Abstract
Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs.
Highlights
Human embryonic stem cells derived from the inner cell mass of human embryos have held great promise for future cell- and tissue-replacement therapy because of their unique capacity to self-renew and to differentiate into any cell type
The G-banding results showed that the normal chHES-3 cells retained a normal karyotype, simple duplication karyotype (SIMP) chHES-3 cells had the karyotype of 46,XX, dup(1)(p32p36) and complex karyotype (COMP) chHES-3 had a consistent and clonal karyotype of 46, XX, dup(1)(p32p36)t(1;6;4)(q25;q23;p16)ins(4;1)(p16;q21q25),der(2) t(2;7)(q35;qter)t(7;8)(q22;q22),inv(10)(p11q21),der(15)t(4;15)(q21;q26), based on the principle of human cytogenetic nomenclature of ISCN 2013 [30]
To further verify the proteomic data, we studied expression levels of 8 proteins by Western blot analysis, among which DNA (cytosine-5)-methyltransferase 3B (DNMT3B), CTNNB1, histone deacetylase 2 (HDAC2), VIM, and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) were upregulated in karyotypically aberrant chHES-3 cells or EC cells, while by contrast NES, HSPA1A and HIST1H1B were downregulated
Summary
Human embryonic stem cells (hESCs) derived from the inner cell mass of human embryos have held great promise for future cell- and tissue-replacement therapy because of their unique capacity to self-renew and to differentiate into any cell type. Concerns have been raised with regard to the safety of hESCs, which commonly undergo adaptive changes during prolonged passaging in vitro, such as increased growth rate, reduced apoptosis and especially karyotypic changes [1,2,3,4,5,6,7,8] With these changes, the culture adaptation of hESCs tends towards a transformed phenotype of tumor stem cells and emphasizes the need for thorough analysis of cells destined for clinical applications [2], [3], [5], [6], [8], [9]. Transformed hESCs may serve as an excellent model for characterizing the initial stages that determine transition of embryonic stem cells into cancerous stem cells
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