RRC ID 52678
Author Tashiro S, Le MNT, Kusama Y, Nakatani E, Suga M, Furue MK, Satoh T, Sugiura S, Kanamori T, Ohnuma K.
Title High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber.
Journal J Biosci Bioeng
Abstract Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis.
Volume 126(3)
Pages 379-388
Published 2018-9-1
DOI 10.1016/j.jbiosc.2018.03.007
PII S1389-1723(17)31150-7
PMID 29681444
MeSH Bone Morphogenetic Protein 4 / pharmacology* Cell Adhesion / drug effects Cell Count Cell Culture Techniques / methods* Cell Differentiation / drug effects* Cells, Cultured Embryo, Mammalian / metabolism Humans Induced Pluripotent Stem Cells / cytology Induced Pluripotent Stem Cells / drug effects Induced Pluripotent Stem Cells / physiology Pluripotent Stem Cells / cytology* Pluripotent Stem Cells / drug effects Pluripotent Stem Cells / physiology* Spatial Analysis Surface Properties
IF 2.366
Times Cited 2
Human and Animal Cells 201B7(HPS0063)