Is stem cell chromosomes stability affected by cryopreservation conditions?

The introduction of the recombinant DNA techniques in the 1970s paved the way to “Gene Therapy” a novel branch of modern medicine. Although gene therapy is still at an experimental stage, it has great potentials since allows to transfer, into organs and tissues, genetic information. However, several technical problems still need to be overcome before this approach takes over conventional pharmacological treatments (Hacein-Bey-Abina et al. 2002; Mullen et al. 1996). Today stem cells, either embryonic or adult, are acquiring a great deal of attention as they promise, and rightly so, to be new vehicles for gene therapy. Unfortunately, the in vivo genetic instability of stem cells, and even more pronounced for embryonic derived stem cells, limits their widespread use. The prototypic example of adult stem cells, the hematopoietic stem cells have already been used in gene therapy (Aiuti et al. 2002) after being isolated from bone marrow or after their mobilization into peripheral blood. Although adult tissues with high turnover rate are maintained by tissue specific stem cells, they themselves rarely divide. Recently, a related stem cell, the multipotent progenitor cells have been isolated from bone marrow and these can differentiate into multiple lineages (Gregory et al. 2005). Other stem cells have been identified, both in the central nervous system and in the heart, but as of today they have been less characterized and are not easily accessible (Stocum 2005). A major drawback in using adult stem cells is that it is very difficult to maintain the stem state during ex vivo manipulations. Adult stem cells tend to loose their stem cell properties and become more specialized since they have the tendency to differentiate. Culturing conditions may influence the differentiating capacity of these cells by acting as signal transmitters (Reya et al. 2003; Willert et al. 2003). On the other hand, embryonic stem cells maintain their capacity to differentiate into derivatives of all three germ layers even after prolonged laboratory growth. They grow rapidly, they are remarkably stable and maintain the in vitro capacity to mature into multiple cell types of the body (Zwaka and Thomson 2005). These characteristics make embryonic stem cells ideal for gene therapy applications. However, while these cells in culturing dishes appear remarkably stable, they may accumulate genetic and epigenetic changes, which may harm the patient. While so much is being learned on how to genetically modify stem cells and how to introduce them into a tissue or organ, relatively little is known on how to cryoprotect them from becoming chromosomically unstable, how to maintain them in toxic free agents and in scaffolds ready to be introduced into patients. Indeed sporadic chromosomal abnormalities in human embryonic stem cells have been reported which appear to be more frequent when they are passaged as bulk populations (Draper et al. 2004). It is then important to optimize culturing conditions, cryostorage and monitoring systems to be applied to the newly derived as well as existing cell lines to decipher any genetic and epigenetic alterations, which may have taken place. Technically, cryobiology is the study of living systems, at any temperature below the standard physiological range. However freezing temperature variations above or below the physiological threshold may be very dangerous if not lethal to all types of cells (Doxey et al. 2006). The therapeutic use of stem and progenitor cells from different organs/systems to treat a variety of human diseases require the development of validated, clinical-grade cellular freeze-thawing procedures to minimize adverse effects or toxicity to patients (Buchanan et al. 2005). To achieve successful results of transplantation and treatment of significant diseases as Parkinson’s disease, Alzheimer’s disease, leukemia, diabetes, stroke, muscular dystrophy, hepatic and renal failure etc., optimum methods need to be developed on how to obtain stem cells, the way in which they are cultivated, how and for how long they are cryopreserved. Essentially, it would be important to develop protocols for freeze-thawing all clinically relevant cells in a state for immediate use on patients and, most of all, establish ways to minimize adverse responses. Today, the scientific community is very concerned with the available cryoprotective agents and their effects on the function of the cells. Indeed, a number of non toxic cryopreservation reagents and protocols have been developed by biotech companies dealing with this aspect of clinical research. Particular attention has been given to hematopoietic stem and progenitor cell conditions since they have been used to treat a number of human diseases. However, one aspect of stem cell research, which has not yet been fully explored, is on the chromosomal stability of these cells as a function of duplication time, cryoprotective reagents and storage conditions. The variation in chromosome number is probably the main type of genomic instability recorded and this is usually manifested as loss or gain of whole chromosomes, generally known as aneuploidy (Pathak and Multani 2006). Chromosome instability is a devastating phenomenon underlying several human diseases. Aneuploidy arises from meiotic errors and is almost always found in cancers, but is also associated with aging. Stochastic or spontaneous chromosomal variations in somatic cells appear as low level mosaic aneuploidy which are usually thought to be insignificant and overlooked probably due to unapparent phenotypic effects. This minireview aims to summarize the present knowledge on the subject.