Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×108 cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×108. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.

Background Fibroblasts are indispensable for the fabrication of cell-sheet–based bioengineered cardiac tissues; however, whether cardiac fibroblasts can improve tissue properties for transplantation or in vitro models compared with other fibroblast types remains unclear. We compared the cell organization and functional properties of cardiomyocyte sheets derived from co-culture with different fibroblast types and investigated the molecular mechanisms for the observed differences. Methods and results Cardiac cell sheets were fabricated by co-culturing mouse embryonic stem cell (ESC)-derived cardiomyocytes with mouse neonatal cardiac fibroblasts (NCFs), mouse adult cardiac fibroblasts (ACFs), and mouse adult dermal fibroblasts (ADFs). Cardiac cell sheets obtained from NCF or ACF co-culture showed numerous uniformly distributed and functional (beating) cardiomyocytes, while cell sheets obtained by co-culture with ADFs showed fewer and aggregated cardiomyocytes. The greater number of cardiomyocytes in the presence of NCFs was because of enhanced cardiomyocyte proliferation, as revealed by protein markers of mitosis and BrdU incorporation. Microarray analysis revealed that NCFs expressed substantially higher levels of vascular cell adhesion molecule-1 (VCAM-1) than ADFs. Treatment of ESC-derived cardiomyocytes in monoculture with soluble VCAM-1 significantly increased the number of functional cardiomyocytes, while the enhancement of cardiomyocyte number by co-culture with NCFs was abolished by anti-VCAM-1 antibodies. Conclusions Cardiac fibroblasts enhance the proliferation of ESC-derived cardiomyocytes through VCAM-1 signaling, leading to an increase in functional myocardial cells in bioengineered tissue sheets. These sheets may be advantageous for cell-based therapy and in vitro heart research.

Cardiovascular diseases are a leading cause of death worldwide. After an ischemic injury, the myocardium undergoes severe necrosis and apoptosis, leading to a dramatic degradation of function. Numerous studies have reported that cardiac fibroblasts (CFs) play a critical role in heart function even after injury. However, CFs present heterogeneous characteristics according to their development stage (i.e., fetal or adult), and the molecular mechanisms by which they maintain heart function are not fully understood. The aim of this study is to explore the hypothesis that a specific population of CFs can repair the injured myocardium in heart failure following ischemic infarction, and lead to a significant recovery of cardiac function. Flow cytometry analysis of CFs defined two subpopulations according to their relative expression of vascular cell adhesion molecule 1 (VCAM1). Whole-transcriptome analysis described distinct profiles for these groups, with a correlation between VCAM1 expression and lymphangiogenesis-related genes up-regulation. Vascular formation assays showed a significant stimulation of lymphatic cells network complexity by VCFs. Injection of human VCAM1-expressing CFs (VCFs) in postinfarct heart failure rat models (ligation of the left anterior descending artery) led to a significant restoration of the left ventricle contraction. Over the course of the experiment, left ventricular ejection fraction and fractional shortening increased by 16.65% ± 5.64% and 10.43% ± 6.02%, respectively, in VCF-treated rats. Histological examinations revealed that VCFs efficiently mobilized the lymphatic endothelial cells into the infarcted area. In conclusion, human CFs present heterogeneous expression of VCAM1 and lymphangiogenesis-promoting factors. VCFs restore the mechanical properties of ventricular walls by mobilizing lymphatic endothelial cells into the infarct when injected into a rat heart failure model. These results suggest a role of this specific population of CFs in the homeostasis of the lymphatic system in cardiac regeneration, providing new information for the study and therapy of cardiac diseases.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Introduction: Cardiac lymphangiogenesis has attracted attention as a therapeutic target after myocardial infarction (MI). Lymphangiogenic remodeling has been observed after MI in adult and fetal hearts; however, reparative lymphangiogenic remodeling is only observed in fetal hearts. The factors that determine the fate of this phenomenon have not been fully elucidated. Hypothesis: We demonstrated that a specific population of VCAM1 + human fetal cardiac fibroblasts (fCFs) restore cardiac function post-MI by lymphangiogenesis. Thus, we hypothesize that adult cardiac fibroblasts (aCFs), compared to fCFs, possess a different distribution of fibroblasts with differing lymphangiogenic potential. Furthermore, we also hypothesize that aCFs can be exogenously manipulated to acquire fCFs-like reparative lymphangiogenic potential, which can be used as a cell therapy for heart failure. Methods: Flow cytometry assessed CD90 and VCAM1 expression of aCFs and fCFs. To shift aCFs towards a fCF phenotype, TNF-α and IL-4 were added to culture medium. aCF subpopulations were intramyocardially injected in nude rats and swine post-MI with subsequent echocardiography. Myocardial tissue staining (Sirius Red, LYVE1) and RNA-seq were performed to identify the molecular mechanism. Results: aCFs and fCFs exhibited different distributions of CD90 and VCAM1 expression, where aCFs showed lower CD90 and VCAM1 expression. The addition of TNF-α and IL-4 shifted the localization of VCAM1 + aCFs towards a fCF distribution by activation of NF-κB. VCAM1 + CD90 + fCFs-like aCFs provided a sustained improvement in left ventricular ejection fraction and showed reduced fibrosis and increased lymphangiogenesis. This effect was recapitulated in a large animal model. In terms of the molecular mechanism, 13 candidate genes were identified. Conclusions: These findings suggest that the heterogenous and plastic polarity of aCFs and fCFs determines the fate of the lymphangiogenic response after MI and that this response can be regulated by 13 genes. This artificial creation of the VCAM1 + fCFs-like fibroblast environment after MI has enabled a clinical trial for a new cell therapy for inducing reparative lymphangiogenesis (clinical trial ID: jRCT2033210078).