Russian Journal of Developmental Biology, Vol. 35, No. 6, 2004, pp. 385–388. Translated from Ontogenez, Vol. 35, No. 6, 2004, pp. 469–472. Original Russian Text Copyright © 2004 by Gordeeva.
CHRONICLES
Human Embryonic Stem Cells: Frontiers in Basic Research and Generation of Cell Technologies (Symposium and Advanced Training Course on Problems of Human Embryonic Stem Cells) O. F. Gordeeva Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 199991 Russia E-mail:
[email protected]
Symposium and training course for specialization in the area of embryonic stem (ES) cells were held in the Pittsburgh Development Center of Mageee-Womens Research Institute, USA, on May 6–23, 2004. The main aim of this course supported by NIH is preparation of qualified specialists for development of fundamental studies and biomedical technologies on the basis of human ES cells. Organization of an international community of scientists, whose efforts are directed at deep and comprehensive investigation of the lines of human ES cells and generation of new original approaches and concepts, becomes an urgent task for development and clinical introduction of effective cell technologies. The intense course in Pittsburgh Development Center is the most lasting and unique by its program and composition of invited lecturers and instructors, who are leaders in the area of studies of ES cells. The organizers of this course, J. Schatten and R. Pedersen, created an original project, which includes theoretical and practical education of specialists planning to work with human ES cells. According to their opinion, significant results and breakthrough in this area cannot be reached without the knowledge and understanding of many chapters of developmental biology and molecular genetics, reproductive biology and transplantology, as well as some aspects of ethics and law. During the intense 3-week course, its participants were given a chance to obtain practical skills in the state-of-art methods of cell and molecular biology. Eighteen participants from USA, Hungary, Russia, India, and Argentine were selected for participation in this course on the basis of their CVs and research projects. The program of course included lectures and training on the following topics: —propagation, passaging, and characterization of lines of human ES cells; —mechanisms of genomic imprinting and germ cells; —technology of somatic nuclear transfer and “cloning of mammals”;
—differentiation and transplantation of stem cells. Maintenance of the pluripotent state of human ES cells is the central problem of fundamental and biotechnological studies and the attention was focused on this problem throughout the course. Well known scientists, who took part in the development of lines of human ES cells and continue to improve the methods of their cultivation, were invited as experts on the first section of the program: M. Pera, Monash Institute of Reproduction and Development, Australia), M. Firpo (University of California, USA), M. Mitalipova (University of Georgia, USA), and A. Krtolica (Lawrence Berkeley National Laboratory, USA). Within the framework of lectures and training, these scientists, who are also instructors at other NIH-supported courses, presented various systems for maintenance of pluripotent human ES cells in vitro, including different composition of culture media, different feeder cells and feeder-free systems, and enzymatic and mechanical methods of passage. Learning of the proposed methods of cultivation of human ES cells allowed the participants to choose the most optimal and effective methods for their own further studies. All lecturers noted the necessity of regular morphological, immunohistochemical, and cytogenetic monitoring of a cultivated population of human ES cells in order to obtain reliable and reproducible results. M. Rao (Geron Corp., USA), M. Firpo. R. Pedersen, and E. Millan (University of Cambridge, UK), and M. Pera and N. Ivanova (Princeton University, USA) paid in their lectures a great attention to the results of studies of the profiles of gene expression and analysis of signal and regulatory pathways in undifferentiated and differentiated human and mouse ES cells with the use of micro-array technology and immunohistochemical methods. Comparative studies have shown that there are significant species-specific differences in the expression of genes, such as vimentin, β-III-tubulin, αfetoprotein, eomesodermin, HEB, ARNT, FoxD3, gp130, STAT3, CD34, etc. At the same time, variations in molecular portraits of different lines of human ES cells, even supported in different system, are less
1062-3604/04/3506-0385 © 2004 MAIK “Nauka /Interperiodica”
386
GORDEEVA
1
2
3
4
Instructors and lecturers of advanced training course at the Pittsburgh Development Center: (1) A. Krtolica and D. Schatten; (2) R. Pedersen; (3) T. Wakayama; (4) P. Donovan.
expressed (this refers to the genes c-kit, Lefty, Smad4, etc.). Several key pathways of the regulation of selfrenewal and maintenance of the pluripotent status of human ES cells can be proposed on the basis of the results obtained by different authors: signal pathways MEK/ERK and pathways induced by factors of TGF-β, Wnt, EGF, FGF, and PDGF families, but precise mechanisms of the genetic control of these processes are still far from understanding. Note that the mechanisms of maintenance of cell pluripotency in the early development and in culture of ES cells also have significant differences. It may well be that because of this, different systems of cultivation are used for production of lines of human ES cells and their further maintenance. The lecture of R. Pedersen was dedicated to the fundamental bases of pluripotency in development: formation of microenvironment (niche) for pluripotent cells and analysis of possible molecular programs for maintenance of pluripotency and commitment of embryonic and ES
cells, and impressed all course participants. An unexpected view on the fate of germline cells in the life cycle and in culture of ES cells affected all the audience. Its essence consists in that with the development of methods of production of new germ cells from ES cells and subsequent fertilization of these cells in vitro, it becomes possible to study the appearance of new generations without the participation of the somatic component. These theoretical and philosophical aspects of developmental biology are essential also for the development of technologies of ES cells. The appearance of germline cells, the fate of these cells in males and females, mechanisms of proliferation, migration, and differentiation of primordial germ cells, as well as methods of isolation and maintenance of lines of embryonic germinative (EG) cells—all these problems were discussed in a brilliant lecture of P. Donovan (John Hopkins University, USA). Isolation of primordial germ cells from mouse gonadal ridges
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 35
No. 6
2004
HUMAN EMBRYONIC STEM CELLS: FRONTIERS IN BASIC RESEARCH
was a topic of training conducted by this pioneer of studies of mouse and human EG cells. Several lectures in the second section of the course were dedicated to the molecular mechanisms of genomic imprinting, which plays important roles in early development of mammals, differentiation of germline cells, and oncogenesis (M. Bartolomei, University of Pennsylvania; R. Chaillet, and R. Jirtle, Duke University). During the lectures and subsequent discussion, a notion was put forward about the importance of studies of epigenetic changes in ES cells during their long-term cultivation, as well as in the nuclear transfer-derived ES cell lines. A special place in the program was given to the technology of production of lines of ES cells by means of transfer of somatic nuclei in the oocyte and generation of parthenogenetic lines of primate ES cells (T. Wakayama, J. Cibelli). The master of this method T. Wakayama (University of Hawaii) presented the results of many year studies into causes of development defects in “cloned” mice arising due to genetic and technical limitations at all stages of this technology. The most significant defects are abnormalities of the spindle formation and damage and incomplete reprogramming of donor nuclei. Despite a progress in this technology, only 2% of embryos are born without pathologies; on the other hand, the efficiency of production of the lines of ES cells using this method is higher by an order of magnitude and reaches 20% in some mouse lines. This suggests that hidden anomalies affect the development until the blastocyst stage to a lesser extent, but they are realized in a subsequent embryonic development. The pattern of methylation of imprinted loci in the “cloned” lines of ES cells, unlike in the standard lines of ES cells, was variable. This method makes it possible to obtain ES cell lines carrying various chromosomal, genetic, and epigenetic defects and these data should be taken into account when developing the technology of co-called “therapeutic cloning.” M. Boiani (University of Pennsylvania, USA) stressed the necessity of a comprehensive investigation of the mechanisms underlying genome reprogramming during cloning. He presented interesting data about the effects of exogenous factors present in various culture media on chromatin remodeling, changes in cell cycle, and gene expression in the cells of “cloned” embryos. During cultivation of normal embryos in various media, the expression of the genes of general metabolism, such as Hprt, Gadph, and Cpt2, varies, while the level of expression of the transcriptional factor Oct4 remains practically unchanged. An opposite trend was described in “cloned” embryos, when different percentages of embryos with normal or abnormal levels of expression were observed in different media. Another important aspect in the regulation of development of the “cloned” mouse embryos is the maintenance of the normal ration of ICM cells to the total number of cells in the blastocyst. Replenishment of RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
387
ICM cells at the expense of integration with normal and tetraploid embryos or several reconstructed embryos with each other leads to an increased survival rate and enhanced developmental potencies. No less intriguing effect of regulation in the early embryogenesis was demonstrated during laboratory training by A. Nagy and K. Vintersten (Mount Sinai Hospital, Canada), who developed the method of aggregation of ES ells with tetraploid embryos, as a result of which the embryos develops at the expense of only donor ES cells. In their lecture, they presented a wide spectrum of the methods of so-called conditional mutagenesis with the use of site-specific Cre/LoxP recombination for studying the role of individual genes in certain cells at different developmental stages. Note that during the course, the participants could learn many methods of cultivation, differentiation, and transfection of ES cells, immunohistochemistry and immunosurgery of embryos, technology of transfer of somatic nuclei into oocytes, isolation and sorting of spermatogonia and bone marrow cells. Wide possibilities of magnetic resonance microscopy, positronic emission tomography, and confocal microscopy in vital studies of behavior and differentiation of stem cells have been demonstrated by E. Ahrens (Carnegie Mellon University, USA), S. Mason (Pittsburgh University, USA), and P. Sammak (Pittsburgh Development Center). The visit was organized to the biotechnological company Cellomics, which produces high technology equipment and software for analysis of proliferation and apoptosis, motility and adhesion, and differentiation and functioning of various signal pathways, including vital studies of cells in a real time. Representatives of the administration of various NIH institutes told in detail about initiatives and grant system of support of stem cell studies, including international programs of cooperation (R. Tasca, National Institute of Child Health and Human Development; T. Beck, National Center for Research Resources; J. Tomas, National Heart, Lung, and Blood Institute). Two symposia were held within the framework of this course: minisymposium “Fertility after Cancer” and symposium “Frontiers in Human Embryonic Stem Cells,” in which well known scientists and representatives of state, religious, social organization took part. The results of sociological studies of public opinion about the development of technology of ES cells in ten countries were presented. A poster communication from the Institutes of Developmental Biology and Molecular Genetics, Russian Academy of Sciences was presented at this symposium: O. Gordeeva, R. Zinovieva, Yu. Smirnova, N. Krasnikova, E. Manuilova, O. Payushina, T. Nikonova, I. Grivennikov, and N. Khrushchov “Differentiation in vivo and Vol. 35
No. 6
2004
388
GORDEEVA
Expression of Pluripotent and Germ Cell-Specific Genes in R1 ES Cells.” Unique creative and well-wishing atmosphere organized at the Pittsburg Development Center by its staff and director D. Schatten helped to learn new methods and open discussion of many ideas and problems of the biology of ES cells. In the end of the course, all participants received with the certificate not only priceless knowledge for further studies of ES cells, but also pos-
sibilities for collaboration and scientific communications. The participation in the Advanced Training Course and Symposium was supported by the Program “Molecular and Cell Bioilogy” of the Presidium of Russian Academy of Sciences, Russian Foundation for basic research, project no. 02-04-48434, and National Institutes of Health, USA.
RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY
Vol. 35
No. 6
2004
