title: Epigenetic analysis and suitability of amniotic fluid stem cells for research and therapeutic purposes.
abstract: Amniotic fluid stem cells (AFSs) are interesting mesenchymal stem cells (MSCs) that are characterized by their great potential for cell proliferation and differentiation compared with other types of MSCs identified to date. However, MSCs in prolonged culture have been found to exhibit defects in genetic stability and differentiation capacity. Epigenetic anomalies have been hypothesized to be a cause of these defects. Here, we investigated the genomic methylation and genetic imprinting in AFSs during prolonged in vitro culture. Four human imprinted genes, insulin-like growth factor 2 (IGF2), H19, small nuclear ribonucleoprotein polypeptide N gene (SNRPN), and mesoderm-specific transcript (MEST), were evaluated for their expression levels and methylation statuses in AFS lines. The data revealed epigenetic instability in high passage number AFS cultures. The real-time polymerase chain reaction analysis showed that the expression levels of the imprinted genes gradually increased with increased time in culture. The loss of parental allele-specific imprinting for at least 1 gene among IGF2, H19, and SNRPN was observed in every AFS line after passage 8 using allelic expression analysis. The imprinting control regions (ICRs) of the IGF2 and H19 genes were assayed for site-specific methylation using bisulfite sequencing. This assay revealed a variable level of methylated CpG sites in the ICRs of IGF2 and H19. This variable level of CpG methylation is related to the aberrant expression of the IGF2 and H19 genes in late-passage AFSs. Our results did not reveal any irregularity in the epigenetic control system in the early-passage AFSs, indicating that the standard in vitro culturing of AFSs used in medical treatments should be limited to 8 passages.
authors.affiliation: Stem Cell Research and Development Unit, Department of Obstetrics & Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. tatsanee.phe@mahidol.ac.th
authors.forename: Tatsanee
authors.initials: T
authors.lastname: Phermthai
authors.forename: Singpetch
authors.initials: S
authors.lastname: Suksompong
authors.forename: Nednapis
authors.initials: N
authors.lastname: Tirawanchai
authors.forename: Surapol
authors.initials: S
authors.lastname: Issaragrisil
authors.forename: Suphakde
authors.initials: S
authors.lastname: Julavijitphong
authors.forename: Suparat
authors.initials: S
authors.lastname: Wichitwiengrat
authors.forename: Decha
authors.initials: D
authors.lastname: Silpsorn
authors.forename: Puttachart
authors.initials: P
authors.lastname: Pokathikorn
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D001483:Base Sequence; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D015870:Gene Expression; D018392:Genomic Imprinting; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D010641:Phenotype; D011247:Pregnancy; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: C094335:H19 long non-coding RNA; C035170:IGF2 protein, human; D062085:RNA, Long Noncoding; D007335:Insulin-Like Growth Factor II