Nitric Oxide and a Conditioned Medium Affect the Hematopoietic Development in a Microfluidic Mouse Embryonic Stem Cell/OP9 Co-Cultivation System.

Kae Sato,Sayaka Ishii,Momoko Maeda,Kanako Yanagisawa,Takahiko Hara,Kenji Kitajima,Eriko Kamata

doi:10.3390/mi11030305

Abstract

A microfluidic co-culture system, consisting of mouse embryonic stem cells (mESCs)/OP9 cells, was evaluated as a platform for studying hematopoietic differentiation mechanisms in vitro. mESC differentiation into blood cells was achieved in a microchannel that had the minimum size necessary to culture cells. The number of generated blood cells increased or decreased based on the nitric oxide (NO) donor or inhibitor used. Conditioned medium from OP9 cell cultures also promoted an increase in the number of blood cells. The number of generated blood cells under normal medium flow conditions was lower than that observed under the static condition. However, when using a conditioned medium, the number of generated blood cells under flow conditions was the same as that observed under the static condition. We conclude that secreted molecules from OP9 cells have a large influence on the differentiation of mESCs into blood cells. This is the first report of a microfluidic mESC/OP9 co-culture system that can contribute to highly detailed hematopoietic research studies by mimicking the cellular environment.

Highlights

In mice, hematopoietic stem cells (HSCs) first appear from the hemogenic endothelium, a specialized subset of vascular endothelial cells between E10.5 and E11.5 in the aorta-gonadmesonephros region of the embryo [1]
OP9 cells were required to be in the culture without medium change for 2 days until Flk-1+ cells were differentiated into blood cells
OP9 cells could not be alive under vascular shear stress levels

Summary

Introduction

Hematopoietic stem cells (HSCs) first appear from the hemogenic endothelium, a specialized subset of vascular endothelial cells between E10.5 and E11.5 in the aorta-gonadmesonephros region of the embryo [1]. Blood flow and shear stress-dependent nitric oxide (NO) play a fundamental role in the emergence and maintenance of HSCs and progenitors [2,3,4,5,6,7]. Treatment with NO synthase (NOS) inhibitors has been reported to reduce intravascular hematopoietic clusters in mouse and zebrafish embryos [2,3]. Mutant mouse and zebrafish embryos lacking heartbeats and, exhibiting reduced blood flow displayed reductions in intravascular hematopoietic clusters [2,3]. The role of blood flow in regulating hematopoietic stem cell fate remains poorly characterized, considerable research efforts have been focused in this area. There are three standard methods for the induction of hematopoietic differentiation from mouse embryonic stem cells (mESCs) [8], namely, the embryoid body formation method [3], direct differentiation methods, in which a monolayer culture is grown in an extracellular-matrix-coated dish [5], and a co-culture method using mESCs with feeder cells, such as a bone marrow stromal cell line [9,10,11]

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