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.

The Impact of Epigenetic Signatures on Amniotic Fluid Stem Cell Fate

“…In 2012, the first study on alterations of imprinted genes (H19, SNRPN, and KCNQ1OT1) in hAFSCs was performed by Peng et al In particular, it was observed that during in vitro hAFSC culture, there are hypermethylation of H19 and KCNQ1OT1 and variable DNA methylation patterns in the SNRPN gene [90]. These results were in agreement with the study by Phermthai et al which demonstrated the aberrant expression of the IGF2 and H19 genes in late passages of AFSCs [91]. On the basis of these preliminary observations, it can be assumed that the epigenetic instability correlates with the loss of differentiation potential in late passages of AFSCs, and due to this reason, the therapeutic use of these cells should be limited to the 8 th passages.…”

Concise Review: Amniotic Fluid Stem Cells: The Known, the Unknown, and Potential Regenerative Medicine Applications

“…Epigenetic anomalies have been hypothesized to be a cause of these defects. Interestingly, previous studies have observed no irregularity in the epigenetic control system in early-passage AFSC, indicating that most likely the standard in vitro culturing of AFSC is safe for clinical application [53].…”

Genetic Stability of Mesenchymal Stromal Cells for Regenerative Medicine Applications: A Fundamental Biosafety Aspect

“…The most commonly used MSC sources are bone marrow (BM) [17] and adipose tissue (AT) [18,19,20,21]. They can also be isolated from a series of other tissues like umbilical cord (UC) or Wharton’s Jelly [22], cord blood [23,24], placenta and amnion [25,26], amniotic fluid [27], chorionic villi [28], dental pulp [29,30], endometrium [31], brain [32], skin [33], synovial membrane and synovial fluid [15,34], sweat glands [35], nasal mucosa [36].…”