Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor.
Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.
Khoo
Melissa L M
ML
Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales 2031, Australia.
McQuade
Leon R
LR
Smith
Murray S R
MS
Lees
Justin G
JG
Sidhu
Kuldip S
KS
Tuch
Bernard E
BE
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000166:cytology*; D005136:Eye Proteins / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005947:Glucose / Q000494:pharmacology*; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics; D051559:Hepatocyte Nuclear Factor 6 / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D064231:Nestin; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D010179:Pancreas / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects
D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D005136:Eye Proteins; D016222:Fibroblast Growth Factor 2; D005947:Glucose; D051542:Hepatocyte Nuclear Factor 3-beta; D051559:Hepatocyte Nuclear Factor 6; D018398:Homeodomain Proteins; D007328:Insulin; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; D064231:Nestin; D050814:Octamer Transcription Factor-3; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; C496502:FOXA2 protein, human; C577484:NES protein, human; C093970:NEUROD1 protein, human; C496605:ONECUT1 protein, human; C000605730:PAX6 protein, human; C494173:POU5F1 protein, human