DEVELOPMENT OF THE MICROFLUIDIC DEVICE FOR DYNAMIC LOAD STIMULATION OF BLOOD VESSEL TO HEMOGENIC ENDOTHELIUMS DERIVED FROM HUMAN IPS CELLS
Masahiro Narazaki1, Susumu Kudo1, Toshihiro Sera1, Kazuhiro Nakashima1, Tani Kenzaburo2, Hiroshi Kohara2
1Kyushu University, Japan; 2University of Tokyo, Japan
Hematopoietic stem cells (HSCs) are the cells which continue producing all blood corpuscles in marrow. Although HSCs transplant is already established, HSCs have been induced and amplified in vitro due to the donor shortage. Recently the human induced pluripotent stem cells (hiPSCs) were proposed, and so the method of inducing multipotent HSCs via hiPSCs has been investigated in vitro. HSCs differentiation from the vascular endothelial cell, such as the aortas of the aorta-gonad-mesonephros (AGM) region (hemogenic endothelium), may be regulated during endothelialhematopoietic transition (EHT) at the midgestation. In EHT, the vascular endothelial cells are exposed to the various stimulations of the shear stress from blood flow and contraction - expansion of vessel wall. We hypothesize that these biomechanical stresses can improve the efficiency of HSCs differentiation in EHT. In this study, we developed the culture device which could load microfluidic flow and extension stimulation independently or simultaneously to investigate the effects of these biomechanical stresses to HSCs differentiation from hemogenic endotheliums derived from hiPSCs. The following specifications are required for culture device, 1) collection of HSCs which are induced from hemogenic endotheliums and carried away by the flow load, 2) study of blood corpuscle colony assay after dynamic load stimulation, 3) live imaging during flow load to evaluate EHT reaction with the difference in time, 4) high-throughput system in small size for molecular analysis. To achieve these requirements, our device consists of the microfluidic culture device and no flow chamber, and particularly, the chamber is used to collect HSCs induced in the microfluidic culture device. After flow load, we analyzed
the kind of the blood corpuscle and the number of the colony forming cell in collected HSCs by flow cytometry analysis and colony assay, respectively.