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Published as doi: 10.1096/fj.08-112847.
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(The FASEB Journal. 2009;23:68-78.)
© 2009 FASEB

Microenvironment drives the endothelial or neural fate of differentiating embryonic stem cells coexpressing neuropilin-1 and Flk-1

Anna Gualandris*,{ddagger},1,2, Alessio Noghero*,{ddagger},1, Massimo Geuna{dagger},{ddagger}, Marco Arese*,{ddagger}, Donatella Valdembri*,{ddagger}, Guido Serini*,{ddagger} and Federico Bussolino*,{ddagger}

* Division of Molecular Angiogenesis,

{dagger} Division of Clinical and Experimental Cytometry, and

{ddagger} Department of Oncological Sciences, Institute for Cancer Research and Treatment (IRCC), University of Turin School of Medicine, Candiolo, Torino, Italy

2 Correspondence: Department of Oncological Sciences, Institute for Cancer Research and Treatment (IRCC), Strada Provinciale 142, km 3,95, 10060, Candiolo (TO), Italy. E-mail: anna.gualandris{at}ircc.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The observation that the architecture of the cardiovascular and nervous systems is drawn by common guidance cues and the closeness between neural progenitors and endothelial cells in the vascular niche strongly suggests the existence of links between endothelial and neural cell fates. We identified an embryonic stem cell-derived discrete, nonclonal cell population expressing the two vascular endothelial growth factor receptors neuropilin-1 (Nrp1) and Flk1 that differentiates in vitro toward endothelial or neural phenotypes depending on microenvironmental cues. When microinjected in the chick embryo, Nrp1+ cells integrate within the host, developing vessels and brain, and acquire endothelial and neural markers, respectively. These results show that precursors of endothelial cells and precursors of neural cells arise from the same pool of differentiating embryonic stem cells and share the expression of Nrp1 and Flk1. These data reinforce the parallelism between vascular and nervous system at the level of cell fate and commitment and open new perspective in regenerative medicine of neurovascular diseases.—Gualandris, A., Noghero, A., Geuna, M., Arese, M., Valdembri, D., Serini, G., Bussolino, F. Microenvironment drives the endothelial or neural fate of differentiating embryonic stem cells coexpressing neuropilin-1 and Flk-1.


Key Words: cell commitment • endothelial differentiation • embryoid bodies


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DURING THE PAST TWO DECADES, the parallelism between the vascular and nervous system has become increasingly evident. By following similar pathways of migration in the developing body, vessels and nerves establish interdependent reciprocal relations (1) . Among the attractive/repulsive molecules that are shared by nerves and vessels there are the semaphorins with their cognate receptors neuropilins (Nrp) and plexins (2) . Besides being localized on axons and growth cones, where it modulates nerves trajectory during embryonic development (3) , the glycoprotein Nrp1 is also expressed by human endothelial cells, where it mediates the activity of VEGF-A165 and VEGF-A121 (4 , 5) . Accordingly, knocking out and overexpressing Nrp1 in engineered mice both lead to lethality because of massive vascular defects (6 , 7) , whereas the specific loss of Nrp1 in endothelial cells decreases arterial marker expression in small arteries (8) . The parallelism between vascular and nervous system is also mirrored at the level of cell commitment and differentiation. Even though the origins from different germ layers make the endothelial and neural lineages restricted to specific progenitors, some data show a plastic correlation between the endothelial and neural commitments. In vitro, in appropriate culture conditions, bone marrow and cord blood cells acquire a neural phenotype (9 , 10) , while neural stem cells (NSCs) acquire the endothelial phenotype (11) . In vivo, bone marrow-derived cells enter the brain and differentiate into neural cells (12 , 13) , whereas murine NSCs can engraft into the hematopoietic system of irradiated hosts to produce blood cells (14) . Furthermore, in the quail-chick chimera model, the avian cranial neuroectoderm originates smooth muscle cells (15) . Considering that Nrp1 and VEGFR-2/Flk-1 are shared by mature neurons and endothelial cells, we wondered whether Nrp1 could be shared also by the respective precursors of both cell types, ultimately marking points of contacts between the two commitments.

To achieve our goal, we used murine embryonic stem cells (ES cells) as a source of precursors of both neural and endothelial cells. ES cells differentiate efficiently in vitro to originate the embryoid bodies (EBs), three-dimensional differentiated cell masses that provide a large number of cells representing an early or primitive stage of development, which is otherwise difficult to access in an embryo (16) . This model has been used in the past to identify the hemangioblasts, the common precursors of both hematopoietic and endothelial cells (17) , and an Flk1+ cell population that could differentiate not only into hematopoietic and endothelial cells, but also into smooth muscle cells, skeletal muscle cells, and cardiomyocytes (18 19 20) . Despite that no proofs have been reported of the existence of such progenitors in vivo and recent studies performed with mouse embryo and tetrachimeric blastocysts argued against it (21 , 22) , the Flk1+ cells isolated from EBs still represent an interesting tool to easily address the topic of cell commitment and differentiation.

On the basis of these observations, we identified an ES-derived population of nondefinitively committed Nrp1+ cells that retains the capacity to embrace the endothelial or the neural commitments. Such potentiality was proved in vitro by culturing the two purified Nrp1+/Flk1+ and Nrp1+/Flk1– populations in cell-type restrictive conditions, and, in vivo, by injecting the cells into different areas of the developing chicken embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
W4 mouse ES cells were kindly provided by Dr. Alexandra L. Joyner (Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA) (23) . R1 ES cells expressing the enhanced yellow fluorescent protein (EYFP) were obtained from Dr. Andras Nagy (Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, ON, Canada) (24) . Both ES cell lines were cultured as described previously (25) . Murine embryonic fibroblasts (MEFs) were purchased from American Type Culture Collection (LGC Standards, Milan, Italy). OP9 and PA6 stromal cell lines, kindly provided by Dr. Timm Schroeder (Institute of Cell Research, Helmholtz Zentrum München–German Research Center for Environmental Health, Neuherberg, Germany), were cultured as described (26) .

In vitro differentiation of ES cells
EBs were obtained by the "hanging drop" procedure, as described previously (25) . The age of the EBs was indicated with progressing numbers starting from the first day of culture in the absence of leukemia inhibitory factor (LIF). For fluorescence immunostaining, the EBs were plated onto Lab-Tek II CC2 chamber slides (Nunc, Naperville, IL, USA).

Cell staining for FACS analysis and for cell sorting
EBs were dissociated into cell suspension by trypsin treatment (0.05%) for 1 min and by a mechanical dissociation through a 25-gauge needle. Cells were stained with 1 µg/106 cells of goat anti-Nrp1 antibody alone or in association with one or two of the following antibodies: anti-Flk1, CD31, ICAM2, CD133, PSA-NCAM, RC2, APC-anti-PDGFR{alpha}, APC-anti-CXCR4, APC-anti-Flk1, and PE-anti-Flk1. Antibody brands and dilutions are given in Supplemental Table 1. The secondary antibodies used were fluoresecin isothiocyanate (FITC) or APC-conjugated-anti-goat to detect Nrp1, PE-conjugated-anti-rat to detect Flk1 and CD133, and FITC-conjugated anti-mouse immunoglobulin G (IgG) or IgM to detect nestin, PSA-NCAM, and RC2 (Southern Biotech., Birmingham, AL, USA). To detect cytoplasmic antigens, the IntraPrep Permeabilization Reagent was used (Beckman Coulter, Marseille, France) by following the manufacturer’s instructions. As a control of intracellular labeling, isotype IgGs were used (data not shown).

To prepare cells for cell sorting, the secondary antibodies used were PE-conjugated anti-rat and biotin-conjugated anti-goat followed by an AlexaFluor 647-PE-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA). Cell sorting was performed with Beckman Coulter Epics Altra. The accuracy of the sorting procedure was confirmed by analyzing representative samples of the two sorted populations through a Becton Dickinson FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA) purposely reserved for routine analysis only. Such analysis revealed that cell populations were sorted at 95–97% of purity.

Culturing sorted cells
Once sorted, cells were seeded onto OP9 monolayers and cultured in EGM-2 medium composed by endothelial cell basal medium-2 and the EGM-2 bullet kit (Bio Whittaker, Walkersville, MD, USA).

For the neural differentiation, sorted cells were seeded onto laminin-1-coated coverslips and grown for the first 2 days in a serum-free Dulbecco modified Eagle medium (DMEM)/F12 medium supplemented with N2 supplement (Invitrogen), 1 µg/ml laminin-1 (Sigma, St. Louis, MO, USA), and 10 ng/ml fibroblast growth factor (FGF-2) (R&D Systems, Minneapolis, MN, USA). FGF-2 was withdrawn from the medium for the following 48 h after that medium was replaced with Neurobasal medium plus B27 supplement, 2% horse serum, 50 ng/ml nerve growth factor (Roche, Mannheim, Germany), and 10 ng/ml brain-derived neurotrophic factor (BDNF; R&D Systems).

Single-cell deposition assay
YFP+ ES cells sorted for Nrp1 and Flk1 expression were seeded as a single cell per well into optical-bottom 96-well plates (Nunc, Rochester, NY, USA) containing confluent monolayers of PA6 feeders and differentiating W4 ES cells. YFP+ clones were derived in N2/B27 medium (27) supplemented with FGF2 20 ng/ml for the first 24 h. Medium was changed every 2 days; after 8 days of coculture, clones were fixed and processed for immunostaining.

RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNAs from the cocultures of YFP sorted cells and OP9 cells or from OP9 cells alone were purified at the times indicated using TriZOL reagent (Invitrogen) and RNase-free DNase I (DNA-free; Ambion, Austin, TX, USA). RNAs were reverse transcribed using SuperscrptII cDNA synthesis kit (Invitrogen). Control reactions without reverse transcriptase were performed for each RNA sample. PCR reactions were carried out with 100 ng of first-strand cDNA using Platinum Taq polymerase (Invitrogen) and optimized to allow semiquantitative comparisons within the log phase of amplification. Comprehensive list of the primer sequences is given in Supplemental Table 2. Images of ethidium bromide-stained gels were acquired with a Molecular Imager Chemidoc XRS (Bio-Rad, Hercules, CA, USA), and densitometric analysis was performed with Quantity One software (Bio-Rad); results were normalized over the expression of the HPRT housekeeping gene and expressed as relative units.

Indirect immunofluorescence
The list of the primary and secondary antibodies used in these experiments is given in Supplemental Table 1. All the images were captured by using a Leica TCS SP2 AOBS confocal microscope and analyzed with Leica Confocal Software (LCS; Leica Microsystems, Wetzlar, Germany).

EBs and cultured sorted cells were fixed with PBS and 4% paraformaldehyde. Incubation with the primary antibodies was carried out for 1 h at 37°C in a moist chamber. Frozen chicken sections were incubated in a moist chamber overnight at 4°C with primary antibodies; only when required, they were incubated with biotinylated sambucus nigra agglutinin (Vector Laboratories, Burlingame, CA, USA) and AlexaFluor-555-streptavidin (Invitrogen).

The immunofluorescence staining of the 96-well plates containing the single-cell deposition assay was performed with a mixture of the following primary antibodies: anti-green fluorescent protein (GFP) with anti-RC2 and with anti-VE-cadherin. Alternatively, anti-O4, anti-{alpha} smooth muscle actin ({alpha}SMA), anti-E-cadherin, or anti-cytokeratin antibody was used.

Immunoprecipitation and Western blot analysis
Cell lysates of W4 undifferentiated ES cells and of 7-day-old EBs were obtained under cold conditions with 10 mM Tris/HCL pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 0.1 mM ZnCl2 supplemented with protease inhibitors cocktail (Sigma). Protein content was measured by BCA assay (Pierce, Rockford, IL, USA). Nrp1 protein was immunoprecipitated with 1 µg of rabbit polyclonal anti-Nrp1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and immunocomplexes were loaded onto 7.5% SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA, USA). The filter was decorated with the goat polyclonal anti-Nrp1 antibody C19 (Santa Cruz). Immunocomplexes were then visualized by enhanced chemiluminescence (ECL) system (Pharmacia-Amersham Biotech., Little Chalfont, Buckinghamshire, UK).

Microinjection experiments on chick embryos
Purified Nrp1+/Flk1+ or Nrp1+/Flk1– cells were microinjected into the beating heart or into the mesencephalic cavity of HH19 chicken embryos by using a manual microinjector (CellTram Oil; Eppendorf, Hamburg, Germany). At different days after injection, the embryo vasculature was labeled with AlexaFluor 405-conjugated wheat germ agglutinin that was injected into an artery of the vitelline sac. Immediately afterward, embryos were sacrificed, fixed with zinc saline formalin (Bio-Optica, Milan, Italy) for 48 h at 4°C or with PBS 4% paraformaldehyde for 24 h at 4°C and cryoprotected in 15% sucrose for 8 h and then in 30% sucrose overnight at 4°C. After embedding and freezing them in Killik compound (Bio-Optica), 12- to 20-µm sections were cut using a Leica CM3050 S cryostat.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Nrp1 expression in differentiating ES cells
ES cells were chosen as an in vitro model to analyze the role played by Nrp1 during cell differentiation. After 5 days of growing ES cells as suspended aggregates in LIF-deprived medium, ES cells were allowed to attach to a substrate (day 5), where they formed EBs (Fig. 1A ). To analyze Nrp1 expression in differentiating ES cells, 6-day-old EBs were fixed and immunostained with anti-Nrp1 antibody. As shown in Fig. 1A , Nrp1 was widely expressed in scattered areas throughout the growing EBs. Furthermore, Nrp1 protein was immunoprecipitated from lysates purified from 7-day-old EBs but not from undifferentiated W4-ES cells (Fig. 1B ).


Figure 1
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  		Figure 1. Nrp1 expression in differentiating ES cells. A) 6 day-old EBs (DIC, differential interference contrast) were analyzed by immunofluorescence with an anti-Nrp1 antibody and by using a Leica DM IRB inverted microscope. Scale bar = 1 mm. B) Lysates from undifferentiated ES cells and 7-day-old EBs were precleared with irrelevant IgG (IP IgG) before Nrp1 immunoprecipitation (IP Nrp1) and Western blot analysis (WB Nrp1). Immunocomplexes were detected only in lysates purified from EBs. C) Single-cell suspensions were prepared from both undifferentiated ES cells and EBs and analyzed by FACS using the anti-Nrp1 antibody or its isotype IgG (gray). Nrp1 is not expressed by ES cells and by the EBs until day 4. The experiment shown is representative of 5 experiments. D) FACS analysis of EB-derived cells immunolabeled with anti-Nrp1 antibody in association with anti-Flk1, CD31, ICAM2, PSA-NCAM, or CD133 antibodies. Each colored bar represents the cells positive for the corresponding marker, whereas the number placed on the top is the percentage value of double-labeled Nrp1+ cells. Data are averages ± SD of 5 different experiments. E) Cells from 7-day-old EBs were labeled with anti-Nrp1, -Flk1, and –PDGFR{alpha} or anti–CXCR4: the distribution of PDGFR{alpha} and CXCR4 among the Nrp1+/Flk1+, Nrp1+/Flk1–, and Nrp1–/Flk1+ cells populations (green, red, and magenta dots respectively) was quantified by FACS respect to a control (gray) and total bulk population (white). Percentage values of cells belonging to each of the four populations are indicated (dot plot, top right).

To quantify Nrp1 expression in the EBs, differentiating ES cells were analyzed by FACS. As shown in Fig. 1C , Nrp1 expression was absent in undifferentiated ES cells and during the first 3 days of differentiation, it became significant only at day 4 to finally stabilize at 45–50% of the total EB cells population in the next days.

Characterization of EB-derived Nrp1+ cells
To characterize the phenotype of Nrp1+ cells in the EBs, we performed a time-course FACS analysis of EB cells double-labeled for Nrp1 and for markers of either endothelial or neural cell precursors (Fig. 1D ). Being the canonical marker of hemangioblasts, Flk1 is expressed from day 4, which is the very first day of appearance of Nrp1 expression. The amount of Nrp1+ cells within the Flk1 component progressively increased over time to almost cover the totality of the Flk1 expressing cells at day 12, time in which the Flk1+ endothelial cells are organized into mature vessels (Fig. 1D , numbers above bars) (25) . At these late time points, ICAM-2 expression and its Nrp1+ fraction became significant, whereas CD133 expression, which identifies both endothelial (28) and neural precursors (29) , maintained a significant percentage of Nrp1-expressing cells throughout the entire EB growth. The polysialylated embryonic form of the neural molecule NCAM (PSA-NCAM) is characteristic of neural progenitors in the forebrain (30) , and its expression was shared almost entirely by Nrp1+ cells at all the time points examined. CD31, which is expressed by undifferentiated ES cells (31) , included a significant component of Nrp1+ cells at its peak of expression (day 7) to later stabilize at a lower level at day 12.

Characterization of EB-derived Nrp1+/Flk1+ and Nrp1+/Flk1– cell populations
Because endothelial cells and neurons share the expression of Nrp1 and VEGFR2/Flk1 (3 , 4) , we focused on the cell populations defined by these two markers, the Nrp1+/Flk1+, Nrp1+/Flk1–, and Nrp1–/Flk1+ cell types (Fig. 1E , green, red, and magenta, respectively; and Supplemental Fig. 1). To better characterize the potential of differentiation of these populations, the expression of the different germ layer markers was evaluated by cytofluorimetry and immunofluorescence. As shown by Fig. 1E , in both Nrp1+/Flk1+ and Nrp1+/Flk1– cell types, more than 70% of the cells express the paraxial mesoderm marker PDGFR{alpha} (32) , whereas only the 44% of the Nrp1+/Flk1+ cells and the 36% of the Flk1– counterpart express the definitive endoderm marker CXCR4 (33) . Similarly, mesoderm orientation prevails on endoderm commitment also in those Flk1+ cells that do not express Nrp1 (35% of PDGFR{alpha}+ cells vs. 14% of CXCR4+ cells in Nrp1–/Flk1+ population) (Fig. 1E , magenta). The orientation toward neuronal lineages was investigated by analyzing the expression of the neuronal markers nestin, sox1, and PSA-NCAM. The colocalization of nestin immunoreactivity with Flk1 and/or Nrp1-expressing cells (arrow and arrowhead, respectively, Fig. 2A ) was quantified as 50% of the cells of both Nrp1+ populations by intracellular FACS analysis (Fig. 2B ). Since the intermediate filament protein nestin is expressed not only by neural precursors but also by the endothelial cells during development (34) , the more specific markers sox1 and PSA-NCAM were next investigated. Sox1 is a SRY-related transcription factor whose expression is characteristic of proliferating neural precursors (35 , 36) . Nrp1+/Flk1+ and Nrp1+/Flk1– cells that expressed sox1 or PSA-NCAM were detected by immunofluorescence in sparse areas of 7-day-old EBs (Fig. 2A , arrow and arrowhead). The expression of PSA-NCAM was quantified at 42% of Nrp1+/Flk1+ cells and at 25% of Nrp1+/Flk1– cells by FACS analysis (Fig. 2B , green and red). Contrary to what was observed with the PDGFR{alpha} molecule (Fig. 1E ), the lack of PSA-NCAM expression in the Nrp1–/Flk1+ population suggested that neural orientation was not contemplated by the differentiation potential of those cells that did not carry Nrp1 (Fig. 2B , magenta). Similar results were obtained by analyzing the expression of RC2, an antigen that identifies neural precursors at the stage of radial glia (37) . The two Nrp1+ cell types were enriched in RC2, whereas the Nrp1– counterpart was not (Supplemental Fig. 2). To summarize, the molecular characterization of the populations defined by Nrp1 and Flk1 revealed an intriguing coexistence of markers of neural precursors with markers of mesoderm/endothelial precursors, which make the fate of Nrp1+ cells worth further investigation.


Figure 2
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  		Figure 2. Characterization of EB-derived Nrp1+ cells. A) Confocal microscopy analysis of 7-day-old EBs immunostained with the anti-Nrp1, anti-Flk1, and anti-nestin or anti-sox1 or anti-PSA-NCAM antibodies. Nrp1+/Flk1+ cells (arrow) and Nrp1+/Flk1– cells (arrowhead) expressing nestin, sox1, and PSA-NCAM are indicated. Scale bars = 50 µm. B) Quantification by FACS analysis of nestin and PSA-NCAM expression by the Nrp1+/Flk1+ cells (green), Nrp1+/Flk1– cells (red) and Nrp1–/Flk1+ cells (magenta) vs. control (white). Percentage values of cells belonging to each of the four detected populations are indicated (dot plot, top right).

In vitro analysis of the potential of differentiation of Nrp1+/Flk1+ and Nrp1+/Flk1– cells: endothelial and neural commitments
To better study the differentiation potential of Nrp1+ cells outside the EB environment, Nrp1+/Flk1+ and Nrp1+/Flk1– cells were isolated at 95% of purity from 7-day-old EBs by flow cytometry sorting and then cultured under different conditions. To support the endothelial differentiation in vitro, Nrp1+/Flk1+ and Nrp1+/Flk1– cells were seeded onto confluent monolayers of OP9 stromal cells, described to be optimal feeders for endothelial differentiation (38) , and grown in the presence of EGM2 medium; the maturation into endothelium was evaluated by analyzing the expression of different endothelial markers by RT-PCR at different days (Fig. 3A ; see also Supplemental Fig. 3A). Immediately after sorting (day 0), the Nrp1+/Flk1+ cells expressed all the endothelial genes tested at levels from 2 to 8 times higher than those of the Nrp1+/Flk1– cells, as shown by the quantification data plotted in Fig. 3A . High expression of CD31 messenger at day 0 in both cell types could reflect an inheritance from undifferentiated ES cells (31) . During the next few days, the levels of expression of the majority of the endothelial markers in the two cell populations became comparable. These last observations indicate that the coculture with OP9 feeders could fill in the initial impairment of the Nrp1+ cells sorted as Flk1–. Nevertheless, such rescue was not complete or fast enough to allow the Flk1– cells to organize tubes on Matrigel, a basement membrane matrix commonly used to evaluate the endothelial phenotype of cell cultures in vitro (Supplemental Fig. 3B). Despite cell sorting procedures guaranteeing 95% purity of the sorted cell populations, the Flk1 mRNA was amplified even in the Nrp1+/Flk1– cell sample at day 0. The possibility that the Nrp1+/Flk1– cells expressed an intracellular inactive Flk1 protein was investigated by performing a FACS analysis on EBs at the same differentiation time of cell sorting (day 7), with the purpose of studying the distribution of both membrane-bound and intracellular Flk1 in the Nrp1+ cells. As shown by Fig. 3B , within the whole Nrp1+ population (blue dots), the presence of 15% of the cells expressing only cytoplasmic Flk1 accounted for both the transcription of the mRNA and for the Flk1-negative phenotype recognized by the cell sorter. Immunofluorescent stainings performed on Nrp1+/Flk1+ cells derived from ES cells expressing the EYFP (24) and cultured for 2 days on endothelial culture conditions proved that CD31 and VE-cadherin mRNAs, previously detected by RT-PCR, were actually translated into the corresponding functional proteins located at cell to cell contacts (Fig. 3C ). Previous data suggested that among the pool of Nrp1– cells, the fraction expressing Flk1 is more mesoderm than ectoderm derived (Figs. 1E and 2B , and Supplemental Fig. 2, magenta). Indeed, when purified by cell sorting and cultured in endothelial medium, the Nrp1–/Flk1+ cells maintained Flk1 and acquired CD31 expression, whereas βIII-tubulin was absent (Supplemental Fig. 4A).


Figure 3
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  		Figure 3. In vitro differentiation of ES-derived Nrp1+ cells toward endothelium. A) Nrp1+/Flk1+ and Nrp1+/Flk1– cells purified by cell sorting from 7-day-old EBs were cultured onto OP9 feeders for 4 days. At different time points, the expression of Flk1, CD31, Flt1, Endoglin, Tie1, Tie2, and VE-cadherin was evaluated by semiquantitative RT-PCR and normalized to the signal given by the housekeeping gene HPRT. After an initial impairment of the Nrp1+/Flk1– cells vs. Nrp1+/Flk1+, the levels of expression of all the endothelial markers, with the exception of Tie1, equalized over time. B) After labeling of cells purified from 7-day-old EBs for the external Nrp1 (FITC) and the external Flk1 (PE), cells were fixed, permeabilized, and incubated with the antibody against cytoplasmic Flk1 (APC). The distribution of both external membrane-bound and intracellular cytoplasmic Flk1 was then analyzed by FACS specifically on the Nrp1+ cells (blue dots). As indicated (second dot plot, top right) 15% of the Nrp1+ cells expressed only cytoplasmic Flk1. C) Immunofluorescent staining of Nrp1+/Flk1+ cells derived from YFP+-ES cells revealed the presence of CD31 and VE-cadherin proteins in the membranes at cell-to-cell contacts. Scale bar = 30 µm.

To support neural differentiation outside the EB environment, purified Nrp1+/Flk1+ and Nrp1+/Flk1– cells were cultured in a neuronal medium and, as control, in endothelial medium. At different days after sorting, Nrp1+/Flk1+ and Nrp1+/Flk1– cells were analyzed by immunofluorescence for the expression of early and late neuronal markers. Sox2, the earliest transcription factor expressed in the neural tube during development (39) , and nestin were expressed by 60–80% of the Nrp1+/Flk1+ population already after 24 h of growth, not only in neuronal medium, but also in the unfavorable endothelial medium (Fig. 4A, B ). If the endothelial medium could not limit the expression of early neuronal markers, it was indeed inhibitory for the late markers, as shown by the almost absent expression of βIII-tubulin (Fig. 4B ). Accordingly, the neuronal maturation was fully accomplished only under the neuron-specific culture conditions: at the latest days of growth, nestin and sox2 expression started to decline, whereas the level of expression of the mature neuronal marker βIII-tubulin increased, suggesting that neuronal differentiation was progressing (Fig. 4A ). Indeed, as soon as FGF-2 was withdrawn from the neural medium (Materials and Methods), Nrp1+ sorted cells underwent a significant change in morphology characterized by cellular βIII-tubulin+ processes, resembling dendrites and axons (Fig. 4C , arrows). Similar results were obtained with Nrp1+/Flk1– cells (data not shown). An analogous analysis was performed on sorted cells to detect the expression of NeuN, a nuclear factor exclusively found in mature neurons (40) : positive nuclei were detected by immunofluorescence when cells were grown only in neuronal medium (Fig. 4E ). The results of the quantitative analysis are shown in Fig. 4D . Similar results were obtained with Nrp1+/Flk1– cells (data not shown). Contrary to the Nrp1+ cell types, the Nrp1–/Flk1+ cells could not survive the restrictions of the neural medium, thus confirming that the presence of Flk1 in the absence of Nrp1 gave these cells a mesoderm-restricted fate. All together, these data demonstrate that in response to the appropriate exogenous microenvironment, the bulk of ES-derived Nrp1+ cell population gives rise to endothelial or neural cells.


Figure 4
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  		Figure 4. In vitro differentiation of ES-derived Nrp1+ cells toward neurons. A, B)Nrp1+/Flk1+ cells purified by cell sorting from 7-day-old EBs were cultured for 7 days in a neuronal medium to favor neural commitment (A) and, as control, in an endothelial-suited medium (B). At different days the expression of nestin, sox2 and of βIII-tubulin was examined by immunofluorescent staining. Positive cells were counted in 5 different fields randomly chosen from the coverslips of 4 different experiments. The number of positive cells was expressed as percentage value of the total cells counted ± SD. C) Expression of βIII-tubulin by Nrp1+/Flk1+ sorted cells. Note the branched cell morphology (arrows). D) Nrp1+/Flk1+ cells were cultured for 7 days in neuronal or in endothelial medium. At different days, the expression of NeuN was examined by immunofluorescent staining. Positive cells were counted over the total cells present in 5 different fields randomly chosen from the coverslips of 4 different experiments. The number of the positive cells was expressed as a percentage value of the total cells counted ± SD. E) Expression of NeuN by Nrp1+/Flk1+ cells cultured in neuronal or endothelial medium. Scale bars = 25 µm (C, E).

Neuropilin-1 and Flk1 identify a discrete, nonclonal population with neurovascular differentiative potential
To analyze the capacity of single YFP+ Nrp1+/Flk1+ cells to differentiate toward endothelium or neurons, the bulk population was purified by cell sorting from 7-day-old EBs, and cells were singularly deposited in wells of 96-well plates. This clonal analysis was specifically performed on Nrp1+/Flk1+ cells because Flk1 favored the endothelial commitment (Fig. 3A and Supplemental Fig. 3B). To support the survival of single-deposited sorted cells, we used a multicellular culture system composed of the stromal PA6 cells as feeders and differentiating ES cells in N2B27 serum-free medium supplemented with growth factors (27 , 41) . The presence of ES-derived neural and endothelial cells progressing in their maturation pathways makes this system a suitable niche to address the neuronal and endothelial commitment of our YFP+ clones. The commitment of the progeny of each single clone was investigated by immunofluorescent staining performed with an anti-RC2 antibody to detect neural precursors (37) and with an anti-VE-cadherin to detect endothelial cells (Fig. 5B, C ). Of the 388 clones obtained and analyzed, 17.5% contained endothelial VE-cadherin+ cells, and 12.9% contained RC2+ cells (Fig. 5A-C ). This analysis indicates that Nrp1 and Flk1 are not markers of a common bipotential precursor able to differentiate into either endothelial or neural cells, but rather they define a restricted population within the EBs containing cells able to differentiate into vessels and cells able to originate neural progenies. There were also some clones, 5.1%, that differentiated into arborized O4-expressing oligodendrocytes (Fig. 5D ), thus reinforcing the number of clones with neuroepithelial-derived progenies. Among the negative clones for endothelial or neural markers, 42% expressed {alpha}SMA, a marker of vascular smooth muscle cells and pericytes (42) , while just few of them contained E-cadherin+ or cytokeratin+ cells, thus confirming that the vascular and the neural commitments are the two main potentials that the ES-derived Nrp1+/Flk1+ cells can alternatively address when cultured under the corresponding culture conditions.


Figure 5
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  		Figure 5. Clonal analysis of Nrp1+/Flk1+ cell population. A) Nrp1+/Flk1+ cells were purified by cell sorting from 7-day-old YFP+ EBs and singularly deposited in 96-well plates, each well containing the multicellular niche described in Materials and Methods. After 8 days of culture, clones containing cells expressing VE-cadherin, RC2, O4, {alpha}SMA, E-cadherin, and cytokeratins were detected by immunofluorescence and counted. B) Confocal images showing RC2+ cells within the progeny of a representative clone distinguishable from all other cell types of the niche because of their YFP expression. The big nuclei counterstained with DAPI reveal the presence of PA6 cell feeders that lay underneath. C) Confocal images showing VE-cadherin+ cells within the progeny of a representative clone. Note that the endothelial cells derived from the single YFP+ founder organize vessel-like structures along with the endothelial cells native of the niche (arrows). D) Oligodendrocytes appear in 5.1% of the clones. Besides the unique arborized morphology (arrowhead), O4 expression was detected by immunofluorescent staining (arrow). Scale bars = 50 µm (B, D); 100 µm (C).

In vivo analysis of the potential of differentiation of Nrp1+/Flk1+ and Nrp1+/Flk1– cells: endothelial and neural commitments
Even with the limits of an in vitro culture system, the data shown so far represented a supporting indication of the capacity of the ES-derived Nrp1+ cells to differentiate into endothelium or neurons depending on the appropriate stimuli. We next wondered whether a more physiological environment, such as a living embryo, could allow the Nrp1+ cells to better accomplish their fate. For that reason, Nrp1+/Flk1+ and Nrp1+/Flk1– cells were purified from 7-day-old YFP+-EBs by cell sorting and microinjected into chicken embryos at stage HH19 of development. To drive the Nrp1+/Flk1+ population toward the developing vascular system, the microinjection was performed in the beating heart of the embryos. Three days after injection, the host vessels were visualized in embryo tissue sections by using sambucus nigra agglutinin (SNA). Figure 6A shows that the Nrp1+/Flk1+-injected cells integrated within the new forming vessels, as indicated by the alignment of the YFP signal with the signal given by the chicken endothelial-specific SNA (43) . Identical results were obtained by using the W4 ES cell line (Supplemental Fig. 5). Of 79 embryos that survived the injection, 34 had integrated the injected YFP+ cells within the vasculature (43% of engraftment). Furthermore, the integrated cells also differentiated into mature endothelium expressing CD31 and VE-cadherin, as demonstrated by immunofluorescent staining of the tissue sections performed with antibodies specific for the murine antigens (Fig. 6B ). Similar results were obtained with the Nrp1+/Flk1– cells, with the exception that the vessel-integrated cells did express CD31 but not the more mature marker VE-cadherin (Fig. 6C ), thus confirming the slight delay in pursuing endothelial maturation by the Nrp-1+ cells sorted as Flk-1– previously suggested by the in vitro data (Fig. 3A ).


Figure 6
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  		Figure 6. In vivo differentiation of ES-derived Nrp1+ cells into endothelial cells. A) Nrp1+/Flk1+ cells purified from 7-day-old YFP+EBs were injected into the heart of HH19-stage chicken embryos; 3 days after injection, tissue sections were analyzed by confocal microscopy. The embryo vasculature was visualized with SNA. Inset: injected cells that integrated within the labeled host vessels. B) Confocal images of tissue sections obtained from chicken embryos injected with YFP+-ES-derived Nrp1+/Flk1+ cells whose vessels were visualized by AlexaFluor 405-conjugated wheat germ agglutinin (WGA). CD31 and VE-cadherin were detected by immunofluorescent staining with murine specific anti-CD31 and anti-VE-cadherin antibodies. Note how the injected cells integrate and differentiate within the host vessels to perfectly match the vessel lumen border (arrow). C) Confocal images showing the integration and CD31 expression of Nrp1+/Flk1– cells within the embryonic vasculature 3 days after injection. Contrary to what observed with the Nrp1+/Flk1+ cells, no immunoreactivity was detected with the anti-VE-cadherin antibody. Scale bars = 30 µm.

When the purpose was to address the injected cells toward a neural fate, the microinjection was performed into the mesencephalic cavity of embryos. Once injected, cells tend to aggregate into spherical clusters that initially make contact with the inner face of the developing cerebral tissues. From this time on, cells leave the clusters and start to invade the host tissues (Fig. 7A ). Figure 7B shows some YFP+ Nrp1+/Flk1– injected cells that have moved from the inner cavity and entered the neurogenesis area of the developing brain, where they started to express MAP2 antigen along with the surrounding MAP2+ chicken neurons, thus becoming part of the developing cerebral tissue. Analogous results were obtained with Nrp1+/Flk1+ cells (Fig. 7C ).


Figure 7
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  		Figure 7. In vivo differentiation of ES-derived Nrp1+ cells into neural cells. A) Nrp1+/Flk1– cells purified from 7-day-old YFP+EB cells were injected into the mesencephalic cavity of HH19-stage chicken embryos; 3 days after injection, tissue sections of the developing brain were analyzed by confocal microscopy by using an anti-MAP2 antibody that recognizes both mouse and chicken neurons. Differential interference contrast (DIC) image shows a cluster of injected cells within the mesencephalic cavity (M) that makes contact with the inner face of the developing tissues: inset shows an enlargement of cells entering the MAP2-labeled neurogenesis area of the developing brain (N). Invading YFP+ cells express MAP2 as well. B) Confocal images of serial optical sections of the same field showing a group of Nrp1+/Flk1– cells 5 days after injection. The YFP+-injected cells also express MAP2 along with the surrounding chicken neurons. C) Confocal images of serial optical sections of the same field, showing Nrp1+/Flk1+ cells 5 days after being injected into the mesencephalic cavity of HH19-stage chicken embryos. Scale bars = 50 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we used Nrp1 and Flk1 to identify a discrete, nonclonal population of uncommitted ES-derived cells that can differentiate into endothelial or neuronal cells depending on the suitable environmental conditions, both in vitro and in vivo. Within the EBs, we identified, characterized, and isolated the Nrp1+/Flk1+ and Nrp1+/Flk1– lineages, and we analyzed their potential for differentiation with respect to endothelial and neural commitments. Nrp1 was selected for its pivotal role in the vascular and neural systems and for its activity on stem cell differentiation. Indeed, Nrp1 was suggested to be involved in neural commitment of adult NSCs (44) and in VEGF-A-mediated endothelial differentiation of bone marrow-derived AC133+ cells (28) . Flk1 is the canonical marker of multipotent mesodermal progenitors that can differentiate into endothelial, hematopoietic, mural cells, and cardiomyocytes (18 , 20) .

The comparative analysis of Nrp1+/Flk1+ and Nrp1+/Flk1– cells indicates that the presence of Flk1 favors the endothelial commitment of the Nrp1+ cells both in vitro, in the Matrigel assay and OP9 cocultures, and in vivo, where we observed that the Nrp1+/Flk1– cells seem to be delayed in integrating within the embryonic host vessels and in differentiating into mature VE-cadherin+ endothelium (Figs. 3A and 6C ). Thus, Flk1 presence matters in terms of endothelial commitment, as previously well documented. The fact that immediately after sorting (day 0), the Nrp1+/Flk1– population expressed Flk1 as mRNA but not as mature protein suggests that these two Nrp1+ populations are very close in the ES differentiation program. The possibility that the Flk1+ type arises from a common Nrp1+/Flk1– ancestor, in which the production of the active membrane-bound molecule involves intermediate steps characterized by the presence of the Flk1-mRNA only and/or of the intracellular inactive form of the Flk1 protein (Fig. 3B ), is more likely than the way around, even though we cannot state which cell types derive from which. If the presence of Flk1 facilitates the endothelial maturation, conversely, the absence of Nrp1 seems to impair the neural differentiation since the Nrp1–/Flk1+ cells did not express neuronal markers as extensively as the two Nrp1+ counterparts both when included in the EB (Fig. 2B and Supplemental Fig. 2) and when isolated as pure cell population (Supplemental Fig. 4).

Even though the presence of markers of neural precursors such as nestin, sox1, sox2 and PSA-NCAM was suggestive of a neural potential, only the presence of the proper neuronal environment allowed both Nrp1+/Flk1+ and Nrp1+/Flk1– cells to definitively mature into βIII-tubulin+ and NeuN+ neurons. In vivo, the Nrp1+-sorted cells integrated into the developing brain of chicken embryos, where they differentiated into MAP2+ neurons independently of their initial production of Flk1.

The purity of ES cell-derived populations is a crucial issue for their use in cell replacement therapy, as well as in the studies of cell lineages. The clonal analysis performed on Nrp1+/Flk1+ cells gave clones whose progenies expressed either markers of vascular (VE-cadherin, {alpha}SMA) or markers of neural cells (RC2, O4). The fact that endothelial and neural cells were never located together within the progeny of the same clone demonstrates that Nrp1 and Flk1 define a population of cells containing precursors restricted to the vascular fate and precursors restricted to the neural fate, thus excluding the existence of a bipotential common precursor. The topic of the bipotentiality or multipotentiality of progenitor cells has recently become controversial. Despite the numerous indirect evidence supporting the existence of the hemangioblast (17 , 20 , 45) , recent in vivo studies argue against its bipotentiality. Cell-lineage tracing of cells from the primitive streak to the yolk sac failed to reveal a common hematopoietic and endothelial progenitor (21) . Moreover, the analysis of tetrachimeras derived from different ES cell lines stably expressing separate fluorescent tracers showed that each blood island has contributions from multiple clonal progenitors (22) . Our results are more in line with these studies, especially considering that a putative neuron-endothelial precursor, by trespassing the ectoderm-mesoderm boundaries, would represent a concept of bipotent progenitor more challenging than the hemangioblast. In this respect, our data are clear: the progeny of a single Nrp1+/Flk1+ cell is only endothelial or neural. The idea of a bipotent progenitor within our Nrp1+/Flk1+ cells was justified by the model used, the ES cells, and by the presence of Flk1. By using the same model with alternative protocols, different groups showed that ES-derived Flk1+ cells could differentiate into multiple mesoderm-derived lineages (18 , 20 , 46) . Regarding these data, the unique presence of Nrp1 along with other neural markers gives more challenges to our model. It was proposed that Flk1 marks a common mesodermal precursor that segregates to successive subsets of Flk1-expressing or nonexpressing cells whose fate is then determined by coexpression of lineage-specific transcription factors (47) . Hence, our Nrp1+/Flk1+ population could represent a transient intermediate pool of cells, in which the committed progenitors arise, thanks to the loss or acquisition of specific molecules. The possibility to purify this pool of cells by using Nrp1 and Flk1 along with the encouraging in vivo transplantation data opens new perspectives for the treatment of disorders characterized by defects of both the neural and vascular systems, including stroke, diabetic neuropathy, and Alzheimer’s disease (48) .


   ACKNOWLEDGMENTS
 
This work was supported by Istituto Superiore di Sanita’ (Programma Nazionale sulle Cellule Staminali, VI Programma nazionale AIDS), Associazione Italiana per la Ricerca sul Cancro, Regione Piemonte (Progetto di Ricerca sanitaria Finalizzata 2006, Ricerca Scientifica 2004, grants A150 and D10), 6FP European Community (LSHM-CT-2003–503254), Fondazione Cassa di Risparmio di Torino and Ministero della Salute (Programma di Ricerca Finalizzata 2006 and Programma Straordinario di Ricerca Oncologica 2006), Ministero dell’Universita’ (PRIN2007), and Telethon, Italy GGP04127. We thank Dr. Alexandra L. Joyner (Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA) for the gift of W4 129SvEv ES cells, Dr. Andras Nagy (Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, ON, Canada) and Anna-Katerina Hadjantonakis (Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA) for R1 EYFP-expressing ES cells, Dr. Timm Schroeder (Institute of Cell Research, Helmholtz Zentrum München–German Research Center for Environmental Health, Neuherberg, Germany) for OP9 and PA6 cells and helpful suggestions, L. Primo for suggestions and comments, and Dr. S. Geuna for assistance with confocal microscopy. We thank Famarco and Susatrasporti S.p.A. for white Leghorn chicken eggs, and the Developmental Studies Hybridoma Bank of the University of Iowa for antibodies.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication May 13, 2008. Accepted for publication August 14, 2008.


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