Embryonic Stem Cell Differentiation to Functional Arterial Endothelial Cells through Sequential Activation of ETV2 and NOTCH1 Signaling by HIF1α.

The generation of functional arterial endothelial cells (aECs) from embryonic stem cells (ESCs) holds great promise for vascular tissue engineering. However, the mechanisms underlying their generation and the potential of aECs in revascularizing ischemic tissue are not fully understood. Here, we observed that hypoxia exposure of mouse ESCs induced an initial phase of HIF1α-mediated upregulation of the transcription factor Etv2, which in turn induced the commitment to the EC fate. However, sustained activation of HIF1α in these EC progenitors thereafter induced NOTCH1 signaling that promoted the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the mouse hindlimb ischemia model. Furthermore, transplantation of aECs in mice showed engraftment in ischemic myocardium and restored cardiac function in contrast to ECs derived under normoxia. Thus, HIF1α activation of Etv2 in ESCs followed by NOTCH1 signaling is required for the generation aECs that are capable of arteriogenesis and revascularization of ischemic tissue.

Production of recombinant proteins in microalgae at pilot greenhouse scale.

Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae.