Stem Cells: Current Advances & Applications

Professor Peter Andrews & Professor Malcolm Alison

Session chaired by: David Montgomery MICR CSci

Reporter: Jane Pelly MICR CSci

Keywords

Cancer, Differentiation, Embryonic stem cells, Pluripotency, Regenerative medicine, Tumour-initiating

History of embryonic stem cell research

The first pluripotent cell lines from preimplantation mouse embryos were established in 1981 by both Martin Evans and Gail Martin, with Jamie Thomson successfully deriving stem cells from human blastocysts in 1998. The ability of these cells to self-renew and also to differentiate into other types of cells generated a great deal of excitement about their potential for therapeutic applications such as Parkinson’s, diabetes and arthritis. However, it was recognised that considerable additional research was needed into the behaviour of the stem cells themselves as well as how to control their differentiation into the type of cells required.

Human embryonic stem (hES) cells are derived from the inner cell mass of a blastocyst 5 days after in vitro fertilisation (IVF). This area of research is strictly regulated by a legal framework. In the UK, the Human Fertilisation Authority (HFEA) licenses work with embryos and the Stem Cell Steering Committee approves hES research and the importation of hES lines. Established hES cell lines must be deposited with the UK Stem Cell Bank. In addition, any hES cell lines intended for clinical use must be generated under GMP conditions to ensure they are free of animal products and viruses; however, the majority of lines derived to date are not to this standard.

Ian Wilmut’s historic research on somatic cell nuclear transfer that resulted in Dolly the sheep has not, so far, been achieved in humans. However, three years ago, Takahashi and Yamanaka¹ succeeded in inducing pluripotency by transfecting genes into mouse fibroblasts, and the same group subsequently reproduced this with human fibroblasts using the same four defined factors.² Thomson also successfully induced pluripotency in human fibroblasts using a different combination of defined factors and these cells resemble hES cells by all measurable criteria.

Applications in regenerative medicine

Peter went on to give examples of current projects in regenerative medicine using hES cells. Although patients with type 1 diabetes have been successfully treated by transplantation of islet cells into the portal vein, there are insufficient donors to make this viable for all sufferers of the condition. Recently, Kroon et al³ have shown that pancreatic endoderm derived from hES cells efficiently generates glucose-responsive insulin-secreting cells in vivo, which would offer an alternative therapy. A project at the University of Sheffield is looking to develop the next generation of coronary stents by using a scaffold to coat them with stem cells differentiated to endovascular cells producing immunosuppressive factors. Age-related Macular Degeneration results from degeneration of the retinal pigment epithelial (RPE) cells and functional RPE cells have been derived from hES cells. Moorfields Eye Hospital and UCL are currently undertaking a collaborative project aimed at using this technology to cure blindness in this group of patients. 

Human embryonic stem cells have several applications in regenerative medicine: drug discovery, toxicology and disease models such as cancer. When hES cells divide, there are three possibilities: self-renewal, differentiation and cell death. However, the cells are subject to mutation and it is possible a mutation could be in a gene critical to the outcome of division. Karyotypic changes have been reported in hES cell lines, with most changes affecting chromosomes 12, 17 and the X chromosome. ‘Culture-adapted’ H7 hES cells demonstrate different plating efficiency and growth characteristics from ‘normal’ H7 hES cells and will also persist in a xenograft. Human embryonic carcinoma (hEC) cells in germ cell tumours, eg, testicular teratoma, commonly gain 17q and 12p, which raises the question as to whether hES cells in culture may be subject to similar selective pressures as hEC cells in tumours. Genetic, epigenetic and metabolic factors induce adaptation in hEC cells and in hES cells in culture and this may affect the outcome of cell division.

Stem cells in cancer

Professor Malcolm Alison then expanded on the theme of stem cells in relation to cancer, looking first at the origins of cancer as a multi-gene defect disease. Early studies on rabbits demonstrated that dimethyl-benzanthracene (DMBA) alone painted onto the skin did not produce tumours, but if tetradecanoyl-phorbol-acetate (TPA) was applied subsequently, even after a long period, a squamous cell carcinoma of the skin was produced. These results were indicative that the potential for cancer development had been initiated by the DMBA in a long-term cell (ie, a stem cell), and promoted by the TPA. The presence of the Philadelphia translocation in chronic myelogenous leukaemia (CML) and development of glial tumours from the sub-ventricular zone astrocytic ribbon in the brain are also suggestive of tumour development from stem cells.

Two models for tumour development have been proposed: one where the tumour cells are heterogeneous and most cells can proliferate to form new tumours, and the hierarchical model where the tumour cells are heterogeneous but only the cancer stem cell subset has the ability to proliferate extensively and form tumours. The hierarchical model is now generally accepted and is supported by evidence that the plating efficiency of most primary tumours is less than 1%⁴ and the existence of two types of squamous cell carcinoma, where the highly differentiated type is a low grade tumour while the poorly-differentiated type is high grade.

Bonnet and Dick⁵ demonstrated that the acute myeloid leukaemia (AML) cells capable of initiating tumours in non-obese diabetic mice with severe combined immunodeficiency (NOD/SCID) comprised only 0.2% of AML cells and were exclusively CD34++ and CD38-, suggesting that normal primitive cells, rather than committed progenitor cells, are the target for leukaemic transformation. Marx⁶ found that CD44+, CD24-/low, Lin- cells are capable of making tumours in NOD/SCID mice and suggested that mutant stem cells may seed cancer, but their resemblance to normal cells might explain why many cancers are so hard to eradicate.

CD133: Molecule of the moment

CD133 was described as the ‘molecule of the moment’.⁷ It is a pentaspan protein representing a marker of tumour-initiating cells in a number of human cancers. Singh et al⁸ found that injection of as few as 100 CD133+ brain tumour cells was capable of producing a tumour that could be serially transplanted in NOD/SCID mice, whereas CD133- cells engrafted but did not cause a tumour. That CD133+ cells only were capable of initiating tumour growth was also demonstrated by Ricci-Vitiani et al⁹ and O’Brien et al¹⁰ in colon carcinomas. However, Shmelkov et al¹¹, using a CD133lacZ/+ mouse, found that CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells could initiate tumours, with the CD133- metastases producing more aggressive tumours.

However, the cancer stem cell ‘bandwagon’ has been questioned by some researchers. Yoo and Hatfield¹² suggest that the majority of malignant cells can support tumour growth while Kelly et al¹³ postulate that the low frequency of tumour sustaining cells in xenotransplantation may reflect the limited ability of human tumour cells to adapt to a mouse environment. Sean Morrison’s group¹⁴ propose that modifications to xenotransplantation assays would increase detection of tumorigenic melanoma cells by several orders of magnitude and demonstrate that they are common in some cancers.

It seems clear that tumours have cancer stem cells and that therapy should target these cancer stem cells, but a number of questions remain unanswered. What is the story with CD133: is its expression restricted or is it ubiquitous in the gut? Are alternative/additional cancer stem cell markers needed; does any one tumour have more than one type of stem cell with different markers? Are cancer stem cells rare or as common as muck? And so the story continues…

References

1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006 Aug 25;126(4):663-76.
2. Takahashi K, Tanabe K, Ohnuki M, Narite M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007 Nov 30;131(5):861-72
3. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin secreting cells in vivo. Nat Biotechnol 2008 Apr;26(4):443-52.
4. Hamburger A, Salmon SE. Primary bioassay of human tumor stem cells. Science 1977 Jul 29;197(4302):461-3.
5. Bonnet D, Dick JE. Human acute myeloid leukaemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997 Jul;3(7):730-7.
6. Marx J. Mutant stem cells may seed cancer. Science 2003 Sep 5;301(5638):1308-10.
7. Mizrak D, Brittan M, Alison MR. CD133: Molecule of the moment. J Pathol 2008 Jan;214(1):3-9.
8. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature 2004 Nov 18;432(7015):281-2.
9. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007 Jan 4;445(7123):111-5.
10. O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007 Jan 4;445(7123):106-10.
11. Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T, et al. CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. J Clin Invest 2008;118:2111-20.
12. Yoo MH, Hatfield DL. The cancer stem cell theory: is it correct? Mol Cells 2008 nov 30;26(5):514-6.
13. Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A. Tumor growth need not be driven by rare cancer stem cells. Sciene 2007 Jul20;317(5836):337.
14. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ. Efficient tumor formation by single human melanoma cells. Nature 2008 Dec 4;456(7222):593-8.