3.1 A modified protocol for generating ECs from human ESCs or iPSCs
A high-efficiency protocol for deriving ECs from ESCs or iPSCs has been published[26], we optimized the protocol according to other protocols [15, 25, 29], we seeded about 3 × 104 cells/well in 12-well plate using 10 µM Y27632 and 10 ng/mL BMP4 for the three days (Fig. 1a, b and c). H1 ESCs generated 92.17% ± 0.42% endothelial cells after 7 days of differentiation (Fig. 1d). Mesoderm cells treated with ROCK inhibitor is efficient to differentiate into skeletal myocytes [30], so we predicted that Y27632 in the process of EC differentiation plays the same role as their protocol. In order to further explore the inhibitor(Y27632)’s function, we designed a serial concentration gradient experiment (Fig. 2c). The result is that a small doses of Y27632 treatment can improve differentiation efficiency in the three days and the differentiation rate increased with the increase of the inhibitor’s concentration, so we predicted that H1 ESC line is very sensitive to Y27632, then, we first built the highly efficient differentiation system.
In addition, thinking of using Y27632 to improve cell survival, we treated cells by Y27632 for one day or three days, and proved that it has a higher differentiation rate for the three days than for the one day (Fig. 2a and b). Then, we repeated the experiment with Y27632 treatment for one day or three days in 202-iPSC line, and the differentiation rate for the three days (41.9% ± 4.78%) is higher than with Y27632 treatment for one day (6.37% ± 1.07%) (Additional file 1: Figure S1a and b). Our results demonstrated that Y27632 has some effect on ESC differentiation, but the efficiency of EC differentiation is still not high as same as the above, so we predicted that there are other influencing factors.
3.2 Optimizing conditions to increase the ESC/iPSC-EC differentiation efficiency
Then we only utilized the protocol[26] and further explore how to promote the efficiency. Firstly, we designed a serial cell density experiment and found that the differentiation efficiency decreased with the increase of the cell density in H1-ESCs (Additional file 1: Figure S2a). Secondly, thinking of the function of two factors: SB431542 which inhibits TGF-β pathway to repress smad signal and BMP4 which promotes smad signal, we chose H9-ESC line to do the next experiments. With the increase of SB431542 concentration (5uM, 10uM and 20uM) in the second stage, the differentiation efficiency increased (Additional file 1: Figure S2b and d). BMP4 has been used both stages (first stage and second stage), here we checked whether adding BMP4 or not affected ESC/iPSC-EC differentiation efficiency in the second stage, the result is that there was a higher differentiation efficiency (Additional file 1: Figure S2c) when we plated 30,000 cells with BMP4; this result is as same as that BMP4 promotes generation of CD31+CD34+ cells[31]. From these results above, we drew a conclusion that seeding cell number has some effect on differentiation, BMP4 and SB431542 in the second stage promote ESC/iPSC-EC differentiation. But, we still didn’t solve the problem of low differentiation efficiency.
3.3 DMSO also plays an important role in Hemogenic Endothelial (HE) and ESC/iPSC-EC differentiation
We also improved the Hemogenic Endothelial differentiation protocol [25]. when we added the inhibitor Y27632 dissolved in DMSO for three days, we found that there is a higher differentiation efficiency in the H1-ESC line: CD31+CD34+ cells occupied 37.70%±1.55% versus 2.39% ± 0.17%, CD43+ cells occupied 21.85%±6.31% versus 9.17% ± 0.65%. It again proved that Y27632 dissolved in DMSO plays an important role in the ESCs-derived HE differentiation (Fig. 3a and b), and the efficiency of differentiation is lower with Y27632 dissolved in ddH2O (Fig. 3c and d). So here, we proved that DMSO also plays an important role in the HE differentiation system.
In order to understand well the function of DMSO, we found several papers referring to ESC differentiation. The efficiency of hPSC differentiation is improved even if hPSCs are treated with DMSO by short time [32]; addition of DMSO also downregulates expression of stemness-related genes such as OCT4 and NANOG, and increases the proficiency of hepatic differentiation [33]; DMSO at 0.01% and 0.1% concentration can act as an inducing agent for the formation of mesodermal phenotypes [34].
Under the condition with adding DMSO in the first step, we confirmed the highly efficient differentiation system using improved protocol again with Y27632 for one day or three days (Fig. 3e and f), and obtained the same result even if we changed another chemically-defined medium (CDM) (Additional file 1: Figure S3). So, the result is that DMSO is an important factor for ESC/iPSC-EC differentiation. From the above, we checked factors of affecting EC differentiation including seeding cell number, BMP4, SB431542, DMSO, Y27632 and treatment time, then we figured out the reason why we can't repeat our improved protocol for a while.
3.4 ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have characteristics of early EPCs
Several articles [16, 35] has reported that they can induce ESCs/iPSCs into ECFCs ( also called late EPCs) with CD31+NRP1+ and maintain these ECFCs in complete endothelial cell medium called EGM2 medium (Lonza). Because peripheral blood-derived EPCs were cultured in EGM2 with 16% FBS [27], ESC/IPSC- ECs also were maintained in the same conditions. To make sure whether ESC/iPSC-ECs belongs to ECFCs or not, we compared cell morphology of ESC/iPSC-ECs with peripheral blood-derived EPCs (Fig. 4a) and detected the elevation of NRP1 gene expression in the differentiation process (Fig. 4e); then, we checked several markers such as CD31(PECAM1), KDR, NRP1 and CD34 through flow cytometry, the result also showed cells with NRP1 or KDR from ESC/iPSC-ECs and EPCs from IPAH patient occupied higher percentage than EPCs from normal persons (Fig. 4b). In addition, we also proved that H1-derived ECs have the ability of tube formation (Fig. 4c) and can uptake acetylated LDL (Fig. 4d). Thus, these results suggest ESC/iPSC-ECs should be like EPCs.
EPCs have two distinct EPC subtypes which have been named as early EPCs and late EPCs which are also known as endothelial colony-forming cells (ECFCs) [35, 36], which can be identified through qRT-PCR by three marker genes containing HLA-DRA, lysozyme(LYZ), and CD14 for the early EPCs, and three marker genes containing caveolin1(CAV1), VE-cadherin(VE-CAD), and vWF for the late EPCs[9]. Next, we tried to isolate the two types of EPCs from 202-iPSC-derived ECs which were induced by day 5 or day 7, and ECs were maintained in complete EGM2 medium with 16% FBS for 2 to 4 passages. Results showed ECs on day 7 with higher expression level of LYZ and lower expression level of VE-CAD have some characteristics of early EPCs (Fig. 5a). In order to further make sure whether ECs belongs to early EPCs or not, we also checked the expression level of an early EPC marker gene CD133 (PROM1) [37] and another two marker gene EFNB2 for arterial endothelium and EPHB4 for vein endothelium by qRT-PCR (Fig. 5b). CD133 and EFNB2 have a higher expression level in 202-iPSC-induced ECs isolated from on day 7, and results of immunofluorescence assay also showed all the three markers have a certain expression level (Fig. 5c). Besides, endothelial cell marker CD146 (MCAM) has an expression level (Fig. 5d). Meanwhile, we conducted bioinformatics analysis on datasets of ESC/iPSC-ECs, normal EPCs, idiopathic pulmonary arterial hypertension (IPAH)-derived EPCs (Fig. 5f) and GSE93511(2D_MG_H1EC and 2D_MG_HUVEC ) (Fig. 5e) [26], these genes including CD43(SPN), CD45 (PTPRC) and PROM1 which express in early-EPC have some expression. Thus, we think conservatively that normal EPCs, IPAH-EPCs and ESC/iPSC-ECs we obtained have characteristics of early EPCs.
3.5 ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have characteristics of mesenchymal stem cells for homing
A lot of homing-related genes have been identified, such as CXCR4 [38], IGF2/IGF2R, CXCL12[39–41]. Besides, mesenchymal stem cells have the ability of homing, whose protein markers including CD73(NT5E), CD44, CD90(THY1), CD105(ENG) [42, 43]. In addition, insulin-like growth factor–binding protein 3 (IGFBP3) may improve vessel repair [44, 45]. Based on these literature reports, we checked all above genes which of all has some expression level in ESC/iPSC-ECs through bioinformatics analysis using datasets of our microarray (Fig. 6a) and 2D_MG_H1EC and 2D_MG_HUVEC from GSE93511[26] (Fig. 6b). From the above, we predicted ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have characteristics of mesenchymal stem cells for homing.
In order to further demonstrate that ESC/iPSC-ECs have the ability of homing, we examined the engraftment potential of ESC/iPSC–ECs in vivo. when ESC/iPSC-ECs were injected into the blood stream of 48 hpf Zebrafish embryos, ESC/iPSC-ECs were capable of integrating into the vascular system that had already developed (Fig. 6c). Besides, we performed cell therapy after sugen5416 treatment, the result is that percentage of zebrafish returning to normal is higher (26.71 ± 5.86) % treated by ESC/iPSC-ECs than (12.06 ± 4.49) % only by medium (EGM2 + 16%FBS) (Fig. 6d). So, ESC/iPSC-ECs we obtained have huge potential applications.
3.6 Microarray analysis reveals dysfunction of IPAH-EPCs
Next, we analyzed the correlation between ESC/iPSC-ECs and normal EPCs (CON-EPCs), and the result showed that the correlation was up to more than 90% (Fig. 7a). In the process of cell culture, we found IPAH-EPCs proliferated faster than ESC/iPSC-ECs, and ESC/iPSC-ECs proliferated a little faster than normal EPCs (data not shown), and from microarray data the relative expression level of MKi67, the cell proliferation marker gene, is the highest in IPAH-EPCs, then in ESC/iPSC-ECs the average of MKi67 expression level is higher than that of CON-EPC (Fig. 7b). In view of the high similarity between ESC/iPSC-ECs and normal EPCs, we performed the Gene ontology biological process (GOBP) analyses of ESC/iPSC-ECs and IPAH-EPCs relative to normal EPCs. Of TOP20 pathways results show that all up-regulated genes of IPAH-EPCs are enriched on the pathways related to cell division, while part genes of ESC/iPSC-ECs are clustered on the pathways related to cell division, extracellular matrix, differentiation and development pathways, etc (Fig. 7c and d). This result again tells us that IPAH-EPCs have a higher proliferation rate, which is consistent with the previous analysis results. Besides, in order to better understand the molecular characteristics of ESC/iPSC-ECs, we continued to do the GOBP analysis of ESC/iPSC-ECs and normal EPCs relative to IPAH-EPCs. Of the TOP20 pathways results showed that up-regulated genes of ESC/iPSC-ECs are enriched in the immune-related pathways, top four of which are related to neutrophils (Fig. 7e); parts of pathways of normal EPCs also were related to immunity (Fig. 7f). This reveals that IPAH-EPCs dismiss immune-related molecular characteristics and primarily enhance proliferation capacity, and suggests that ESC/iPSC-ECs, with early EPC characteristics, have greater cellular therapeutic potential.