Human mid-trimester amniotic fluid stem cells cultured under embryonic stem cell conditions with Valproic acid acquire pluripotent characteristics.

Human mid-trimester amniotic fluid stem cells (AFSC) have promising applications in regenerative medicine, being broadly multipotent with an intermediate phenotype between embryonic (ES) and mesenchymal stem cells (MSC). Despite this pro-pluripotent phenotype, AFSC are usually cultured in adherence in a serum-based expansion medium and how expansion in conditions sustaining pluripotency might affect their phenotype remains unknown. We recently showed that early AFSC from first trimester amniotic fluid, which endogenously express Sox2 and Klf4, can be reprogrammed to pluripotency without viral vectors using the HDAC inhibitor Valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogeneously expressing telomerase, CD24, OCT4, C-MYC, and SSEA4, but low/null levels of SOX2, NANOG, KLF4, SSEA3, TRA-1-60 and TRA-1-81, with cells unable to form embryoid bodies or teratomas. In contrast, AFSC cultured under hESC conditions were smaller in size, grew faster, formed colonies, upregulated OCT4 and C-MYC, and expressed KLF4 and SOX2 but not NANOG, SSEA3, TRA-1-60 and TRA-1-81. Supplementation with VPA for 5 days further upregulated OCT4, KLF4 and SOX2, and induced expression of NANOG, SSEA3, TRA-1-60 and TRA-1-81, with cells now able to form embryoid bodies and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethical restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this media-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of pre-pluripotent cells.