Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame - Cellular and Molecular Life Sciences

Lineage-specific differentiation and phagocytic activity in h-Muse cells. (A, B, D, F, H, J) (A) qPCR of human-cardiac markers in naïve-Muse cells (n-Muse) and h-Muse cells after co-culture with intact m-cardiomyocytes from D3 to D21 (mean ± SEM). qPCR of human-cardiac (B, D), -neural (F, H), and -hepatic (J) markers in naïve h-Muse cells (n-Muse) and h-Muse cells after incubating with apoptotic fragments derived from m-cardiomyocyte, r-neural cells, and r-hepatic cells from D3 to D21 (mean ± SEM). In D and H, D7 (P + T) indicates h-Muse cells incubated with apoptotic-cell fragments for 3 days and then co-cultured with a damaged tissue slice for 7 days (mean ± SEM). Confirmed species-specific primers were used in qPCR. Apoptotic cell fragments were used as a negative control (Nega). For the positive control, human fetal heart total RNA (A, B), h-adult heart (D), and h-fetus whole total RNA (F, H, J) were used (Posi). *p < 0.05, **p < 0.01, ***p < 0.001. C, G, K Immunocytochemistry in h-Muse cells after incubating with apoptotic fragments. C Non-labeled h-Muse cells were incubated with apoptotic GFP-m-cardiomyocyte fragments. Arrows: GFP-positive fragments/particles in the cytoplasm. Inset arrowheads: striated-like pattern of troponin-I in h-Muse cells. G, K h-Muse cells were transduced with GFP-lentivirus for identification. Apoptotic fragments were from non-labeled cells. Low magnification images of (G) are shown in Fig. S2B. E, I Intracellular calcium dynamics in green fluorescent protein (GFP)-based Ca calmodulin probe (GCaMP)-h-Muse cells after biochemical depolarization with 70 mM (E) and 50 mM (I) KCl, respectively (added at 20 s) (see Movies 1). The change in the fluorescence intensity over time was demonstrated. L–N Laser confocal microscopy of GFP-h-Muse cells incubated with mCherry-m-Hepa1 DDCs. L Live imaging at 20 h (see Movie 2) shows Z-series 3D construction. M Analysis of (L) by Bitplane Imaris software revealed phagocytosed fragments in h-Muse cells. Inset: magnification of the box area. M Transition of large mCherry-fragments to smaller particles after phagocytosis (see Movie 2). N GFP-h-Muse cells in the post-infarct rat heart tissue at D5 contained LAMP-1( +) phagosomes (red, arrowheads). Bars: C, G, K = 25 μm; E, I, M–O = 50 μm; L = 100 μm

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