Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies.
Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation.
Pekkanen-Mattila
Mari
M
Regea Institute for Regenerative Medicine, University of Tampere, Tampere University Hospital, Biokatu 12, 33520, Tampere, Finland.
Pelto-Huikko
Markku
M
Kujala
Ville
V
Suuronen
Riitta
R
Skottman
Heli
H
Aalto-Setälä
Katriina
K
Kerkelä
Erja
E
D017209:Apoptosis / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D004475:Ectoderm / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004707:Endoderm / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005326:Fetal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008648:Mesoderm / Q000378:metabolism; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D046888:Tissue Array Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism; D000098908:Brachyury Protein
D015415:Biomarkers; D053148:Caspase 3; D005136:Eye Proteins; D005326:Fetal Proteins; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D055748:SOXB1 Transcription Factors; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D020107:Troponin T; D000509:alpha-Fetoproteins; D000098908:Brachyury Protein; C095552:NKX2-5 protein, human; C000605730:PAX6 protein, human