The ability to generate human vascular endothelium from iPSC

The ability to generate human vascular endothelium from iPSCs is an enabling platform. Cell-based assays of endothelial function can be combined with ECs derived from iPSC lines produced from patients with genetic diseases, creating powerful model systems of human disease or associations where the pathological molecular mechanisms and their functional consequences are poorly understood (Adams and García-Cardeña, 2012; Grskovic et al., 2011; Kiskinis and Eggan, 2010). Extension of these disease iPSC-based assays to high throughput may enable the new therapeutic discovery for genetic diseases and permit the personalization of drug toxicity testing, an unmet need because vascular toxicity is a significant cause of attrition in drug development.
Experimental Procedures
Acknowledgments
Introduction Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in distinct areas of the adult mammalian brain, including the subventricular zone (SVZ) lining the lateral ventricles from which newborn cells migrate toward the olfactory bulb and the hippocampal dentate gyrus (DG) (Zhao et al., 2008). Due to the relative sparseness of NSPCs and their progeny in relation to pre-existing neural structures, there is a need to selectively manipulate gene activity in NSPCs and their progeny to address their functional significance during the course of development from a dividing NSPC to a fully mature and synaptically integrated neuron (Dhaliwal and Lagace, 2011). One approach to test the functional significance of genes/pathways is to use transgenic mice carrying floxed conotoxin manufacturer of genes of interest, together with Tamoxifen (TAM)-regulatable Cre-recombinase controlled by NSPC- or immature neuron-selective promoters, to genetically recombine and delete genes (Ihrie et al., 2011; Sahay et al., 2011). In addition, retroviral vectors derived from Moloney murine leukemia viruses have proved to be an important tool to visualize newborn cells through the expression of fluorescent proteins, as well as to manipulate gene expression using both gain- and loss-of-function strategies (Ge et al., 2006; van Praag et al., 2002). The use of retroviruses has the advantage that it is highly selective for dividing cells (i.e., neurogenic cells when injected into the SVZ or DG) and is a very fast method because extensive breeding to obtain correct genotypes, as is the case for classical transgenesis, is not required (Zhao and Gage, 2008). However, retroviral vectors do not target bona fide NSPCs that are largely quiescent but integrate into highly proliferative neural progenitors, which restrict virus-mediated genetic manipulations to later steps of neurogenesis. Furthermore, current vectors do not allow inducible or temporally controlled expression of the virus-driven transgene. Temporal control of transgene expression would be advantageous for studying gene function during distinct steps in the course of neuronal development. This is especially true for transcription factors (TFs) that may exert stage-specific functions depending on the age of a given newborn cell (Iwano et al., 2012). We reasoned that fusion of a TAM-regulatable estrogen receptor (ERT2) motif (Indra et al., 1999; Jiang et al., 2010) to expression constructs of TFs involved in neurogenesis would enable TAM-induced transgene expression, allowing for temporal control of virus-mediated gene expression (Figures 1A and 1B; Supplemental Experimental Procedures available online).
Results It was previously shown that Ascl1 overexpression in cultured adult NSPCs results in robust neuronal differentiation (Jessberger et al., 2008). To analyze whether fusion of Ascl1 to the ERT2 motif leads to functional ASCL1 expression upon TAM treatment, we transduced NSPCs isolated from adult mice with Ascl1-ERT2-IRES-GFP (hereafter called Ascl1-ERT2)-expressing retroviruses in vitro. To induce translocation of the ASCL1-ERT2 fusion protein, we treated the cells with hydroxy-TAM (OH-TAM) and analyzed them 7 days after the onset of differentiation. Whereas Ascl1-ERT2-expressing cells cultured without OH-TAM did not show any difference in their rate of neuronal differentiation compared with cells transduced with a control retrovirus (expressing IRES-GFP), treatment of Ascl1-ERT2-expressing cells with OH-TAM resulted in a dramatic increase in neuronal differentiation (Figures 1C, 1D, S1A, and S1B). These findings suggest that OH-TAM treatment resulted in the robust and efficient transcriptional activation of ASCL1 target genes to induce neuronal differentiation in vitro. To confirm that this approach is applicable to other TFs and not restricted to ASCL1, we fused the basic helix-loop-helix TF NEUROD1 to an ERT2 motif (Figure 2D) and infected NSPC cultures with NeuroD1-ERT2-IRES-GFP-expressing retroviruses. As before, we observed the almost complete neuronal differentiation of NSPCs upon OH-TAM treatment (Figures 2A–2C). This finding is consistent with the induction of NEUROD1 transcriptional activity, as it was previously shown that NEUROD1 overexpression promotes neuronal differentiation of NSPCs (Gao et al., 2009). Furthermore, we were able to confirm that the subcellular localization of ASCL1-ERT2 and NEUROD1-ERT2 was indeed controlled by OH-TAM-dependent translocation of the fusion proteins from the cytoplasm to the nucleus of transduced NSPCs (Figures S2A and S2B).