Osteogenic differentiation within intact human embryoid bodies result in a marked increase in osteocalcin secretion after 12 days of in vitro culture, and formation of morphologically distinct nodule-like structures.
Osteogenic lineages derived from human embryonic stem cells hold much promise for clinical application in bone regeneration, in addition to providing a useful research model in developmental biology, and for pharmacological and cytotoxicity screening of bone-related biomaterials and drugs in vitro. Previously, osteogenic differentiation of human embryonic stem cells was achieved through dissociation of embryoid bodies by trypsinization, prior to culture with osteogenesis-promoting medium. This study therefore attempted a new approach: that is to achieve osteogenesis within intact human embryoid bodies. After 22 days of culture in osteogenesis-promoting medium comprising a cocktail of ascorbic acid, beta-glycerophosphate and dexamethasone, the attached embryoid bodies exhibited much cellular outgrowth and migration, and formed morphologically distinct nodule-like structures. These were somewhat similar to osteogenic nodules formed by mesenchymal stem cells, as reported by previous studies. Immunohistochemical staining and RT-PCR analysis confirmed the presence of osteogenic cells within these nodule-like structures. Additionally, the quantitative assay of osteocalcin secretion demonstrated a rapid sharp increase in osteocalcin expression on day 12 of in vitro culture, which could suggest the appearance of differentiated osteoblasts from day 12 onwards. Future work will attempt to investigate whether other cytokines, growth factors and chemical compounds could further enhance osteogenesis within intact human embryoid bodies.
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore, Singapore. dencaot@nus.edu.sg
Tong
T
Cao
Boon Chin
BC
Heng
Chao Peng
CP
Ye
Hua
H
Liu
Wei Seong
WS
Toh
Paul
P
Robson
Pin
P
Li
Yun Han
YH
Hong
Lawrence Walter
LW
Stanton
D001205:Ascorbic Acid / Q000494:pharmacology; D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000096:biosynthesis / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
D015415:Biomarkers; D015675:Osteocalcin; D003907:Dexamethasone; D001205:Ascorbic Acid