Objectives
Extraembryonic endoderm (XEN) cells are isolated from primitive endoderm (PrE) of blastocysts. Just like PrE, XEN cells have the ability to differentiate into parietal endoderm (PE) and visceral endoderm (VE), and therefore, they are useful tools for studying mechanisms of PrE cells development and differentiation. Pig is an ideal model for studying human cardiovascular and metabolic diseases and a potential organ source for allotransplantation, while no XEN cell has been obtained from porcine embryos.
Materials and Methods
Using a serum‐free culture system, we directly derived porcine extraembryonic endoderm‐like cells (pXEN‐like cells) from day 6‐7 blastocysts, which could maintain self‐renewal for at least 30 passages.
Results
The pXEN‐like cells resembled mouse XEN cells with large and flat clone morphology and expressed XEN marker genes but not pluripotent genes. Upon in vitro induction, the cells could differentiate into VE and PE. FGF/MEK signalling was not only essential for the maintenance of pXEN‐like cells, but also the induction of pXEN‐like cells from porcine embryonic stem (pES) cells.
Conclusions
We directly obtained cell lines with XEN characteristics from porcine embryos for the first time. The cells will be helpful tools for studying embryonic development and cell differentiation, which also represent promising cell sources for human regenerative medicine.
The stimulatory G-protein alpha subunit (Gsα), a ubiquitously expressed protein, mediates G-protein receptor-stimulated signal transduction. To investigate the functions of Gsα in cardiomyocytes. We developed transverse aortic constriction (TAC)-induced heart failure mouse models and tamoxifen-inducible transgenic mice with cardiac-specific Gsα disruption. We detected alterations in Gsα expression in TAC-induced heart failure mice. Moreover, we examined cardiac function and structure in mice with genetic Gsα deletion and investigated the underlying molecular mechanisms of Gsα function. We found that Gsα expression increased during the compensated cardiac hypertrophy period and decreased during the heart failure period. Moreover, cardiac-specific Gsα disruption deteriorated cardiac function and induced severe cardiac remodeling. Mechanistically, Gsα disruption decreased CREB1 expression and inhibited the Bmp10-mediated signaling pathway. In addition, we found that Gsα regulates Bmp10 expression through the binding of CREB1 to the Bmp10 promoter. Our results suggest that fluctuations in Gsα levels may play a vital role in the development of heart failure and that loss of Gsα function facilitates cardiac remodeling.
Fetal bovine serum (FBS) supplementation has beneficial effects on invitro porcine embryonic development, but the underlying mechanisms are unclear. In the present study we found that the addition of FBS to PZM-3 increased the number of cells in porcine blastocysts and hatching rate invitro primarily by promoting proliferation of the inner cell mass and further differentiation. Moreover, based on the following results, we surmise that FBS benefits blastocyst development by activating Rho-associated kinase (ROCK) signalling: (1) the ROCK signalling inhibitor Y-27632 decreased the blastocyst rate and the number of cells in blastocysts, whereas FBS rescued the developmental failure induced by Y-27632; (2) the mRNA levels of two ROCK isoforms, ROCK1 and ROCK2, were significantly increased in blastocysts derived from medium containing FBS; and (3) FBS increased RhoA/Rho-kinase expression in the nucleus of embryonic cells. These results indicate that FBS promotes the invitro development of porcine embryos by activating ROCK signalling in a chemically defined medium.
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