eXtraembryonic ENdoderm (XEN) Stem Cells Produce Factors that Activate Heart Formation

Abstract

BackgroundInitial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.Methodology/Principal FindingsHere, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.Conclusions/SignificanceThese studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

Induction of Cardiomyogenesis in Human Embryonic Stem Cells by Human Embryonic Stem Cell-Derived Definitive Endoderm

“…Hence, although we cannot compare the relative efficacies of hDE and END-2 cells, these both appear to be less potent than embryonic AL endoderm. The relatively modest inductive effect of hDE reported here may indicate that DE has less cardiomyogenic potency than VE, as recently reported using the mouse system [12,16]; we are currently preparing VE from pluripotent cells to examine this possibility in the human system. Although AL endoderm explanted from embryos appears to be more cardiomyogenic than that of cultured endoderm cells, it must be kept in mind that, with the exception of our recent study using mouse EBs [7], studies employing AL endoderm addressed its effect on gastrulation-stage target tissues [21,22], in contrast to this and other studies wherein inductive effects of cultured hDE and mouse endoderm cell-lines [13] were assessed on pluripotent cells.…”

“…Previous studies have shown that endoderm explanted from embryos [7], or endoderm-derived murine cell lines, can induce cardiomyogenesis in pluripotent ESCs [12][13][14][15][16]. The goal of this study was to extend this principle by establishing an all-human system, using endoderm cells that can be generated in large numbers under defined cell culture conditions.…”

“…The concept that DE must be regionally specialized to induce cardiomyogenesis is also consistent with the temporo-spatial expression pattern of known cardiomyogenic molecules, including, Cerberus, hex, and BMP2-4/FGF8 [25][26][27][28], all of which are expressed in AL endoderm adjacent to the heartforming region in the embryo. It is therefore possible that, similar to the differential effects recently ascribed to DE and VE [12,16], the modest cardiomyogenic potency of hDE, in comparison with AL endoderm, reflects the relatively nonspecialized status of hDE when generated in homogeneous cell culture. Other factors may contribute to the relatively modest induction efficiency of hDE described here; for example, these determinations were performed in defined medium consisting of RPMI/B27, the B27 component of which contains a large quantity of insulin, which was recently shown to inhibit cardiomyocyte differentiation in pluripotent cells [29].…”

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states

“…2B). Moreover, cXEN and embryo-derived XEN cells similarly express genes associated with parietal endoderm (Fst, Snai1, Pth1r, Pdgfra, Dab2, Emp2, Fxyd3, Atp6v0a1, Elf1, Foxq1, Col4a1, Lama1, Ttr and Stra6) (Kunath et al, 2005;Brown et al, 2010). Pearson correlation analysis confirms that cXEN and embryo-derived XEN cells have stronger correlation (R 2 value0.97±0.00) than the correlation between either mESCs and cXEN or embryo-derived XEN cells (R 2 value0.90±0.00) (supplementary material Fig.…”

“…qRT-PCR was performed using Quantace Sensimix on an Applied Biosystems 7500 machine (Life Technologies Corporation, CA, USA). Primer pairs were designed using Primer3 software or previously published (Molkentin et al, 1997; Fujikura et al, 2002;Niwa et al, 2005;Brown et al, 2010) and are listed in supplementary material Table S4. …”