Research On How To Overcome The Immune Barrier Of Human Embryonic Stem Cell Transplantation

Human embryonic stem cells (hESCs), which are derived from the inner c ell mass of the blastocyst, can proliferate indefinitely in culture, have a normal karyotype, and are able to differentiate into all cell types under the appropriate conditions. Therefore, they represent both a valuable tool for the study of human development and a highly promising source of cells for future regenerative medicine technologies. However, a major challenge for the development of these therapeutic technologies is that transplantation of hES-cell derivatives can lead to allograft rejection due to major histocompatibility complex (MHC) incompatibility. A key issue for safe therapy from hESCs and their derivatives is how to overcome the immune barrier to stem cell transportation. In our study, we estimate two strategies for avoiding immunological rejection. One is to establish a hESCs bank with a relatively large number of hES cell lines for HLA-matching, the other is to produce parthenogenetic embryonic stem cells that is benefit for improving the HLA-matching rate of our stem cell bank and is also personalized pluripotent stem cell line for female patients. The thesis consists of two parts as following: Chapter 1:HLA-matching potential of an established human embryonic stem cell bank in ChinaIntroduction:It was suggested by theoretical calculation that establishment of 150-170 human embryonic stem (hES) cell lines with various human leukocyte antigen (HLA) genotypes will help to prevent allograft rejection in hES cell based transplantation therapy. However, whether embryos from In Vitro Fertilization treatment could provide sufficient HLA diversity for matching is unclear and ethical debate will arise considering destroying large number of normal human embryos for derivation.Methods:Blastocysts with various abnormalities, including abnormal pronuclear zygotes, poor quality day 3 embryos after fertilization and abnormal embryos after pre-implantation genetic diagnosis were used to establish a hES cell bank. Detailed characterizations were conducted on most of the established hES cell lines. Then the HLA and ABO blood group genotypes were analyzed and further matched to that of 5236 randomly selected local individuals from China bone marrow bank.Results:From 692 blastocysts used,188 hES cell lines were established and 156 (83%) have euploidy karyotype. These cell lines showed typical hES characters in performed analysis.174 cell lines with diploid HLA genotype were used for HLA matching and provide a beneficial match for 24.94%-56.26% of the 5236 presumed patients, regardless of ethnic difference. Eight hES cell lines with homozygous HLA genotypes from the bank contribute most to the matching rate in a range of 50-80% in different mismatch level.Conclusion:A hES cell bank with great HLA-matching value could be established using only the clinical invaluable embryos.Chapter 2:Establishment of human parthenogenetic embryonic stem cells and comparison with normal embryonic stem cellsIntroduction:To establish personalized pluripotent stem cell lines through parthenogenesis, and then to estimate whether the clinical application potential of pESCs is equal to that of normal hESCs through comparing the biological characterization with normal hESCs.Methods:pESCs were derived from embryos by parthenogenetic activation of human oocytes or naturally parthenogenetic activation of 1PN embryos in IVF-ET process, and were fully characterized in stem cell property. Furthermore, pESCs were compared with normal hESCs in four aspects, including:the expression pattern of imprinted genes, the analysis of differential genes expression in genome, genome-wide SNP genotyping, and the key genes expression related with the development of main organs from all three germ layers.Results:pESCs only expressed maternal imprinted genes and the paternal imprinted gene expression was absent, while both paternal and maternal imprinted genes were expressed in normal hESCs. For genome-wide SNP genotyping, chHES-32, which is derived from 1PN embryos, is a homozygous diploid parthenote due to duplication of a single haploid pronucleus; unlike the chHES-69, which was created by prevention of second-polar-body extrusion, resulting in partial heterozygosity due to the failure to segregate the rearranged chromosomes at meiosis II. In contrast, the normal hESC lines-chHES-8 and chHES-10 are highly heterozygous and do not exhibit pericentromeric homozygosity pattern on the chromosomes, as observed in pESCs. pESCs and normal hESCs were detached to grow as aggregates in suspension for 21 days to start up differentiate, and then both two kinds of EBs were tested the expression of the key genes expression related with the development of main organs from all three germ layers by RT-PCR and Real-time PCR. The results showed that no common differentiation defects are found in pESCs. We did clustering analysis of gene-chip results and found that there are no distinct differentiation between pESCs and normal hESCs.Conclusion:pESCs can maintain the expression pattern of imprinted genes in culture, the genome-wide SNP genotyping is obviously different between the normal hESCs and pESCs from different parthenogenetic activation methods; no distinct differentiation defects are found in pESCs.