Transgenic embryonic stem cells in research and later clinical application - new perspectives of the European CRYSTAL program

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Stem cells are at the centre of biomedical research: besides advancing the basic understanding of human development and cellular differentiation processes, stem cells hold the unique potential for novel therapies of degenerative diseases such as ischemia of the heart, Parkinson's disease, diabetes, and certain types of tumours. Because of their ability to reproduce the embryological differentiation of nearly all different cellular phenotypes, embryonic stem (ES) cells represent an ideal tool to to study processes of embryogenesis under in vitro conditions, in particular the signalling cascades and genes involved in the functional development (functional genomics) as well as to provide a new source for cellular replacement therapy. We have cultivated ES cells in three dimensional cell aggregates, where they differentiate into derivatives of all three germ layers. To demonstrate the ability of ES cells for regenerative medicine and tissue repair, cardiomyocytes were differentiated from ES cells were injected into the infarcted left ventricular wall of adult wild type mice. Immunological cross reactions were avoided by using the same inbred mouse strain. To allow identification of the transplanted cells transgenic ES cells were used carrying an IRES vector with two cloning sites for EGFP and an antibiotics resistance for selective selection both under the a-МНC promoter. EGFP positive transplanted cardiomyocytes could be easily detected in the native heart at different intervals after operation. The cells were found to engraft and differentiate into adult-like cardiomyocytes as confirmed by cross striation after immunostaining with a- actinin. These data were corroborated by patch clamp experiments on isolated EGFP positive cardiomyocytes at different time points after operation. The transplanted cells displayed ventricular action potentials and Я-adrenergic- as well as muscarinic regulation. When survival was investigated in a large colony, the control group had an almost double mortality rate as compared to the transplanted mice. Our data show engraftment and differentiation of embryonic cardiomyocytes after transplantation into cryoinfarcted areas of heart. Before a clinical use of human ES cells for therapeutic trials in humans two major prerequisites must be fulfilled: (i) they must be safe, i.e. the development of tumors because of the high proliferative potential must be omitted and (ii) the rejection of the transplanted cells must be prevented. For the criterion (i) the technology of «lineage selection» has been developed, strategies in order to only allow the needed cell to differentiate from ES cells but all other cells are prevented to survive. This can be obtained by modifying the culture conditions and/or adding a combination of various growth factors and signalling molecules which preferentially supports the growth of a specific cell type but prevents the development of other types. Нowever, up to now, the transgenic drug selection approach has proven to be the most specific and effective for reliable purification of ES cell derived cells. For example, IRES- vectors encoding EGFP and the puromycin resistance gene under specific promoters are used. Application of puromycin (optimal time point will be seen from the EGFP expression) will eliminate all other cells except those containing the resistance gene. In our experiments using cardiac specific promoters we found an extremely efficient purification of over 99%. For the criterion (ii) several suggestions have been made which will be discussed in the oral presentation.

Partnering on a European Level to advance Banking of Stem Cells

CRYSTAL - «Cryo-banking of stem cells for human Therapeutic Application» assembles distinguished scientists from 6 European countries as part of a Research Project funded through the 6^th Framework Programme of the European Union. Future human stem cell therapy will have to build on a readily available, safe and reliable supply of high-quality human stem cells or stem-cell derived progeny. This supply must be assured by cell banking. Today's banking approaches still rely on storing sources of stem cells such as umbilical cord blood, rather than on banking of defined, well-characterised stem cell populations (both adult and embryonic), which is still in the stage of infancy. Isolation, identification and culture of stem cells are not standardised between laboratories, and reproducibility of protocols is limited. Culture of human embryonic stem cells routinely requires the use of animal products or cells, thus currently ruling out their therapeutically use. Cryopreservation of stem cells itself is not yet optimised and validated for the different cell types, and multiple cell biological and biophysical challenges remain to be addressed in order to define optimised cryopreservation protocols. These problems currently limiting the routine application of stem cell banking in a therapeutic perspective will be addressed by the CRYSTAL project. The consortium will carry out focused research to develop tools and procedures to enable cryopreservation of different stem cell types for generation of sufficient numbers of high-quality cells suitable for future safe stem cell therapy. Five stem cell research laboratories providing different sources of human adult (from cord blood, bone marrow and placenta) and embryonic stem cells have teamed up with two partners specialising in applied banking and fundamental cryobiological research. After 3 years of collaborative research, CRYSTAL will deliver a set of optimised, validated protocols covering the core aspect of stem cell banking and achieving significant innovation in the three areas of preparation and cultivation methods, preservation methods and validation methods. These optimised, validated methods and tools will be made available to the scientific community to underpin initiatives on stem cell banking and to advance therapeutic stem cell research in Europe.

About the authors

J. Hescheler

Author for correspondence.
Email: redaktor@celltranspl.ru
Germany, Robert-Koch-Str. 39, 50931 Köln, BRD

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