Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer

Abstract

Because stem cells are often found to improve repair tissue including heart without evidence of engraftment or differentiation, mechanisms underlying wound healing are still elusive. Several studies have reported that stem cells can fuse with cardiomyocytes either by permanent or partial cell fusion processes. However, the respective physiological impact of these two processes remains unknown in part because of the lack of knowledge of the resulting hybrid cells. To further characterize cell fusion, we cocultured mouse fully differentiated cardiomyocytes with human multipotent adipose-derived stem (hMADS) cells as a model of adult stem cells. We found that heterologous cell fusion promoted cardiomyocyte reprogramming back to a progenitor-like state. The resulting hybrid cells expressed early cardiac commitment and proliferation markers such as GATA-4, myocyte enhancer factor 2C, Nkx2.5, and Ki67 and exhibited a mouse genotype. Interestingly, human bone marrow-derived stem cells shared similar reprogramming properties than hMADS cells but not human fibroblasts, which suggests that these features might be common to multipotent cells. Furthermore, cardiac hybrid cells were preferentially generated by partial rather than permanent cell fusion and that intercellular structures composed of f-actin and microtubule filaments were involved in the process. Finally, we showed that stem cell mitochondria were transferred into cardiomyocytes, persisted in hybrids and were required for somatic cell reprogramming. In conclusion, by providing new insights into previously reported cell fusion processes, our data might contribute to a better understanding of stem cell-mediated regenerative mechanisms and thus, the development of more efficient stem cell-based heart therapies.

Figure 1. Formation of GATA-4⁺ hybrid cells after fusion of hMADS with cardiomyocytes

(A) Detection of hybrid cells (blue) by Xgal staining on day 2 of co-culture of Rosa26R cardiomyocytes with Cre-human multipotent adipose-derived stem cells (hMADS) cells (arrowhead). (B) Negative Xgal staining on co-culture with non-transduced hMADS cells (arrowhead). (C–D) Co-immunostaining for β-galactosidase (β-gal, white) and GATA-4 (red) on day 7 of co-culture of Rosa26R cardiomyocytes and (C) Cre- or (D) non-transduced hMADS cells. Arrow: dead cardiomyocyte bodies. Nuclei were counterstained with Hoechst 33342 (blue). (A–D) Scale bars, 50 μm.

Figure 2. Phenotypic characterization of hybrid cells

Immunohistochemistry on day 7 of co-culture showed that colony-derived GATA-4⁺ cells expressed (A) Nkx2.5 and (B) myocyte enhancer factor 2C (MEF-2C) but rarely (C) desmin and (D) cardiac troponin I (cTnI) and were negatively stained for (E) α-sarcomeric actinin, (F) α-smooth actin and (G) prolyl 4 hydroxylase subunit beta (P4HB) fibroblastic markers. HMADS cells were positive for P4HB staining (arrow). (H) Day-7 co-culture-derived GATA-4⁺ cells also expressed Ki67. (A–H) Antibodies were conjugated with FITC (green), except for GATA-4, which was conjugated with Cy3 (red). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm.

Figure 3. Cell-fusion induction of somatic reprogramming of mouse cardiomyocytes

(A–B) Mouse and human gene expression changes during co-culture assessed by quantitative RT-PCR from day 0 to 7 of co-culture. (C–D) Immunocytochemistry of mouse phenotype of colony GATA-4⁺ cells at day 7 of co-culture. (C) A GATA-4⁺ (Cy3, red) colony expressing murine (Cy5, white) but not human (FITC, green) Lamin A/C. Arrow: hMADS cells expressing human Lamin A/C but not GATA-4. (D) Co-culture with GFP⁺ mouse cardiomyocytes leading to GATA-4⁺ (Cy3, red)/GFP⁺ (FITC, green) cells. (C–D) Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. (E–F) RT-PCR showing no modification of mouse- or human-cardiac gene expression when human and mouse cells were separated by permeable membrane. (G–J) Real-time PCR analysis of mouse and human gene expression on co-culture with (G–H) human bone-marrow–derived stem cells or (I–J) human MRC5 fibroblasts. (A, B, E–J) Data represent the mean±SD of at least 3 independent experiments *P<0.05; ** P<0.01; ***P<0.001. MEF-2C: myocyte enhancer factor 2C; cTnT: cardiac troponin T

Figure 4. Analysis of nuclear fusion events during co-culture

(A–B) FISH with mouse (FITC) and human (Cy3) COT-1 DNA probes. (A) Right panel: detection of synkaryons containing human (red) and mouse (green) DNA (arrows) 2 days after co-culture initiation. Arrowhead: hMADS cells labeled with only human COT-1 probe. Left and middle panels: double FISH performed on mouse cardiomyocytes or hMADS alone, respectively. Star: cardiomyocyte body aggregate. (B) Nuclei of colony cells expressing mouse but not human COT-1 DNA. Arrow: human nucleus (red). (C–D) Immunocytochemistry on day 2 of co-culture with GATA-4 combined with FISH labeling with FITC-conjugated human or mouse all centromere probes (green) confirmed that GATA-4⁺ nuclei (Cy3, red) contained (C) mouse but (D) not human DNA. Human nuclei devoid of GATA-4 staining are also shown. (A–D) Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 20 μm.

Figure 5. Occurrence of partial cell fusion events during co-culture

(A–B) Live cell imaging at 6 hr after co-culture initiation revealed thin membranous channels (arrowheads) connecting hMADS cells (arrow) to cardiomyocytes (stars). Scale bars, 20 μm. (C–G) Electron microscopy images illustrating various types of intracellular interactions between hMADS cells and mouse cardiomyocytes (CM) in 24-hr co-cultures. (C–E) Adherent long slender protrusions connecting hMADS to distant cardiomyocytes. (C) Partial view of a long hMADS protrusion surrounding a cardiomyocyte membrane (arrowheads). Scale bar, 378 nm. (D) Terminal portion of a protrusion. Scale bar, 644 nm. (E) Higher magnification of the contact area shown in (D) with blurring (arrowhead) and suspected focal loss (arrow) of the 2 plasma membranes. Scale bar, 275 nm. (F) Close membrane apposition between stem cells and cardiac cells. Scale bar, 863 nm. (G) Higher magnification of the contact site shown in (F) suggesting membrane disruption (arrows). Scale bar, 410 nm.

Figure 6. Involvement of f-actin and microtubule filaments in cardiomyocyte reprogramming

(A) Six hours after co-culture initiation, thin membrane channels interconnecting stem (arrow) to cardiac (arrowhead) cells contained both f-actin (rhodamine-phalloidin staining, red) and microtubules (FITC-conjugated α-tubulin, green). (B) Close membrane apposition between stem and cardiac cells showing concentration of microtubule (green) and f-actin (red) filaments at the contact site. Scale bars (A, B), 10 μm. (C–D) Impaired somatic reprogramming by inhibitors of f-actin (cytochalasin D 0.5 μM, latrunculin A 0.25 μM) or microtubule polymerization (nocodazole 1 μM) assessed by real-time PCR at 7 days of co-culture. No statistical differences between inhibitory effects of nocodazole, cytochalasin D or latrunculin A were observed. Relative expression was compared to day-7 untreated co-cultures. Data represent the mean±SD of at least 3 independent experiments *P<0.05; ** P<0.01; ***P<0.001.

Figure 7. Role of functional stem cell mitochondria transfer towards cardiomyocytes for somatic reprogramming

(A) Microscopy live-cell imaging in a 6-hr co-culture showing hMADS mitochondria (MitoTracker Green FM) in cardiomyocyte (arrowhead). Arrow points to an hMADS cell. (B) Presence of human mitochondria (MitoTracker Green FM) inside a tunneling nanotube-like structure connecting a stem cell to a cardiac cell and into cardiomyocyte cytoplasm (arrowhead; 12-hr co-culture, paraformaldehyde-fixed cells). (C) Despite their mouse phenotype, β-gal⁺ (Cy5, white)/GATA-4⁺ (Cy3, red) hybrid cells show human stem-cell mitochondria in their cytoplasm (FITC, green). (D) Human mitochondria antibody used in (C) stained human but not mouse cells (left and right panels, respectively. (E) MitoTracker green staining showing mitochondrial DNA depletion in hMADSρ⁰ (ethidium bromide-treated, right panel) compared to untreated hMADS cells (left panel). (F) Day 7 co-culture of Cre-hMADSρ⁰ cells and Rosa26R mouse cardiomyocytes shows a marked decrease in number of β-gal⁺/GATA-4⁺ hybrid cells (Cy5, white and Cy3 red staining, respectively) as compared with (G) untreated co-cultures. Scale bars, (A–D) 20 μm, (C–G) 50 μm. (H) Real-time PCR assays showing human (right panel) and mouse (left panel) cardiac gene expression on co-culture with hMADSρ⁰ cells compared to untreated co-cultures. Data represent the mean±SD of at least 3 independent experiments. MEF-2C: myocyte enhancer factor 2C; cTnT: cardiac troponin T