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Keywords:

  • Human embryonic stem cells;
  • Endothelial differentiation;
  • BMP4;
  • Endothelial cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133+KDR+ population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144+KDR+ population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144+KDR+ peak was shifted to day 7. Most of these cells, however, were CD31, becoming CD31+ in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144+KDR+CD31 population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. STEM CELLS 2009;27:1750–1759


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

In the embryo, the formation of the vascular network is governed by two distinct processes, vasculogenesis and angiogenesis. Vasculogenesis occurs during early morphogenesis and is defined as the de novo development of a vascular tree from intrinsic endothelial cells [1]. Angiogenesis is defined as the development of the pre-existing vascular network by a remodeling process [2, 3]. Comprehension of the mechanisms involved in vessel formation during development has numerous applications, including the treatment of cancer and ischemic disease [4, 5]. However, the human embryo is too complex to permit the study of the fine-tuning mechanisms controlling these processes. In this context, human embryonic stem cells (hESCs) represent a unique model for studying successive events occurring during human development [6]. ESCs have the capacity to self-renew and to differentiate into the three germ layers, mesoderm, endoderm, and ectoderm [7–9]. Different studies have described the differentiation of hESCs into endothelial cells using various culture conditions. The first hESC-derived endothelial cells were isolated from embryoid bodies (EBs) by CD31 cell sorting. These cells expressed endothelial markers in culture and were functional in vitro [10]. Further studies established that this differentiation occurs in early mesoderm, from a common stem cell, the hemangioblast, which gives rise to both hematopoietic and endothelial cells [11, 12]. In a recent study, Wang et al. have shown that endothelial cells produced by hESCs are fully functional, as assessed in vivo where they have been observed to be incorporated into mouse vessels in a model of ischemia [13]. Several studies have pointed to the critical role of bone morphogenetic protein 4 (BMP4) in the induction of both hematopoietic and endothelial differentiation from hESCs. The BMPs are transforming growth factor β related. BMP4 induces ventral mesoderm, suppresses induction of dorsal mesoderm by activin, and inhibits dorsoanterior development of embryos, suggesting that it is a ventralizing factor [14].

Treatment of hESCs with BMP4 yields different effects according to the duration of the treatment. Long-term exposure of hESCs to BMP4 results in trophoblast and extraembryonic endoderm differentiation, whereas short-term treatment promotes early mesoderm induction. It has been shown that members of the BMP family regulate the proliferation and differentiation of primitive human hematopoietic stem cells. High levels of BMP4 increase hematopoietic stem cell survival from hESCs, whereas low levels of BMP4 induce differentiation and proliferation of these stem cells [15–18].

In this study, we analyzed the influence of BMP4 on endothelial cell appearance. We show that a short induction with a high-dose of BMP4 accelerates the rate of appearance and the number of cells committed to the endothelial lineage during the course of EB differentiation. Endothelial cells emerging after a BMP4 “boost” display an immature phenotype, and mature into fully functional endothelial cells in culture.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

hESC Culture

hESCs (H9; WiCell Research Institute, Madison, WI, http://www.wicell.org) were cultured according to the supplier's instructions. Cells were maintained on a mitomycin C inactivated mouse embryonic fibroblast feeder layer. The medium—Dulbecco's modified Eagle's medium/F12 supplemented with 20% Knock Out serum replacer, 1 mM L-glutamine, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 10 ng/ml recombinant human basic fibroblast growth factor (all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com)—was changed daily.

Differentiated EB Formation

For EB formation, H9 colonies were harvested and cultured under different conditions. We first used the human cytokine cocktail described by Chadwick et al. [15]. Briefly, every 4 days, 100 ng/ml human stem cell factor (hSCF) (AbCys s.a., Paris, France, http://www.abcysonline.com), 100 ng/ml human Flt3 ligand (hFlt3L) (AbCys s.a.), 10 ng/ml human interleukin (hIL)-3 (AbCys s.a.), 10 ng/ml hIL-6 (AbCys s.a.), 50 ng/ml hG-CSF (AbCys s.a.), 10 ng/ml human vascular endothelial growth factor (hVEGF) (PromoCell Biosciences, Heidelberg, Germany, http://www.promocell.com), and 10 ng/ml hBMP4 (AbCys s.a.) were added to the culture medium. These conditions are hereafter referred as “standard conditions.” To analyze the effect of cytokines, EBs were cultured under standard conditions, with one cytokine removed at a time (except for VEGF and BMP4). We then analyzed the effect of a 1- or 3-day BMP4 induction using different doses of BMP4 (10, 50, and 100 ng/ml) in the presence of 100 ng/ml hSCF, 100 ng/ml hFlt3L, and 50 ng/ml hVEGF. After 1 or 3 days, new medium supplemented with hSCF, hFlt3L, and hVEGF was used. Conditions in which BMP4 at 50 ng/ml was added for 24 hours are hereafter referred to as “boost conditions.”

To block the effect of BMP4, EBs were cultivated for 3 days with 150 ng/ml Noggin (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). At day 3, new medium supplemented with 100 ng/ml hSCF, 100 ng/ml hFlt3L, and 50 ng/ml hVEGF was used.

RNA Extraction and Quantitative Polymerase Chain Reaction

At day 0, day 7, and day 12, EBs were dissociated with 1 mg/ml collagenase IV for 2 hours at 37°C. Total RNA was extracted using the RNeasy mini-kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) and cDNA was prepared with the cDNA Archive kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Quantitative polymerase chain reaction (PCR) was performed with TaqMan Gene Expression Assays on an ABI7000 system (Applied Biosystems) in the presence of 10 ng of RNA. GATA2, OCT3/4, RUNX1, and TATA box binding protein (endogenous control) were investigated (references, respectively, Hs00231119_m1, Hs00742896_s1, Hs01021970_m1, Hs99999910_m1). The 2-[/delta][/delta]Ct method was used and results were expressed as fold increase in expression of each gene relative to day 0.

Flow Cytometry

EBs at day 7 and day 12 were dissociated by collagenase IV treatment. Cells were then labeled with control isotypes—mouse IgG1-phycoerythrin (PE), IgG1-fluorescein isothiocyanate (FITC), and IgG1-allophytocyanin (APC) (Beckman Coulter, Villepinte, France, http://www.beckmancoulter.com) or CD133/1-PE (Miltenyi Biotec, Paris, France, Germany, http://www.miltenyibiotec.com), kinase insert domain-containing receptor (KDR)-APC (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), or CD45-PE (Beckman Coulter) antibodies. Cells were analyzed using a fluorescence-activated cell sorting (FACS)Calibur (BD Pharmingen, San Jose, CA, BD Pharmingen, http://www.bdbiosciences.com/index_us.shtml) with Weasel Software (WEHI, Melbourne, Australia, http://www.wehi.edu.au).

For cell sorting, dissociated EB cells were labeled with control mouse isotypes or with anti-CD144-PE (Beckman Coulter), anti-KDR-APC, or anti-CD31-FITC (R&D Systems). Cells were sorted using a FACSDiva cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and plated on 48 fibronectin-coated plates (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in 80% Medium-199 (Invitrogen), 20% fetal bovine serum, 50 μg/ml bovine pituitary extract, 10 IU/ml heparin, 5 ng/ml VEGF, and antibiotics. After the first passage, cells were cultured in EGM-2 (Lonza, Paris, France, http://www.lonza.com).

Cultured cells were analyzed with monoclonal antibodies (CD144-PE, VEGFR-2-APC, CD133/1-PE, CD34-FITC, CD34-PE, CD31-FITC, CD43-FITC, CD14-PE, or CD45-FITC) or with control mouse isotypes.

CD144+KDR+ sorted cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS). When required, cells were permeabilized with 0.1% Triton X100 PBS for 10 minutes at room temperature. Cells were incubated with the primary antibodies stage-specific embryonic antigen (SSEA)-3, tumor rejection antigen (TRA)1-60, and OCT3/4 (Invitrogen) and labeled with secondary antibodies (Alexa Fluor 488 goat anti-mouse IgM, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG; Invitrogen).

Immunohistochemistry Analysis

Cells were fixed in 4% paraformaldehyde/PBS for 15 minutes at room temperature and rinsed with PBS/0.025% Tween. For intracellular staining, cells were permeabilized with 0.1% Triton X100/PBS for 10 minutes at room temperature.

CD144+KDR+ cells were incubated for 60 minutes with control isotype (mouse IgG1-Alexa Fluor 488) or with monoclonal antibodies—anti-CD144 (Beckman Coulter), anti-CD31 (Dako, Trappes, Francehttp://www.dako.com), anti-von Willebrand factor (vWF) (Dako)—and labeled with secondary antibodies—Alexa Fluor 488 goat IgG (Invitrogen), Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen). Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined with a fluorescence microscope (Leica DMS, Wetzlar, Germany, http://www.leica.com).

Endothelial Function Assays

Tumor Necrosis Factor α Treatment.

Cells were incubated for 18 hours with 10 ng/ml TNF-α (R&D Systems) and incubated with monoclonal antibodies (intercellular adhesion molecule [ICAM]-1; R&D Systems) or with control isotypes (mouse IgG; Beckman Coulter) and labeled with secondary antibody: Alexa Fluor 488 goat IgG. Cells were analyzed using a FACSCalibur cell sorter.

Vascular Tube Formation.

Twelve-well plates were coated with Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com). Cells (200,000) were plated onto Matrigel in EGM-2 with 50 ng/ml VEGF and incubated for 18 hours at 37°C. Photographs were taken every 2 hours.

Endothelial Cell Migration.

Cells were trypsinized and suspended in 100 μl EGM-2 without VEGF and seeded onto Costar Transwell inserts (BD Biosciences) precoated with type I rat tail collagen (BD Biosciences). Inserts were placed in a 12-well plate containing 600 μl EGM-2 without VEGF. After 24 hours, 50 ng/ml VEGF was added to the lower chamber. Cells that had migrated to the lower side of the insert after 12 hours were fixed in 4% paraformaldehyde/PBS and stained with DAPI. The insert, cut and mounted in Glycergel, was examined with a fluorescence microscope. Migrated cell numbers were counted in three different representative high-power fields per insert.

Wound-Healing Assays.

Cells were plated onto fibronectin in EGM-2. A scratch was made using a pipette cone and photographs were taken before, just after, and 24 hours after the scratch.

Population Doubling Assay.

The experiment started at passage 3, 20 days after the initial seeding of CD144/KDR sorted cells. At confluence, cells were detached and counted. The cumulative population doubling was estimated using the following equation: (Ln(number of cells counted/number of cells at the beginning of the assay)/Ln2). Population doubling of boosted day 7 and standard culture day 12 endothelial cells was compared by analyzing the number of population doublings at day 66 (n = 6). The statistical analysis was performed using Student's t-test. These population doubling curves were compared with those obtained with human umbilical vein endothelial cells (HUVECs) seeded at passage 1.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

Effect of Hematopoietic Cytokines and BMP4 on Endothelial Differentiation

The standard culture conditions described in Materials and Methods were designed for obtaining both hematopoietic and endothelial cells from a common progenitor, the hemangioblast. For this reason, a panel of hematopoietic cytokines, including Il-3, IL-6, G-CSF, Flt3L, and SCF, was used in addition to VEGF and BMP4 at a low concentration (10 ng/ml). In the present study, we first analyzed if any of these hematopoietic cytokines was also critical for endothelial differentiation and proliferation. EBs were cultured either in the standard medium containing all the cytokines or under conditions in which one cytokine was removed at a time. The percentage of the CD144+KDR+ population, which corresponds to our definition of endothelial cells, was analyzed at day 12, a stage at which endothelial cells were shown to be present under these culture conditions [21]. When EBs were cultured without SCF or Flt3L under standard conditions, the percentage of CD144+KDR+ cells at day 12 was more than twofold lower (p < .005) than with the standard conditions. The other cytokines (IL-3, IL-6, G-CSF) did not induce any change in the percentage of CD144+KDR+ cells (Fig. 1A, 1B).

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Figure 1. Effect of cytokines and BMP4 on the number of CD144+KDR+ cells. (A): CD144/KDR expression at day 12 in the presence or absence of IL-3, IL-6, G-CSF, SCF, and Flt-3. (B): CD144/KDR expression during differentiation at various times and concentrations of BMP4. (C): CD144/KDR expression under BMP4 boost conditions in the presence or absence of Flt-3 and SCF. Abbreviations: BMP4, bone morphogenetic protein 4; IL, interleukin; KDR, kinase insert domain-containing receptor; SCF, stem cell factor; VEGF, vascular endothelial growth factor.

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To determine whether BMP4 had an effect on the appearance of endothelial cells during the course of EB differentiation, we added this factor on day 1 at a concentration of 10, 50, or 100 ng/ml and cultured for 24 hours or 3 days. We analyzed the effect of this BMP4 boost on the kinetics of appearance of the CD144+KDR+ population. These conditions were compared with the standard conditions. The CD144+KDR+ population was analyzed by flow cytometry at different time points during EB differentiation. When EBs were cultured for 24 hours with BMP4 at 50 or 100 ng/ml, the percentage of CD144+KDR+ cells at day 7 was 2.2-fold higher than with the standard conditions (n = 3; p < .005) and lower by half at day 12 (n = 3; p < .005). A longer duration of BMP4 induction (3 days) at the same concentration (50 or 100 ng/ml) did not yield any variation in the percentage of the CD144+KDR+ population at either day 7 or day 12 as compared with the standard conditions (Fig. 1B). Moreover, treatment of EBs with BMP4 (10 ng/ml) for 24 hours led to a decrease in the CD144+KDR+ population at day 12 but not at day 7 (p < .005). A longer duration of BMP4 treatment (10 ng/ml for 3 days) did not modify the kinetics of CD144+KDR+ cells. Finally, we show that SCF and Flt3L, the two cytokines that have been shown to be critical for maximal CD144/KDR expression, were also required in the BMP4 boost culture conditions (BMP4, 50 ng/ml for 24 hours) (Fig. 1C).

Effect of BMP4 on the Kinetics of Expression of Endothelial Markers

To determine if a 24-hour BMP4 boost modified the kinetics of appearance of endothelial/progenitor cell markers, we followed the expression of these markers, either individually (KDR, CD31, CD144, CD34, and CD133) (Fig. 2A) or in combination (CD133/KDR and CD144/KDR) (Fig. 2B) (n = 5). Expression of KDR and CD133 was detected in undifferentiated hESCs. The BMP4 boost induced a significant increase in KDR+ cells at day 7. The BMP4 boost resulted in a significantly lower expression of CD133 at day 9 and day 12, compared with noninduced cells. CD144 expression peaked at day 7 upon BMP4 stimulation, whereas the peak in CD144 expression was detected at day 12 under standard conditions. Differences between the BMP4-induced and standard conditions were significant at day 7 and day 12 (Fig. 2A). When CD133 and KDR expression was measured in combination, three peaks in expression were observed: at day 2 under standard conditions, at day 7 after BMP4 stimulation, and at day 9 under standard conditions. At day 2, CD133/KDR expression was significantly higher under standard condition (p < .005), at day 7 this population was significantly higher in BMP4-boosted cells (p < .005 ), and at day 9 expression was significantly higher under standard conditions (p < .005) (Fig. 2B). CD144/KDR combined expression yielded two peaks: the first at day 7 after BMP4 boost (p < .005) and the second at day 12 under standard conditions (p < .005) (Fig. 2B). To make sure that the differences observed between standard conditions and BMP4 stimulation were a result of a direct effect of BMP4, BMP4 was blocked by adding Noggin (an antagonist of BMP4) to the culture during the first 3 days of EB differentiation. Noggin treatment abolished the effect of BMP4 on CD144/KDR expression during EB differentiation (Fig. 2C) (n = 3). Similar results were observed with the CD133+KDR+ population (data not shown).

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Figure 2. Effect of BMP4 on the kinetics of endothelial marker expression. Show in the effect of BMP4 induction on the expression of individual endothelial markers (A) or a combination of endothelial markers (B). (C): Inhibition of CD144/KDR BMP4-boosted expression by Noggin during the course of EB differentiation. Abbreviations: BMP4, bone morphogenetic protein 4; EB, embryoid body; KDR, kinase insert domain-containing receptor.

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The population of cells present in the different peaks was sorted and analyzed in culture. Because CD133 and KDR are expressed in undifferentiated hESCs, CD133+KDR+ cells at day 2 were not further analyzed. At day 9, CD133+KDR+ sorted cells did not proliferate and were not analyzed further. Finally, CD133+KDR+ cells sorted at day 7 proliferated in culture, but these cells did not express any endothelial markers (data not shown).

Sorting and Characterization of Endothelial Cells in Differentiated EBs at Day 12

When EBs were cultured under standard conditions, the expression of CD144/KDR peaked at day 12 (Fig. 1C). We then analyzed this population after sorting. These CD144+KDR+ cells represent an average of 10% of the total cells (±2, n > 5) (Fig. 3A), and they express CD31 (97%) and CD34 (97%), confirming their endothelial phenotype (Fig. 3B). In contrast, these CD144+KDR+ sorted cells expressed neither CD133 nor the hematopoietic marker CD43 (Fig. 3B). Also, it was observed that CD144+KDR+ cells from EBs cultured under BMP4 boost conditions and sorted at day 12 expressed endothelial markers, including CD31, immediately after sorting (data not shown). CD144+KDR+ day 12 sorted cells from EBs cultured under standard conditions gave rise to a homogeneous population of cells with the morphology of endothelial cells. At day 30 of culture, the cells were still positive for the endothelial markers KDR and CD144. The culture did not contain any hematopoietic cells, as attested by the absence of expression of CD14, CD45, and CD133 (Fig. 4A). Immunofluorescent microscopy indicated that these cells expressed vWF in the cytoplasm and CD31 and CD144 on their membranes (Fig. 4B). These data confirm that cells derived from CD144+KDR+ day 12 EBs have the phenotypic features of endothelial cells.

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Figure 3. Characterization of CD144+KDR+ cells after sorting at day 12 of EB differentiation. (A): Flow cytometry analysis of the CD144+KDR+ population before sorting. (B): Phenotype of sorted CD144+KDR+ cells. Expression of CD31, CD34, CD43, and CD133 was analyzed by flow cytometry just after sorting. Abbreviations: EB, embryoid body; FSC, forward scatter; KDR, kinase insert domain-containing receptor; SSC, side scatter.

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Figure 4. Characterization of day 12 CD144+KDR+ sorted cells after 30 days of expansion. (A): Flow cytometry analysis of endothelial cell surface marker expression in CD144+KDR+ sorted cells. (B): Immunodetection of CD31, vWF, and CD144 on hES-derived endothelial cells. (C): TNF-α induced upregulation of ICAM-1. (D) Population doubling of hES EC and HUVECs. (E): Vascular tube formation on Matrigel. Abbreviations: FSC, forward scatter; hES EC, human embryonic stem endothelial cell; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule 1; KDR, kinase insert domain-containing receptor; TNF-α, tumor necrosis factor α; vWF, von Willebrand factor.

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These cells were activated in response to the proinflammatory factor TNF-α, as shown by the upregulation of ICAM-1 (Fig. 4C).

We compared the cumulative population doubling curve of endothelial cells derived from CD144+KDR+ day 12 EBs to that of HUVECs, taken as a reference of mature vascular wall endothelial cells. As shown in Figure 4D, hESC-derived endothelial reached a plateau at day 47 (21 days after passage 3) and HUVECs reached a plateau at day 60 (n = 6).

When CD144+KDR+ cells were placed onto a Matrigel film, they formed vascular-like network structures (Fig. 4E). These endothelial cells take up diacetylated low-density lipoprotein (data not shown).

Finally, CD144+KDR+ sorted cells did not display features of progenitor cells, because we did not observe any colonies when they were seeded under limiting dilution conditions, suggesting that these cells are mature.

Phenotypic and Functional Properties of BMP4-Induced Endothelial Cells at Day 7

Under BMP4 boost conditions, the expression of CD144/KDR peaked at day 7 (Fig. 1C). The CD144+KDR+ population was sorted by flow cytometry at day 7 (n > 5). Sorted cells did not express the hematopoietic markers CD45 (Fig. 5A) and CD43 (data not shown). The majority of the cells (73%) expressed CD34 (Fig. 5A). The CD144+KDR+ population contained a major population (65%) that did not express CD31 and a minor population (35%) that did express this endothelial marker.

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Figure 5. Phenotypic characterization of BMP4-boosted endothelial cells. (A): Flow cytometric analysis of a set of markers (CD31, CD34, CD45) in CD144+KDR+ cells just after sorting. Expression of CD31 after 21 days of culture. (B): Population doubling. (C): Expression of ICAM-1 after TNF-α treatment. (D): Migration assays. (E): Wound-healing assay. Abbreviations: BMP4, bone morphogenetic protein 4; FSC, forward scatter; hES EC, human embryonic stem endothelial cell; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule 1; TNF-α, tumor necrosis factor α.

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To characterize the CD144+KDR+CD31 population, these sorted cells were analyzed at day 21 of culture. They had the typical morphology of endothelial cells and acquired CD31 expression (98% of the cells) (Fig. 5A, right panel). This suggests that the initial CD144+KDR+CD31 sorted cells displayed an immature endothelial phenotype and that they differentiated into mature endothelial cells in culture (Fig. 5A). The cumulative population doubling curve of the day 7 CD144+KDR+ cells was linear until day 66 (42 days after passage 3) (Fig. 5B). At day 66, the cumulative population doubling obtained with these cells was significantly higher (n = 6; p < .005) than that obtained with the day 12 CD144+KDR+ cells cultured under standard conditions (Fig. 4D). This curve was comparable with that obtained with HUVECs (day 60). As shown in Figure 5C, the CD144+KDR+CD31-derived endothelial cells are activated by TNF-α, as assessed by the induction of ICAM-1 by flow cytometry. Migration of CD144+KDR+CD31-derived endothelial cells in response to a VEGF gradient was compared with that of HUVECs and with CD144+KDR+-derived endothelial cells cultured under standard conditions sorted at day 12 (n > 5). We found that CD144+KDR+CD31-derived endothelial cells had a migration capacity comparable with that of HUVECs. Moreover, this migration capacity was significantly higher than that observed with late (day 12) CD144+KDR+-derived endothelial cells (Fig. 5D) (p < .005).

We then measured the migratory rates of these CD144+KDR+ cells in an in vitro wound-healing assay. We showed that the cell-free wound gaps healed almost completely after 24 hours (Fig. 5E) (n > 3). Finally, CD144+KDR+CD31 sorted cells were seeded under limiting dilution conditions. These cells, like CD144+KDR+ cells sorted at day 12, were unable to generate any colonies, suggesting that even if they are immature, they are not fully competent endothelial progenitors.

Expression of Transcriptional Factors in hESCs and During EB Differentiation

GATA2 and RUNX1/AML1 are transcription factors involved in the emergence of hematopoietic/endothelial stem cells (hemangioblasts) from mesodermal progenitors. We analyzed their transcriptional level at day 7 and day 12 of EB differentiation, either under standard or BMP4-boost conditions, and compared these levels of expression with those measured at day 0 (expressed as fold increase in expression). At day 12, the increase in GATA2 expression was higher under standard conditions than under BMP4-boost conditions (n = 3; p < .005). GATA2 expression was greater at day 7 than at day 0 (ninefold), but no significant difference in expression level was observed between standard and BMP4-boost conditions (Fig. 6A, upper left). A relative decrease in GATA2 expression was observed between day 7 and day 12 under BMP4-boost conditions, whereas greater expression of this factor was observed under standard conditions (Fig. 6A, upper right).

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Figure 6. Expression of transcriptional factors during EB differentiation. (A): Quantitative RT-PCR analysis of GATA2, RUNX1, and OCT3/4 in day 7 and day 12 BMP4-stimulated and unstimulated EBs. (B): Flow cytometry analysis of OCT3/4, TRA1-60, and SSEA-3 cell surface markers on CD144+KDR+ sorted cells. Abbreviations: BMP4, bone morphogenetic protein 4; EB, embryoid body; FSC, forward scatter; KDR, kinase insert domain-containing receptor; RT-PCR, reverse transcription-polymerase chain reaction; SSEA, stage-specific embryonic antigen; TRA-1-60, tumor rejection antigen 1-60.

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RUNX1 expression was higher than at day 0 (n = 3). However, this increase was less important after BMP4 boost than under standard conditions (p < .005). The increase in RUNX1 expression between day 7 and day 12 was significant under standard conditions but nonsignificant under BMP4-boost conditions (Fig. 6A, lower left).

We also measured OCT3/4, a marker of undifferentiated hESCs (Fig. 6A lower right) (n = 3). We show that expression of this factor decreased after day 7 and this decrease was significantly more prominent after BMP4 boost than under standard conditions. After BMP4 boost, expression of OCT3/4 was dramatically lower at day 12 relative to day 0 (144-fold). This decrease was less important under standard conditions (27-fold). We then analyzed the expression of OCT3/4 and two other hESC markers, SSEA-3 and TRA1-60, in the CD144+KDR+ boosted population sorted at day 7 (Fig. 6B). We show that, in this population, none of these factors was detected either in the cytoplasm (OCT3/4) or at the surface of the cells (SSEA-3 and TRA1-60).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

hESCs recapitulate the sequential events giving rise to endoderm, mesoderm, and ectoderm [7] in a manner similar to what occurs during human development. Because these cells can be expanded almost indefinitely, they represent a useful model of human development, allowing molecular studies that are difficult to perform in human embryos. In this study, we have further defined the steps of commitment and differentiation of ESCs into endothelial cells. We first used culture conditions that were previously defined as inducing the formation of hemangioblastic cells by the Bathia group [15, 19]. Because these conditions were designed for obtaining both hematopoietic and endothelial cells, the culture medium contained a panel of cytokines acting on the hematopoietic system (IL-3, IL-6, G-CSF, Flt-3, SCF). We checked if all these cytokines were also required for endothelial differentiation. We show that IL-3, IL-6, and G-CSF did not affect the number of endothelial cells, but removal of SCF and Flt-3L yielded a significant lower number of these cells. We then investigated if BMP4 had an effect on the endothelial differentiation process. BMP4 has been shown to be required for mesoderm formation in mouse ESCs [20]. BMP4 also plays a crucial role in the commitment of mesoderm to the hematopoietic and endothelial lineages [15, 19, 21]. It has been shown that exposure of hESCs to a short BMP4 treatment induces endothelial differentiation [18]. We have confirmed this observation and extended the analysis of its effect on the sequential emergence of endothelial cells during the differentiation of H9 hESC-derived EBs. We first defined the optimal BMP4 concentration and showed that 50 ng/ml was the minimal dose yielding the maximal effect. Time of exposure to BMP4 was also critical to reach the maximal effect. We show that exposure of EBs to a 24-hour BMP4 (50 ng/ml) induction was optimal.

We show that, similar to standard conditions, SCF and Flt3L are also required in BMP4 boost conditions. This observation is consistent with the role of these factors in the formation of hemangioblastic cells and further differentiation of these cells into endothelial cells [22–24]. CD133 was first considered as a marker of hematopoietic progenitors [25], but it was further described as being expressed by stem/progenitor cells in different tissues [26]. A number of studies have shown that endothelial progenitor cells (EPCs) could be defined by a combination of stem cell (such as CD133) and endothelial (such as KDR, CD144) markers [26, 27]. In this study, we thus considered two populations during EB differentiation: the CD144+KDR+ population, for the identification of all cells with an endothelial phenotype, and the CD133+KDR+ population, for the identification of stem/progenitor cells with potential endothelial differentiation capacity.

We measured the number of CD144+KDR+ cells at day 7 obtained under BMP4-boost conditions, comparing different times of exposure with the standard, a continuous low level of BMP4 (10 ng/ml). We found that BMP4 delivered on day 1 of EB differentiation at 50 ng/ml (for only 1 day) yielded a peak in the population of CD144/KDR-expressing cells. We thus used these induction conditions in further experiments. Complete inhibition of induction of the CD144+KDR+ population upon BMP4 boost in the presence of Noggin confirms the direct involvement of BMP4 in this induction (Fig. 2C).

We then analyzed the kinetics of expression of KDR, CD144, and CD133, both individually and in combination, under standard and BMP4 boost conditions during the EB differentiation process. CD144 expression peaked at day 7 under BMP4-boost conditions and at day 12 under standard conditions, suggesting that the BMP4 boost shortens the onset of mesodermal cell commitment to the endothelial lineage. KDR was expressed from day 0, in both the BMP4-boost and standard conditions, confirming that KDR is expressed on cells other than endothelial cells during development. It has been suggested that early KDR+ cells could be a common mesodermal precursor that later gives rise to hemangioblastic cells [28]. At day 7, KDR expression was, however, significantly higher after the BMP4 boost. Similar to KDR, CD133 is expressed on native hESCs, but surprisingly, in contrast to the KDR+ population, BMP4 boost reduced the number of CD133+ cells at every time point, and this reduction was significant at day 9 and day 12.

When CD133 and KDR expression were analyzed in combination, a dramatically lower expression level of this population was observed after BMP4 boost, which was not observed when these two markers were analyzed individually. This indicates that this early CD133+KDR+ population is rapidly induced to differentiate after BMP4 boost. Because day 2 EBs did not express endothelial markers other than KDR, this population was not further analyzed.

The CD133+KDR+ population peaked at day 7 under BMP4 boost conditions and at day 9 under standard conditions. Aiming at identifying a population of endothelial progenitors, we sorted the CD133+KDR+ cells corresponding to these two peaks. Cells corresponding to the day 9 peak were unable to proliferate in culture and were not further analyzed. In contrast, the BMP4-boosted CD133+KDR+ population sorted at day 7 represents 7% of the total cells and gives rise to a highly proliferative population. These cells did not express any endothelial markers, and therefore they were not considered within the present study.

We then analyzed the CD144+KDR+ population that peaks at day 12 under standard culture conditions. The phenotype of these cells (CD144+KDR+CD31+CD34+) corresponds to that of endothelial cells, and they represent 10% of the total cells in these cultures. No hematopoietic cells were detected, because neither CD45+ nor CD43+ cells were detected. The absence of CD133 expression suggests that no progenitor cells were included in the sorted CD144+KDR+ population. In culture, sorted cells expressed KDR, CD144, and CD31 on the surface of the cells and vWF in the cytoplasm.

The CD144+KDR+ population sorted at day 7 under BMP4-boost conditions represents approximately 5% of the total cells. Interestingly, the majority of sorted cells did not express CD31, but after a few days, expression of this marker increased, and by day 21, all cells were CD31+. This indicates that the initial CD144+KDR+ population contained immature endothelial cells (early) that differentiated in vitro. The immature phenotype of the initial CD144+KDR+CD31 population was confirmed by the analysis of their cumulative proliferation curve. These cells remained in the linear phase of their proliferation longer than day 12 sorted CD144+KDR+ cells that were cultured under standard conditions (day 66 versus day 47).

Expression of ICAM-1 in the CD144+KDR+CD31 cells was also induced by TNF-α, but the basal level of ICAM-1 expression was much lower in these early cells than in late endothelial cells derived from day 12 EBs. Early, day 7 EB-derived endothelial cells also displayed a more prominent migration capacity than the late, day 12 EB-derived ones. Finally, early, day 7 EB-derived endothelial cells have the capacity to fill the gap or wounded area in an in vitro model of wound healing. Taken together, these data suggest that endothelial cells derived from day 7 BMP4-boosted EBs or from day 12 EBs cultured under standard conditions have all the phenotypic and functional features of endothelial cells. However, the former displayed a globally more immature phenotype than the latter.

EPCs have the capacity to generate secondary and tertiary colonies in culture [29]. EPC-like cells have been identified during avian development [30]. In contrast, even early endothelial cells isolated at day 7 under BMP4 conditions failed to form colonies in limiting dilution assays. This indicates that, under our culture conditions, hESC-derived endothelial cells do not correspond to endothelial stem/progenitor cells comparable with EPCs. One of the major differences between EPCs and hESC-derived endothelial cells is the microenvironment of these cells. In the bone marrow, these cells are probably in close contact with the hematopoietic niche, and, when blood outgrowth EPCs are seeded in culture to generate colonies, the initial adhesion step is performed in the presence of blood cells, which represent >95% (CD45+ cells) of the total cells. In contrast, hematopoietic cells appear later during hESC differentiation, and at day 7 under BMP4 induction conditions no CD45+ cells were detected inside EBs. hESC-derived endothelial cells may thus be unable to form colonies because they have not been instructed by an inducible niche, of which hematopoietic cells may represent an essential component.

Finally, the mRNA level of three transcription factors, GATA2, RUNX1, and OCT3/4, was analyzed by quantitative reverse transcription-PCR at different stages of EB differentiation, either under standard or BMP4-boost conditions. GATA2 is a transcription factor expressed in both hematopoietic and endothelial cells. It is also highly expressed in hemangioblasts [31], and interaction between BMP4 and GATA2 has been shown in different studies [17, 32]. We saw, however, a lower level of GATA2 expression after BMP4 boost (relative to that observed under standard conditions). Such a decrease was also observed with RUNX1, another hematopoietic/endothelial transcription factor involved in the development of hemangioblasts. The lower expression level of GATA2 and RUNX1 was more prominent at day 12 than at day 7. This indicates a restriction in GATA2/RUNX1-expressing cells following BMP4 boost, probably linked to the commitment of hemangioblasts to the endothelial lineage, and a disappearance of cells with hematopoietic potential, which cannot survive in a culture medium lacking hematopoietic growth factors. This indicates that, not only are these factors required for hemangioblastic cell appearance, they are also essential to hematopoietic commitment, voiding the development of endothelial cells. BMP4 also accelerates the decrease in OCT3/4 expression, consistent with the effect of BMP4 on EB differentiation.

In conclusion, our paper not only confirms that functional endothelial cells can be obtained from hESCs but also defines superior conditions for their development. Under our standard conditions, these endothelial cells represented up to 12% of the total EB cells at day 12, a yield significantly better than in other studies [10, 13, 15]. After BMP4 boost, functional endothelial cells were detected earlier than under standard conditions, suggesting that this factor accelerates the rate of endothelial differentiation from mesoderm. This factor also increases the number of endothelial cells, suggesting that it forces commitment of mesoderm into the endothelial lineage. Our results thus provide optimized conditions for obtaining functional endothelial cells from hESCs. Endothelial cells at different maturation stages could also be obtained, which would represent a useful model for testing molecules involved in endothelial development and differentiation. The cell populations obtained in our different culture and sorting conditions are illustrated in Figure 7.

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Figure 7. Model of BMP4-induced endothelial differentiation from hES cells. Sorted CD144+KDR+ cells at day 7 after BMP4 boost show an immature endothelial phenotype. Sorted CD144+KDR+ cells from day 7 to day 12 in standard culture conditions exhibited a mature endothelial phenotype. Abbreviations: AP, phosphatase alcalin; BMP4, bone morphogenetic protein 4; EB, embryoid body; hES, human embryonic stem; KDR, kinase insert domain-containing receptor; SSEA, stage-specific embryonic antigen; vWF, von Willebrand factor.

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We thus propose a model of endothelial differentiation that could be followed in culture. This model could be useful for analyzing the mechanisms controlling the endothelial maturation steps.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

This work was supported by a research grant (IngeCell) from the Ile de France region. We acknowledge Vanina Jodon de Villeroché, Jérôme Avouac, and Valérie Vanneaux for their helpful advice in experimental design, and Christophe Desterke for his help with the statistical analyses.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES

The authors indicate no potential conflicts of interest.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES