Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11.
Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.
Hwang
Yu-Shik
YS
Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA 02139, USA.
Chung
Bong Geun
BG
Ortmann
Daniel
D
Hattori
Nobuaki
N
Moeller
Hannes-Christian
HC
Khademhosseini
Ali
A
D000818:Animals; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D000071818:Wnt-5a Protein
D034741:RNA, Small Interfering; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C508957:Wnt11 protein, mouse; C438615:Wnt5a protein, mouse; D049452:Green Fluorescent Proteins