ABSTRACT
Cardiomyocytes have been induced from various pluripotent cells, such as embryonic stem cells and myeloid stem cells; however, the generation of cardiac tissues beyond two-dimensional cell-sheets has not been reported. Creating higher order, three-dimensional structures that are unique to heart is the long-awaited next step in realizing cardiac regenerative medicine. We have previously shown that cardiomyocytes can be induced in vitro from undifferentiated cells (animal caps) excised from Xenopus embryos. Cardiomyocytes were induced by first dissociating the animal caps and then reaggregating them following treatment with activin. Here, we describe an interesting method for creating a complete ectopic heart in vivo, involving the introduction of in vitro-created tissue during early embryogenesis. Thus, animal cap reaggregates were transplanted into the abdomen of late-neurula-stage embryos, resulting in two-chambered hearts being formed. The dual-heart larvae matured into adult animals with transplanted hearts intact. Involvement of transplanted hearts in systemic circulation was demonstrated. Moreover, the ectopic hearts possessed higher order structures such as atrium and ventricle, and were morphologically, histologically, and electrophysiologically identical to original hearts. This system should facilitate the study of heart organogenesis and may promote a shift from tissue to organ engineering for clinical applications.
Subject(s)
Embryo, Nonmammalian/physiology , Heart/physiology , Myocardium/cytology , Xenopus laevis/physiology , Action Potentials/drug effects , Activins/pharmacology , Animals , Atrial Natriuretic Factor/analysis , Echocardiography, Doppler , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/transplantation , Heart/embryology , Heart Transplantation/methods , Immunohistochemistry , Microscopy, Electron , Myocardium/metabolism , Myocardium/ultrastructure , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Myocytes, Cardiac/transplantation , Xenopus laevis/embryology , Xenopus laevis/metabolismABSTRACT
Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.
Subject(s)
Bone Marrow Cells/physiology , Multipotent Stem Cells/physiology , Neurons/physiology , Nuclear Proteins/metabolism , Oncogene Proteins, Viral/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Stromal Cells/physiology , Telomerase/metabolism , Aged , Aged, 80 and over , Animals , Bone Marrow Cells/cytology , Calcium/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation/physiology , Cell Lineage , Cells, Cultured , DNA-Binding Proteins , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Karyotyping , Mice , Multipotent Stem Cells/cytology , Neurons/cytology , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Viral/genetics , Polycomb Repressive Complex 1 , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Stromal Cells/cytology , Telomerase/genetics , Telomere/metabolismABSTRACT
BACKGROUND: Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell-cycle-associated genes can differentiate into cardiomyocytes in vitro. METHODS: We attempted to prolong the life span of hMSCs by infecting retrovirus encoding bmi-1, human papillomavirus E6 and E7, and/or human telomerase reverse transcriptase genes. To determine whether the hMSCs with an extended life span could differentiate into cardiomyocytes, 5-azacytidine-treated hMSCs were co-cultured with fetal cardiomyocytes in vitro. RESULT: The established hMSCs proliferated over 150 population doublings. On day 3 of co-cultivation, the hMSCs became elongated, like myotubes, began spontaneously beating, and acquired automaticity. Their rhythm clearly differed from that of the surrounding fetal mouse cardiomyocytes. The number of beating cardiomyocytes increased until 3 weeks. hMSCs clearly exhibited differentiated cardiomyocyte phenotypes in vitro as revealed by immunocytochemistry, RT-PCR, and action potential recording. CONCLUSIONS: The life span of hMSCs was prolonged without interfering with cardiomyogenic differentiation. hMSCs with an extended life span can be used to produce a good experimental model of cardiac cell transplantation and may serve as a highly useful cell source for cardiomyocytic transplantation.
