Stem Cell Research Initiative Research
Professor Kidson formed the UCT Stem Cell Initiative with Dr Robea Ballo and Professor Jacquie Greenberg from Human Genetics, UCT. Their expertise in disease modelling “in a dish” using human induced pluripotent stem (hiPS) cells, mesenchymal stem cells and mouse embryonic stem cells is shared with researchers at UCT and further afield who wish to investigate and understand specific diseases at a cellular and molecular level.
iPS cell technology can be used to identify tissues that are critical for modelling disease in a culture dish and which would ultimately help define the physiological phenotype being investigated. Reprogramming a somatic cell into an iPS cell and back to a differentiated cell requires relatively few manipulations (Figure 1). The induction of pluripotency in a somatic cell is brought about by the uptake of reprogramming factors, such as Oct3/4, Sox2, Klf4 and c-Myc, which leads to a number of changes, including changes in physical structure, gene expression and proliferative ability. The differentiation process requires the introduction of factors required to force the iPS cells along a lineage of a specific target cell type. The target cells can be used to create disease models in a cell culture dish.
Dr Ballo spent time in the laboratories of two collaborators at the University of Oxford (Dr Sally Cowley and Professor Matthew Wood) where she developed her expertise in generating iPS cells. iPS cell work is routinely conducted in the UCT facility and the technology has been and is currently the basis of a number of PhD theses in both human genetics (Dr Lauren Watson and Dr Danielle Smith) and cell biology (Ms Dimakatso Gumede and Ms Sylvia Kamanzi-wa). Thus far, the spectrum of cells that have been generated by our students have been neurons, photoreceptors and retinal pigment epithelium for spinocerebellar ataxia type 7 and iPS cell-derived mesenchymal cells, adipocytes and cartilage for conditions such as hereditary fibrosing poikiloderma and Mseleni Joint Disease.
A. iPS cell modelling for Spinocerebellar ataxia type 7, hereditary fibrosing poikiloderma and Mseleni Joint disease.
Spinocerebellar ataxia type 7: Collaboration with Prof Jacquie Greenberg, Dept of Human Genetics, UCT
i) Disease modelling of the Retinal pigment epithelium using iPSc technology (Mrs S Kamanzi-wa, Walter Sisulu University)
ii) Genotype correlation with non-invasive imaging of the retina (Drs R Rautenbach and M Naude – Tygerberg Hospital, US)
Hereditary familial fibrosing poikiloderma: Collaboration with Prof Bongani Mayosi, Dept of Medicine, UCT
i) iPSc technology to investigate the role of FAMIIIB in hereditary fibrosing poikiloderma
Mseleni Joint Disease: Collaboration with Dr Victoria Gibbon, Dept of Biological Anthropology, UCT and Dr Victor Fredlund (Mseleni Hospital, KZN)
i) An iPS cell model for investigating cartilage degeneration in Mseleni Joint disease
B. Animal Models for eye defects, cataracts and glaucoma.
Glaucoma, one of the leading causes of blindness in South Africa, is characterised by irreversible retinal ganglion cell (RGC) death and optic nerve damage. Early identification and treatment would improve the quality of life for many individuals. An individual's susceptibility to glaucoma is modified by many factors, highlighting the multi-causal nature of this condition. The cellular and molecular basis of most forms of glaucoma and the nature of susceptibility factors is poorly understood and few causative genes have been identified. There is thus an urgent need for animal models to help investigate the aetiology of the different forms of glaucoma and to identify causal and modifier genes. The anatomical and functional similarities between mouse and human eyes have encouraged researchers to search for suitable mouse models for glaucoma. Our aim is to understand the genetic basis of glaucoma and to explain the abnormalities seen in the eye when glaucoma occurs. Our approach is to make use of normal and mutant mice (knockouts for Foxc1 and Bmp4, singly and in combination and to focus on the development and abnormalities in the cornea, trabecular network, ciliary body, iris and retina. In addition, have been carrying out studies on the mole-rat, a unique and unusual African creature, and show that this is a very useful model animal for investigating blinding diseases. It is particularly interesting because it is blind from birth, and has early onset cataracts.
1. Morphology and development of Schlemm's canal in murine eyes. Three-dimensional reconstruction of the aqueous drainage vessels in the irido-corneal angle of the mouse eye.
The trabecular meshwork, Schlemm's canal and associated aqueous drainage vessels are located in the corneo-scleral junction at the irido-corneal angle. These structures encircle the outer perimeter of the iris at its junction with the cornea and sclera. Together they form the major exit route of the aqueous humour from the ocular anterior chamber. FoxC1 and BMP4 heterozygous mice display anterior segment dysgenesis that includes various degrees of structural aberrations in the irido-corneal angle, notably the Schlemm's canal and the trabecular meshwork (Van der Merwe and Kidson, in preparation). The aim of our next study is to reveal the entire Schlemm's canal and associated drainage vessels in order to evaluate how haplo-insufficiency of FoxC1 and BMP4 affects all drainage structures in the irido-corneal angle. In revealing these vessels we are hoping to determine whether or not their patterning could indicate possible downstream genes affected by FoxC1 and BMP4 during eye development and also indicate clinical phenotype.
2. Functional analysis of downstream targets of Foxc1 by microarray analysis.
Microarray analysis was used to subtract the transcriptitomes of Foxc1+/+ and Foxc1-/- mice in order to discover potential downstream targets of Foxc1. RNA was extracted from E12.5 and E13.5 whole heads of Foxc1+/+ and Foxc1-/- mice, reverse transcribed into cDNA and indirectly labelled with flourophores. The labelled cDNA was hybridised to both a small candidate cDNA array and a 7K oligo array. The data was normalised using LOWESS and genes differing significantly between wild-type and mutant conditions identified by t-tests and SAM (p<0,05). These genes were classified according to Gene Ontology using PathFinder. Significant differences in expression between wild-type and mutant conditions were verified using both northern blotting and real-time PCR. (Napier and Kidson, 2005, IN PRINT; Sommer, Napier and Kidson, in preparation). Expression patterns of these genes are currently being mapped using in situ hybridisation and functional studies will be carried on each of the identified targets.
The African mole-rat as a potential model for degenerative eye diseases.
The naked mole-rat (Heterocephalus glaber) is a subterranean rodent whose eyes are thought to be visually non-functional and is an ideal animal with which to pursue questions in evolutionary developmental biology. We have made substantial progress in our study of the mole-rat as a potential model for cataract formation and degenerative eye diseases (Nikitina and Kidson, 2004). In our original proposal, we indicated that we planned to study the eye and assess it as a model for anterior segment dysgenesis, and retinal degeneration. Our analyses have revealed that in addition to these potentials, it is clear that the animal develops early cataracts and that the lens is very abnormal. We have now embarked in a detailed analysis of lens development and differentiation.
C. Melanocytes in disease
Melanocyte differentiation and oncogenesis:
When melanocytes transform into metastatic melanomas, they go through an intermediate phase of immortalisation. In order to study how melanocytes proliferate, migrate and differentiate during this transformation process, two lines of investigation have been followed. Firstly, to study the molecular and cellular events that initiate melanocyte transformation into melanoma), we transformed mouse melanoblasts with a conditional allele of the SV-40 oncogene, derived six cell lines, characterised the derived immortal cell lines and compared their gene expression profiles during proliferation versus differentiation (Prince et al, 2001). We then used them to explore the role of the transcription factor, Mitf, as a central governor in the cell differentiation – cell proliferation equation (Prince et al, 2003). At the same time, we have developed methods to track the process of metastasis in humans. Our focus has been the early stages of metastases, where micro-metastases are clinically undetectable: our rationale being that accurate early detection would improve treatment regimens. A RT-PCR based method has been development thoroughly tested for accuracy and reliability, and applied to a cohort of patients being treated in melanoma clinics. (Hanekom et al, 1997, 1999, 2002, Davids et al, 2003a, 2003b, Davids 2002). This line of investigation has now been expanded into studies on human breast cancer and we are now converting the assays for use with real time quantitative PCR: (Hussein et al, publication submitted).
Vitiligo is a depigmenting skin disorder that affects about 1-5 % of the general population. Affected individuals display a gradual loss of pigmentation in patches of the skin, most commonly on the hands and face, causing cosmetic disfigurement and often resulting in severe psychological and social distress, particularly in children. Current treatments are not very efficacious or permanent and there are severe phototoxic side effects, including erythema, oedema, and sunburn. (Carlie, Ntusi, Hulley and Kidson, 2003). Despite extensive investigations, the fate of melanocytes in vitiliginous skin remains unresolved. (Gottschalk and Kidson, 2005 – submitted to Brit J Derm). Furthermore, it is not known how the skin re-pigments when treated with standard photochemotherapy agents (for example, by psoralens or khellin which is photoactivated by UVA light). The answer to this question of melanocyte fate is central to the improvement of the treatments for vitiligo. We are carrying out cellular and molecular studies on melanocyte and keratinocyte interactions in vitiligo. In addition, we are currently following up work in which embryonic stem cells have been induced to differentiate into melanocytes. This work is exciting because it opens up the possibilities of using stem cells to treat human pigment cells disorders in the skin and in the eye (such as in retinitis pigmentosa).