Embryo Biotechnology for Cell Regenerative Medicine

Stem cells (SCs) are primitive cells with a regulated capacity for self-renewal and differentiation. Precise control of these processes is essential for development and the renewal of tissues to maintain physiological homeostasis, or repair damage inflicted by disease or injury. Conversely, perturbation of this control can lead to cancer and death. In this context, the isolation and manipulation of human stem cells for the repair of diseased or degenerating organs is widely regarded as the next frontier in curative medicine. It provides an alternative to the current transplantation of donated organs; whose supply is frequently limited, or future proposals to rely on animal sourced organs for xenotransplantation, with a potential to directly transmit unknown animal (ie. zoonotic) pathogens to the human species. Historically, zoonosis has accounted for some of the most virulent diseases known to man, including anthrax, hantavirus, Q-fever, and more recently variant Creutzfeldt-Jakob disease arising from bovine spongioform encephalopathy.

Human stem cells can be sourced from a range of tissues including those of embryonic, fetal, neonatal, or adult origin. Of these, those from embryo-derived tissue arguably prompt the greatest moral concern owing to the necessary destruction and non-reproductive use of a conceptus before its implantation in a female uterus. Also concerning is the intentional creation of embryonic tissue, such as for example through somatic cell nuclear transfer (ie. “cloning”) in order to produce embryo stem cells of a specific genetic constitution. Despite this, the unparalleled potential that embryo stem cells have to provide the quantity and specificity of tissue required for most treatments cannot be discounted lightly. In addition, basic research on such tissue provides unique opportunities to acquire an understanding and mastery of genetic mechanisms controlling cell function and disfunction that ultimately could lead to the in situ manipulation of endogenous cells without the requirement for cellular transplantation at all. This is exemplified by the potential to use cloning to create embryo stem cell models of congenital diseases whose genetic basis remains poorly understood, such as for example motorneuron disease.

Using mouse embryo derived stem cells (mESC) several studies have provided evidence for the therapeutic promise of this kind of tissue. Transplantation of un- or partly differentiated mESC cells into rat models of nerve demyelination or spinal cord contusion, results in oligodendrocyte replacement and gain of function. Similarly, transplantation of un- or partly differentiated mES cells into a rat model for Parkinson’s disease also results in the development of functional dopaminergic neurons and gain of function. In addition, undifferentiated insulin secreting mES cell clones isolated by a recombinant cell trapping strategy have been reported to normalise glycemia after transplantation into the spleen of chemically induced diabetic mice. In another study transplantation of insulin-secreting structures differentiated from mES cells in vitro did not result in a sustained correction of hyperglycemia but did contribute to maintenance of body weight and improved survival of recipients.

To realise the therapeutic promise of human stem cells and their derivatives, significant challenges must be overcome in culture systems mediating their isolation, expansion and differentiation. First, these systems must produce sufficient quantities of functionally normal cells that are tolerable, or can be made tolerable to the recipient. Secondly, cells produced by these systems must be free of contaminants that could harm the individual. Reducing the potential for contamination requires isolation and manipulation of cells according to quality assured and regulator approved practices. This includes the implementation of measures to ensure trace-ability of transplanted tissue in the event of future complications. It also requires minimising or eliminating dependence on poorly defined human or animal tissues or cells or cell products in culture systems supporting stem cell isolation or manipulation. That is the future application and safety of stem cell technology is critically dependent on not only their production in clinically appropriate facilities but also the evolution of completely defined culture systems for handling of cells in vitro.

Seven years on from the first successful derivation of human embryo stem cells (hESCs) by Prof. Jamie Thomson’s laboratory in Wisconsin (1998), all efforts to isolate and expand these cells have continued to involve direct exposure to one or more animal sourced reagents first described. This includes the use of; immune compliment to recover embryo inner cell mass (ICM), mitotically inactivated mouse embryonic fibroblasts (MEF) for embryo cell attachment and outgrowth, and supplementation of media with bovine serum. Empirical evaluation of alternatives has shown that at least some of these factors can be replaced. Thus, hESC derivation from whole embryos without animal immune compliment to isolate ICM has recently been shown. In addition, human fibroblasts derived from neonatal foreskin and adult placenta have been exemplified as a useful feeder to support outgrowth of embryonic cells. Most recently, extracellular matrix extracted from mouse embryo fibroblasts was shown capable of supporting hESC isolation. However, even these accomplishments have still involved supplementation of media with bovine sourced serum or serum replacement (SR) containing lipid-enriched serum albumin.

In our own research we have produced several new hESC lines isolated without the use of immune compliment, and with a transient reliance on a human fibroblast substrate. This includes one line isolated in a completely defined media with no serum or serum replacement supplement. Arguably this makes this cell line amongst the most therapeutically safe of those isolated to date. Furthermore this work has been accomplished in a facility working to the spirit of Good Manufacturing Practice, whose accreditation by the Medicines Health Care Products Regulatory Agency is under review. In this regard our ambition is to be amongst the first in the world to create therapeutically suitable hESC lines. Other research includes the production of alternative embryos stem cell models for the study of development and disease including those derived from parthenogenetic or cloned embryos. Whereas the former would be composed entirely of maternal genetic information (ie. from the egg), the latter would be derived from a somatic cell donor, in our case, specifically from sufferers of congenital but unknown forms of motorneuron disease.

Dr Paul de Sousa, University of Edinburgh, March 2006