TRPV-1 activation through thermal and agonist treatment in the process of scalable cardiac differentiation and tissues fabrication is the novel strategy to eliminate undifferentiated iPS cells in the bioengineered cardiac tissues

The replacement of injured heart tissues with the bioengineered cardiac tissues is expected as a possible therapeutic strategy for heart failure. Currently pluripotent stem cells are the most potent cell source for cardiac cells. However the risk of tumor formation due to the remaining undifferentiated stem cells in the bioengineered tissues remains resolved. We previously developed the scalable three-dimensional suspension bioreactor for human iPS cells and also the culture strategy for cardiac differentiation. Since some conditions including temperature, agitation rate, pH and dissolved oxygen concentration are continuously monitored and regulated in the bioreactor, it might be ideal to eliminate undifferentiated iPS cells in the process of cardiac differentiation with the optimization of these conditions. In the present study, we show that TRPV-1 activation through transient culture at 42 °C or with agonists is a simple and useful strategy to eliminate iPS cells from bioengineered cardiac cell sheet tissues. When feeder free human iPS cells were cultured at 42 °C, almost all cells disappeared by 48 hours through apoptosis. Furthermore when iPS cells were co-cultured with iPS cell-derived cardiac cells at 42 °C for 2 days, the number of Oct4 expressing iPS cells was significantly decreased. Conversely, in spite of cultivation at 42 °C, the number of iPS cell-derived cardiomyocytes and fibroblasts was maintained, and cardiac cell sheets were fabricated after reducing the temperature. TRPV-1 expression in iPS cells was upregulated at 42 °C, and the expression levels were significantly higher than that in cardiomyocytes, suggesting that iPS cells might be more sensitive to TRPV-1 activation than cardiomyocytes, which lead to eliminate iPS cells effectively without affecting cardiomyocyte viability. When cardiac cell sheets were cultured at 42 °C or with TRPV-1 agonist for 2 days, the expression of Lin28 and the number of Lin28 expressing cells were significantly decreased. Furthermore when 42 °C cultivation was applied to the later stage of cardiac differentiation in bioreactor, Lin28 expression was also significantly decreased. These findings suggest that the difference in tolerance to TRPV-1 activation between iPS cells and iPS cell-derived cardiac cells could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues, which will further reduce the risk of tumour formation. Please click Additional Files below to see the full abstract

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

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

A Crucial Role of Activin A-Mediated Growth Hormone Suppression in Mouse and Human Heart Failure

Infusion of bone marrow-derived mononuclear cells (BMMNC) has been reported to ameliorate cardiac dysfunction after acute myocardial infarction. In this study, we investigated whether infusion of BMMNC is also effective for non-ischemic heart failure model mice and the underlying mechanisms. Intravenous infusion of BMMNC showed transient cardioprotective effects on animal models with dilated cardiomyopathy (DCM) without their engraftment in heart, suggesting that BMMNC infusion improves cardiac function via humoral factors rather than their differentiation into cardiomyocytes. Using conditioned media from sorted BMMNC, we found that the cardioprotective effects were mediated by growth hormone (GH) secreted from myeloid (Gr-1(+)) cells and the effects was partially mediated by signal transducer and activator of transcription 3 in cardiomyocytes. On the other hand, the GH expression in Gr-1(+) cells was significantly downregulated in DCM mice compared with that in healthy control, suggesting that the environmental cue in heart failure might suppress the Gr-1(+) cells function. Activin A was upregulated in the serum of DCM models and induced downregulation of GH levels in Gr-1(+) cells and serum. Furthermore, humoral factors upregulated in heart failure including angiotensin II upregulated activin A in peripheral blood mononuclear cells (PBMNC) via activation of NFκB. Similarly, serum activin A levels were also significantly higher in DCM patients with heart failure than in healthy subjects and the GH levels in conditioned medium from PBMNC of DCM patients were lower than that in healthy subjects. Inhibition of activin A increased serum GH levels and improved cardiac function of DCM model mice. These results suggest that activin A causes heart failure by suppressing GH activity and that inhibition of activin A might become a novel strategy for the treatment of heart failure

Functional Thyroid Follicular Cells Differentiation from Human-Induced Pluripotent Stem Cells in Suspension Culture

The replacement of regenerated thyroid follicular cells (TFCs) is a promising therapeutic strategy for patients with hypothyroidism. Here, we have succeeded in inducing functional TFCs from human-induced pluripotent stem cells (iPSCs) in scalable suspension culture. Differentiation of iPSCs with Activin A treatment produced Sox17- and FoxA2-expressing definitive endodermal cells that also expressed thyroid transcription factors Pax8 and Nkx2-1. Further treatment with thyroid-stimulating hormone (TSH) induced TFCs expressing various types of thyroid proteins including TSH receptor, sodium–iodide symporter, thyroglobulin, and thyroid peroxidase. Interestingly, differentiated cells secreted free thyroxine in vitro. These results indicate successful differentiation of human iPSCs to functional TFCs that may enable us to fabricate thyroid tissues for regenerative medicine and disease models