Induction of osteogenic markers in differentially treated cultures of embryonic stem cells.
BACKGROUND
Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation.


METHODS
Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and beta-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR.


RESULTS
ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17.


CONCLUSIONS
Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.
Handschel
Jörg
J
Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University Düsseldorf, Moorenstr, 5, 40225 Düsseldorf, Germany. handschel@t-online.de
Berr
Karin
K
Depprich
Rita A
RA
Kübler
Norbert R
NR
Naujoks
Christian
C
Wiesmann
Hans-Peter
HP
Ommerborn
Michelle A
MA
Meyer
Ulrich
U
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D001205:Ascorbic Acid / Q000494:pharmacology; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D053595:Embryonic Stem Cells / Q000187:drug effects*; D015870:Gene Expression; D005994:Glycerophosphates / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D015675:Osteocalcin / Q000096:biosynthesis; D010012:Osteogenesis / Q000187:drug effects*; D053495:Osteopontin / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D018952:Antigens, CD34; D015415:Biomarkers; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005994:Glycerophosphates; D053605:Leukemia Inhibitory Factor; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D053495:Osteopontin; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid