The Biology of Human Pluripotent Stem Cell Fate Determinations
Human embryonic stem cells (HESCs) are derived from the inner cell mass of the early embryo; the cells are genetically normal and retain in vitro both the ability to self renew and also to differentiate into each of the cell types found in the adult body. Such a system offers an in vitro model of human embryogenesis. Thus, a powerful resource now exists for the investigation of human developmental biology, the testing of candidate drugs for toxicology and efficacy and ultimately, tissue engineering for regenerative medicine.
The sensitivity of HESCs to biologically active factors and their developmental plasticity are attributes that confer upon them such great potential, but also present significant obstacles to their exploitation.
Many of these issues derive from an inadequate understanding of how HESCs regulate their self renewal and commitment to differentiation, and how these processes may be affected by the environment in which the cells are maintained.
Adaptation of Human embryonic Stem Cells to in vitro culture
In human ES cells, after prolonged culture, repetitive karyotypic changes have been noted (1, 2). These repetitive karyotypic changes are strikingly similar to the karyotypic abnormalities observed in Embryonal Carcinoma (EC) cells, which are the malignant stem cells of germ cell tumours.
Culture adapted ES cells may also show higher proliferative and cloning ability than their `normal´ early passage counterparts. Pluripotent ES cells in culture are subject to continual selection for genetic variants that increase the probability of `self-renewal´ and decrease the probability of differentiation or apoptosis. It also seems likely that EC cells in germ cell tumours are subject to similar selection pressures. Thus understanding the nature of the karyotypic and other genetic changes that occur in human ES cells in culture may give insights, not only into the genetic and molecular mechanisms that control self-renewal and commitment in these cells, but also give insights into the processes of cancer development and progression in germ cell tumours.
Embryonal Carcinoma (EC) Cells and Germ Cell Tumours
EC cells are the malignant stem cells of teratocarcinomas, a subset of germ cell tumours which occur predominantly as testicular cancers and are the most common cancers of young men. (3). EC cells are malignant counterparts of ES cells derived from human blastocysts. Human EC cells share some features in common with mouse ES and EC cells (e.g. expression of the transcription factors Oct4 and Nanog, and high levels of alkaline phosphatase) but they differ in other respects such as their distinct patterns of surface marker antigen expression. Neverthelesss, just as mouse EC and ES cells resemble the inner cell mass cells of the early mouse embryo at the blastocyst stage, so human EC cells resemble human ES cells and inner cell mass cells of early human blastocysts (4).
Over many years we have used EC cell lines as surrogates for the study of plurotent human ES cells and have used them to define a series of marker antigens for defining the undifferentiated EC phenotype (5) and for monitoring differentiation (6). These antigens are now widely used in the study of human ES cells (7). Human EC cell lines also remain relatively simple models for further studies with ES cells, since they can readily be grown in large numbers without the need for feeder cells or expensive media additives. Widely used lines include relatively nullipotent lines such as 2102Ep, useful in defining the phenotype of undifferentiated human EC and ES cells, and NTERA2 which differentiates extensively into neurons and other cells types when induced by agents such as retinoic acid (8). Studies in NTERA2 provided the first evidence of the dosage dependence of retinoic acid induction of HOX gene expression, in a way that appears to reflect the pattern of HOX gene expression along the anterior posterior axis of the developing embryo (9). In a different area of research, differentiatial susceptibility of undifferentiated and differentiated NTERA2 EC cells to replication of human cytomegalovirus (HCMV) and human immunodeficiency virus (HIV) provides tools for investigating the interaction of these pathogenic viruses with human cells (10, 11).
Signalling pathways that contribute to the self-renewal and differentiation of human embryonic stem cells
Multiple signalling pathways contribute to maintenance of the undifferentiated state of ES cells and to controlling their commitment to differentiation. Relatively little is known about how specific pathways interact with each other in ES cells, but it is becoming evident that the relative importance of particular pathways may differ substantially between mouse and human ES cells. We are currently using a variety of approaches, including comparative studies of `normal´ and `adapted´ ES cells, as well as studies of human EC cells to identify particular signalling systems that contribute to self renewal and differentiation in human ES cells. Our specific areas of interest include the Notch and Wnt pathways as well as the role of TGFbeta superfamily members. Our techniques include the use of RNAi to modulate gene expression Using the latter technique , for exampole, we demomnstrated that, indeed, human EC and ES cells require the expression of Oct4 to maintain their undifferentiated state and that if its expression is knocked-down, differentiation towards trophectoderm ensues (12).
Differentiation of Human ES cells
Pluripotency of human ES cells is their property that offers such great potential for future applications. Human ES cells will spontaneously differentiate into a wide range of cell types but this process is difficult to control. Eventual applications will depend on understanding the mechanisms that control lineage selection once the cells commit to differentiate and developing protocols to make use of that knowledge. In our group we are specifically focussing on the mechanisms that regulate neural differentiation and differentiation toward pancreatic cells. Eventually these cell types could be used in regenerative medicine to treat a range of neurodegenerative diseases and diabetes. However, it is important to recognise that considerable research and development will be necessary before these goals can be realised.
The Centre for Stem Cell Biology
The Centre for Stem Cell Biology (CSCB), directed by Professors Peter W Andrews and Harry D Moore, brings together researchers in the University of Sheffield interested in the biology of human ES cells and in developing applications that involve these cells. Within the CSCB, we have derived six new human ES cell lines and are building a new cGMP facility that will enable us to derive new lines in the future to meet standards required for their future clinical applications. The CSCB also runs an annual training course in the culture of human ES cells, and can provide assistance to others who wish to establish human ES cell research in their own laboratories. For this we have specific funding from the MRC to operate a `Human ES Cell Resesource Centre´ to assist development of human ES cell research throughout the UK. As well as a variety of human ES cell lines, we also maintain various facilities such as a recently installed state-of-the-art Dakocytomation cell sorter.
The International Stem Cell Initiative (ISCI)
The ISCI is a study being carried out under the auspices of the International Stem Cell Forum. It includes a comparative study of surface antigen markers and gene expression patterns of up to 75 human ES cell lines from some 15 laboratories and 10 countries worldwide (13). It is expected to complete its work and issue a report by the end of 2005.
Funding
- European Union
- UK Regenerative Medicine
- MRC/International Stem Cell Forum
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