A 3D"Sandwich" Co-culture System with hPPSCs and hUVECs Supports Mouse Embryo Development from E3.5 to E7.5 In Vitro

Abstract

Background A variety of methods for ex-utero culture systems have been explored, but there remain limitations in terms of in vitro culture platforms used prior to the implantation of mouse embryos, and the ability of mouse blastocysts to develop normally in vitro has not been established. Whether vascular niche support mouse embryo development from embryonic day (E) 3.5 to E7.5 in vitro is unknown.Methods We established a three-dimensional (3D) “sandwich” vascular-niche culture system with in vitro culture medium (IVCM) with human placenta perivascular stem cells (hPPSCs) and human umbilical vein endothelial cells (hUVECs), as supportive cells, which were seeded into the bottom layer of Matrigel to test mouse embryos from E3.5 to E7.5 in vitro. Mouse embryos from E3.5 to E7.5 development rates and greatest diameters at each stage were quantitatively determinated using statistics of SPSS software. Pluripotent markers and embryo transplantation in vivo were used to monitor mouse embryo quality and function.Results Embryos in the IVCM + Cells (hPPSCs + hUVECs) showed higher development rates and greatest diameters at each stage than those in the IVCM group. Embryos in the IVCM + Cells group cultured to E5.5 resembled natural egg cylinders in morphology and expressed specific embryonic cell markers, including Oct4 and Nanog, which were features similar to embryos developed in vivo. After transplantation, the embryos could be re-implanted in the internal uterus and continue to develop to a certain stage.Conclusions Therefore, the 3D in vitro culture system enabled the development of embryos from E3.5 to E7.5, and the vascularization microenvironment constructed by Matrigel, hPPSCs, and hUVECs significantly promoted the development of implanted embryos. This system allowed us to further study the physical and molecular mechanisms of embryo implantation in vitro.