The Primary Researches About The Tumor Progression From The Aberrant Chromosomal Karyotypic Human Embryonic Stem Cells To Cancer

Human embryonic stem (hES) cells are derived from inner cell massof human blastocyst and maintain an indefinite growth in culture while,like its origin, keep the pluripotency to differentiate into most of the celltypes in the body. Human embryonic stem cells hold great promise infuture research and regenerative medicine. Recent reports indicated thathES cells acquired chromosomal and genomic alterations in long-termculture similar to those observed in human cancer and raises concernsabout the safety of future clinical use of cultured hES cells. But as yetnone has shown direct evidence of malignant tumor formation. Here, weanalyszed dynamically the chromosomal changes of chHES3 during along term culture, identified its biological characteristics and phenotypeat differential stage in the process and then reported firstly that geneticinstability of aberrant chromosome human embryonical stem cellsinduced malignant transformation, at last, we explored the molecularmechanism in the transformation procession at the level of gene throughthe gene chip. Systematic analyses from the cytogenetic, cellularbiological phenotype and molecular genetic indicated that the malignanttransformation of the aberrant chromosome human embryonic stem cellshad a tendency of progression into tumor or tumor stem cells.Chapter 1Occurrence, identification and cytogenetic analysis of theaberrant chromosomal karyotypic human embryonicstem cellsObjective:To explore rule of the chromosomal changes and detect the cellular biological and cytogenetic characteristics of the aberrantkaryotypic human embryonic stem cells.Methods: The chromosome analysis were performed every 5-10passage during long term culture and the karyotype were furtheridentified by the CGH and FISH. The aberrant karyotyic HES cells usedin our experiments were performed to detect three aspects: 1 theundifferentiated characteristics of HES cells, such as surface marker,alkaline phosphatase (AKP) activity; 2 the identification of the capacityof differentiation in vivo and in vitro, such as the power of embryonicbody formation, gene expression related to three layer differentiation,teratoma formation in vivo and so on; 3 detection and ascertainment ofcelluar karyotype with the passage by the G-bing, Comparative GenomicHybridization and Fluorescent in situ Hybridization and exploration aboutthe regularity of the karyotypic changes.Results: A new cell line with karyotypic aberration come from thenormal karyotypic chHES-3 cells during long culture were identified, andfound that the karyotype became more complex with the passaging. Atearly stage, about passage 30-40, the chHES3 exhibited the singlekaryotype and was 46,XX,dup(1)(p32pter); At the passage 44-99, thestructural and complex rearrange involved many chromosomes appeared,with the passage, the karyotype become more and more complex anddisorder, the karyotype were 46, XX, dup(1)(p32p36) t(1;6;4) (q25;q23;p16) ins(4;1)(p16;q21q25),t(7;8)(q32;q13); at passage 114, a mosaickaryotype was observed with three kinds of novel chromosomalre-arrangments beside the above chromosomal karyotype, the other threekaryotype were 46,XX,dup(1)(p32p36) t(1;6;4) (q25;q23;p16)ins(4;1)(p16; q21q25), t(7;8)(q32;q13), mar(27) 46,XX,dup(1)(p32p36)t(1;6;4)(q25;q23;p16)ins(4; 1)(p16;q21q25),t(7;8)(q32;q13),mar(2),mar(6?), mar(127)47,XXX,dup(1)(p32p36)t(1; 6; 4)(q25; q23; p16)ins(n; 1)(p16; q21q25), t(7;8) (q22;q22). But the cells from P142 to P153 only demonstrated thekaryotype of 47,XXX,dup(1)(p32p36)t(1;6;4) (q25;q23;p16) ins(4;1)(p16;q21q25),t(7;8)(q22;q22). From P188 to P197, A novel markerchromosome appeared but the trisomy X was restored to disomy, Thekaryotype changed again were 46, XX, dup(1) (p32p36) t(1;6;4)(q25;q23;p16)ins(4;1)(p16;q21q25),t(7;8)(q22;22),mar(15?). The biolo-gical characteristics of chHES3 used in our experiments were accord withthe international standard about the human embryonic stem cells. Thecells expressed the special markers of HES cells such as SSEA3, SSEA4,TRA-1-60, TRA-1-81, Oct4 and exhibited the alkfluoremeterphosphatase(AKP) activity; The ultrastructure of the two kinds of stem cells wereanalysed by ETM, the cell apparatus in the cytoplasm developed better inthe abnormal chHES-3 cells than in the normal chHES-3 cells; chhES-3formed embryonical body in suspension culture after isolation from thefeeder cells and EBs expressed germ layer related genes, such as AFP,transferring, KDR, nestin and soxl with high levels, on the contrary, themultipopent related genes, such as OCT4 and nanog were in a low level.The aberrant karyotypic chhES3 cells were injected into the SCID miceunder the thigh and gave the teratoma growth. In the teratoma cells comefrom three differential layers contained in it.Conclusions: The aberrant karyotypic chhES-3 cells, like thenormal karyotypic chhES-3 could maintained the undifferentiated stateand had pluripotency; The aberrant karyotypic chhES-3 cells had thenormal ES cells characteristics and were embryo-derived cells; thekaryotypic changes in the suboptimal culture were from the simplicity tothe complexity and its change rules were accord with chromosomalchange in tumor. Chapter 2Phenotypic characteristics of the aberrant karyotypic humanembryonic stem cellsObjective: To investigate biological phenotype characteristics of theaberrant karyotype human embryonic stem cells.Methods: The normal and aberrant karyotype human embryonicstem cells were cultured in the suboptimal culture and observed thedifferentiation state in vitro culture; The distribution of cell cycle aboutthe normal and aberrant karyotype human embryonic stem cells wereanlysed by FACS; The apoptosis of the normal and aberrant karyotypehuman embryonic stem cells were detected through anexin-V; thecapacity of the single clone formation in the normal and aberrantkaryotype human embryonic stem cells were detected in the differentbatch feeder cells and transplanted density; The CD133, TRA-1-60 weredeteced by FACS and by the result we analysed the subline distributioncharacteristics in the normal and aberrant karyotypic human embryonicstem cells. At last, we analysed the genes related with cell proliferation,cell cycle regulation and tumor progression in the ampliferation region of1p32-36 and detected the expression of such genes in the normal andaberrant karyotypic human embryonic stem cells.Results: In the suboptimal cultures the normal karyotype HES cellsdifferentiated seriously and had higher differentiated ratio, but theaberrant karyotype HES cell could maintain easy the undifferentiatedstate in the edge or centre of the clone. The percentage of S phage in thenormal karyotype HES ceils was 34.12±0.8054%, in the earlier period ofthe complex rearrange group (p70-110), the percentage of S phage was35.08±3.96%, G1 phage was 52.16±8.38%; in the advanced stage, thepercentage of S phage 47.6±7.54%, but the G1 phage was decreased to 37.2±3.09%, with the passage, the percentage of S phage become moreand more higher. The ratio of apoptosis in the normal karyotype was10.27±0.45%, but the aberrant karyotype HES cells had lower ratio ofapoptosis than the normal and with the passage had a tendency ofdecrease in the apoptosis ratios. In the experiments of the single cloneformation, in the earlier stage of the complex rearrange chHES-3 cells(157—163), the efficiency of the single clone formation was 0.034%, inthe advanced stage, the efficiency was 0.044%. We found that theefficiency of the single clone formation was related with the density ofcellular transplantation, the batch of the feeder cells and the aberrantkaryotype HES cells had higher single clone formation efficiency than thenormal. There were less TRA-1-60, CD133 positive cells in theabnormal karyotypic chHES-3 cells (51.6%, 60.4%) than in the normalkaryotypic chHES-3 cells (65.4%, 75.4%). In the ampliferation region of1p32-36, the expression of oncogene HKR3,LCK upregulated in theaberrant karyotypic HES cells.Conclusions: The aberrant karyotype chHES-3 had more strongerpower of self-renewal, anti-apoptosis and more higher single cloneformation efficiency; The Cellular hetero-genicity and differentiationhierarchies existed in the normal and aberrant karyotype HES; Thenormal karyotype HES, aberrant karyotype HES and EC cells weredifferent differentiation stage during the malignant transformation.Chapter3Phenotypic characteristics of tumor progression about theaberrant karyotypic human embryonic stem cellsObjective: to compare the histological different in teratoma comefrom the normal, the aberrant karyotypic HES and EC cells and to evaluate the capacity of the tumor formation.Methods: The tumor progression process of the aberrant karyotyeHES cells were mimicked by the teratoma formation experiments. TheHES cells were divided to three group: 1, the normal karyotype group; 2,the complex karyotye group; 3, embryonal carcinoma group. HES and ECcells with good states were injected into the thigh of SCID mice, theweigh and size were observed with the time. After 10 weeks, the SCIDmice were killed and the teratoma were prepared the histology slide. Thehistological characteristics and primary neural tube-like structure wereobserved and compared with the clinic sample. To understand therelationship between the development time and the tissue immature level,the teratoma sample at the different time in the SCID mice werecompared by the HE staining. At last, we detected the related geneexpression in the teratoma by the immunohistochemical method.Results: The teratoma come from the normal and aberrantkaryotype HES cells contained different amounts of ectodermal,endodermal and mesodermal tissues, showing immature, sub-mature andcompletely mature differentiation. Tumor components were arrangedrandomly. Majority were immature mesenchymal tissue and nerve tissue,and some were mature bone, cartilage, squamous epithelium, glandularepithelium and liver tissue etc. Most of the immature nerve tissuesshowed the primitive neuroectodermal differentiation, such as rosettestructure, tubular structure, dispersively arranged in the glial fiber. Someof the structures were like astrocytoma, neuroblastoma, ependymblastomaand the retina epithelium with pigmentation. The tumor contained moreimmature tissues and showed atypia. According to the diagnostic criteriadefined by the World Health Organization, the tumor come from theaberrant karyotype HES cells was classified as immature teratoma ofgrade 3 and fit for chemotherapy in clinic. The cells in the-teratoma come from the normal karyotypic HES cells were normal karyotype and thedifferentiated degree was more and more higher with the developmenttime. After growth 10 weeks in the SCID mice, the normal and complexkaryotyic HES cells formed immature teratoma and the tumor did notmetastasize. The immature teratoma come from the complex karyotypeHES cells had histological similarity with the clinic patho-sample and hadmore and more rosset-like structure, these structures and cells expressedthe Oct4.Conclusion: The complex karyotypic HES cells could formteratoma. There were lots of undifferentiated tissue and cells in theteratama. The teratoma was classified as immature teratoma of grade 3.The cells in it had the characteristics of stem cell and the power of highproliferation, and had a tendency to progress into cancer or cancer stemcells.Chapter 4Primary study of the molecular mechanism of the tumorprogression about the aberrant karyotypic human embryonicstem cellsObjective: To investigate the molecular mechanism of the tumorprogression about the aberrant karyotypic human embryonic stern cells.Methods: The normal, duplication in chromosome 1, complexkaryotypic HES cells and embryonal carcinoma cells were collected. Afterhybridization with gene chip U133 plus 2.0, the gene expression profileanalysis, gene function analysis, chromosomal location analysis, hierarchiescluster analysis, cell signal transduction pathway analysis and expressionratio of these different genes in every chromosome were performed.Results: In our four samples, 12378, 11001, 11375 and 11096 genesexpressed in the normal HES cells, duplication in the chromosome 1, complex HES cells and embryonical carcinoma cells, respectively. 1176genes were uniquely expressed in the normal HES cells compared with theduplication in chromosome 1 and the complex karyotype HES cells. 280genes were uniquely expressed in the duplication in chromosome 1compared with the normal HES cells and the complex karyotype countparts.359 genes were uniquely expressed in the complex karyotype countpartscompared with the normal HES cells and the duplication in chromosome 1.As the abnormal karyotypic HES cells were duplication in the chromosome1, we mapped these differentially expressed genes to their chromosomallocations. In the 158 genes above 4-fold expression, there were 23 genes inthe chromosome 1. Visual inspection and initalstatistical analysis revealedthat the distribution of downregulated genes is even across all chromosomes,whereas the upregulated genes were core concentrated along the short armof the chromosome 1, especially in the 1p32-36. However, no obviousabnormalities associated with the chromosome were detected in thecomplex karyotype HES cells; The POU5F1, SOX2, Nanog, LDB2,GABRB3, FGFR4, LEFTY2, DNMT3B, CD9 in 18 stringent pluripotencyand candidate genes all expressed in all samples and inreased grudually.FN1,MSI2,NEDD1,SEPT2,NEFH,NEDD4,PAX6,OTX2,MCFD2,SOX3 in 21 ectodern development-related genes, EOMES,FLT1,GATA2,HLA-C,KDR,LMO2,MYL4,PECAM1,PITX2,RUNX1,T,THBS1,THBS2,VCAM1 in 28 mesoderm development-related genes, AFP,CER1,FOXA2,GATA4,GATA6 and HNF4A in 7 endoderm development-relatedgenes, all have tendency to decrease; Comparsion of the global geneexpression profiles of the important signature transduction molecular in thedifferent subsets of normal,abnormal HES cells and embryonical carcinomacells. In the all differentially expressed genes related with the Wnt signalpathway, the effector GSK3-beta had a decrease in the chromosome 1aberration and complex karyotype HES cells; Comparison of the global gene expression profiles of the oncogenes and suppress tumor genes in thedifferent subsets of normal and abnormal and embryonical carcinoma. In theoncogene group, ERBB2, TPM3,TPM4, FGFR1, BCL2, CDK4 allexpressed in the four samples, such genes could be the potentially stem cellgenes, they might play an important roles in the maintain of the stem cell,especially the TPM3, which was related to the cytoskeleton and the muscledevelopment. MET gene did not express in the normal HES cells but in theembryonical carcinoma, it might be the key molecular in the tumorprogression. In the suppress tumor genes group, PTPRG, PTCH, SMAD4,PTEN, RERE, RPL10A, TIMP1, CDH1, APC, TP53, RERE, BRCA1,MSH2, NME1 all expressed in the four samples, especially the NME1 andCDH1 expressed at the high level. They were critical in keeping the balancein prolieration and differntiation and maitainning the monostate in cells.RERE, VHL, TP53, CDH1, NF1 all expressed in the normal HES cells butnot in the EC cells; Among the genes highly expressed in the the group ofthe duplication in the chromosome 1 versus normal group, we noticed 43genes which over-expressed 16-fold. These genes included the HNRPD,NCAM2, GRB2, MALAT1, THRAP3 which could confer stronger power ofcell proliferation. In the complex karyotype group versus the duplication inthe chromosome 1, 29 genes over-expressed 16-fold, some genes, such asCDK6, FL(?)21148, DIP13B, MAD1L1, DIP13B regulated the cell cycle andcell proliferation; some genes, such as NRCAM, CELSR1, CUGBP2,SMURF1 positively regulated of neuron differentiation and were involvedin neuropeptide signalling pathway development or nervous systemdevelopment.; By comparison of the apoptosis analysis of the normal andthe chromosome 1 duplication HES cells, we extracted a list of 34 genesrelated with the apoptosis significantly differentially expressed. In theup-regulated 11 genes, there were 6 genes which were anti-apoptosis andnegative regulated apoptosis. In the down-regulated 23 genes, there were 15 genes which conferred with induction of the apoptosis and positive regulatedapoptosis, other 8 genes were related with apoptosis, but the function aboutnegative or positive regulated apoptosis was not clear. In the whole, thechromosome 1 duplication HES cells have stronger power of anti-apoptosisand negative-regulated apoptosis. In same way, we extracted a list of 31genes related with the apoptosis significantly differentially expressed. In theup-regulated 11 genes, there were 3 genes which were anti-apoptosis andnegative regulated apoptosis, other genes were related with the apoptosis,but the function is not clear. In the down-regulated 20 genes, there were 9genes which conferred with induction of the apoptosis and positive regulatedapoptosis, 4 genes was anti-apoptosis, but the function of other genes wasnot clear.Conclusion: The magnitude of the increase in related geneexpression to maintain the cellular biological phenotype was due to theirincreased genomic copy number such as the chromosome 1p32-36, andresulted from some gene over-expression in "hot" chromosome 12, 17.The complex karyotypic chHES3 cells did not progress to the stage of ECcells and were in the process to the cancer stem cell or cancer. The Wnt,TGF signal transduction pathway dis-regulated in the aberrant karyotypicHES cells and might result in cellular biological characteristics. Theaberrant karyotypic HES cells owned the stronger self-renewal andweaker differentiation capacity to three layer and its was at expense of thecellular death or differentiation. The oncogenes, suppress tumor genes,apoptosis related gene, cell cycle regulation related gene disregulated inthe aberrant karyotypic HES cells and were main reasons of the cellularphenotype.