Using human pluripotent stem cell differentiated endothelial cells to study the differential regulation in PAH

Aims Endothelial cells (ECs) have been applied in clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, lack of enough transplantable cells before the deterioration of patient condition is current limitation to apply cell therapy in cardiovascular diseases. So, we thought it necessary to continue to differentiate embryo stem cells (ESCs)/induced-pluripotent stem cells (iPSCs) into endothelial cells (ECs) and identify their characteristics. Methods and results Comparing previous reported methods of human pluripotent cell differentiation toward vascular cells/ Hemogenic Endothelial cells/ endothelial colony-forming cells, we established a highly ecient differentiation protocol to get cells that match phenotype of isolated ECs from health donors. This protocol, including two stages, early mesoderm endothelial progenitor stage and EC marker expression stage. In the rst stage, Rock inhibitor Y27632 and DMSO plays an important role in inducing-APLNR + mesoderm and promotes EC differentiation potential; later on SB431542 and BMP4 drives cells toward EC lineage. Meanwhile, an improved protocol with chemically-dened medium (CDM) has similar differentiation eciency,again demonstrating that a large number of clinically needed cells could be obtained with simple factors. ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have the characteristics of early EPCs marked by CD133 and mesenchymal stem cells. Microarray analysis further revealed IPAH-derived EPCs features of rapid proliferation and weak immune regulation. At last, a model Zebrash xenograft was utilized to assess the functionality of differentiated ESC/iPSC-ECs. Conclusions We established a highly ecient differentiation protocol to get ESC/iPSC-ECs with characteristics of phenotype and molecular matched with early-EPCs from health donors, and revealed the molecular pathogenesis in PAH.


Introduction
During hypoxia environment, endothelial cell (ECs) have an ability to modulate vascular tone and induce vascular remodeling and angiogenesis [1], and endothelial progenitor cells (EPCs) have been demonstrated that it can promote ischemic tissue angiogenesis [2], so they both are capable of facilitating vascular repair in different ischaemic tissues, such as acute myocardial infarction, unstable angina, stroke, diabetic micro vasculopathies, pulmonary arterial hypertension, atherosclerosis, and ischaemic retinopathies [3][4][5][6][7]. ECs and EPCs, which have limited expansion potential, are rare in peripheral or umbilical cord blood, comparing with other blood cells, so it's a huge challenge to repair vascular in clinical treatment [8][9][10] .
Human ESCs/iPSCs can be induced to produce scalable endothelial cells and endothelial progenitor cells for vascular modeling [9,[11][12][13][14][15][16], and now there have been two methods of inducing ESCs or iPSCs differentiate to vascular cell, which are embryoid body formation [12,17] and monolayer-directed differentiation [18,19]. In the former method, the cells need to be transferred into ultra-low-attachment plates to get embryoid bodies (EBs) and produce various types of cells, the cost is large and the e ciency is low [7,12,20,21]; besides, embryoid body differentiation is often time consuming [22]. Monolayer differentiation methods have a higher e ciency [15], but it is necessary to explore further to have a better understanding of complicated factors.
Endothelial dysfunction has been thought to be the main cause of PAH/IPAH. Some articles have reported that numbers of CD133 + cells in peripheral blood increased in PAH/IPAH patients as compared with controls [23,24]. Toshner et al also demonstrated PAH patient-derived endothelial progenitor cells have a hyperproliferative phenotype and a reduced capacity to form vascular networks [24]. But further investigation is necessary to continue at gene expression pro le level.
In this study, we aim to explore the factors affecting vascular cell differentiation and to establish a chemical de nition and cost-effective system of generating human ESC/iPSC-ECs through monolayerdirected differentiation, which is more e cient comparing with three other protocols [15,25,26]; meanwhile, we want to reveal the molecular features of ESC/iPSC-ECs, IPAH-derived EPCs and normal EPCs. We found that Rock inhibitor Y27632 and DMSO markedly accelerated vascular mesoderm generation from ESCs/iPSCs and improved the differentiation e ciency. Then, the functions of isolated cells have been jointly tested through tube formation assay, LDL uptake assay, cell transplantation assay, Microarray and zebra sh assay. Thus, the improved system can offer a simple, cost-effective platform to produce ESC/iPSC-ECs from ESCs/iPSCs for vascularization research and clinical application.

EPCs from adult peripheral blood samples
EPCs were isolated from human peripheral blood (PB). Fresh human PB (20 mL) was obtained under full ethical approval; then mononuclear cells (MNCs) were isolated from PB by density gradient centrifugation and cultured in EGM2(Lonza) supplemented with 16% FBS.

Flow cytometry
At day 3 or 7 of differentiation, adherent cells were harvested using 0.25% TrypleE with EDTA and made into a single-cell suspension in PBS with 0.2% BSA. Mouse Anti-Human APJ APC-conjugated

In vitro capillary network formation assay on Matrigel
Endothelial cells were trypsinized and resuspended in EGM-2 media with 16% FBS. Cells were plated at a density of 1.0 × 10 4 cells per well in triplicate in 96-well plates coated with 50 µl of growth factor-reduced Matrigel (BD Biosciences). Plates were incubated overnight at 37 °C. After 6 h of incubation, photomicrographs were taken from each well at 10x magni cation using an Olympus CKX41 microscope with a 10x objective.

RNA extraction, cDNA synthesis and RT-qPCR
Total RNA from human cell lines was extracted with Trizol (Life Technologies). RNA yield was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Total RNA (1 µg) was converted to cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA). Quantitative PCR (qPCR) was done using TransStart Tip Green qPCR SuperMix (TransGen Biotech) and detection was achieved using the CFX Connect™ Real-Time System (BIO-RAD). Primer sequence are listed in Additional le 1: Table S1. Expression of target genes was normalized to reference gene GAPDH.

Cell transplantation and therapy in Zebra sh
Zebra sh, the transgenic line Tg (Flk: GFP) were maintained according to standard procedures in compliance with local approval. After 48 hours post fertilization (hpf), embryos were used to inject ESC/iPSC-ECs stained with CM-Dil at approximately 60 µm above the ventral end of the duct of Cuvier, then the embryos were maintained at 30℃ for 48 hours. Fluorescent image acquisition was performed using a Leica MZ16FA stereo-microscope. Zebra sh embryos were treated with sugen5416 for 24 hours at 4 hpf, then 20-30 sh were respectively therapied by ESC/iPSC-ECs /medium (EGM2 + 16%FBS) and maintained at 30℃ for 48 hours, the number of normal zebra sh were recorded. The experiments were repeated three times. Euthanasia of all zebra sh was performed by exposure to bleach or rapid freezing followed by maceration.

Statistical analysis
Statistical analyses were performed using Student's t-test and data were reported as mean ± s.d. or standard error of the mean, Figure legends show the number of biological repeats for each experiment (n), the experiments were repeated three or more than three times. Statistical signi cance was assumed when P < 0.05.

A modi ed protocol for generating ECs from human ESCs or iPSCs
A high-e ciency 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 × 10 4 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 e cient 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 e ciency 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 rst built the highly e cient 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 le 1: Figure S1a and b). Our results demonstrated that Y27632 has some effect on ESC differentiation, but the e ciency of EC differentiation is still not high as same as the above, so we predicted that there are other in uencing factors.

Optimizing conditions to increase the ESC/iPSC-EC differentiation e ciency
Then we only utilized the protocol[26] and further explore how to promote the e ciency. Firstly, we designed a serial cell density experiment and found that the differentiation e ciency decreased with the increase of the cell density in H1-ESCs (Additional le 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 e ciency increased (Additional le 1: Figure S2b and d). BMP4 has been used both stages ( rst stage and second stage), here we checked whether adding BMP4 or not affected ESC/iPSC-EC differentiation e ciency in the second stage, the result is that there was a higher differentiation e ciency (Additional le 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 e ciency.

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 e ciency 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 e ciency 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 e ciency 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 pro ciency 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 rst step, we con rmed the highly e cient 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-de ned medium (CDM) (Additional le 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 gured out the reason why we can't repeat our improved protocol for a while.

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 ow 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 identi ed 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 immuno uorescence 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)
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 Zebra sh 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 zebra sh 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.

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.

Discussion
As a matter of fact, DMSO plays a role in the whole ESC/iPSC-EC differentiation, which we just ignored, because normally small chemical molecules are dissolved in DMSO, and in our study, we used two chemical molecules Y27632 and SB431542; some research used CHIR99021 to promote EC differentiation [14,26]. According to our experience, DMSO treatment need a separate 37℃ and 5% CO2 incubator, which avoids to induce the differentiation of other cells, so we guess that DMSO is very powerful, and suggest that we should use DMSO with care.
The role of the inhibitor Y27632 was usually neglected in the process of ESC/iPSC-EC and HE differentiation, because robust differentiation protocols using single cell have to make cell survive by the inhibitor Y27632 which can phosphorylate and activate the myosin II pathway and inhibit an E-cadherindependent apoptotic pathway [46]. Some articles have reported that Y27632 enhances differentiation of human term placenta-derived trophoblasts in vitro [47], differentiation induction [48] and mesendodermal differentiation [49], and in another article prolonged Tbx6 expression-induced mesoderm differentiated into skeletal myocytes with the Rock inhibitor Y27632 [30]. Besides, the extension of treatment time to 3 days in the rst stage for mesoderm induction, consistent with another research [14], may be able to produce mesodermal cells substantially.
ESC/iPSC-ECs have the characteristics of EPCs or ECFCs. Hematopoietic and endothelial progenitor cells share a number of cell-surface markers, because they may originate from a common precursor, the hemangioblast [50]. It has been reported that CD34 + CD133 + VEGFR2 + cells are haematopoietic and may not actually be true EPCs [9], so we chose the endothelial marker CD31 + to isolate CD31 + cells, then identi ed their characteristics as ESC/iPSC-ECs through GOBP analysis of microarray data and so on; moreover, the expansion ability of isolated CD31 + cells is as the same as normal person's EPCs, which of both are less proliferative than EPCs from IPAH-patients. At last, gene expression analysis from our microarray data showed that ESC/iPSC-ECs expressing CD40, CD90 and CD105 are similar to mesenchymal stem cells, and the experiment of zebra sh transplantation proves their potential application value.
In conclusion, to verify the system and reduce the cost, we tried another CDM medium and repeated the protocol, the results proved that's good. On the one hand, the function of each factor in the differentiation process is better to understand, and on the other hand, it lays a foundation for future application. ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have characteristics of early-EPCs and mesenchymal stem cells for homing. Meanwhile, we demonstrate IPAH-EPCs have a higher proliferation again than normal EPC, and further reveal IPAH-EPCs with lack of immune-related genes.  Characteristics of ESC/IPSC-ECs (H7EC, 202EC, H1EC) and the comparation with peripheral blood derived

Abbreviations
EPCs. a Comparation of cell morphology with peripheral blood derived EPCs (scale bars 50um). b Using Selected markers to identify cell characters by ow cytometric analysis. c Uptake assay of Dil-acetylated LDL(scale bars 50um). d Tube formation assay of H1-derived EPCs(scale bars 500um). e qRT-PCR of NRP1 which was reported promotes ECFC proliferation. Gapdh was used as an internal control. Data are represented as mean ± SD, n=3, the experiments were repeated three times. Student's t test (*p < 0.05,***p < 0.001).  Student's t test (*p < 0.05).