BSS PBMC iPS F expressed the endogenous pluripotent transcri

BSS1-PBMC-iPS4F4 expressed the endogenous pluripotent transcription factors OCT3/4, SOX2, REX1 and NANOG, assessed by RT-PCR (Fig. 2A) and the protein pluripotent markers SSEA3, SSEA4, Tra1–60, Tra1–81 and Oct3/4, evaluated by flow cytometry analysis (Fig. 2B). To demonstrate the capacity of BSS1-PBMC-iPS4F4 to differentiate into the three germ layers in vivo, teratoma formation assays were accomplished (6). As shown in Fig. 2C, teratomas derived from this line showed expression of representative markers of ectoderm (β3-Tubulin), mesoderm (Vimentin) and endoderm (Cytokeratin CK AE1–AE) (Fig. 2C).
Materials and methods
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Resource Details The Notch ligand DLL4 is essential for the correct formation of the embryonic vasculature in mouse. DLL4 knock-out is embryonic lethal, and heterozygous animals present severe vascular defects, such as reduced dorsal pyruvate dehydrogenase kinase caliber, abnormal branching of vessels and vascular remodeling (Duarte et al., 2004; Gale et al., 2004; Krebs et al., 2004). In order to investigate if DLL4 has a similar function in human embryonic development we have generated human pluripotent stem cell lines (hPSCs) that stably express a combination of short hairpin RNAs (shRNA) targeting DLL4 mRNA. We transduced the iPSC cell line PBMC1-iPS4F1 (Montes et al., 2015) and the hESC cell line H9 (Thomson et al., 1998) with lentiviruses expressing either a control, non-targeting shRNA with a scrambled sequence (shSCR), or a cocktail of 5 different shRNAs targeting the human DLL4 mRNA (shDLL4). After selection with Puromycin for 10days we obtained resistant colonies that were expanded, leading to the establishment of the following four cell lines: PBMC1-iPS4F1 shSCR, PBMC1-iPS4F1 shDLL4, H9 shSCR and H9 shDLL4. We confirmed the identity of the transgenic cell lines by Short Tandem Repeat (STR) profiling (Table 1). They are routinely tested for mycoplasm and remain mycoplasm-free. The four cell lines present the typical morphology of pluripotent stem cells, growing in compact colonies, with tightly packed cells (Fig. 1A). They express the pluripotency markers SSEA4, SSEA3, TRA-1-81, TRA-1-60 and OCT3/4, assessed by flow cytometry (Fig. 1B), and OCT4, REX1, NANOG and SOX2, assessed by PCR (Fig. 1C). They are also positive for alkaline phosphatase activity (Fig. 1D). Therefore, neither the expression of shSCR nor shDLL4 constructs has affected the pluripotency of the cell lines. The transgenic cell lines were capable of forming embryoid bodies (EBs) (Fig. 2A). We harvested EBs after 21days of spontaneous differentiation, and analyzed by PCR the expression of makers of the three germ layers: PAX6 for ectoderm, HNF3A for endoderm, and KDR for mesoderm. The four cell lines expressed the three markers (Fig. 2B), demonstrating that the expression of shSCR and shDLL4 does not affect the ability of these pluripotent stem cells to differentiate to the three germ layers. DLL4 is expressed at very low levels in human pluripotent stem cells, and its expression increases as the cells are differentiated towards the hematopoietic lineage (Ayllón et al., 2015). In fact, the DLL4 protein located at the plasma membrane only becomes detectable by flow cytometry in a subpopulation of hemato-endothelial progenitors (HEPs) (Ayllón et al., 2015) (Fig. 2C). These HEPs are bipotent progenitors that can give rise to either hematopoietic or endothelial cells, and they express the surface markers CD34, KDR, VE-cadherin and CD31 (Ayllón et al., 2015; Uenishi et al., 2014) (Fig. 2C). To confirm that the expression of DLL4 was indeed suppressed in our transgenic cell lines we differentiated them towards hemato-endothelial progenitors and assessed the levels of DLL4 expression by flow cytometry. At day 8 of differentiation DLL4 is only expressed in CD34+KDR+VE-CAD+CD31+ cells (HEPs) in control shSCR cell lines, and its expression is reduced down by 75–90% in shDLL4 cell lines (Fig. 2D).