ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells
© Lindgren et al.; licensee BioMed Central. 2015
Received: 2 October 2014
Accepted: 9 December 2014
Published: 7 January 2015
Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.
We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.
Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.
KeywordsHuman embryonic stem cells Endothelium ETV2 Differentiation
Embryonic stem cells (ESCs) are pluripotent and thus have the ability to differentiate into all cell types of the body. Previous groups have derived endothelial cells, the cells that line blood vessels, from mouse ESCs (mESCs) and human ESC (hESCs) and shown that they are functional in in vivo assays [1-7]. In many differentiation protocols, only a small percentage of cells differentiate into endothelial cells; however, large quantities of cells are required for potential downstream bioengineering applications [1,2].
Expression of Etv2 is required for the proper formation of the vasculature in fish and mammals [8-10]. Overexpression of Etv2 in differentiating mESCs has been shown to be effective at increasing the number of endothelial cells [9,11]. Inducible expression of Etv2 over a short period of time concurrent with endogenous expression is sufficient to increase the population of endothelial cells from 8% to 70% . Recent work infecting hESCs with ETV2 expressing virus showed that roughly 40% of the infected cells could become endothelial-like under modified culture conditions that also support hESC self-renewal .
To determine the percentage of endothelial-like cells, we analyzed the surface expression of VE-CADHERIN/CDH5, CD31, FLK1/KDR, and CD34 on day 7 of differentiation by flow cytometry. The greatest number of cells expressed KDR (40.4%) (Figure 1E,F). This agrees with previous reports in the mouse and human systems where KDR marked endothelial cells as well as a large population of mesodermal precursors and undifferentiated hESCs [15,16]. VE-CADHERIN (8.5%), CD31 (4.8%), and CD34 (13.8%) were expressed on similar-sized populations of cells and the majority of these cells showed overlap with the three markers (Figure 1E,F). To determine if the cells differentiated in clusters or from scattered single cells, we stained the cells in situ on day 7 of differentiation. Clusters of CD31 and VE-CADHERIN cells were seen (Figure 1G,H).
Previous studies in the murine system have examined the effect of exogenous Etv2 on differentiating mESCs and demonstrated that up to 70% of the differentiating cells were responsive to exogenous Etv2 . mESCs were induced to transiently express exogenous Etv2 concurrently with endogenous Etv2 in differentiating embryoid bodies . Experiments in hESCs have tested the ability of exogenous ETV2 to induce endothelial cells from undifferentiated hESCs and from a FLK1+ subset of differentiated hESCs [12,17]. We wanted to examine whether the addition of ETV2 at different stages of hESC differentiation would result in differing yields of endothelial-like cells. We infected cells either before differentiation (−7i/+7a) (Figure 2B) or at day 4 (+4i/+7a) (Figure 2C) of differentiation and analyzed the cells 7 days after the initiation of differentiation by flow cytometry for YFP and VE-CADHERIN expression. The infection efficiency of −7i/+7a cells was low in comparison to that of +4i/+7a, despite using a higher multiplicity of infection (MOI), 20 vs 2, respectively (Figure 2B,C,E). This agrees with previous publications demonstrating the low infection efficiency of hESCs [18,19]. In control virus-infected cells at each time point, a similar percentage of VE-CADHERIN positive cells (~10%) were observed compared to that in uninfected cells (Figure 2B,C,F). In the population infected with the ETV2-mCherry virus before differentiation, 22.9% of the cells were positive for VE-CADHERIN after differentiation, a twofold increase over the control (Figure 2B,C,F). This did not result in an absolute increase in endothelial-like cells as the slight increase in VE-CADHERIN-positive cells infected with ETV2-mCherry virus was counteracted by the decreased infection efficiency of these cells (Figure 2B). The apparent lack of response to ETV2 in cells infected before differentiation may be due to the loss of cells that responded to ETV2. Cells that gained an endothelial-like phenotype in the 7 days after infection would have been excluded during the EB generation step of the differentiation protocol. ETV2 administered during differentiation had a much more pronounced effect. Of the cells infected during differentiation, 60.4% were VE-CADHERIN positive on day 7 resulting in a sixfold increase over control cells (Figure 2B,C,F). This is comparable to what was seen in mESC culture . The absolute number of cells that expressed VE-CADHERIN was significantly higher following ETV2-mCherry infection, increasing from an average of 645/10,000 cells in control conditions to 4,141/10,000 cells in ETV2 overexpression conditions (Figure 2C).
Our previous work in zebrafish found that only a few cell types are responsive to exogenous etv2 after 24 h of differentiation and unresponsive by 48 h . Work performed in vivo in conditionally transgenic Etv2 mice showed that cells committed to the somitic or neural lineages were unresponsive to exogenous Etv2 . Infection of adult human fibroblast and mesenchymal cells with an ETV2 virus in combination with FLI1 and ERG1 viruses only modestly induced endothelial genes . Based upon this knowledge, we anticipated that infection of differentiating hESCs with an ETV2-expressing virus at time points later than the peak of endogenous expression would result in decreasing percentages of cells responding to the exogenous signal. The cells were infected at day 12 of differentiation and analyzed for VE-CADHERIN expression 3 days after infection. Surprisingly, the surface expression of VE-CADHERIN was not significantly different in +12i/+15a ETV2-mCherry-infected cells from +4i/+7a ETV2-infected cells demonstrating that the cells had not lost responsiveness to ETV2 as predicted (Figure 2D,F).
Sorted YFP+ VE-CADHERIN+ cells were grown on gelatin in endothelial media supplemented with 20 ng/ml bFGF, 25 ng/ml VEGF165, and 10 μg/ml TGFβ inhibitor SB431542. The ETV2-infected cells proliferated faster than the control cells and maintained a uniform morphology (data not shown). An increased level of proliferation has been shown previously in cells constitutively expressing exogenous ETV2 . After 7 days of growth, the vast majority of ETV2-infected cells all maintained CD31 and VE-CADHERIN expression by immunostaining and flow cytometry (Figure 3C,D,F). These cells formed networks within 3 h of plating on Matrigel with optimal tube network formation at 8 h (Figure 3E).
Practical and ethical reasons prevent us from examining the timing and role of expression of ETV2 in early human fetal samples. We have examined the timing of ETV2 expression in differentiating human ESCs and found that its expression in relation to other developmental genes is similar to that found in the developing mouse in vivo and in vitro. In our method of infection during mesoderm-directed differentiation, 60% of the ETV2 expressing cells were able to respond. This is similar to the level found in mESCs .
Clinical and preclinical trials are being performed that use endothelial cells from various sources to aid in the recovery from many vascular and cardiac conditions [22-24]. A common roadblock in these procedures is a lack of source allogeneic endothelial cells. Other clinical work is being performed to generate vascular structures that can then be seeded with endothelial cells prior to transplant into patients . Use of induced pluripotent cells derived directly from the patient would allow for histocompatible graft material being available for transplant. Addition of ETV2 is a viable strategy for increasing the yield of endothelial-like cells from these sources. Any cells transplanted into a patient would need to be derived through non-integrating viral or non-viral methods. These methods would result in a pulse of gene expression corresponding to the time of treatment. Using these methods would generate transient ETV2 expression. This might allow the endothelial-like cells generated during ETV2 expression, demonstrated in this report, to more fully differentiate into arterial and venous endothelial cells upon loss of ETV2 expression. Recent work has shown that administration of ETV2 and GATA2 mRNA is able to convert hESCs to hematopoietic cells. Addition of ETV2 mRNA alone may be able to generate endothelial cells with an unaltered genome that could be used for transplantation into patients.
Human embryonic stem cells
Mouse embryonic stem cells
Multiplicity of infection
We thank the UCLA BSCRC flow cytometry core for cell sorting and the UCLA vector core for the preparations of the viral particles. The UCLA vector core is supported by JCCC/P30 CA016042 and CURE/P30 DK041301. The cells were supplied through the UCLA BSCRC stem cell core laboratory. This work was supported by funds from the California Institute for Regenerative Medicine (CIRM RB3-02165).
- James D, Nam HS, Seandel M, Nolan D, Janovitz T, Tomishima M, et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nat Biotechnol. 2010;28:161–6.PubMed CentralPubMedView ArticleGoogle Scholar
- Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R. Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A. 2002;99:4391–6.PubMed CentralPubMedView ArticleGoogle Scholar
- Nourse MB, Halpin DE, Scatena M, Mortisen DJ, Tulloch NL, Hauch KD, et al. VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering. Arterioscler Thromb Vasc Biol. 2010;30:80–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Tan JY, Sriram G, Rufaihah AJ, Neoh KG, Cao T. Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation. Stem Cells Dev. 2013;22:1893–906.PubMed CentralPubMedView ArticleGoogle Scholar
- Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H, et al. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat Biotechnol. 2007;25:317–8.PubMedView ArticleGoogle Scholar
- Watabe T, Nishihara A, Mishima K, Yamashita J, Shimizu K, Miyazawa K, et al. TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells. J Cell Biol. 2003;163:1303–11.PubMed CentralPubMedView ArticleGoogle Scholar
- Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, Yurugi T, et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature. 2000;408:92–6.PubMedView ArticleGoogle Scholar
- Kataoka H, Hayashi M, Nakagawa R, Tanaka Y, Izumi N, Nishikawa S, et al. Etv2/ER71 induces vascular mesoderm from Flk1 + PDGFRalpha + primitive mesoderm. Blood. 2011;118:6975–86.PubMedView ArticleGoogle Scholar
- Lee D, Park C, Lee H, Lugus JJ, Kim SH, Arentson E, et al. ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specification. Cell Stem Cell. 2008;2:497–507.PubMed CentralPubMedView ArticleGoogle Scholar
- Sumanas S, Lin S. Ets1-related protein is a key regulator of vasculogenesis in zebrafish. PLoS Biol. 2006;4:e10.PubMed CentralPubMedView ArticleGoogle Scholar
- Koyano-Nakagawa N, Kweon J, Iacovino M, Shi X, Rasmussen TL, Borges L, et al. Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression. Stem Cells. 2012;30:1611–23.PubMed CentralPubMedView ArticleGoogle Scholar
- Elcheva I, Brok-Volchanskaya V, Kumar A, Liu P, Lee JH, Tong L, et al. Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators. Nat Commun. 2014;5:4372.PubMed CentralPubMedView ArticleGoogle Scholar
- Herrmann BG. Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos. Development. 1991;113:913–7.PubMedGoogle Scholar
- Wilkinson DG, Bhatt S, Herrmann BG. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature. 1990;343:657–9.PubMedView ArticleGoogle Scholar
- Sone M, Nakao K. Vascular research using human pluripotent stem cells and humoral factors. Endocr J. 2013;60:397–402.PubMedGoogle Scholar
- White MP, Rufaihah AJ, Liu L, Ghebremariam YT, Ivey KN, Cooke JP, et al. Limited gene expression variation in human embryonic stem cell and induced pluripotent stem cell-derived endothelial cells. Stem Cells. 2013;31:92–103.PubMed CentralPubMedView ArticleGoogle Scholar
- Ginsberg M, James D, Ding BS, Nolan D, Geng F, Butler JM, et al. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFbeta suppression. Cell. 2012;151:559–75.PubMed CentralPubMedView ArticleGoogle Scholar
- Jang JE, Shaw K, Yu XJ, Petersen D, Pepper K, Lutzko C, et al. Specific and stable gene transfer to human embryonic stem cells using pseudotyped lentiviral vectors. Stem Cells Dev. 2006;15:109–17.PubMedView ArticleGoogle Scholar
- Xiong C, Tang DQ, Xie CQ, Zhang L, Xu KF, Thompson WE, et al. Genetic engineering of human embryonic stem cells with lentiviral vectors. Stem Cells Dev. 2005;14:367–77.PubMedView ArticleGoogle Scholar
- Veldman MB, Zhao C, Gomez GA, Lindgren AG, Huang H, Yang H, et al. Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2. PLoS Biol. 2013;11:e1001590.PubMed CentralPubMedView ArticleGoogle Scholar
- Hayashi M, Pluchinotta M, Momiyama A, Tanaka Y, Nishikawa S, Kataoka H. Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice. Exp Hematol. 2012;40:738–50. e711.PubMedView ArticleGoogle Scholar
- Amato B, Compagna R, Della Corte GA, Martino G, Bianco T, Coretti G, et al. Peripheral blood mono-nuclear cells implantation in patients with peripheral arterial disease: a pilot study for clinical and biochemical outcome of neoangiogenesis. BMC Surg. 2012;12 Suppl 1:S1.PubMed CentralPubMedView ArticleGoogle Scholar
- Friis T, Haack-Sorensen M, Mathiasen AB, Ripa RS, Kristoffersen US, Jorgensen E, et al. Mesenchymal stromal cell derived endothelial progenitor treatment in patients with refractory angina. Scand Cardiovasc J. 2011;45:161–8.PubMedView ArticleGoogle Scholar
- Sevestre MA, Larghero J, Castier Y, Nugent HM, Visonneau S, Alsac JM. Pilot safety study of perivascular injection of tissue-engineered allogeneic aortic endothelial cells in patients undergoing minimally invasive peripheral revascularization. J Vasc Surg. 2014;59:1597–606.PubMedView ArticleGoogle Scholar
- Weymann A, Schmack B, Okada T, Soos P, Istok R, Radovits T, et al. Reendothelialization of human heart valve neoscaffolds using umbilical cord-derived endothelial cells. Circ J. 2013;77:207–16.PubMedView ArticleGoogle Scholar
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