Self-Renewal Requirements of Human Embryonic Stem Cells and Their Engraftment Potential in Mouse Blastocysts

Human embryonic stem cells (hESCs) are a unique population of cells derived from a 6 day old human embryo that can be maintained indefinitely in vitro and have the ability to differentiate to all adult cell types. In addition to their potential for cell based therapies in the treatment of disease and injury, the broad developmental capacity of hESCs offers potential for studying the origins of all human cell types. Embryonic stem cells were first derived from mouse embryos (mESCs), and years of work have demonstrated their utility to developmental research, but relatively little is known about human ESCs. The experiments described below address two fundamental questions in hESC biology: First, what are the molecular signaling pathways that are relevant to hESC sternness ? And second, can hESCs contribute to specialized human cell types in the context of mouse embryogenesis? Because it plays a prominent role in the early cell fate decisions of embryonic development, we examined the role of TGFP superfamily signaling in hESCs. W e found that, in undifferentiated cells, the TGFp/activin/nodal branch is activated (through the signal transducer Smad2/3) while the BMP/GDF branch (Smadl/5) is only active in isolated mitotic cells. Upon early differentiation, Smad2/3 signaling is decreased while Smadl/5 signaling is activated. We next tested the functional role of TGFp/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extended these findings to show that Smad2/3 activation is required downstream of Wnt signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, Smad2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a critical role for TGFP signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFP and Wnt signaling in these contexts. To date, the emergence of specialized cells from hESCs has commonly been studied in tissue culture or upon injection into adult mice, yet these methods have stopped short of demonstrating the potential exhibited by mESCs, which can give rise to every cell type when combined with embryos at the blastocyst stage. Due to obvious barriers precluding the use of human embryos in similar cell mixing experiments with hESCs, human/non-human chimeras may need to be generated for this purpose. In order to define the developmental potential of hESCs in the context of embryogenesis, we explored the ability of hESCs to engraft into mouse blastocysts. In advance of these cell mixing experiments, we derived a new hESC line, RUES1, and characterized its marker expression, functional characteristics and gene expression profiles. Using this new line, we showed that hESCs engrafted into mouse blastocysts, where they, proliferated and differentiated in vitro and persisted in mouse/human embryonic chimeras that implanted and developed in the uterus of pseudopregnant foster mice. Embryonic chimeras generated in this way offer the opportunity to study the behavior of specialized human cell types in a non-human animal model. Our data demonstrate the feasibility of this approach, using mouse embryos as a surrogate for hESC differentiation

Contribution of human embryonic stem cells to mouse blastocysts

AbstractIn addition to their potential for cell-based therapies in the treatment of disease and injury, the broad developmental capacity of human embryonic stem cells (hESCs) offers potential for studying the origins of all human cell types. To date, the emergence of specialized cells from hESCs has commonly been studied in tissue culture or in teratomas, yet these methods have stopped short of demonstrating the ESC potential exhibited in the mouse (mESCs), which can give rise to every cell type when combined with blastocysts. Due to obvious barriers precluding the use of human embryos in similar cell mixing experiments with hESCs, human/non-human chimeras may need to be generated for this purpose. Our results show that hESCs can engraft into mouse blastocysts, where they proliferate and differentiate in vitro and persist in mouse/human embryonic chimeras that implant and develop in the uterus of pseudopregnant foster mice. Embryonic chimeras generated in this way offer the opportunity to study the behavior of specialized human cell types in a non-human animal model. Our data demonstrate the feasibility of this approach, using mouse embryos as a surrogate for hESC differentiation

New Dimensions in Vascular Engineering: Opportunities for Cancer Biology

Angiogenesis is a fundamental prerequisite for tissue growth and thus an attractive target for cancer therapeutics. However, current efforts to halt tumor growth using antiangiogenic agents have been met with limited success. A reason for this may be that studies aimed at understanding tissue and organ formation have to this point utilized two-dimensional cell culture techniques, which fail to faithfully mimic the pathological architecture of disease in an in vivo context. In this issue of Tissue Engineering, the work of Fischbach-Teschl's group manipulate such variables as oxygen concentration, culture three-dimensionality, and cell–extracellular matrix interactions to more closely approximate the biophysical and biochemical microenvironment of tumor angiogenesis. In this article, we discuss how novel tissue engineering platforms provide a framework for the study of tumorigenesis under pathophysiologically relevant in vitro culture conditions

Notch hyper-activation drives trans-differentiation of hESC-derived endothelium

During development, endothelial cells (EC) display tissue-specific attributes that are unique to each vascular bed, as well as generic signaling mechanisms that are broadly applied to create a patent circulatory system. We have previously utilized human embryonic stem cells (hESC) to generate tissue-specific EC sub-types (Rafii et al., 2013) and identify pathways that govern growth and trans-differentiation potential of hESC-derived ECs (James et al., 2010). Here, we elucidate a novel Notch-dependent mechanism that induces endothelial to mesenchymal transition (EndMT) in confluent monolayer cultures of hESC-derived ECs. We demonstrate density-dependent induction of EndMT that can be rescued by the Notch signaling inhibitor DAPT and identify a positive feedback signaling mechanism in hESC-ECs whereby trans-activation of Notch by DLL4 ligand induces elevated expression and surface presentation of DLL4. Increased Notch activation in confluent hESC-EC monolayer cultures induces areas of EndMT containing transitional cells that are marked by increased Jagged1 expression and reduced Notch signal integration. Jagged1 loss of function in monolayer hESC-ECs induces accelerated feedback stimulation of Notch signaling, increased expression of cell-autonomous, cis-inhibitory DLL4, and EndMT. These data elucidate a novel interplay of Notch ligands in modulating pathway activation during both expansion and EndMT of hESC-derived ECs

Generation of Stable Co-Cultures of Vascular Cells in a Honeycomb Alginate Scaffold

Scaffold-guided vascular tissue engineering has been investigated as a means to generate functional and transplantable vascular tissue grafts that increase the efficacy of cell-based therapeutic strategies in regenerative medicine. In this study, we employed confocal microscopy and three-dimensional reconstruction to assess the engraftment and growth potential of vascular cells within an alginate scaffold with aligned pores. We fabricated honeycomb alginate scaffolds with aligned pores, whose surface was immobilized with fibronectin and subsequently coated with matrigel. Endothelial cells were seeded into aligned pore scaffolds in the presence and absence of human smooth muscle cells. We showed that endothelial cells seeded into alginate scaffolds attach on the surface of aligned pores in vitro, giving rise to stable co-cultures of vascular cells. Moreover, the three-dimensional alginate depots containing the cells were exposed to laminar flow in order to recapitulate physiological shear stress found in the vasculature in vivo. After the flow exposure, the scaffold remained intact and some cells remained adherent to the scaffold and aligned in the flow direction. These studies demonstrate that alginate scaffolds provide a suitable matrix for establishing durable angiogenic modules that may ultimately enhance organ revascularization

Prominin 1/CD133 endothelium sustains growth of proneural glioma.

In glioblastoma high expression of the CD133 gene, also called Prominin1, is associated with poor prognosis. The PDGF-driven proneural group represents a subset of glioblastoma in which CD133 is not overexpressed. Interestingly, this particular subset shows a relatively good prognosis. As with many other tumors, gliobastoma is believed to arise and be maintained by a restricted population of stem-like cancer cells that express the CD133 transmembrane protein. The significance of CD133(+) cells for gliomagenesis is controversial because of conflicting supporting evidence. Contributing to this inconsistency is the fact that the isolation of CD133(+) cells has largely relied on the use of antibodies against ill-defined glycosylated epitopes of CD133. To overcome this problem, we used a knock-in lacZ reporter mouse, Prom1(lacZ/+) , to track Prom1(+) cells in the brain. We found that Prom1 (prominin1, murine CD133 homologue) is expressed by cells that express markers characteristic of the neuronal, glial or vascular lineages. In proneural tumors derived from injection of RCAS-PDGF into the brains of tv-a;Ink4a-Arf(-/-) Prom1(lacZ/+) mice, Prom1(+) cells expressed markers for astrocytes or endothelial cells. Mice co-transplanted with proneural tumor sphere cells and Prom1(+) endothelium had a significantly increased tumor burden and more vascular proliferation (angiogenesis) than those co-transplanted with Prom1(-) endothelium. We also identified specific genes in Prom1(+) endothelium that code for endothelial signaling modulators that were not overexpressed in Prom1(-) endothelium. These factors may support proneural tumor progression and could be potential targets for anti-angiogenic therapy

Direct differentiation of human pluripotent stem cells into vascular network along with supporting mural cells

During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro

Prom1⁺ cells are present throughout the brain of an eight-week old <i>Prom1^lacZ/+</i> mouse model.

<p>(A) X-gal staining of Prom1 cells in sagittal section of the brain. (B–F, left panel) Low-power and respective magnified images (C-G, right panel) of anteroposterior coronal sections showing the distribution of ß-galactosidase activity in the hippocampus (B, C), occipital cortex (D, E) and cerebellum (F, G). Abbreviations: CPu, caudate/putamen; Ent, enthorinal cortex; fi, fimbria; GrDG, granular layer dentate gyrus; GrL, granular layer cerebellum; hipp, hippocampus; LV, lateral ventricle; MolDG, molecular layer dentate gyrus; MoL, molecular layer cerebellum; OB, olfactory bulb; OC, occipital cortex; PcL, Purkinje cell layer; RMS, rostral migratory stream; Tc, temporal cortex; Th, thalamus; WM, white matter; 3V, third ventricle.</p