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 propluripotent 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 histone deacetylase inhibitor valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogenously 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 (EBs) or teratomas. In contrast, AFSC cultured under human ESC 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 EBs and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethics restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this medium-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of prepluripotent cells.
The Institute of Reproductive and Developmental Biology, Imperial College London, London, UK.
Dafni
D
Moschidou
Sayandip
S
Mukherjee
Michael P
MP
Blundell
Gemma N
GN
Jones
Anthony J
AJ
Atala
Adrian J
AJ
Thrasher
Nicholas M
NM
Fisk
Paolo
P
De Coppi
Pascale V
PV
Guillot
D000653:Amniotic Fluid / Q000166:cytology*; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D051379:Mice; D009374:Neoplasms, Experimental / Q000473:pathology; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology / Q000637:transplantation; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000378:metabolism; D013724:Teratoma / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects; D014635:Valproic Acid / Q000494:pharmacology*
D000943:Antigens, Differentiation; D003470:Culture Media; D056572:Histone Deacetylase Inhibitors; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014635:Valproic Acid