Neuropilin-2 Identifies Cardiovascular Precursor Cells and is Required for Vascular Differentiation in Murine Embryonic Stem Cells System

Research Article

J Stem Cell Res Transplant. 2015; 2(1): 1017.

Neuropilin-2 Identifies Cardiovascular Precursor Cells and is Required for Vascular Differentiation in Murine Embryonic Stem Cells System

Ding S1,2, Ma M¹, Negro A¹, Berry C1,3, Kovacic JC1,4, Cimato TR5 and Boehm M¹*

¹Center for Molecular Medicine, National Heart Lung and Blood Institute, National Institutes of Health, USA

²Institute of Stem Cell, H&B Tech. Inc. Being, China

³Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK

4Wiener Cardiovascular Institute, Mount Sinai School of Medicine, USA

5Clinical and Translational Research Center, SUNY at Buffalo School of Medicine and Biomedical Sciences, USA

*Corresponding author: Boehm M, Center for Molecular Medicine, National Heart Lung and BloodInstitute, National Institutes of Health, Bethesda,Maryland, USA

Received: November 26, 2014; Accepted: February 19, 2015; Published: February 24, 2015


Neuropilins are cell surface receptors that bind Class III semaphorins and Vascular Endothelial Growth Factor (VEGF). While Neuropilin-1 is essential for vascular development, the role of Neuropilin-2 is less well defined. The objective of this study was to determine the role of Neuropilin-2 in early cardiac and vascular differentiation in embryonic stem cells. We report that expression of Neuropilin-2 coincides temporally with Brachyury expression in murine embryonic stem cells and that Neuropilin-2 expression occurs in the majority of Brachyury positive cells. In contrast to Neuropilin-1, we found very few Neuropilin-2 positive cells that co-express the hemangioblast marker Flk-1. Both Neuropilin-2 positive and Brachyury positive cells express mesoderm markers that can differentiate into cardiomyocyte, endothelial and smooth muscle cell types with equivalent efficiency. However, inhibition of Neuropilin-2 expression impairs the differentiation of cardiovascular precursors to endothelial cells and smooth muscle cells, but not cardiomyocytes. The onset of Neuropilin-2 expression identifies a sub-population of Brachyury positive cardiovascular precursors that can differentiate to endothelial, smooth muscle and cardiomyocyte cell types. Furthermore, Neuropilin-2 is functionally required for differentiation of vascular cell types but not cardiomyocytes.

Keywords: Neuropilins; Cardiovascular differentiation, Vascular cells


Neuropilins (NRP) -1 and -2 are transmembrane receptors for class III semaphorin and Vascular Endothelial Growth Factors (VEGFs) [1,2]. NRPs are implicated in embryonic neurogenesis and vasculogenesis [3-5], with NRP-1 gene deletion leading to embryonic lethality due to cardiovascular malformations between E10.5 and E13.5 [4]. Embryos lacking NRP-1 display abnormal yolk sac vascular development and subsequent formation of the cardiac outflow tract and aortic arches is also disturbed [2,6]. In contrast, NRP-2 is a receptor for semaphorin IV and VEGF-C, and is required for embryonic vessel development [6-8]. While NRP-2 is expressed in embryonic blood islands in a pattern distinct from NRP-1, NRP-2 is preferentially expressed in the lymphatics and veins of more mature embryos [9]. NRP-2 knockout embryos exhibit defective vascular development with disordered small vessel formation [10], but does not result in early embryonic lethality, as found in VEGF and VEGFR-2 knockout embryos. Knockout of both NRP-1 and NRP-2 results in an even more pronounced phenotype, with severe impairment of yolk sac vascular development and early embryonic lethality [4,6]. These observations suggest a role for NRP-2 in embryonic vascular differentiation although the mechanisms involved underlying this process remain to be defined.

Brachyury (Bry), one of the most important mesoderm inducing factors (25) is a member of the T-box transcription factor gene family and is required for posterior mesoderm and notochord formation during vertebrate development [11]. In murine embryonic stem cells, Bry expression is an important tool for tracking mesendodermal precursors [12]. To facilitate the use of Bry as a mesendodermal marker, the GFP gene was targeted to the Bry locus (Bry-GFP) in Murine Embryonic Stem Cells (mESC), with GFP expression correlating with endogenous Bry transcription. Consequently, developing mesendodermal progenitors can be identified and separated based on GFP expression [12].

Recently, cardiovascular progenitors were shown to differentiate from Bry+/Flk-1- cells acquired from EBs of Bry-GFP mESCs [13]. We previously reported that NRP-1 expression identifies Bry+ and Flk-1+ vascular precursors in both murine and human embryonic stem cells [14]. However, the relationship between Bry and NRP- 2, if any, are unknown. Since NRP-2 and Bry are involved in early aspects of cardiovascular development, we hypothesized that NRP-2 expression may temporally correspond to Bry expression, and may have an important role in the formation of cardiovascular tissues including cardiomyocytes, endothelial cells and smooth muscle cells. In this study, using Bry-GFP mESCs, we determined that the timing of NRP-2 expression coincides with Bry expression during mesoderm differentiation, and that NRP-2 is functionally required for the differentiation of mesodermal precursors into vascular cell types but not cardiac precursors.

Materials and Methods

mESC Culture

Bry/GFP knock-in murine embryonic stem cells (Bry- GFP mESCs) [12] were initially grown on mitomycin-c-treated primary murine embryonic fibroblasts in ES Cell Qualified DMEM (EmbryoMax®, CHEMICON) supplemented with 1.5 x 10-4 M monothioglycerol (Sigma, St. Louis, MO), 15% Fetal Calf Serum (FCS) (Hyclone, Thermo Fisher Scientific, Waltham, MA), 10 mg/ ml leukemia inhibitory factor (# L5158-5UG, Sigma), 10 mg/ml recombinant human Bone Morphogenetic Protein (BMP) 4 (#314- BP, R&D Systems, Minneapolis, MN), and transferred to feeder-free, serum-free conditions in N2B27 medium (Invitrogen, Carlsbad, CA) supplemented with 2 mM L-glutamine (Sigma), 0.1 mM β-mercaptoethanol (BDH), 10 mg/ml LIF and 10 mg/ml BMP4 as previously showed [15].

Mesodermal differentiation

Bry-GFP mESCs were differentiated as EBs by trypsinization of mESCs and plating at a density of 1.5 x 103 cells/cm2 in 150 x 25 mm suspension culture dishes in N2B27 medium supplemented with BMP4 and basic fibroblast growth factor (bFGF), both at 5 mg/ml (# 233FB, R&D Systems).

Cardiovascular Differentiation: Day 3.5-EBs derived from Bry- GFP mESCs were dissociated to single cells by treatment with 0.25% trypsin (Invitrogen) and stained with antibodies for flow cytometry analysis as described below. NRP-2+/Flk-1- cells and Bry+/Flk-1- cells were sorted using a BD FACSAria and reaggregated for 20 hours at a density of 4 x 105/ml in ultra low attachment 24-well plates (Costar, Thermo Fisher Scientific) containing N2B27 media supplemented with 5 mg/m rhVEGF (# 293VE, R&D Systems) and 30 mg/ml bFGF. Reaggregated cells were then trypsinized and plated in 24-well cell culture plates (Costar) at density of 2 x 104 /cm2 , and cultured for an additional 5 - 7 days in N2B27 medium. For cardiomyocyte differentiation, medium was supplemented with 10 mg/ml rhBMP4 and 10 mg/ml rhVEGF; for endothelial cell differentiation, the medium was supplemented with 10 mg/ml rhVEGF; for smooth muscle differentiation, the medium was supplemented with 10 mg/ ml rhPDGF-BB (# 220BB, R&D Systems).

Flow cytometry analysis

Day 3.5-EBs derived from Bry-GFP mESCs were dissociated to single cells by treatment with 0.25% trypsin. Cells were incubated with the following antibodies: APC conjugated anti-mouse Flk-1 IgG (# 17-5821-81, eBioscience, San Diego, CA) or co-incubated with goat polyclonal anti-NRP-2 IgG (# AF2215, R&D Systems) and Flk- 1-APC for 40 minutes at 4°C. Cells were washed with 0.2 mM EDTA-, 1% FCS-PBS, centrifuged at 500g for 5 minutes then incubated with swine anti-goat IgG-phycoerythrin (# MBS674933, MYBIOSOURCE, San Diego, CA) for 30 minutes at 4°C. The quantity of Bry-GFP, Flk- 1 and NRP-2 expressing cells was determined by flow cytometry analysis using a BD FACSCalibur.

Immunofluorescence microscopy

Reaggregated NRP-2+/Flk-1- and Bry+/Flk-1- cells were washed with 1X PBS and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature. The following primary antibodies were applied to cells overnight at 4°C: cardiac lineage differentiation, Nkx2.5 (#sc-74692, Santa Cruz biotechnology), Islet-1 (#AF1837, R&D Systems), Troponin I (#sc-8118, Santa Cruz biotechnology) and Connexin 43 (#35-5000, Invitrogen); endothelial and smooth muscle cell differentiation, VE-Cadherin (#550548, BD Pharmingen, San Diego, CA), CD31 (#14-0311-82, eBioscience) and SM-22 (#ab14106 Abcam, San Francisco, CA), α-smooth muscle actin (#A2547, Sigma). Cells were washed with 0.5% Tween 20 in PBS, and detected with donkey anti-goat, anti-rabbit, and antirat IgG conjugated Alexa fluor 488 or 555 (Invitrogen). Cells were imaged with an Olympus IX71 fluorescence microscope.

Reverse transcription-polymerase chain reaction (RTPCR)

Total RNA was extracted from sorted NRP-2+ cells and Bry+ cells using RN easy Mini Kit (#74104, QIAGEN, Valencia, CA). Firststrand cDNA was synthesized using M-MLV Reverse Transcriptase (#9PIM170, Promega, Madison, WI), followed by PCR using PuReTaq Ready-To Go PCR Beads (#27955701, GE Healthcare, Pittsburgh, PA). PCR cycle conditions were: 1 cycle of 94°C for 5 minutes; 35 cycles of 94°C for 20 seconds, 60°C for 20 seconds, 72°C for 40 seconds; and 1 cycle of 72°C for 7 minutes. GAPDH was used as an internal control. The sequences of primers used were (forward and reverse): PDGFR-β, GTCTGGTCTTTTGGGATCCT and AAGGCTGGTTACAGTTTGGC;





shRNA Knockdown of NRP-2 in Bry-GFP mESCs: Murine NRP- 2 shRNA plasmid vectors (#sc-36041-SH, Santa Cruz biotechnology) were isolated from E.Coli using Endo Free DNA Maxi Kit (#12362, QIAGEN). NRP-2 shRNA vectors were then transfected into HEK293 cells plated at a density of 2.5 x 105 cells/well on 6-well plates. Transfection was performed using DNA/Arrest-In reagent according to the manufacturer’s instructions. Lent viral particles were collected from the cell supernatant and used for transfection of Bry- GFP mESCs. Bry-GFP mESCs were cultured at density of 1.5 x 104 / cm2 in N2B27 medium for 24 hours. The medium was then replaced with 1 ml of the supernatant medium obtained from the transfected 293 cell supernatant medium, and after 48 hr another 1 ml N2B27 medium was added with culture at 37°C, 20% Oxygen, 5% CO2. Transfected cells were selected by culture for 6 additional passages in N2B27 medium μg/ml containing puromycin1.

Statistical analysis

Experimental data were analyzed by unpaired 2-tailed t-test. Results are expressed as mean ± SEM. Differences were deemed significant when P < 0.05. Statistical analyses were performed using Prism, Version 4.00 (GraphPad Software, La Jolla, CA, USA).


NRP-2 identifies Bry+ mesodermal precursor cells

Bry is an important regulator of early mesodermal cell commitment during development and in the differentiation of murine ESCs [16]. Consistent with previous findings [12,15], flow cytometry analysis of GFP expression in EBs derived from Bry-GFP mESCs showed that Bry expression was up-regulated at 2.5 days, peaked at 3.5 days, decreased dramatically at 4.5 days, and was undetectable at 5.5 days (Figure 1A). To determine if NRP-2 was co-expressed in Bry+ cells, we performed flow cytometry analysis of dissociated EBs 3.5 days after differentiation in mesoderm-inducing conditions. The majority of Bry+ cells co-expressed NRP-2 (> 80%) at day 3.5 (Figure 1B). Immunofluorescence staining of NRP-2 of day 3.5 EBs derived from Bry-GFP mESCs confirmed that NRP-2 positive cells co-expressed Bry+ cells at that stage of differentiation (Figure 1C). Collectively, these results indicate that NRP-2 identifies the majority of Bry+ differentiated mESCs under mesoderm-inducing conditions.