title: A three dimensional anchorage independent in vitro system for the prolonged growth of embryoid bodies to study cancer cell behaviour and anticancer agents.
abstract: We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior.
authors.affiliation: Department of Obstetrics & Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 119074.
authors.forename: Chui-Yee
authors.initials: CY
authors.lastname: Fong
authors.forename: Li-Ling
authors.initials: LL
authors.lastname: Chak
authors.forename: Arjunan
authors.initials: A
authors.lastname: Subramanian
authors.forename: Jee-Hian
authors.initials: JH
authors.lastname: Tan
authors.forename: Arijit
authors.initials: A
authors.lastname: Biswas
authors.forename: Kalamegam
authors.initials: K
authors.lastname: Gauthaman
authors.forename: Mahesh
authors.initials: M
authors.lastname: Choolani
authors.forename: Woon-Khiong
authors.initials: WK
authors.lastname: Chan
authors.forename: Ariff
authors.initials: A
authors.lastname: Bongso
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis
chemical_list: D000970:Antineoplastic Agents