Embryonic development, epigenics and somatic cell nuclear transfer - The science and its social implications - Moshe Yaniv Unité d Expression Génétique et Maladies, Institut Pasteur, Paris, France September 2005 1
Major advances were made in the last three decades in the understanding of the genetic, molecular and cellular mechanisms that govern embryonic development. At the same time, we have also learnt how to manipulate embryonic stem cells and how to generate genetically modified animals. These advances have raised immense hopes in developing new approaches for biotherapies. However, the advent of animal cloning has raised also vast worries and ethical concerns about the possibility to clone human beings. In the following paragraphs, I will first discuss some principles of embryonic development in mammals and issues related to epigenetic regulation of development. I will next review the promise and difficulties in the use of human embryonic stem cells for biotherapies. Finally, I will relate to the report of the French Academy of Sciences on biotherapy in humans. I- Embryonic development, epigenetics and nuclear transfer 1 - Epigenetic events in early embryonic development The genetic program for the development of the embryo is controlled by the maternal and paternal genomes that are contributed by the oocyte and the sperm respectively. DNA composing the maternal and paternal chromosomes carry different epigenetic signatures comprising of DNA methylation, a covalent modification of cytosine residues and chromosomal proteins, histones and non-histone proteins. Many but not all (see below) of these epigenetic markers are erased during blastocyst formation and are reprogrammed later in the different cell types of the embryo, including the future germ cells. Each cell type carries a distinct epigenetic signature. The success of somatic cell nuclear transfer into enucleated oocytes and the development of viable progeny demonstrate that the epigenetic program of the zygote can be reset and reprogrammed. However, the limited success rate in development to term suggests that epigenetic resetting and reprogramming of somatic cell chromosomes is not very efficient. It is obvious that further research of this reprogramming process is essential for improving the efficiency of post nuclear transfer development. One the of the exciting findings in the last two decades was the observation that some genes are expressed only from the maternal genome while others are expressed from the paternal genome only. This selectivity is controlled by epigenetic markers, which differ between the male and female chromosomes. These markers (e.g. DNA methylation) escape the general reprogramming occurring in the blastocyst. Defined as imprinting, this system is very 2
important for the coordination of the development of the embryo and its interaction with the maternal placenta. The loss of imprinting due to mutations or stochastic events is associated with developmental abnormalities and postnatal disease One of the major set backs for somatic cells nuclear transfer into oocytes and the development of a normal organism may stem from the failure to imprint correctly the maternal and paternal chromosomes. This is related to the epigenetic reprogramming process described above. Very early development can proceed for several days in a test tube. However, further growth, patterning and organogenesis of the embryo requires an extensive interaction with the maternal placenta. I believe that we are very far away from finding a replacement for this contribution by the maternal womb.. Recent studies raise the possibility that assisted reproduction technologies (ART) may, sometimes, perturb the epigenetic imprinting and may give rise to imprinting disease syndrome. A more careful monitoring of epigenetics effects of the currently used techniques as well as novel future technologies is essential. 2- Embryonic stem (ES) cells. The developing blastocyst is a spherical structure composed of two cell types. Trophoblastic cells form the outer layer of the blastocyte and a mass of undifferentiated cells or inner cell mass that occupies part of the interior of the sphere. These last cells give rise to all the cell types of the adult organism. Protocols for the isolation and culture of such cells from mouse blastocytes were developed in the last decades. These cells called ES, or Embryonic Stem cells, are frequently used in protocols to generate genetically modified mice. In essence, foreign DNA can be introduced in these cells by an experimental procedure called electroporation. The foreign DNA can undergo homologous recombination with the cellular genome if it contains sequence of homology with the genome. This can be used to repair defective genes or, on the contrary, to inactivate specific genes. After identification and expansion of the cells carrying the desired genomic modification these cells can be injected into normal blastocytes and contribute to the formation of progeny. The newborn animals are usually chimeric, containing cells derived from the normal blastocyte and from the genetically modified ES cells. A fraction of these animals incorporate the modified genome in their germ cells and can transmit the genetic modification(s) to their progeny. These animals are genetically modified or transgenic and can be used in crossed to generate animals with homozygous genetic modifications in which both genes are replaced with the desired genetic 3
modification. In a certain percentage of cases, such animals are non viable since the genetic modification involved a gene essential for prenatal development. To study the function of such essential genes in specific cell types, organs or specific stages of development, mouse geneticists elaborated techniques for conditional inactivation or activation of genes in specific cell types and at specific time points or development or postnatal life. Contrary to the approaches involving the modification and introduction of ES cells, somatic cells nuclear transfer into enucleated oocytes followed by implantation gives rise to genetically homogenous progeny. The donor somatic cells can be also modified before their transfer. Such modifications can permit the repair of a genetic defect or on the contrary the generation of a mutant phenotype for future studies (see below). 3- In vitro or in vivo differentiation of embryonic stem (ES) cells. Under certain culture conditions, ES cells can differentiate into specific cell types. Neuronal cells, blood cells, muscle, insulin producing cells etc can be obtained under specific conditions. Initially done with ES cells derived from the mouse, recent studies suggest that such differentiation protocols can be also developed for human ES cells. These experiments raise the hope that ES derived cells will be a valuable tool in biotherapies of diseases like diabetes, neuronal degeneration, spinal cord injury, heart disease etc. Defective cells will be replaced by healthy cells of matching HLA or histo-compatibility antigens. These will be derived from human ES cells with identical HLA markers. A perfect immunological match and full escape of host versus graft rejection can be obtained only with immunologically identical donor cells. In this context, somatic cell nuclear transfer followed by the isolation of ES cells can permit the generation of the perfect cells for biotherapies. Such cells will be isolated from in vitro developing blastocytes that form several days after the induction of zygotic development. Such ES cells will be differentiated in vitro and used for therapy. Our capacity to modify genetically mouse ES cells suggest that inborn genetic defects in the genome of the donor cells could be the subject for repair before preparing specific cells for autologous biotherapy. Another advantage of such an approach can be an unlimited supply of cells and the possibility for repeated treatments. However, one should be aware that these are future possible scenarios and scientists and physicians have to overcome many conceptual and experimental difficulties before such biotherapies could become a reality. Much hope arose in this field with the reports by Hwang et al. on the very high frequency of the generation of human ES cells after somatic cells 4
nuclear transfer in human oocytes. Needless to say that the realization that most (or all) of these claims were false, caused a terrible disappointment and a serious drawback to this field. I do believe that progress will be made in this field but certainly slower than what was anticipated several months ago. Other difficulties that we will still face concern the development of medium/culture conditions that permit the isolation of pure populations of differentiated cells to use in biotherapies. In addition, one will have to eliminate the residual ES cells that can be tumorogenic in the receiving patients. 4- Human ES cells for research A much more rapid use of human ES cells and differentiated derivatives can be foreseen in research applied to human diseases. The possibility to generate human ES cells by somatic cells nuclear transfer opens new avenues for the isolation of human cells carrying diseases causing mutations. Such cells will be differentiated in vitro and used for better characterization of their biological and physiological defects and for the development of drugs or other therapies that will overcome these defects. Such experiments will certainly advance our knowledge on a number of diseases and open new avenues for their cure. II- The position of the French Academy of Science on somatic cell nuclear transfer and human cloning The cloning of Dolly and its very broad coverage by the media initiated intense discussions in the French society on human cloning and somatic cell nuclear transfer. As can be imagined, scientists took an active part in this discussion. The French Academy of Sciences decided to set up a specific panel to study issues ranging from animal transgenesis to biotherapies for humans. After discussing the state of the research and its potential application, the committee drew a number of recommendations that were endorsed by the quasi totality of the members of the Academy 1. 1 Full record was published: De la trangenèse animale à la biothérapie chez l homme Animateur : Moshe Yaniv. Académie des Sciences, RST n 14 Février 2003, Edition Tec & Doc. 5
The general recommendations were as follows: The laboratory mouse, an essential tool of the post-genomic era The near completion of the sequence of the mouse and human genomes opens many possibilities for understanding the normal and pathological development and function of the human body. In the present post-genomic era, it is essential that France increase its research effort on mice as a tool both for understanding mammalian development, genetics and physiology and for creating animal models of human diseases. Transgenic and cloning of animals The adaptation of transgenesis procedures to mammals other than the mouse and the use of the nucleus transfer system for cloning animals open new opportunities for producing organs for xenotransplants, for the production of protein drugs and for creating research models for human diseases. The increased research effort in animal transgenesis and cloning based on somatic nuclei transfer is necessary for better understanding the difficulties and the possibilities of these techniques. The focus on larger mammals as models of human pathologies and their prevention will aid the development of gene and cellular therapies in humans. Research on larger mammals that are likely to become cell and organ donors (in particular, the pig) should be promoted. The use of stem cells of human and animal origins The isolation and experimentation on embryonic stem cells, whether of fetal or adult origin, in mice and more recently in humans, open new possibilities for a novel approach in human medicine called biotherapy or regenerative medicine. The development of research on animal totipotent and multipotent stem cells will lead to defining the conditions of how they will be used. Research on human embryonic stem cells should be legalized, as well as the possibility of using supernumerary embryos hat are currently stocked in fertility clinics in order to isolate new lines of such cells. In parallel, research aimed at identifying multipotent stem cells in adults and at defining their proliferative and differentiation capacities in vitro and in vivo is essential. Therapeutic nuclei transfer The worldwide ban on human reproductive cloning based on the transfer of somatic nuclei into ovocytes, followed by their implantation, must be maintained and should become a universal law for humanity. The transfer of nuclei into ovocytes and their in vitro culture, for 6
a limited period of time, allows the isolation of pluripotent stem cells that are immunologically identical to the donor. Potentially, these cells present a considerable advantage for directed and repeated biotherapies that are aimed at treating degenerative diseases and debilitating damage. A legal framework should be created for authorizing a limited number of French laboratories to undertake somatic nuclei transfers into human ovocytes and cultivate these cells in vitro for purposes strictly confined to therapeutic research. Science and society A certain lack of understanding persists between scientists and the public as to the benefits and risks linked to biological research. We recommend that an effort be made to reduce this gap by making the teaching of biology more attractive, at middle and high school levels, and by including a biology course at the undergraduate level open to all students. Different ways must also be found to increase public understanding of the concepts, theoretical risks and advantages of the methods for modifying living organisms. Researchers must play a more important role in this dialogue between science and society while respecting the different and opposing sensibilities. 7