Proliferation and Differentiation Characteristics of Double-Trisomic Embryonic Stem Cells
ZHANG Meili*, XIAO Rong, JIA Yuyan, HUANG Yue*
This study aims to investigate the proliferation, differentiation, and teratoma formation characteristics of double-trisomic ESCs (embryonic stem cells), and discover the relationships between aneuploidy and tumorigenesis. Two lines of autosomal double-trisomic mouse ESCs were established. Array CGH (array comparative genomic hybridization) and FISH (fluorescence in situ hybridization) were used to determine the chromosome copy number variations and the karyotyping of the two double-trisomic ESC lines. Cell growth curves were made to evaluate the proliferation abilities of the double-trisomic ESCs. Flow cytometry was used to detect the cell cycle distributions and the levels of apoptosis in double-trisomic ESCs. Colony-forming assays were performed to evaluate the colony formation efficiencies of double-trisomic ESCs. qRT-PCR and immunofluorescence analyses were conducted to determine whether the pluripotency markers were normally expressed in double-trisomic ESCs. Moreover, LIF (leukemia inhibitory factor) withdrawal and EB (embryoid body) formation assays were performed to evaluate the in vitro differentiation status of these double-trisomic ESCs. Teratoma assays were conducted by using SCID (severe combined immunodeficiency) mice to determine the effects of double-trisomies on teratoma formation and the differentiation capacities in vivo. Array CGH and FISH experiments showed that one cell line gained extra chromosome 3 and chromosome 6 but lost chromosome Y, which was named as DTs-3+6. Another cell line had extra chromosomes of 6 and 8, which was named as DTs-6+8. Double-trisomic ESCs exhibited rapid proliferation characteristics when compared with wild-type ESCs. They expressed stem cell markers OCT4, SOX2 and NANOG when cultured under normal ESC culture conditions. Upon LIF withdrawal, wild-type ESCs mostly went to total differentiation, while double-trisomic ESCs formed many partially differentiated or undifferentiated clones, which were positive for AP (alkaline phosphatase) staining. In the early stage of EB differentiation, the expression of genes related to three-germ layers such as Fgf5, T, and Foxa2 in double-trisomic EBs were significantly lower than those in wild-type EBs, indicative of delayed differentiation of double-trisomic ESCs. Once injected into SCID mice subcutaneously, double-trisomic ESCs showed enhanced teratoma formation efficiencies compared with wild-type ESCs. Teratomas derived from double-trisomic ESCs were comprised of many undifferentiated regions. Thus, double-trisomic ESCs had increased proliferation capacities and promoted teratoma formation by impairing cellular differentiation. Double-trisomic ESCs are important models for investigating the roles of aneuploidy in tumorigenesis.
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