The salient findings of this study are: 1) ECs differentiated from hiPSCs are heterogenous in nature, displaying arterial, venous or lymphatic markers, 2) Specification of arterial or venous subtype is influenced by the concentration in the media of VEGF 3) High VEGF concentration and 8Br-cAMP induce arterial CD31+ hiPSCECs, in association with increased expression of the Notch pathway, 4) High VEGF-A and VEGF-C concentration with supplementation of Ang-1 in the medium promotes the specification to lymphatic CD31+ hiPSC-ECs. 5) CD31+ hiPSC-artECs form more extensive and mature capillary networks in vivo.
EC heterogeneity is a well-established concept. It is known that ECs are extraordinarily diverse in their function and gene-expression profile [15
]. Morphologically, they can be different in size, shape and thickness depending on the vascular beds in which they reside. Specification of arterial or venous phenotype occurs even before blood circulation during embryonic development. In this regard, the Notch signaling pathway is known to be a crucial pathway in specifying the EC phenotype [12
Heterogeneity of phenotype develops in part through interactions of ECs with the microenvironment such as growth factors, extracellular matrix and mechanical forces. Several growth factors are known to be important in arterial and venous specification during development. BMP-4 is part of the transforming growth factor-beta superfamily and plays a crucial role in the commitment of pluripotent stem cells into mesoderm lineage and following that to the hematopoietic and endothelial lineages. It was previously shown that exposure of pluripotent stem cells to a short BMP-4 treatment induces EC differentiation [18
] and hence we have incorporated a short induction of high dosage of BMP-4 in our differentiation protocol. VEGF on the other hand is a widely known key growth factor involved in the development of ECs in vivo
, it thus used for robust differentiation toward EC lineage [19
Standard protocols for generating ECs from human pluripotent stem cells use either CD31 or CD144 to isolate the EC population. CD31 is a 130kDa transmembrane molecule of the immunoglobin gene superfamily that is responsible for cell-cell adhesion in ECs [22
]. On the other hand, CD144 is a 130kDa transmembrane adhesion molecule implicated in the regulation of endothelial tube formation during vasculogenesis and angiogenesis [24
]. Since CD31 and CD144 are expressed in ECs of all subtypes, one of the implications of this study is that these two markers alone may not be sufficient for purifying ECs of specific subtypes.
One of the aims of this study was to analyze further the properties of human pluripotent stem cell derived ECs. By observation of morphology, our differentiation protocol () generated homogenous ECs with cobblestone morphology. However, gene expression analysis showed that these cells expressed varying levels of arterial and venous markers indicating the heterogenous nature of these hiPSC-derived ECs. Previous studies reported differentiation protocols that were used for specific arterial and venous EC induction in mouse ESCs [10
] or iPSCs [26
]. Yurugi-Kobayashi et al reported that addition of cyclic AMP was able to enhance mouse EC specification into arterial EC subtype [10
]. Cyclic AMP is known to be an important secondary messenger in mediating many physiological functions including cell growth and differentiation. They have shown that in the mouse ESC system, cyclic AMP promotes arterial EC specification through upregulation of Flk1 and Nrp1 during differentiation and through dual activation of Notch and β-catenin signaling [10
]. The same group repeated their mouse ESC system differentiation procedure on mouse iPSCs and was able to generate the three EC subtypes from mouse iPSCs. Our differentiation system differs from theirs as we initiated the various EC subtypes differentiation from the beginning of the differentiation and without any enrichment for any mesodermal progenitors. Lanner et al
. on the other hand, reported that high VEGF concentration resulted in upregulation of arterial markers in the differentiated mouse ECs [25
We found that the combined effect of BMP4, high VEGF concentration and cyclic AMP was to enhance arterial specification of hiPSC-ECs, as indicated by the expression of EphrinB2. Furthermore, in these ECs, the expression of venous markers such as EphB4 and CoupTFII was downregulated. Activation of the Notch pathway is a hallmark for arterial EC identity [13
]. With the arterial specification protocol we observed an upregulation of Notch family members including the receptors Notch 1 and 4 and the ligands, Jag-1 and Dll-4. Unlike arterial EC specification, the molecular mechanisms that govern venous EC specification are still largely unknown. EC differentiation and specification depends highly on growth factor environmental cues which expose ECs to various signaling pathways. Such environmental influence is observed during development in the dorsal aorta and cardinal vein [13
]. The ECs in the dorsal aorta are close to the notochord which is a Sonic hedgehog source (Shh). Shh induces high local concentrations of VEGF which in turn induces Notch in the adjacent dorsal aorta. Notch is upstream of the arterial marker EphrinB2, and reinforces the arterial fate of the ECs in the dorsal aorta. Shh signaling has also been shown recently to induce arterialization by repressing the venous EC fate [28
]. However, for specification of venous fate, it is possible that lower Shh levels act on the cardinal vein to induce low levels of VEGF, which may induce Coup-TFII expression. Coup-TFII inhibits Notch signaling and thus blocks arterial differentiation [29
]. Furthermore, the greater distance of the posterior cardinal vein as compared to the dorsal aorta from the VEGF-expressing somites may play a part in the specification of EC into the venous fate [31
]. In vitro
exposure of mouse ESCs to low VEGF concentrations strongly upregulates expression of CoupTFII [25
]. In our study, we primed EC differentiation by using high concentration of BMP4 and VEGF for the first 4 days of differentiation before using a low VEGF concentration for venous specification. VEGF dose dependency in determining arterial-venous EC subtypes seems to be operative in hiPSCs, with low VEGF expression inducing significant expression of EphB4 and CoupTFII.
Lymphatic differentiation of pluripotent stem cells is not well characterized. During embryonic development, lymphatic ECs are thought to arise from ECs of the cardinal vein that acquire a lymphatic phenotype through the expression of transcription factors Sox18 and Prox1 [32
]. VEGF-C, which acts on the VEGFR3 receptor, promotes migration of lymphatic endothelial progenitors from the vein [32
]. Another modulator of lymphatic phenotype is Ang1, which acts through the receptor tyrosine kinase, Tie-2 in regulating lymphatic vessel formation, sprouting, and lymphatic endothelial proliferation [36
]. Due to the role of VEGF-C and Ang1 signaling during lymphatic vessel formation, we sought to enhance lymphatic EC specification using these growth factors. Based on gene and protein expression data, there was a marked increase in the expression of lymphatic markers podoplanin and LYVE-1 in hiPSC-ECs with the use of supplemental VEGF-C and Ang1 (). In the absence of these lymphatic-promoting factors the levels of lymphatic gene expression was even less than that of hiPSCs (). Other groups have also utilized VEGF-C or Ang1 to stimulate lymphatic differentiation of murine ESCs in the presence of other microenvironmental factors such as OP9 stromal feeder cells [37
], EB aggregates [38
], or in extracellular matrices [39
]. Together our results suggest that lymphatic EC specification can be achieved by VEGF-C or Ang1 signaling.
Based on our findings, we have demonstrated that modulation of soluble factors can enrich for specific EC subtypes. Furthermore, we observed that hiPSC-ECs enriched for arterial phenotype generated a more extensive and mature vascular network in vivo than did hiPSC-ECs of a more heterogenous population. However, it is unknown whether sustained delivery of the soluble factors is necessary to retain arterial phenotype, and whether hiPSC-ECs might undergo phenotypic changes upon growth factor withdrawal. It is likely that other microenvironmental factors such as extracellular matrix proteins or mechanical cues are necessary to augment or maintain subtype specification. Furthermore, it remains to be determined whether the enriched arterial subtype is more favorable for therapeutic angiogenesis or if the hiPSC-LECs are superior for treatment of lymphedema. These experiments are interesting and warranted, but beyond the scope of this study.
In summary, this study demonstrates the heterogeneity of hiPSC-ECs and the methods to enrich for subtype specific cells using soluble factors. Furthermore, arterial-enriched hiPSC-ECs enhanced neovascularization compared to heterogeneous cells, suggesting a potential therapeutic benefit in transplanting enriched subtypes.