Evidence for Transfer of Membranes from Mesenchymal Stem Cells to HL-1 Cardiac Cells

Abstract

This study examined the interaction of mouse bone marrow mesenchymal stem cells (MSC) with cardiac HL-1 cells during coculture by fluorescent dye labeling and then flow cytometry. MSC were layered onto confluent HL-1 cell cultures in a 1 : 4 ratio. MSC gained gap junction permeant calcein from HL-1 cells after 4 hours which was partially reduced by oleamide. After 20 hours, 99% MSC gained calcein, unaffected by oleamide. Double-labeling HL-1 cells with calcein and the membrane dye DiO resulted in transfer of both calcein and DiO to MSC. When HL-1 cells were labeled with calcein and MSC with DiO, MSC gained calcein while HL-1 cells gained DiO. Very little fusion was observed since more than 90% Sca-1 positive MSC gained DiO from HL-1 cells while less than 9% gained gap junction impermeant CMFDA after 20 hours with no Sca-1 transfer to HL-1 cells. Time dependent transfer of membrane DiD was observed from HL-1 cells to MSC (100%) and vice versa (50%) after 20 hours with more limited transfer of CMFDA. These results demonstrate that MSC and HL-1 cells exchange membrane components which may account for some of the beneficial effect of MSC in the heart after myocardial infarction.

Figure 1

Flow cytometry of unlabeled MSC and CMFDA/calcein labeled HL-1 cells after 4 and 20 hours of coculture. (a–c) Four hours of coculture. (d–f) 20 hours of coculture. (a) and (d) Unlabeled MSC (M) prior to coculture. (b) and (e) HL-1 cells (H) double labeled with CMFDA and calcein prior to coculture. (c) and (f) Unlabeled MSC and CMFDA/calcein labeled HL-1 cells after coculture.

Figure 2

Scrape loading of H9c2 cells. (a) Scrape loading with Lucifer yellow in the absence of oleamide. (b) Scrape loading with Lucifer yellow in the presence of oleamide. (c) Scrape loading with dextran-Rhodamine B in the absence of oleamide. Arrow: edge of scrape.

Figure 3

Flow cytometry of unlabeled MSC and DiO/calcein labeled HL-1 cells after 4 hours of coculture. (a) Unlabeled MSC (M) prior to coculture. (b) HL-1 cells (H) double labeled with DiO and calcein prior to coculture. (c) Unlabeled MSC and DiO/calcein labeled HL-1 cells after 4 hours of coculture.

Figure 4

Flow cytometry of DiO labeled MSC and calcein labeled HL-1 cells after 4 and 20 hours of coculture. (a) HL-1 cells (H) labeled with calcein prior to coculture. (b) MSC (M) labeled with DiO prior to coculture. (c-4) DiO labeled MSC and calcein labeled HL-1 cells after 4 hours of coculture. (c-20) DiO labeled MSC and calcein labeled HL-1 cells after 20 hours of coculture.

Figure 5

Flow cytometry of Sca1-PEcy5 labeled MSC and DiO or CMFDA labeled HL-1 cells after 20 hours of coculture. (a) MSC (M) labeled with Sca1-PEcy5 prior to coculture. (b) DiO: HL-1 cells (H) labeled with DiO prior to coculture. (c) DiO: Sca1-PEcy5 labeled MSC and DiO labeled HL-1 cells after 20 hours of coculture. (b) CMFDA: HL-1 cells (H) labeled with CMFDA prior to coculture. (c) CMFDA: Sca1-PEcy5 labeled MSC and CMFDA labeled HL-1 cells after 20 hours of coculture.

Figure 6

Flow cytometry of DiD/CMFDA labeled MSC and unlabeled HL-1 cells or unlabeled MSC and DiD/CMFDA labeled HL-1 cells after 20 hours of coculture. (a) MSC (M) labeled with DiD and CMFDA prior to coculture. (b) Unlabeled HL-1 cells (H) prior to coculture. (c) DiD/CMFDA labeled MSC and unlabeled HL-1 cells after 20 hours of coculture. (d) Unlabeled MSC (M) prior to coculture. (e) HL-1 cells (H) labeled with DiD and CMFDA prior to coculture. (f) Unlabeled MSC and DiD/CMFDA HL-1 cells after 20 hours of coculture.

Figure 7

Percent cells that gained membrane or cytoplasm after 4 and 20 hours of coculture of MSC and HL-1 cells. MSC and HL-1 cells were labeled, cultured, and analyzed as in Figure 6. Data was calculated as mean percent ± s.e.m. of cells that gained membrane (DiD label) or cytoplasm (CMFDA label) during coculture as determined by flow cytometry. MSC→HL-1 = transfer from MSC to HL-1 cells (open bars). HL-1→MSC = transfer from HL-1 cells to MSC (solid bars).