self-renewal and multilineage differentiation potential is well advanced. In fact such cells were recognised operationally back in 1961 by Till and McCulloch as cells that gave rise to multilineage haematopoietic colonies in the spleen (colony forming units – spleen [CFU-S]). In the human bone marrow the sialomucin CD34 is a haematopoietic cell surface antigen that has been extensively exploited for the selection of long-term repopulating cells with multilineage potential, though not all HSCs express this marker. In the mouse HSCs are known as KLS cells (c-kit⁺lin^-Sca-1⁺). An alternative method of enriching for HSCs exploits the fact that some cells have evolved a cellular protection mechanism against toxic metabolites and xenobiotics. This mechanism involves the expression of efflux pumps that belong to the ATP-binding cassette (ABC) superfamily of membrane transporters, and such cells are able to efflux a combination of Hoechst 33342 and Rhodamine123, thus appearing at the bottom left corner of a dual parameter FACS analysis – hence called the side population (SP). There are SP cells in many other tissues that might well correspond to their multipotential stem cells (15).

6) Adult stem cell plasticity: A large body of evidence now supports the idea that certain adult stem cells, particularly those of bone marrow origin, can engraft alternative locations (e.g. non-haematopoietic organs), particularly when the recipient organ is damaged and transdifferentiate in to cell types that function appropriate to their new location (Figure 3). Hence there is considerable excitement in using haematopoietic stem cells (HSCs) in cell based therapies and as vectors to deliver therapeutic genes for the correction of diseases such as haemophilia. This is particulary attractive to the clinician because bone marrow stem cells can be readily obtained from patients by simply mobilizing HSCs into the peripheral circulation by injection of a cytokine such as G-CSF. Moreover, if a patient’s own stem cells were taken for ex vivo expansion and directed differentiation in, to say, liver cells or brain cells, no immune rejection problems would need combating.

However before the Pro-Life lobby call for an end to ES cell research, seeing adult stem cells as the panacea for all ills, it is worth noting that not everyone is convinced of their versatility (16). The reason for this is twofold, 1) certain instances of so-called plasticity have now been attributed to cell fusion between bone marrow cells (or their macrophage descendants) and cells of the recipient organ, and 2) several remarkable claims have not been able to be confirmed, most recently the inability to show that HSCs directly injected into the heart can contribute to the healing of a myocardial infarction in experimental animals. This last observation is particularly perplexing given the number of Medical Centres worldwide, including some in the UK, who are claiming a significant clinical benefit results from the injection of autologous bone marrow to patients with congestive heart failure or who are recovering from myocardial infarction.

There are also claims that adult cells can be retrodifferentiated and then redifferentiated. Dr Ilham Abuljadayel, part of a husband and wife team from TriStem UK Ltd whose endeavours were widely publicized in The Sunday Times Colour Magazine (1^st February 2004), claims to be able to turn normal white blood cells into pluripotent stem cells with little more than exposure to a monoclonal antibody against the MHC class II region (17). A considerable furore erupted very recently after an online physics journal published with unseemly haste a ‘breakthrough’ in adult stem cell technology (18,19). Critics argued that the paper was not rigorously peer reviewed, while the claims made (that pluripotent stem cells can be isolated from adult pancreas) would strengthen opposition to ES cell research.

The resurgence in interest in stem cells has reaped dividends in terms of how we understand other diseases. Often accompanying chronic inflammation and tissue damage is metaplastic change - a major switch in tissue differentiation (a sort of plasticity), and it is reasonable to suppose that this switch occurs at the level of stem cells rather than between terminally differentiated cells. Examples include squamous metaplasia in the conducting airways of smokers, various types of intestinal metaplasia in the stomach often associated with gastritis, and gastric metaplasia in the intestine affected by inflammatory bowel diseases such as Crohn’s colitis.

There may be a ‘dark side’ to adult stem cells. HSCs in particular appear to contribute to fibrogenesis in both pulmonary and hepatic scarring (20) and may also be responsible for allograft failure by contributing to transplant arteriosclerosis. It is also likely that many cancers, particularly those of continually renewing tissues (blood, gut, skin), are in fact a disease of stem cells since these are the only cells that persist in the tissues for a sufficient length of time to acquire the requisite number of genetic changes for malignant development. Moreover, tumours are heterogeneous populations in which many cells are terminally differentiated (reproductively sterile) or transit amplifying cells with limited division potential, and only tumour stem cells are capable of ‘transferring the disease’. For example, in human acute myeloid leukaemia, only the CD34+CD38- cells are capable of propagating the disease in immunodeficient NOD/SCID mice, while in human breast cancer, the CD44+ESA+CD24^-/low fraction has a similar potential (21). Thus, successful cancer chemotherapy is critically dependent upon eradicating all the stem cells within a cancer.

In this article we have reviewed the potential benefits of ES cells and the present legislative framework. We have shown that adult stem cells are involved in almost all aspects of tissue homeostasis, they are responsible for normal cell renewal and recent observations suggest that some stem cells, notably HSCs, may have a role in regenerative medicine by virtue of their ability to transdifferentiate. According to recent statistics (February 2004) from the UK Transplant Registry, 5,717 patients are on the waiting list for organ transplantation; stem cell therapies using either embryonic or adult stem cells may be the solution to the long waiting lists worldwide. Furthermore we have illustrated that if ES cells are created by nuclear transfer from the patient or the patient’s own adult HSCs are used, then the horrors of lifelong immunosuppression may be avoided. On the other hand, cells from the bone marrow may also contribute to organ scarring as seen in liver cirrhosis. Stem cells also exist in tumours, and further characterization and identification of stem cells in normal tissues will aid in targeting stem cells in cancer.

3. Dayton L. (2002) Australia agreement allows new lines. Science 296, 238.

4. Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JM, Jeon HY, Lee BC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY. (2004) Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 303, 1669-1674. (First demonstration of human ES cells derived from nuclear transfer)

5. Wilmut I. (2002) Are there any normal cloned mammals? Nature Medicine 8, 215-216.

6. Tamashiro KLK, Wakayama T, Akutsu H, et al. (2002) Cloned mice have an obese phenotype not transmitted to their offspring. Nature Medicine 8, 262-267.

7. Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Manuel-Garcia Verdugo J, Berger MS, Alvarez-Buylla A. (2004) Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 427, 740-744. (First demonstration of multipotentiality of astrocytes derived from sub-ventricular layer of human brain)

8. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. (2003) Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114, 763-776. (First demonstration of multipotentiality of cardiac stem cells derived from human heart)

9. Alison MR, Vig P, Russo F, Bigger BW, Amofah E, Themis M, Forbes S. (2004) Hepatic stem cells: from inside and outside the liver? Cell Proliferation 37, 1-21. (Critical review of the role of HSCs in liver regeneration)

10. Gonzalez-Reyes A. (2003) Stem cells, niches and cadherins: a view from Drosophila. J Cell Sci 116, 949-954. (Beautifully illustrated review of the stem cell niche in the fly ovarioles)

11. Yamashita YM, Jones DL, Fuller MT. (2003) Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301, 1547-1550. (Superb account of the control of stem cell number in the fly testis)

12. Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L. (2003) Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425, 836-841.

13. Potten CS, Owen G, Booth D. (2002) Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Science 115, 2381-2388. (Shows that label retaining cells do proliferate but still retain their immortal DNA strands)

14. Braun KM, Niemann C, Jensen UB, Sundberg JP, Silva-Vargas V, Watt FM. (2003) Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis. Development 130, 5241-5255. (Beautiful illustration of label retaining cells in the mouse tail)

15. Alison MR (2003) Tissue-based stem cells: ABC transporter proteins take centre stage. J Pathol 200, 547-550. (Review of ABC proteins and their role in stem cell biology)

16. Alison MR, Poulsom R, Otto WR, Vig P, Brittan M, Direkze NC, Lovell M, Fang TC, Preston SL, Wright NA. (2004) Recipes for adult stem cell plasticity: fusion cuisine or readymade? J Clinical Pathology 57, 113-120. (Critical review of claims for adult stem cell plasticity)

18. Kruse D, Birth M, Rothwedel J, Assmuth K, Goepel A, Wedel T (2004) Pluripotency of adult stem cells derived from human and rat pancreas. Applied Physics A, Published online 26 May 2004.

20. Forbes SJ, Russo FP, Rey V, Burra P, Rugge M, Wright NA, Alison MR. (2004) A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis. Gastroenterology 126, 955-963. (First demonstration that bone marrow can contribute to human organ scarring)

21. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100, 3983-3988. (Illustrates that only human tumour cells with a stem cell phenotype can propagate the tumour in immunodeficient mice)

Address for correspondence: Professor Malcolm R. Alison DSc, FRCPath, Histopathology Unit, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX. E-mail: m.alison@qmul.ac.uk.