Katsuhisa Matsuura

G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo

Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.

Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice

Cardiac progenitor cells are a potential source of cell therapy for heart failure. Although recent studies have shown that transplantation of cardiac stem/progenitor cells improves function of infarcted hearts, the precise mechanisms of the improvement in function remain poorly understood. The present study demonstrates that transplantation of sheets of clonally expanded stem cell antigen 1-positive (Sca-1-positive) cells (CPCs) ameliorates cardiac dysfunction after myocardial infarction in mice. CPC efficiently differentiated into cardiomyocytes and secreted various cytokines, including soluble VCAM-1 (sVCAM-1). Secreted sVCAM-1 induced migration of endothelial cells and CPCs and prevented cardiomyocyte death from oxidative stress through activation of Akt, ERK, and p38 MAPK. Treatment with antibodies specific for very late antigen-4 (VLA-4), a receptor of sVCAM-1, abolished the effects of CPC-derived conditioned medium on cardiomyocytes and CPCs in vitro and inhibited angiogenesis, CPC migration, and survival in vivo, which led to attenuation of improved cardiac function following transplantation of CPC sheets. These results suggest that CPC transplantation improves cardiac function after myocardial infarction through cardiomyocyte differentiation and paracrine mechanisms mediated via the sVCAM-1/VLA-4 signaling pathway.

Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.

Regenerative therapies have currently emerged as one of the most promising treatments for repair of the damaged heart. Recently, numerous researchers reported that isolated cell injection treatments can improve heart function in myocardial infarction models. However, significant cell loss due to primary hypoxia or cell wash-out and difficulty to control the location of the grafted cells remains problem. As an attempt to overcome these limitations, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cells (two-dimensional), cell sheets, to construct three-dimensional cell-dense tissues. Cell sheet transplantation has been able to recover damaged heart function. However, no detailed analysis for transplanted cell survival has been previously performed. The present study compared the survival of cardiac cell sheet transplantation to direct cell injection in a rat myocardial infarction model. Luciferase-expressing neonatal rat cardiac cells were harvested as cell sheets from temperature-responsive culture dishes. The transplantation of cell sheets was compared to the direct injection of isolated cells dissociated with trypsin-ethylenediaminetetraacetic acid. These grafts were transplanted to infarcted rat hearts and cardiac function was assessed by echocardiography at 2 and 4 weeks after transplantation. In vivo bioluminescence and histological analyses were performed to examine cell survival. Cell sheet transplantation consistently yielded greater cell survival than cell injection. Immunohistochemistry revealed that cardiac cell sheets existed over the infarcted area as an intact layer. In contrast, the injected cells were scattered with relatively few cardiomyocytes in the infarcted areas. Four weeks after transplantation, cardiac function was also significantly improved in the cell sheet transplantation group compared with the cell injection. Twenty-four hours after cell grafting, significantly greater numbers of mature capillaries were also observed in the cardiac cell sheet transplantation. Additionally, the numbers of apoptotic cells with deterioration of integrin-mediated attachment were significantly lower after cardiac cell sheet transplantation. In accordance with increased cell survival, cardiac function was significantly improved after cardiac cell sheet transplantation in comparison to cell injection. Cell sheet transplantation can repair damaged hearts through improved cell survival and should become a promising therapy in cardiovascular regenerative medicine.

Critical Roles of Muscle-Secreted Angiogenic Factors in Therapeutic Neovascularization

The discovery of bone marrow–derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 “no-option” patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1&bgr; (IL-1&bgr;). We then examined an experimental model of limb ischemia using IL-1&bgr;–deficient mice. Implantation of IL-1&bgr;–deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1&bgr;–deficient mononuclear cells increased expression of IL-1&bgr; and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1&bgr; markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle

The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle–derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.

Cell sheet approach for tissue engineering and regenerative medicine.

After the biotech medicine era, regenerative medicine is expected to be an advanced medicine that is capable of curing patients with difficult-to-treat diseases and physically impaired function. Our original scaffold-free cell sheet-based tissue engineering technology enables transplanted cells to be engrafted for a long time, while fully maintaining their viability. This technology has already been applied to various diseases in the clinical setting, including the cornea, esophagus, heart, periodontal ligament, and cartilage using autologous cells. Transplanted cell sheets not only replace the injured tissue and compensate for impaired function, but also deliver growth factors and cytokines in a spatiotemporal manner over a prolonged period, which leads to promotion of tissue repair. Moreover, the integration of stem cell biology and cell sheet technology with sufficient vascularization opens possibilities for fabrication of human three-dimensional vascularized dense and intact tissue grafts for regenerative medicine to parenchymal organs.

Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes

Cell transplantation could be a potential therapy for heart damage. Skeletal myoblasts have been expected to be a good cell source for autologous transplantation; however, the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses the stem cell population that is distinct from myoblasts. To elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes, we cocultured skeletal muscle cells isolated from transgenic mice expressing green fluorescent protein with cardiomyocytes of neonatal rats. Skeletal muscle‐derived cells expressed cardiac‐specific proteins such as cardiac troponin T and atrial natriuretic peptide as well as cardiac‐enriched transcription factors such as Nkx2E (formerly called Csx/Nkx2.5) and GATA4 by coculture with cardiomyocytes. Skeletal muscle‐derived cells also expressed cadherin and connexin 43 at the junctions with neighboring cardiomyocytes. Cardiomyocyte‐like action potentials were recorded from beating skeletal muscle‐derived cells. Treatment of nifedipine or culture in Ca2+‐free media suppressed contraction of cardiomyocytes and inhibited skeletal muscle cells to express cardiac‐specific proteins. Cyclic stretch completely restored this inhibitory effect. These results suggest that some part of skeletal muscle cells can transdifferentiate into cardiomyocytes and that direct cell‐to‐cell contact and contraction of neighboring cardiomyocytes are important for the transdifferentiation.

Creation of human cardiac cell sheets using pluripotent stem cells

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.

Creation of mouse embryonic stem cell-derived cardiac cell sheets

Research on heart tissue engineering is an exciting and promising area. Although we previously developed bioengineered myocardium using cell sheet-based tissue engineering technologies, the issue of appropriate cell sources remained unresolved. In the present study, we created cell sheets of mouse embryonic stem (ES) cell-derived cardiomyocytes after expansion in three-dimensional stirred suspension cultures. Serial treatment of the suspension cultures with noggin and granulocyte colony-stimulating factor significantly increased the number of cardiomyocytes by more than fourfold compared with untreated cultures. After drug selection for ES cells expressing the neomycin-resistance gene under the control of the α-myosin heavy chain promoter, almost all of the cells showed spontaneous beating and expressed several cardiac contractive proteins in a fine striated pattern. When ES-derived cardiomyocytes alone were seeded onto temperature-responsive culture dishes, cell sheets were not created, whereas cocultures with cardiac fibroblasts promoted cell sheet formation. The cardiomyocytes in the cell sheets beat spontaneously and synchronously, and expressed connexin 43 at the edge of adjacent cardiomyocytes. Furthermore, when the extracellular action potential was recorded, unidirectional action potential propagation was observed. The present findings suggest that stirred suspension cultures with appropriate growth factors are capable of producing cardiomyocytes effectively and easily, and that ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium.