Embryonic Stem Cells – a cure for old age (4)
(Continued. The beginning of the article is here.)
IV. MODERN PROBLEMS OF THERAPEUTIC USE
HUMAN EMBRYONIC STEM CELLS
AND POSSIBLE SOLUTIONS
Cell therapy methods involving the use of cESC are currently under development and are just beginning to be tested in clinical trials. The International Stem Cell Conservation Initiative (ISCBI) was established by the International Stem Cell Forum (a group of American and international organizations funding stem cell research) to develop a set of optimal methods and principles for the conservation, testing and distribution of CESCS for subsequent clinical use [74]. In the USA, the FDA also monitors the guidelines being developed and has formulated recommendations for experts considering proposals for conducting clinical trials using stem cells [75]. It is important to note that these recommendations do not ensure either the quality or the effectiveness of cells obtained from cESC used in clinical practice. Instead, the recommendations ensure that the cells used in therapy are capable of reproduction and meet special criteria that ensure patient safety (Table 2). The main safety issues related to the use of CESCS are described in the following sections.
A. Cultivation without the use of xenobioticsMost of the currently used cESC lines were exposed to animal products during isolation and cultivation in vitro.
Under such conditions, CESCS can become infected with animal viruses and other unknown substances that can cause the development of destructive immune reactions in the recipient's body. Currently, according to the strict requirements of the ISCBI, cESC lines created for clinical use are undergoing intensive microbiological testing. The FDA officially requires the submission of documents containing information about the source, as well as about the possible content of genetically modified components and pathogenic agents in each line of cESC intended for clinical use. Thus, efforts are currently being made to eliminate the possible effects of xenobiotics. Recently, substitutes for nutrient media have been developed that allow the cultivation of cESC in conditions that do not contain xenobiotics. These include xenobiotic-free serum substitutes, such as Knockout Serum Replacer (Invitrogen), and xenobiotic-free culture media, such as HESGRO (Millipore) and TeSR (STEMCELL).
Currently, in order to reduce the risk of contamination by foreign agents during the cultivation of CESCS on feeder layers of cells, the development of feeder-free cultivation systems is underway. To date, cultivation media that do not require the use of feeder layers and do not contain xenobiotics, which are mixtures of recombinant factors capable of suppressing differentiation and preserving cESC in a pluripotent state, are already presented on the market. However, some publications have data according to which the rejection of feeder layers during the cultivation of cESC is associated with an increase in chromosome instability and an increased risk of reproduction of genetically modified cells [76]. For this reason, most of the laboratories working with cESC practice a program for monitoring the stability of the genome of cultured cell lines [28, 49].
Data on the creation of cESC lines using human feeder cells were also published. For example, cESC lines were successfully obtained on a substrate of human fibroblasts isolated from the foreskin of a newborn [77, 78] and adult skin fibroblasts [79]. Some laboratories engaged in the creation of new cell lines have completely switched to the use of xenobiotic-free cultivation conditions [80]. The ability to isolate and maintain new lines of CESCS using a feeder layer of human fibroblasts is a significant achievement on the way to creating suitable for clinical use of CESCS.
B. Genetic anomalies in cESC culturesThe most well-characterized cESC lines to date are the lines highlighted among the first.
However, they may not be the best candidates for use in clinical practice, since animal products were used when working with many of them. Chromosomal and genomic instability, characterized by loss of heterozygosity or changes in the number of copies of genes associated with the development of cancer, was registered in several cESC lines [81, 82]. Considering that these changes were not registered in the cells of early passages, most likely, many of these mutations are induced by prolonged cultivation. It was suggested that such karyotypic aberrations arose during the adaptation of cells to the initial conditions of cultivation, which were used in the isolation and reproduction of the first few lines [83]. These observations emphasize the need to obtain detailed characteristics of cESC lines, especially the effects of long-term cultivation, as well as to create guidelines for evaluating the characteristics of cESC suitable for clinical use.
C. Enrichment, directed differentiation and purification of cells obtained from cESCThe main safety issue associated with the use of CESCS is their ability to form tumors consisting of germ sheets.
As described above, in vivo transplantation of undifferentiated cescs into mouse models leads to the formation of teratomas. Evidence of teratoma formation was also obtained during in vivo transplantation of differentiated cESC derivatives [8, 85]. Thus, an extremely important point is the absence of oncogenic cells in cESC derivatives intended for transplantation. Another problem is the possibility of differentiation of cells obtained from cESC into cells of undesirable types. For example, the insertion of "wrong" muscle cells into a damaged heart muscle can disrupt the electrical activity of the recipient's myocardial tissue and cause arrhythmia [86]. Thus, the development and further optimization of differentiation and purification protocols is necessary to minimize the likelihood of the appearance of undesirable cell types during preclinical experiments on transplantation and clinical therapy.
As described earlier, the enrichment of populations of specific cell types can be achieved by introducing certain molecules into the culture at critical times. However, many of these methods provide only moderate enrichment, insufficient for the use of cells in clinical practice. It may be advisable to enrich the population with partially differentiated proliferating intermediate derivatives of cESC, which are characterized by a certain orientation of differentiation. Such cells can subsequently be multiplied until the final differentiation into the cells necessary for therapy. For example, the expression of CD133 cell surface antigen on proliferating CESCS indicates that their differentiation into neuroectoderm is predetermined [26]. CD133-positive cells isolated from cultures of undifferentiated CESCS in vitro and in vivo predominantly demonstrated differentiation into neuroectodermal cells [26].
In the absence of specific surface antigens, such as CD133, allowing the identification of tissue-specific progenitor cells, molecular probes are used to isolate specific subpopulations of CESCS. King et al. [25] demonstrated for the first time the suitability of this system for the isolation of viable pluripotent cescs expressing Oct4 using a specific high-performance method. Molecular probes are single-stranded oligonucleotides that emit a fluorescent signal when binding to an mRNA target. This makes it possible to register and isolate cells by the fluorescence-activated sorting method. More importantly, molecular probes have a very short lifetime inside the cell and do not disrupt the functioning and structure of the cESC genome. Thus, this method can be used to enrich target populations of cells derived from CESCS, with the removal of undesirable cell types, such as undifferentiated cescs capable of forming tumors [25].
D. Prevention of immune rejection of transplanted cells obtained from cESCTransplanted CESCS cause the development of an immune rejection reaction [87], since both proliferating and differentiated CESCS express HLA type I and II antigens, as well as secondary histocompatibility antigens in quantities sufficient to activate the immune system [87, 88].
Another potential barrier to engraftment may arise due to the mismatch of blood group antigens of the AB0 system on the cells of the donor and recipient [89-91]. Although studies to identify the effect of AB0 incompatibility on the success of cESC transplantation have not yet been conducted, this indicator has long been a criterion for the success of organ transplantation and, accordingly, the incompatibility of donor cells and the recipient according to the AB0 system is most likely also capable of triggering an immune rejection reaction.
The optimal method of preventing immune rejection is the selection of genetically identical donor cells and the recipient. Therefore, specialists are interested in developing and using the method of nuclear transfer to create patient-specific cESC lines. When using this method, DNA extracted from the skin cells or muscles of the patient is injected into an unfertilized egg, from which its own genetic material is previously removed. After that, the egg is artificially fertilized and allowed to develop until it reaches the blastocyst stage suitable for the extraction of cESC. The cells of the resulting line have an immunological profile corresponding to the immunological profile of the patient and, accordingly, are suitable for use in cell therapy. The described method has been successfully tested on animals using species-specific ESCs, but it has not yet been possible to obtain ESCs by transferring somatic cell nuclei.
Other strategies for creating cESC lines that are maximally compatible with a potential recipient include the creation of "universal donor CESCS", cells with reduced expression of HLA antigens carrying blood group 0 antigen, or chimeric hematopoietic cells derived from cESC and capable of suppressing immune responses during simultaneous transplantation with cells derived from cESC [92]. As an alternative, the creation of banks of cESC lines carrying combinations of HLA/AB0 antigens suitable for transplantation to most patients is considered. A number of studies have been conducted, the purpose of which was to estimate the number of cESC lines needed to meet the needs of a certain population. Taylor et al. [93] estimated that approximately 150 cESC lines are sufficient to provide the majority of UK residents with HLA-compatible transplants. Alternatively, to meet the needs of the majority of the population, approximately 10 lines of HLA-homozygous CESCS obtained as a result of parthenogenetic fertilization can be used. According to estimates by Nakajima et al. [94], approximately 170 full-fledged cESC lines or 55 cESC lines homozygous for HLA antigens are sufficient to provide 80% of the Japanese population with transplants. These figures confirm the expediency of creating and functioning of cESC banks, the contents of which will solve the problem of a large number of patients. However, to meet the needs of the ethnically and genetically diverse population of countries such as the United States, a much larger number of cESC lines will be required. Given the ethical issues and limitations applied to cESC research, as well as the small number of currently approved cESC lines, the creation of a bank containing an extensive collection of various cESC lines will invariably be associated with very serious difficulties.
End: Therapeutic benefits of human embryonic stem cells compared to stem cells from other sources.
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19.12.2011