Figure 1.
Schematic of the laser-patterning procedure.
An rMSC is pumped out of the hollow fiber, trapped by the focused laser beam, and guided into the right side of a contact-preventive microwell (dotted pink circle) containing a cardiomyocyte (left side of the microwell). (pink: rMSCs; green: cardiomyocytes).
Figure 2.
Portions of laser-patterned biochips shown with (A) contact-promotive microwells and (B) contact-preventive microwells. After 48 hours culturing, the heterotypic cells (C, D) formed broad cellular contact in contact-promotive microwells; the heterotypic cells (E, F) did not form broad cellular contact in contact-preventive microwells. (α-actinin positive: cardiomyocytes, α-actinin negative: rMSCs, DAPI: nuclei).
Figure 3.
Junction formation between rMSCs and cardiomyocytes.
Junctional distribution of (A, B) connexin 43 and (C, D) N-cadherin expressed at the contact area between rMSCs and cardiomyocytes in the contact-promotive microwells. (White arrows point to the junctional distribution of protein expression), (E) tetra-stained confocal image of cardiomyocyte (CM) and mesenchymal stem cell (MSC) coculture. The cell membranes of CM (red) and MSC (green) were labeled by different markers. Connexin43 was expressed at the junctional area between the CM and MSC membranes.
Figure 4.
Cell fusion between rMSCs and cardiomyocytes.
Full cell fusion (A–D) was observed by a DiO-labeled cardiomyocyte (A, green) fused with a DiI-labeled MSC (B, red) into double-nuclei, mixed labels (observed in fluorescent image, C), and membrane reorganization (observed in bright-field image, D). Partial cell fusion (E, F) was observed by rMSC mitochondrial transfer; labeled mitochondria (red) migrate across the membrane at contact area and accumulate near the cardiomyocyte nucleus.
Figure 5.
Tunneling nanotube extension from one rMSC.
Time-lapse images (A–D) show the nanotube extension from an rMSC making contact with the cardiomyocyte in 180 minutes within the same microwell. (Red arrows point to the nanotube).
Figure 6.
Long-distance communication between rMSCs and cardiomyocytes.
Long distance connections between rMSCs and cardiomyocytes in contact-preventive microwells through either (A, B) rMSC-origin tunneling nanotube (determined by MSC surface marker, CD105) or (C, D) cardiomyocyte-origin filopodium-like structure (determined by cardiac marker, α-actinin). (E): Mitochondria transfer from one rMSC to its contacting cardiomyocyte. (F): Mitochondria transfer through the cardiomyocyte-origin filopodia and accumulate around the cardiomyocyte's nucleus. (White arrows point to the nanotubes).
Figure 7.
Mitochondria propagation through a nanotube.
Time-lapse images (Zeiss Fluorescent Microscope, 63×, NA = 1.4) of mitochondrial propagation with a time interval of 15 minutes (A–D); red arrows point to a dot-shape (small) mitochondria, and yellow arrows point to a rod-shape (large) mitochondria. (E): The transfer velocities are highly dependent on mitochondrial size and shape; larger mitochondria have lower velocities. (F): rMSC and cardiomyocyte cell bodies connected by a nanotube, which is enlarged in the left column (A–D).