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Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes.

ABSTRACT: BACKGROUND: Human aneuploidy is the leading cause of early pregnancy loss, mental retardation, and multiple congenital anomalies. Due to the high mortality associated with aneuploidy, the pathophysiological mechanisms of aneuploidy syndrome remain largely unknown. Previous studies focused mostly on whether dosage compensation occurs, and the next generation transcriptomics sequencing technology RNA-seq is expected to eventually uncover the mechanisms of gene expression regulation and the related pathological phenotypes in human aneuploidy. RESULTS: Using next generation transcriptomics sequencing technology RNA-seq, we profiled the transcriptomes of four human aneuploid induced pluripotent stem cell (iPSC) lines generated from monosomy × (Turner syndrome), trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome), and partial trisomy 11:22 (Emanuel syndrome) as well as two umbilical cord matrix iPSC lines as euploid controls to examine how phenotypic abnormalities develop with aberrant karyotype. A total of 466 M (50-bp) reads were obtained from the six iPSC lines, and over 13,000 mRNAs were identified by gene annotation. Global analysis of gene expression profiles and functional analysis of differentially expressed (DE) genes were implemented. Over 5000 DE genes are determined between aneuploidy and euploid iPSCs respectively while 9 KEGG pathways are overlapped enriched in four aneuploidy samples. CONCLUSIONS: Our results demonstrate that the extra or missing chromosome has extensive effects on the whole transcriptome. Functional analysis of differentially expressed genes reveals that the genes most affected in aneuploid individuals are related to central nervous system development and tumorigenesis.

SUBMITTER: Zhang R

PROVIDER: S-EPMC3852284 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes.

Zhang Ruosi R Hao Lili L Wang Lingping L Chen Meili M Li Wen W Li Rujiao R Yu Jun J Xiao Jingfa J Wu Jiayan J

BMC genomics 20131016

<h4>Background</h4>Human aneuploidy is the leading cause of early pregnancy loss, mental retardation, and multiple congenital anomalies. Due to the high mortality associated with aneuploidy, the pathophysiological mechanisms of aneuploidy syndrome remain largely unknown. Previous studies focused mostly on whether dosage compensation occurs, and the next generation transcriptomics sequencing technology RNA-seq is expected to eventually uncover the mechanisms of gene expression regulation and the ...[more]

PMID: 24564826

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Project description:In recent years, regenerative medicine research using human somatic and induced pluripotent stem cells has advanced considerably, promoting clinical applications. However, it is essential that these cells are cryopreserved safely and effectively. Most cryopreservation solution agents contain dimethyl sulfoxide (DMSO), which exhibits strong toxicity and can potentially promote cell differentiation. Hence, it is important to explore substitutes for DMSO in cryoprotectant solutions. One such alternative is StemCell Keep (SCK), a DMSO-free solution that has been reported to effectively cryopreserve human induced pluripotent stem cells (hiPS cells). To clarify the effect of cryopreservation agents on cells, DNA microarray analysis is useful, as it can identify a large number of gene expression differences in cryopreserved cells, as well as functional increases in gene groups. In this study, we performed gene expression analysis of SCK-cryopreserved hiPS cells using a DNA microarray gene chip. The hiPS cells vitrified with SCK or DMSO-based vitrification solutions were thawed and cultured on Matrigel under feeder-free conditions, and RNA was extracted for DNA microarray analysis. Genes obtained from DNA microarray data were classified by the keywords of Gene Ontology Biological Process Term, and their relationships were analyzed using DAVID or the GeneMANIA database. SCK-cryopreserved hiPS cells expressed several anti-apoptotic genes, as well as genes related to cell adhesion or proliferation at levels that were nearly equivalent to those of non-frozen hiPS cells. Gene enrichment analysis with selected genes of SCK-cryopreserved hiPS cells whose expression differences were superior to those of DAP-cryopreserved showed strong interactions of negative regulation of apoptotic process, cell adhesion and positive regulation of cell proliferation in DAVID analysis. We demonstrated that SCK successfully maintained the key functions of hiPS cells, including anti-apoptosis, cell adhesion, and cell proliferation, during cryopreservation.

Project description:BackgroundEuploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost.MethodsFluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids.ResultsIn both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region.ConclusionThe inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.

Project description:BackgroundIn mammals, specification of primordial germ cells (PGCs) is established in the early post-implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-β-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRINα6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in non-rodent mammals remains to be clarified.MethodsWe differentiated human induced pluripotent stem cells (hiPSCs) into PGCLCs in vitro in response to Activin A and BMP4. The percentage of EpCAM/INTEGRINα6 double-positive cells (PGCLCs) was analyzed by flow cytometry. The expression of PGC genes was evaluated by qRT-PCR and immunofluorescence. The expression dynamic of multi-lineage genes during the differentiation process was evaluated by qRT-PCR.ResultsUnder the stimulation for PGCLC induction, the embryoids derived from hiPSCs initiated significant upregulation of the early PGC genes (BLIMP1, TFAP2C, and NANOS3), but maintained low or no levels of DPPA3 and late PGC genes (DAZL and DDX4). The percentage of EpCAM/INTEGRINα6 double-positive PGCLCs reached the highest at day 6 of induction. After pre-induction, the incipient mesoderm-like cells (iMeLCs) upregulated most of the mesoderm genes (EOMES, T, MSXI, RUNX2, and MIXL1). The differentiating embryoids showed high levels of key pluripotency genes, OCT4 and NANOG, but became negative for SOX2. In contrast to iMeLCs, the differentiating embryoids downregulated mesoderm genes RUNX2 and EOMES, and ectoderm gene PAX6, but increased the expression of endoderm gene SOX17.ConclusionsDuring PGCLC induction process in vitro, the differentiating embryoids not only activated the PGC-related genes, but also displayed complex regulation of pluripotency genes and multi-lineage genes. These results would be meaningful for future research investigating the regulation of human early germ line development.

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Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes.

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Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes.

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