Introduction: We have established a crystal phagocytic analysis method using human induced pluripotent stem cell (iPSC)-derived macrophages (Mfs) in order to construct a drug discovery screening model. The aim of this study is to differentiate human iPSC-derived Mfs into inflammatory type (M1) and anti-inflammatory type (M2), and compare their phagocytosis and processing ability of fluorescent calcium oxalate crystals.
Methods: The six reprogramming factors (Oct4, Sox2, Lin28, L-Myc, Klf4, mp53DD) were introduced into peripheral blood cells of healthy subjects to prepare iPSCs. The primitive linear-like cells were induced with BMP4; vascular stem cell-like cells were induced with VEGF, basic FGF, and SCF; hematopoietic cells were prepared using hematopoietic cytokines (SCF/FL3/IL-3/TPO/M-CSF); and specific differentiation of a monocyte lineage was performed using Flt-3 ligand, GM-CSF, and M-CSF. Thereafter, the CD14+ monocyte lineage specific cell fraction was differentiated into Mfs using M-CSF. Furthermore, M1s and M2s were induced by IFN-? and IL-4, respectively. Alexa-Fluor 488-combined calcium oxalate monohydrate (f-COM) crystals were exposed to M1s / M2s, and the amount of phagocytic fCOM were measured with InCell Analyzer 6000 for 15 hours.
Results: The human iPSC-derived Mfs were able to differentiated into Ms and M2s labeled with CD11c and CD163, respectively, and could phagocytose f-COM crystals. The M2s differentiated from iPS-derived Mfs increased intracellular fluorescence intensity about 1.3 times about 2 hours after fCOM exposure, but M1s did not change. On the other hand, the fluorescence intensities of M1s and M2s gradually decreased by half over 15 hours. However, the fluorescence intensity of M2s rapidly decreased from the 10th hour and dropped below M1 after about 12 hours.
Conclusions: iPS cell-derived M2s was thought to be more capable of phagocytosing and processing crystals than M1s. This result showed the possibility that the subtype of M2s differentiated from the human iPSCs contributes to the novel prevention of stone formation. Source of