Brief report: VGLL4 is a novel regulator of survival in human embryonic stem cells.

Human embryonic stem cells (hESCs) are maintained in a self-renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes using a gain-of-function screen of a large collection of human open reading frames. We identified Vestigial-like 4 (VGLL4), a cotranscriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain-of-function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process. Stem Cells 2013;31:2833-2841.

Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart.

The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.

Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11-30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.

Live Cell Monitoring and Enrichment of Stem Cell-Derived ß Cells Using Intracellular Zinc Content as a Population Marker.

Our laboratory and others have developed protocols to generate glucose-responsive stem cell-derived ß cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell-based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell-derived ß cells and gene-editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell-derived ß cells. This purification of SC-ß cells is based on intracellular zinc content and is a simple method to complement other approaches for generating and assaying these cells. © 2019 The Authors. Basic Protocol: Fluorescent labeling and isolation of stem cell-derived ß cells.

Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium.

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.

Developmental bias in cleavage-stage mouse blastomeres.

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.

Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells.

The proepicardial organ is an important transient structure that contributes cells to various cardiac lineages. However, its contribution to the coronary endothelium has been disputed, with conflicting data arising in chick and mouse. Here we resolve this conflict by identifying two proepicardial markers, Scleraxis (Scx) and Semaphorin3D (Sema3D), that genetically delineate heretofore uncharacterized proepicardial subcompartments. In contrast to previously fate-mapped Tbx18/WT-1-expressing cells that give rise to vascular smooth muscle, Scx- and Sema3D-expressing proepicardial cells give rise to coronary vascular endothelium both in vivo and in vitro. Furthermore, Sema3D(+) and Scx(+) proepicardial cells contribute to the early sinus venosus and cardiac endocardium, respectively, two tissues linked to vascular endothelial formation at later stages. Taken together, our studies demonstrate that the proepicardial organ is a molecularly compartmentalized structure, reconciling prior chick and mouse data and providing a more complete understanding of the progenitor populations that establish the coronary vascular endothelium.

Modulating Notch signaling to enhance neovascularization and reperfusion in diabetic mice.

Diabetes can diminish the responsiveness to angiogenic factors (e.g., VEGF) important for wound healing and the treatment of ischemic diseases, and this study investigated the hypothesis that this effect can be reversed by altering Notch signaling. Aortic endothelial cells (ECs) isolated from diabetic mice demonstrated reduced sprouting capability in vitro, but adding a Notch inhibitor (DAPT) led to cell-density and VEGF-dose dependent enhancement of proliferation, migration and sprouting, in both 2-D and 3-D cultures, as compared to VEGF alone. The in vivo effects of VEGF and DAPT were tested in the ischemic hind limbs of diabetic mice. Combining VEGF and DAPT delivery resulted in increased blood vessel density (∼150%) and improved tissue perfusion (∼160%), as compared to VEGF alone. To examine if DAPT would interfere with vessel maturation, DAPT was also delivered with a combination of VEGF and platelet derived growth factor (PDGF). DAPT and PDGF did not interfere with the effects of the other, and highly functional and mature networks of vessels could be formed with appropriate delivery. In summary, modulating Notch signaling enhances neovascularization and perfusion recovery in diabetic mice suffering from ischemia, suggesting this approach could have utility for human diabetics.

Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field.

A hallmark of heart-valve development is the swelling and deposition of extracellular matrix in the heart-valve region. Only myocardium overlying this region can signal to underlying endothelium and cause it to lose cell-cell contacts, delaminate, and invade the extracellular space abutting myocardium and endocardium to form endocardial cushions (EC) in a process known as epithelial to mesenchymal transformation (EMT). The heart-valve myocardium expresses bone morphogenetic protein-2 (Bmp2) coincident with development of valve mesenchyme. BMPs belong to the transforming growth factor beta superfamily (TGF-beta) and play a wide variety of roles during development. We show that conditional ablation of Bmp2 in cardiac progenitors results in cell fate changes in which the heart-valve region adopts the identity of differentiated chamber myocardium. Moreover, Bmp2-deficient hearts fail to induce production and deposition of matrix at the heart-valve-forming region, resulting in the inability of the endothelium to swell and impairing the development of ECs. Furthermore, in collagen invasion assays, Bmp2 mutant endothelium is incapable of undergoing EMT, and addition of BMP2 protein to mutant heart explants rescues this phenotype. Our results demonstrate that Bmp2 is both necessary and sufficient to specify a field of cardiac progenitor cells as the heart-valve-inducing region amid developing atria and ventricles.

Development of heart valves requires Gata4 expression in endothelial-derived cells.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions, the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form the mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in the formation of portions of the atrial and ventricular septae, and the generation of thin, pliable valves. Gata4, which encodes a zinc finger transcription factor, is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3 downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in a decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.