Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.
Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.
Department of Stem Cell Biology, School of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea.
Sung-Hwan
SH
Moon
Department of Biomedical Engineering, College of Health Science, Korea University, Jeongneung 3-dong, Seongbuk-gu, Seoul 136-703, Republic of Korea.
Jongil
J
Ju
Department of Stem Cell Biology, School of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea.
Soon-Jung
SJ
Park
CHA Bio & Diostech Co., Ltd., Seoul 135-081, Republic of Korea.
Daekyeong
D
Bae
Department of Stem Cell Biology, School of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea. Electronic address: hmchung@kku.ac.kr.
Hyung-Min
HM
Chung
Department of Biomedical Engineering, College of Health Science, Korea University, Jeongneung 3-dong, Seongbuk-gu, Seoul 136-703, Republic of Korea. Electronic address: dbiomed@korea.ac.kr.
Sang-Hoon
SH
Lee
D019149:Bioreactors; D002460:Cell Line; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D058732:Embryoid Bodies / Q000145:classification / Q000166:cytology* / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000145:classification* / Q000166:cytology / Q000502:physiology; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*
Concave microwells; Embryoid body; Human embryonic stem cell; Optimize differentiation; Size screening