In Vivo Generation and the Mechanism of Bone Marrow- and Cord Blood-Derived Cardiomyocytes.
Authors: Ishikawa Fumihiko , Shultz Leonard D. , Fukata Mitsuhifo , Yoshida Shuro , Yasukawa Masaki , Harada Mine 
Published: April 2020

Abstract
(Purpose) We tested the capacity of mouse bone marrow (BM) and human cord blood (CB) cells to give rise to cardiomyocytes in vivo. In the generation of murine BM-derived cardiomyocytes, structural- and functional differences between donor BM- and host-derived myocytes were examined. In the xenogeneic transplantation system, we examined the contribution of cell fusion as mechanism underlying generation of CB-derived cardiomyocytes. (Methods) For syngeneic transplantation, 5x105 lineage-antigen negative (Lin-) BM cells of mice with ubiquitous expression of green fluorescence protein (GFP) were intravenously injected into irradiated neonatal C57BL/6 recipients. For xenogeneic transplantation, 2 x 106 Lin- CB cells were transplanted into NOD/SCID/b2-microglobulinnull (NOD/SCID/b2mnull) mice. Morphological analysis was performed by immunostaining for myocyte-specific antigens (connexin 43, troponin I, and dystrophin). Transmission electron microscopic analysis with immunostaining for GFP was performed to examine the structural difference between GFP+ and GFP− myocytes. In xenogeneic transplantation, double FISH analysis using species-specific probes was performed to identify the origin of each myocyte. (Results) During the maturation of recipient mice, donor-derived GFP+ striated cells were generated and displayed characteristic phenotypic features of cardiomyocytes. Nuclear staining revealed the presence of GFP+ mononucleate- and binucleate cardiomyocytes. The transmission electron microscopic analyses on GFP+ myocytes revealed the presence of abundant mitochondria, which would be needed for continuous contraction. More importantly, the presence of gap junction between GFP+ and GFP− myocytes indicated that BM-derived cardiomyocytes formed functional network with host-derived cardiomyocytes. Mechanical injury on neonatal cardiac apex accelerated the migration of BM-derived cells and generation of GFP+ cardiomyocytes. The kinetic analysis of freshly isolated myocardium revealed that GFP+ myocytes contracted efficiently at the single cell level. In recipient cardiac tissues, GFP+ myocytes, vimentin+ fibroblasts, and smooth muscle cells persisted until 15 months post-transplantation. Finally, human CB Lin- cells generated connexin 43+ cardiomyocytes, following transplantion into immune-deficient neonatal mice. Double FISH analysis using species-specific probes demonstrated that the majority of human chromosome-containing cardiomyocytes in recipient cardiac tissue possessed murine chromosome in their nuclei, while human chromosome+ murine chromosome- cardiomyocytes were also present. (Conclusion) Murine BM and human CB cells can give rise to cardiomyocytes, which show similar morphological and structural characteristics to those of host-derived cardiomyocytes. In conclusion, hematopoietic tissue-derived cells can contribute to the generation of cardiomyocytes mainly by fusion-dependent and partly by fusion-independent mechanism even across a xenogeneic histocompatibility barrier.