By Orla Dunne
Big pharma and biotech have put significant investment into developing high throughput screening (HTS) technologies to improve the efficiency of early drug discovery. Immortalised cells, sourced from tumours or from transformed oncogenes, are readily engineered to express target proteins and reporters in a fashion that minimizes background noise, thus making them a popular candidate for many HTS systems. Immortalised cells may be grown on an almost unlimited scale; however, over time their phenotype may alter due to genetic abnormalities.
Additionally, immortalised cells lack full complement signalling pathways and over express target protein as they are devoid of physiological context, consequently if an effective compound is trialled it may be unnoticed if it activates an absent signalling pathway in the immortalized cell.
Primary cells derived from human populations express target protein and associated signalling pathways at physiologically relevant levels and are thus presumed to be optimal for HTS. Unfortunately, numerous disadvantages are associated with primary cells such as; a short life-span, inability to withstand freezing, exhibition of unstable phenotypes in culture, a limited supply of donor tissues and due to difficulties in cell isolation there is generally an insufficient supply of cells to support HTS. Currently, the most common use of primary cells are in tissue specific toxicology assessments and secondary functional assays.
The liabilities of immortalized cells and limitations of primary cells may be overcome by the use of Stem Cell (SC)-based HTS in drug discovery. Adult sourced SC’s reconditioned to an embryonic SC phenotype, known as induced pluripotent stem cells (iPSC’s), may potentially become a large scale renewable source of disease-specific SCs with the prospect of innovating HTS. (National Institutes of Health, US Department of Health and Human Services., 2009).
Embryonic Stem Cells
Pluripotent ESC’s used in research are commonly derived from the inner cell mass of blastocysts. (National Institutes of Health, US Department of Health and Human Services., 2009). Advantageously, mouse ESCs can withstand freezing, may be directed into specific cell types, can grow undifferentiated in culture for a prolonged period of time and can be produced in sufficient quantities to support HTS. Human ESCs have a slower growth rate, need unique culture conditions to remain undifferentiated and are difficult to genetically engineer. Legal and ethical restrictions impede the use of hESCs in medical research and hindering the use of hESCs in the near future. As a result, current pharmaceutical HTS depends on mouse derived ESCs. The need for animal based-toxicity models can possibly be reduced with the use of human SCs as opposed to mouse SCs. A reliable source of homogenous populations of cardiomyocytes and hepatocytes that reproducibly predict toxicity in humans are essential for the use of HTS and secondary assays that reliably predict clinical cardiac and hepatic safety during drug discovery; with the aim of shortening time-lines and increasing clinical success rates. An additional goal is the use of homogeneous human hepatocytes as there are considerable differences between murine and human liver physiology.
Adult Stem Cells
Self-renewing SCs localized in adult tissues and organs with the ability to differentiate into a confined number of tissue-specific cell types are described as multipotent. Hematopoietic, mesenchymal, neural, epithelial or skin cells can all therefore be derived from multipotent adult SCs depending on the type of cell generated during differentiation. The availability of multipotent SCs are limited due to the small numbers present in adult tissues and the finite capacity of adult SCs to grow in culture.
The prospect of an unlimited supply of human SCs that no longer rely on the availability of embryonic or foetal tissue may be possible due to the discovery of pluripotent SCs derived from adult somatic cells. Presently, two techniques may be used to induce pluripotency in adult somatic stem cells. One method involves the transfection of four oncogenic transcription factors, Oct3/4, Klf4, Sox2 and c-Myc into somatic cells of human or murine origin. Alternatively, blastocyst induction may occur from the transfer of a mouse somatic cell nucleus to an enucleated oocyte. The methods of conversion require improvement before iPSCs can be used extensively in clinical trials. SC-based assays that represent human disease may be created as iPSCs can be derived from individuals with inherited disease.
Opportunities & Challenges
The greatest opportunity to improve the drug development process lies in the replacement of currently used murine SCs with commercially sourced reproducible human SC populations. Early identification of compounds exhibiting undesirable and desirable effects may be possible through SC based HTS detection of biomarkers thus increasing pharmaceutical research efficiency. The use of disease models from engineered ESCs is an alluring goal for HTS in drug development.
For the aspiration of human SCs to be used in place of mouse SCs in HTS to be realised, improvements in differentiation protocols must be made and new sources of human SCs found.
Orla Dunne is a summer intern at Kelly Scientific. Orla is a final year BSc student majoring in Physiology at University College Dublin. Orla has previously undertaken research projects in both U.C.D and Trinity College, Dublin. Orla has particular interest in differential splicing patterns in CD1d glycoproteins and the effects of hypercapnia on immune cell function.