Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

OBJECTIVES: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. MATERIALS AND METHODS: Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. RESULTS: Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although ß-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than ß-AR signalling. In addition, selective activation of α1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α2 - or ß-AR signalling induced no or less differentiation, respectively. EPI- and α1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. CONCLUSIONS: These results demonstrate that activation of ARs, particularly of α1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling.

The long-term differentiation of embryonic stem cells into cardiomyocytes: an indirect co-culture model.

BACKGROUND: Embryonic Stem Cells (ESCs) can differentiate into cardiomyocytes (CMs) in vitro but the differentiation level from ESCs is low. Here we describe a simple co-culture model by commercially available Millicell™ hanging cell culture inserts to control the long-term differentiation of ESCs into CMs. METHODOLOGY/PRINCIPAL FINDINGS: Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with 0.1 mmol/L ascorbic acid to induce the differentiation of ESCs into CMs. In the indirect co-culture system, EBs were co-cultured with epidermal keratinocytes (EKs) or neonatal CMs (NCMs) by the hanging cell culture inserts (PET membranes with 1 µm pores). The molecular expressions and functional properties of ESC-derived CMs in prolonged culture course were evaluated. During time course of ESC differentiation, the percentages of EBs with contracting areas in NCMs co-culture were significantly higher than that without co-culture or in EKs co-culture. The functional maintenance of ESC-derived CMs were more prominent in NCMs co-culture model. CONCLUSIONS/SIGNIFICANCE: These results indicate that NCMs co-culture promote ESC differentiation and has a further effect on cell growth and differentiation. We assume that the improvement of the differentiating efficiency of ESCs into CMs in the co-culture system do not result from the effect of co-culture directly on cell differentiation, but rather by signaling effects that influence the cells in proliferation and long-term function maintenance.

Collagen/ß(1) integrin interaction is required for embryoid body formation during cardiogenesis from murine induced pluripotent stem cells.

BACKGROUND: The interactions between stem cells and extracellular matrix (ECM) mediated by integrins play important roles in the processes that determine stem cell fate. However, the role of ECM/integrin interaction in the formation of embryoid bodies (EBs) during cardiogenesis from murine induced pluripotent stem cells (miPSCs) remains unclear. RESULTS: In the present study, collagen type I and ß(1) integrin were expressed and upregulated synergistically during the formation of miPSC-derived EBs, with a peak expression at day 3 of differentiation. The blockage of collagen/ß(1) integrin interaction by ß(1) integrin blocking antibody resulted in the production of defective EBs that were characterized by decreased size and the absence of a shell-like layer composed of primitive endoderm cells. The quantification of spontaneous beating activity, cardiac-specific gene expression and cardiac troponin T (cTnT) immunostaining showed that the cardiac differentiation of these defective miPSC-derived EBs was lower than that of control EBs. CONCLUSIONS: These findings indicate that collagen/ß(1) integrin interaction is required for the growth and cardiac differentiation of miPSC-derived EBs and will be helpful in future engineering of the matrix microenvironment within EBs to efficiently direct the cardiac fate of pluripotent stem cells to promote cardiovascular regeneration.

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.

Controlling the in vitro differentiation of embryonic stem cells for myocardial tissue engineering applications.

We studied the differentiation of embryonic stem cells (ESCs) and developed a novel protocol for generating functional cardiomyocytes (CMs) from ESCs by co-culturing these with live cardiac cells. We then evaluated the structural and functional properties of these ESC-derived CMs (ESCMs). An acellular matrix obtained from rabbit heart tissues was used as a scaffold. Then ESCMs were seeded onto the acellular matrix for preliminary tissue engineering applications. We found that by mimicking the cardiac microenvironment, the percentage of beating embryoid bodies (EBs) was much higher and the homogeneity of EBs were significantly improved over that seen in the control group (p<0.001). ESCMs in EBs acquired almost the same structural and functional properties as typical CMs. After implantation, the cells in the EBs rapidly grew and expanded in the extracellular matrix. These results indicate that the differentiation of ESCs can be controlled in a cardiac mimicking microenvironment and that ESCs can be used as an ideal cell source for large-scale tissue engineering applications for the procurement of cardiac muscle.

Methods of maintaining human embryonic stem cells continues to be worth studying.

This article gives a brief comment on the culture of human embryonic stem cells (hESCs) with the aim to maintain the potency of hESCs in well state and produce more homogenous cell clones.

Using embryonic stem cells to form a biological pacemaker via tissue engineering technology.

Biological pacemakers can be achieved by various gene-based and cell-based approaches. Embryonic stem cells (ESCs)-derived pacemaker cells might be the most promising way to form biological pacemakers, but there are challenges as to how to control the differentiation of ESCs and to overcome the neoplasia, proarrhythmia, or immunogenicity resulting from the use of ESCs. As a potential approach to solve these difficult problems, tissue-engineering techniques may provide a precise control on the different cell components of multicellular aggregates and the forming of a construct with-defined architectures and functional properties. The combined interactions between ESC-derived pacemaker cells, supporting cells, and matrices may completely reproduce pacemaker properties and result in a steady functional unit to induce rhythmic electrical and contractile activities. As ESCs have a high capability for self-renewal, proliferation, and potential differentiation, we hypothesize that ESCs can be used as a source of pacemaker cells for tissue-engineering applications and the ambitious goal of biological cardiac pacemakers may ultimately be achieved with ESCs via tissue-engineering technology.