title: Selective isolation of nanog-positive human amniotic mesenchymal cells and differentiation into cardiomyocytes.
abstract: Adult cardiomyocytes have little ability to regenerate, thus cardiac regeneration therapy represents a potential method for treating severe heart failure. Human amniotic mesenchymal cells (hAMCs) have the potential to be a useful cell source for cardiac regeneration therapy. We attempted to isolate stem cells from hAMCs and differentiate them into cardiomyocytes. Nanog promoter-Cre plasmid and cytomegalovirus (CMV) promoter-loxP-STOP-loxP-Red-puro(r) plasmid were co-transfected into immortalized hAMCs (iHAMs). Nanog-positive iHAMs were treated with 5-azacytidine (5-aza), trichostatin A (TA), activin A (AA), and bone morphogenetic protein-4 (BMP-4), or co-cultured with murine fetal cardiomyocytes for cardiomyocytes differentiation. Isolated Nanog-positive iHAMs were analyzed by quantitative RT-PCR and immunofluorescent staining before and after differentiation. Expression of Nanog, Oct3/4, Sox2, and Klf4 was significantly higher in Nanog-positive than in Nanog-negative iHAMs. Nanog-positive iHAMs were stained for Nanog and Oct3/4 in the nucleus. Nanog-positive iHAMs treated with 5-aza expressed Nkx2.5, GATA-4, human atrial natriuretic peptide (hANP), cardiac troponin T (cTnT), myocin light chain (Mlc)-2a, Mlc-2v, β-myosin heavy chain (β-MHC), hyperpolarization-activated cyclic nucleotide gated channels (HCN)-4, and inwardly rectifying potassium channels (Kir)-2.1. Although Nanog-positive iHAMs treated with TA, AA, or BMP-4 expressed several cardiac markers, no contraction was observed. Co-cultured Nanog-positive iHAMs with murine fetal cardiomyocytes spontaneously contracted in a synchronized manner and expressed the cardiac markers. In conclusion, Nanog-positive hAMCs with characteristics of stem cells were isolated and differentiated into cardiomyocyte-like cells, suggesting that these isolated hAMCs could be a useful cell source for cardiac regeneration therapy.
authors.affiliation: Department of Regenerative Medicine, University of Toyama, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Otaka
authors.forename: Saori
authors.initials: S
authors.lastname: Nagura
authors.forename: Chika
authors.initials: C
authors.lastname: Koike
authors.forename: Motonori
authors.initials: M
authors.lastname: Okabe
authors.forename: Toshiko
authors.initials: T
authors.lastname: Yoshida
authors.forename: Moustafa
authors.initials: M
authors.lastname: Fathy
authors.forename: Kentoku
authors.initials: K
authors.lastname: Yanagi
authors.forename: Takurou
authors.initials: T
authors.lastname: Misaki
authors.forename: Toshio
authors.initials: T
authors.lastname: Nikaido
mesh_terms: D000650:Amnion / Q000166:cytology / Q000378:metabolism; D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation; D018920:Coculture Techniques; D006801:Humans; D000090062:Kruppel-Like Factor 4; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D000943:Antigens, Differentiation; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors