Natriuretic peptides in ectopic myocardial tissues originating from mouse embryonic stem cells

Abstract

In a previous report we described the survival and contractile function of mouse embryonic stem cell-derived cardiomyocytes in the host retroperitoneum. To further understand the nature of embryonic stem cell-derived cardiomyocytes, the study assessed the synthesis of natriuretic peptides in ectopic myocardial tissues of embryonic stem cell origin. Cardiomyocytes formed in embryoid body outgrowths were transplanted into the retroperitoneum of adult nude mice, and the myocardial tissues that developed were characterized by RT-PCR and immunohistochemistry concerning atrial and brain natriuretic peptides (ANP, BNP). In the outgrowths of embryoid bodies in vitro, gene expression of ANP and BNP was detected by RT-PCR and granules positive for the peptides were identified in a few cardiomyocytes by light and electron microscopic immunocytochemistry. Seven days after transplantation the transplants exhibited multidifferentiated teratoma tissues. Developing chamber myocardial tissues positive for cardiac troponin I, cadherin, and connexin 43 were evident in the transplants, which contained ANP-positive cardiomyocytes. Transplants with beating bundles were observed 30 days after transplantation, in which gene expression of both natriuretic peptides was detected. Myocardial tissues with abundant ANP-immunoreactivity, as well as with BNP-immunoreactivity to a lesser extent, were evident in the transplants. Also, myocardial tissues without immunoreactivity for natriuretic peptides were observed. Immunoelectron microscopy showed discernible secretory granules containing ANP and/or BNP in the cardiomyocytes. These results showed that part of the cardiomyocytes in embryonic stem cell-derived ectopic myocardial tissues are capable of producing natriuretic peptides, which suggests that they may be used as an endocrine source for cardiac hormones.

Structural differentiation, proliferation, and association of human embryonic stem cell-derived cardiomyocytes in vitro and in their extracardiac tissues

“…The serial sections from the beating areas were cut to a thickness of 5 m. After deparaYnization and rehydration, the antigen retrieval procedure already described was also performed prior to immuno-staining. Then the sections were doubly stained for cardiac troponin I and one of the followings: connexin 43, pan-cadherin (clone CH-19, mouse IgG1, Sigma), atrial natriuretic peptide (ANP; rabbit polyclonal, Chemicon) (Johkura et al, 2005), and PCNA. After labeling with the appropriate Xuorescence secondary antibodies, and nuclear counterstain with DAPI, specimens were observed by CLSM.…”

Localization of Liv2 as an Immature Hepatocyte Marker in EB Outgrowth

“…Pre-and postembedding methods were performed to determine the microlocalization of Liv2. Livers of E11.5 embryos and EB outgrowths were fixed in 4% paraformaldehyde/0.1% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) at 4°C for 1 h and embedded in Lowicryl K4M at −20°C [7]. Ultrathin sections were immunostained with anti-Liv2 antibody and a protein-G gold solution containing 15-nm gold particles (ICN Biomedicals, Aurora, OH).…”