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First published online March 1, 2007
Stem Cells Vol. 25 No. 6 June 2007, pp. 1423 -1430
doi:10.1634/stemcells.2006-0556; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Identification of High Proliferative Potential Precursors with Hemangioblastic Activity in the Mouse Aorta-Gonad- Mesonephros Region

Huiyu Yaoa, Bing Liua, Xiaoyan Wanga, Yu Lanb, Ning Houb, Xiao Yangb, Ning Maoa

aDepartment of Cell Biology, Institute of Basic Medical Sciences, Beijing, China;
bGenetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China

Key Words. Hemangioblast • Aorta-gonad-mesonephros • High proliferative potential–colony-forming cell • Definitive hematopoiesis

Correspondence: Ning Mao, M.D., Department of Cell Biology, Institute of Basic Medical Sciences, Tai Ping Road 27, Beijing 100850, China. Telephone: 8610-68173543; Fax: 8610-68213039; e-mail: maoning{at}nic.bmi.ac.cn

Received on August 31, 2006; accepted for publication on February 20, 2007.

First published online in STEM CELLS EXPRESS  March 1, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hemangioblast, a precursor possessing hematopoietic and endothelial potential, is identified as the blast colony-forming cell in the murine gastrulating embryos (E7.0–E7.5). Whether hemangioblast exists in the somite-stage embryos is unknown, even though hemogenic endothelium is regarded as the precursor of definitive hematopoiesis in the aorta-gonad-mesonephros (AGM) region. To address the issue, we developed a unique three-step assay of high proliferative potential (HPP) precursors. The AGM region contained a kind of HPP precursor that displayed hematopoietic self-renewal capacity and was able to differentiate into functional endothelial cells in vitro (i.e., incorporating DiI-acetylated low-density lipoprotein, expressing von Willebrand factors, and forming network structures in Matrigel). The clonal nature was verified by cell mixing assay. However, the bilineage precursor with high proliferative potential—the HPP-hemangioblast (HA)—was not readily detected in the yolk sac (E8.25–E12.5), embryonic circulation (E10.5), placenta (E10.5–E11.5), fetal liver (E11.5–E12.5), and even umbilical artery (E11.5), reflective of its strictly spatial-regulated ontogeny. Expression of CD45, a panhematopoietic marker, distinguished hematopoietic-restricted HPP–colony-forming cell from the bipotential HPP-HA. Finally, we revealed that basic fibroblast growth factor, other than vascular endothelial growth factor or transforming growth factor-β1, was a positive modulator of the HPP-HA proliferation. Taken together, the HPP-HA represents a novel model for definitive hemangioblast in the mouse AGM region and will shed light on molecular mechanisms underlying the hemangioblast development.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic hematopoiesis in the mouse is categorized into two phases: the primitive hematopoiesis is characterized by nucleated erythrocytes (large and yielding of embryonic hemoglobin) in the extraembryonic yolk sac (YS) [1, 2], and the definitive hematopoiesis is profoundly marked by generation of the first adult type hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) region at embryonic day 10.5 (E10.5) and maturation of enucleated erythrocytes (smaller size and production of adult hemoglobin) in the fetal liver (FL) [3, 4]. Different in hematopoietic nature, both waves closely intertwine with endothelial lineage in two forms as supposed: the hemangioblast (a common precursor for hematopoietic and endothelial lineages) and the hemogenic endothelium (dedifferentiation of mature endothelium into hematopoietic lineage) [5–7].

The term of the hemangioblast has been raised for a long time by early observations that, in the YS, the mesodermal cells simultaneously give rise to primitive erythrocytes and surrounding endothelial cells in close proximity, forming a distinct structure called blood island [8, 9]. Recently, couples of evidence further support the hypothesis, including the surface markers (CD34, CD31, Tie2, vascular endothelial [VE]-cadherin, and Flk-1) and the transcriptional programs (SCL, LMO2, and Runx1) shared by the two lineages [6, 7]. As the most convincing proof, Choi and Keller et al. defined the clonal blast colony-forming cells (BL-CFCs) in descendants of embryonic stem (ES) cells as an in vitro counterpart of the hemangioblast [10], and, most recently, they revealed that the BL-CFC (Brachyury+/Flk-1+) also arises in the primitive streak at the midstreak stage (E7.0) embryos [11]. Instead, Nishikawa et al. used an adherent differentiation method of ES cells to illustrate the development pathway of the hemangioblast (Flk-1+VE-cadherin+CD45–) [12]. Alternatively, Li and Yoder et al. demonstrated that Tie2+Flk-1+ endothelial cells lining the nascent capillaries of the YS are capable of forming CD41+ hematopoietic cells, providing a novel paradigm of hemogenic endothelium in the stage of primitive hematopoiesis [13].

Compared with the YS, delineation of the cellular origin of definitive hematopoiesis has been more challenging. It seems that the precursor of definitive hematopoiesis cannot be the hemangioblast because the aorta and vitelline arteries form at least 1 day earlier than hematopoietic clusters. In contrast, the hemogenic endothelium is more likely to be a direct precursor [4, 5]. Thus, the key question, whether the hemangioblast arises during the onset of definitive hematopoiesis, remains unsolved.

High proliferative potential–colony-forming cell (HPP-CFC) is the earliest multipotential hematopoietic progenitor that can be cultured in vitro without stromal support. It was first identified as bone marrow cells that can give rise to macroscopic colonies greater than 0.5 mm in diameter [14]. Also, the developmental dynamics of the HPP-CFC have been elegantly determined both spatially (YS, bloodstream, embryo proper, and FL) and temporally (E8.25–E11.5) [15]. Here, we found that a clonal HPP-CFC in the AGM region demonstrated self-renewal hematopoiesis and endothelial activity (incorporating DiI-acetylated low-density lipoprotein [DiI-Ac-LDL] and forming extensive tube-like structures in Matrigel), pointing to its authentic hemangioblast identity. However, it is strikingly spatial-regulated, since such bipotential precursor was not detected in the YS, placenta, FL, embryonic circulation, and even umbilical arteries.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryo Dissections
C57BL/6 or BALB/c mice were mated overnight. Noon of the day when a vaginal plug was detected was designated as E0.5. At the time point as indicated, mice were killed, and embryos were dissected free of decidual tissues and Reichter's membrane. Somite-stage embryos were staged by counting somite numbers. The embryonic tissues were treated with 0.1% type I collagenase (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 37°C for 0.5–1 hour and then dispersed into a single-cell suspension. Cell counts and viability were estimated after trypan blue staining. Magnetic cell sorting with CD45 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) was performed according to the manufacture's protocol.

HPP-CFC Assay
HPP-CFC culture medium is composed of 0.9% methylcellulose (Sigma), 2 mM glutamine (HyClone, Logan, UT, http://www.hyclone.com), 2 mM penicillin/streptomycin (HyClone), 5% protein-free hybridoma medium II (Gibco, Grand Island, NY, http://www.invitrogen.com), 200 µg/ml iron-saturated transferrin (Sigma), 1% bovine serum albumin (BSA) (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 0.45 mM monothioglycerol (MTG, Sigma), 15% fetal bovine serum (FBS) (HyClone), and a cytokine cocktail including recombinant mouse stem cell factor (rmSCF) (50 ng/ml), rm interleukin (IL)-3 (10 ng/ml), recombinant human IL-11 (10 ng/ml), rm granulocyte macrophage–colony-stimulating factor (GM-CSF) (10 ng/ml), recombinant human thrombopoietin (rhTpo) (20 ng/ml), recombinant human erythropoietin (rhEpo) (3 U/ml), rm vascular endothelial growth factor (VEGF) (5 ng/ml), rh basic fibroblast growth factor (bFGF) (10 ng/ml), and rh insulin-like growth factor (IGF)-1 (10 ng/ml). rmVEGF and rhbFGF are products of R&D Systems Inc. (Minneapolis, http://www.rndsystems.com), and Epo was obtained from Kirin Brewery Co. (Tokyo, http://www.kirin.co.jp/english). The other cytokines were purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com). At the beginning, 1 x 105 cells from indicated hematopoietic tissues were plated in each 35-mm Petri dish in 2 ml of HPP-CFC medium. Following 2 weeks of inoculation, compact colonies (>0.5 mm in diameter) or more diffuse colonies (>1.0 mm in diameter) were scored as HPP-CFC. Of note, after 10 days of culture, colonies that met the criterion of HPP-CFC were used for further experiments.

Nested Reverse Transcriptase-Polymerase Chain Reaction for Individual Colonies
The cell samples in a volume of 1 µl were placed into 9 µl of prechilled lysis buffer containing 2 µl of 5x reverse transcriptase (RT) buffer (Takara, Otsu, Japan, http://www.takara.co.jp), 0.25 mM deoxynucleoside-5'-triphosphate (dNTP) (Takara), 0.4% NP40 (Fluka, Buchs, Switzerland, http://www.sigmaaldrich.com), 2,000 U/ml RNase inhibitor (Takara), and 1 µM oligo(dT) primer (Takara), incubated for 15 minutes on ice, and followed by addition of 5 U of avian myeloblastosis virus reverse transcriptase. Samples were then incubated at 42°C for 1 hour. First-round polymerase chain reaction (PCR) was performed in 10 µl of PCR reaction mixture containing 0.5 µl of reverse transcription products, 1 µl of 10x Taq buffer (Takara), 0.25 mM dNTP (Takara), 0.6 µM gene-specific primers, and 0.5 U of Taq DNA polymerase (Takara) for 35 cycles. One µl of aliquot of the first-round PCR reactions was then used as template in a new PCR reaction using nested primer sets. Finally, the PCR products were gel electrophoresed and visualized after ethidium bromide staining. The specific primer sequences for nested RT-PCR are listed in supplemental online Table 1.

Flow Cytometry
Cells were stained with monoclonal antibodies against Sca-1 (fluorescein isothiocyanate [FITC]-conjugated, eBioscience [San Diego, http://www.ebioscience.com]), c-kit (FITC-conjugated, eBioscience), CD34 (FITC-conjugated, eBioscience), H-2kb (FITC-conjugated, BD Pharmingen [San Diego, http://www.bdbiosciences.com/index_us.shtml]), H-2kd (phycoerythrin [PE]-conjugated, BD Pharmingen), CD31 (BD Pharmingen), and VE-cadherin (BD Pharmingen) in phosphate-buffered saline (PBS) containing 1% BSA at 4°C for 30 minutes. CD31- and VE-cadherin-labeled cells were washed twice in PBS and incubated with PE-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com) for an additional 30 minutes at 4°C. Then the samples were analyzed by a BD Pharmingen FACSCalibur.

Immunofluorescent Staining
Cells were fixed in 4% paraformaldehyde for 10 minutes and were incubated with primary antibodies against CD31 (BD Pharmingen), Mac-1 (eBioscience), and CD45 (eBioscience) for 40 minutes at 4°C. To detect von Willebrand factor (vWF), cells were permeabilized with 0.1% Triton X-100 for 10 minutes before incubation with primary antibody against vWF (Chemicon, Temecula, CA, http://www.chemicon.com). Then they were washed three times and incubated with FITC or PE-conjugated goat anti-rat IgG (SouthernBiotech) for an additional 40 minutes at 4°C.

In Vitro Angiogenesis Assay
Individual HPP colonies were plucked and transferred to single wells with endothelial expansion medium containing Iscove's minimal essential medium with 10% FBS, 10% horse serum, VEGF (5 ng/ml), IGF-1 (10 ng/ml), bFGF (10 ng/ml), L-glutamine (2 mM), and 0.45 mM MTG and incubated for 5 days. The adherent cells were then trypsinized and replated into single wells of 48-well plates precoated with Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com). Tube-like and network structures were documented after 12 hours of culture. For DiI-Ac-LDL uptake assay, adherent cells were cultured in the presence of 10 µg/ml DiI-Ac-LDL (Biomedical Technologies Inc., Stoughton, MA, http://www.btiinc.com) for 4 hours.

Phase Contrast and Fluorescence Imaging
Phase contrast and fluorescence images were collected using a Nikon TE2000-U (Tokyo, http://www.nikon.com) or an Olympus CK2 inverted microscope (Tokyo, http://www.olympus-global.com). Images were acquired using a Cool SNAP 5.0 (Roper Scientific, Tucson, AZ, http://www.roperscientific.com) or Nikon Coolpix 995 camera. Confocal image was collected by a Nikon TE300 inverted microscope and the Bio-Rad Radiance 2100 (Hercules, CA, http://www.bio-rad.com) and was acquired using a Bio-Rad LaserSharp 2000. Composite images were assembled in Adobe Photoshop version 7.0 (San Jose, CA, http://www.adobe.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Developmental Kinetics of HPP-CFC in the Bilineage Culture System
HPP-CFCs are usually grown in semisolid culture containing only hematopoietic cytokines, including SCF, IL-3, IL-11, GM-CSF, Tpo, and Epo. To retain or stimulate the endothelial potential of HPP-CFC, we modified the culture system and included the endothelial expansion cytokines VEGF, bFGF, and IGF-1 (referred to as bilineage medium). Typical macroscopic hematopoietic colonies emerged in the presence of either hematopoietic or bilineage cocktails but not the endothelial cytokines alone. Under the bilineage condition, the spatial and temporal distribution of HPP-CFC within various hematopoietic and/or hemogenic tissues was in line with that reported previously [15]. Prior to E8.25, no HPP-CFC was detected in the whole conceptus. HPP-CFC was first found exclusively in the YS at E8.25, then in the intraembryonic para-aortic splanchnopleura (P-Sp) at E8.5, and in the FL at E11.5 (Fig. 1). A significant number of HPP-CFC was also detected in the placenta between E10.5 and E12.5, which is consistent with recent findings that hematopoietic stem cells are present in the placenta [16, 17].


Figure 1
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  		Figure 1. Spatial and temporal distribution of HPP-CFC during mouse embryogenesis (E8.25–E12.5). Results are expressed as mean ± SEM. ND means that the analysis on the placenta at E9.5 was not done. Abbreviations: FL, fetal liver; HPP-CFC, high proliferative potential–colony-forming cell; No., number; P-Sp/AGM, para-aortic splanchnopleura/aorta-gonad-mesonephros; Pla, placenta; YS, yolk sac.

 
The AGM Region Contained the HPP Precursor with Both Hematopoietic and Endothelial Potential
As shown in Figure 1, the first intraembryonic HPP-CFC was detected at E8.5 in the P-Sp, which further develops into the AGM region (E10.5–E12.5) with a moderately expanded number of HPP precursors (13.8 at E10.5 and 29.4 at E11.5 per tissue). Upon an initial assessment, the well circumscribed HPP colonies derived from the P-Sp/AGM region (Fig. 2A) were individually picked for mRNA profiling on several hematopoietic/endothelial related genes by nested RT-PCR. As known, Runx1 is expressed by all the HSC in the midgestation embryos, and its absence leads to defective HSC generation in dorsal aorta but leaves the YS hematopoiesis largely undisturbed [18–20]. GATA-1 is a transcriptional factor critical for erythropoiesis in the YS, and its deletion results in erythrocyte arrest at the proerythroblast stage [21]. Flk-1, also known as VEGFR2, is indispensable for normal development of the blood island and marks BL-CFC together with SCL [22, 23]. Expression of Tie2, the receptor for angiopoietins, defines virtually all the HSC and endothelial lineage during mouse embryogenesis [24, 25]. As shown in Figure 2B, all the HPP colonies from the E11.5 AGM region expressed erythrocyte-specific β-major globin and Runx1, whereas a significant proportion contained transcripts for GATA-1. Furthermore, the colonies positive for Flk-1 and Tie2 appeared at a low frequency. Next, the colonies were collected in bulk for surface marker identification by flow cytometry. As shown in Figure 2C, 9.4% and 18.3% were respectively positive for Sca-1 and c-kit, the molecules expressed in the early hematopoietic progenitors. In consistence, single primary HPP colonies were able to produce secondary macroscopic colonies (at a frequency of 7/21, Fig. 2D, left) and hundreds of low proliferative potential colonies (at a frequency of 21/21, Fig. 2D, right), suggestive of their self-renewal property and primitiveness in the hematopoietic hierarchy. With respect to the common markers for hematopoietic and endothelial lineages, the ratio of CD31+ cells within the primary HPP colonies was relatively higher (33%) than that of VE-cadherin+ (2.3%) and CD34+ cells (1.5%).


Figure 2
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  		Figure 2. High proliferative potential–colony-forming cells from the aorta-gonad-mesonephros region demonstrated hematopoietic self-renewal capacity and differentiated to adherent cells expressing endothelial-related markers. The expression of hematopoietic/endothelial-related genes was analyzed by nested reverse transcription-polymerase chain reaction (B) and flow cytometry (C). Adherent cells from seven individual HPP colonies express endothelial-specific genes (E). vWF and Ac-LDL (red); CD31, CD45, and Mac-1 (green); DAPI (blue). Original magnification: x40 (A, D), x200 (Fa, Fb, Fd, Fe), and x1,000 (Fc). Abbreviations: Ac-LDL, acetylated low-density lipoprotein; DAPI, 4,6-diamidino-2-phenylindole; HPRT, hypoxanthine phosphoribosyltransferase; VE, vascular endothelial; vWF, von Willebrand factor.

 
To further verify whether the HPP-CFC was capable of producing endothelial cells, the cells within single HPP colonies were replated to the wells with endothelial expansion medium. In nearly half of the wells, dramatically proliferating adherent layers were observed after 5-day incubation (Fig. 2Fa). Immunocytochemistry analysis revealed that some adherent cells were vWF+ but CD45– (Fig. 2Fb). Meanwhile, they expressed CD31 and incorporated DiI-Ac-LDL without CD45 and Mac-1 staining, indicating they were most likely endothelial cells other than monocytes or macrophages (Fig. 2Fc–2Fe). Furthermore, a significant proportion of the wells with adherent populations, evidenced by seven representative colonies in Figure 2E, were strengthened in the expression of molecules essential for endothelial progression, such as Flk-1, VE-cadherin, and vWF. In particular, they all acquired Flk-1 expression that was almost absent in the primary HPP colonies, correlating well with their amplification in the medium containing VEGF. To determine whether functional endothelial cells were generated in vitro, the adherent cells derived from a single HPP colony were digested and transferred to Matrigel. Within 12 hours, 46 from a total of 99 colonies were capable of generating extensive tubule and network structures (Fig. 3A, 3B). As shown in Figure 3C, CD31+ cells lining the tube were easily seen. Alternatively, the adherent cells were labeled with DiI-Ac-LDL 4 hours prior to replating. The DiI-Ac-LDL positive cells elongated in shape and participated in the tube and network formation in Matrigel (Fig. 3D–3F). Upon close examinations, some of the DiI-Ac-LDL-labeled cells, presumably macrophages, as well as nonlabeled round hematopoietic cells, scattered or gathered around the tube-like structures, suggestive of the bilineage potential of the HPP-CFC. Thus, the capillary structure expressing CD31 and incorporating DiI-Ac-LDL strongly suggested that the adherent cells were authentic endothelial cells other than fibroblasts or macrophages. Of note, if prior adherent expansion was omitted, the HPP colonies failed to form tubule structures in Matrigel. Almost no endothelial potential was detected if HPP colony was generated with hematopoietic cytokines only, implying that the endothelial cytokine was essential for maintaining the dual potential. The result was also reproducible with BALB/c strain (data not shown).


Figure 3
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  		Figure 3. In vitro angiogenesis assay. Adherent cells from single high proliferative potential colonies generate capillary-like structures expressing CD31 ([C], red) and incorporating DiI-Ac-LDL ([F], red) in Matrigel. (E) and (F) represent a higher magnification of the rectangle region in (D). Original magnification: x40 (A, B), x100 (D), and x200 (C, E, F). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DiI-Ac-LDL, DiI-acetylated low-density lipoprotein; h, hours.

 
To distinguish between the bipotential and the conventional hematopoietic HPP-CFC, we referred to the former as the HPP-hemangioblast (HPP-HA) and the latter as the HPP-hematopoietic (HPP-H). Temporally, a significant number of the HPP-HA could be detected as early as E9.5 (2.4 ± 0.57), peaked at E11.5 (17.4 ± 3.50), and thereafter dropped evidently (Fig. 4A, 4B). Next, spatial distribution was investigated by separating the AGM region of E11.5 embryos into dorsal aorta with its surrounding mesenchyme (AoM) and the gonad/mesonephros (GM). As shown in Figure 4C, nearly all the HPP-CFC colonies, including the HPP-HA, were included in the AoM but not the GM. Subsequently, cell sorting was used to determine the surface marker of the HPP-HA. As expected, expression of CD31 could define all the HPP colonies in the AGM region but failed to distinguish the HPP-H from the HPP-HA (data not shown). However, the CD45– subpopulation contained all the HPP-HA in the AGM region of E11.5 embryos with the CD45+ cells generating only HPP-H colonies (Fig. 4D).


Figure 4
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  		Figure 4. Characterization of HPP-HA distribution in the para-aortic splanchnopleura/aorta-gonad-mesonephros region. Temporal (percent or number) and spatial localization of HPP-HA is displayed in (A), (B), and (C). Magnetic cell sorting reveals that the HPP-HA is exclusively included in CD45– population (D). Abbreviations: AoM, dorsal aorta with surrounding mesenchyme; CFC, colony-forming cell; GM, gonad/mesonephros; HA, hemangioblast; HPP, high proliferative potential; No., number.

 
Thereafter, two steps were taken to validate the clonal nature of the HPP colonies. First, cells from the AGM region were plated at different concentrations (0.5 x 105, 1 x 105, and 2 x 105 cells). As shown in Figure 5A, the number of HPP-CFC colonies was directly proportional to that of AGM cells input, indicating that a single cell corresponded to the growth of a HPP colony. Cell mixing analysis was also carried out as reported but with some modification [10, 11]. An equal number of cells from the AGM region of C57BL/6 (H-2Kb) and BALB/c (H-2Kd) strain were mixed and cultured under conditions appropriate for HPP colony growth. After 10 days of culture, HPP colonies were picked out individually to determine their major histocompatibility complex I type by flow cytometry. Among 12 representative colonies analyzed, five displayed exclusively immunophenotype of H-2Kb resembling the C57BL/6 control without any contamination from H-2Kd positive cells, and the other seven showed the staining pattern identical to the BALB/c control (Fig. 5B). This experiment strongly suggested that each HPP colony originated from a single cell.


Figure 5
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  		Figure 5. The clonal nature of HPP-CFC in the aorta-gonad-mesonephros region. The relationship between HPP colony and cell dose is calculated in (A). Moreover, cell mixing assay (B) was carried out to prove that HPP colony was clonal. Abbreviations: CFC, colony-forming cell; HPP, high proliferative potential; No., number.

 
HPP-HA Was Not Detected Within Yolk Sac Even Cocultured with AGM Cells
To obtain an overall picture of the spatial distribution of the HPP-HA, multiple hematopoietic tissues were further evaluated for their hemangioblastic properties. The AGM region was used as a positive control in all the experiments described below (Fig. 6A). As the YS gave rise to HPP-CFC 6 hours earlier than the P-Sp region, 131 HPP colonies from the extraembryonic tissue of 56 embryos (E8.25–E12.5) were investigated on their endothelial properties. The data shown in Figure 6B were obtained from E11.5 YS. After the colonies were individually transferred to the endothelial expansion medium, the majority of the cells assumed nonadherent, hematopoietic cell phenotype, and the small portion of scattered adherent cells did not display the morphology of canonical endothelium after 5 days of culture (data not shown). Then, the adherent cells from a single well were collected and replated to Matrigel for verification of their endothelial function in vitro. Nevertheless, they failed to form any typical tube-like structures (Fig. 6B). The absence of dual potential precursors in the YS was not strain specific, because the result was the same when using BALB/c embryos. Given the paracrine roles of stromal cells in the AGM region, we cocultured YS cells of green fluorescent protein-transgenic mouse with wild-type AGM cells. However, none of the green fluorescent HPP colonies individually plucked displayed endothelial potential in Matrigel (data not shown).


Figure 6
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  		Figure 6. Anatomical mapping of the high proliferative potential (HPP)-hemangioblast in the midgestation embryos. HPP colonies from the yolk sac (B), embryonic bloodstream (C), placenta (D), fetal liver (E), and umbilical arteries (F) fail to form tube-like structures in Matrigel, in contrast to those from the AGM region (A). Original magnification is x100. Abbreviations: AGM, aorta-gonad-mesonephros; BL, bloodstream; FL, fetal liver; PL, placenta; UA, umbilical arteries; YS, yolk sac.

 
The fully functional circulation in the mouse embryo is established at E10, a time point slightly earlier than that of hematopoietic cluster protruding from the ventral-lateral side of dorsal aorta [26]. For this reason, 20 HPP colonies were obtained by inoculation of cells from embryonic bloodstream at E10.5 and analyzed for their HPP-HA activity. Unexpectedly, none displayed any endothelial potential (Fig. 6C), implying that the HPP-HA presumably did not emigrate from the dorsal aorta. Recently, the placenta has been reported to be an important organ for embryonic hematopoiesis, almost paralleling the AGM region and later the FL in the generation and expansion of HSC [16, 17]. Therefore, we analyzed 71 HPP colonies from the placenta (E10.5–E11.5) and 76 from the FL (E11.5 and E12.5), among which none was capable of forming capillary-like structure in Matrigel (Fig. 6D, 6E). The vitelline and umbilical arteries are analogous to dorsal aorta in that they are major vasculatures in nature and can autonomously generate HSC almost concomitantly [27, 28]. However, we could not detect any HPP-HA after testing 65 HPP colonies in three independent experiments (Fig. 6F). Thus, the anatomical mapping suggested that the HPP-HA was much more spatially than temporally regulated, implying that microenvironment elements, including stromal cells, extracellular matrix, and cytokines, might be critical in its generation, maintenance, and expansion.

bFGF Is a Positive Regulator of the HPP-HA Development
Little is known about the regulation of hemangioblast development during mouse definitive hematopoiesis. The HPP-HA described here may represent an in vitro quantifiable equivalent of the presumed hemangioblast. We next studied the influence of VEGF, bFGF, and transforming growth factor (TGF)-β1, which are involved in the embryonic hematopoiesis and angiogenesis, on the development of the HPP-HA. VEGF, the ligand for Flk-1 or KDR, is indispensable for hemangioblast migration, blood island emergence, and blast colony formation [29, 30]. By comparison, bFGF may act earlier, as evidenced by its critical roles in mesoderm induction and patterning. Deletion of the receptor for bFGF leads to decreased number of BL-CFC [31]. TGF-β1 and its receptors are pivotal in embryonic angiogenesis and/or hematopoiesis [32–34]. We have previously shown that disruption of Smad5, a candidate downstream signaling molecule of TGF-β1, resulted in an elevated number of BL-CFC [35]. Here, 3 x 105 cells of the AGM region at E10.5 were plated on irradiated embryonic fibroblast feeders for 12 or 24 hours in the absence or presence of VEGF (10 ng/ml), bFGF (10 ng/ml), or TGF-β1 (5 ng/ml) followed by the HPP-HA assay. As compared with the control, a significant amplification of HPP-CFC (p = .0013) together with HPP-HA (p = .0097) only occurred in the presence of bFGF, and the expansion became more evident after 24 hours of culture (Fig. 7), implying that bFGF could either accelerate the hemangioblast specification from early precursor or directly amplify the hemangioblast pool. In contrast, TGF-β1 treatment for 12 hours suppressed both HPP-CFC and HPP-HA development (p < .01). In particular, the 24-hour incubation with TGF-β1 abolished the HPP-HA, whereas a significant number of HPP-H was spared, suggesting that the bipotential precursor was more sensitive to the inhibition by TGF-β1. In contrast to bFGF and TGF-β1, adding VEGF to the primary culture for 24 hours did not significantly affect the HPP-HA proliferation. Thus, the HPP-HA assay could be used to delineate the precise roles of different cytokines in hemangiopoiesis by classifying bi-/unipotential hematopoietic precursors.


Figure 7
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  		Figure 7. bFGF is a positive regulator of HPP-HA development. Aorta-gonad-mesonephros cells were incubated for 12 or 24 hours in the absence or presence of VEGF, bFGF, and TGF-β1 followed by the HPP-HA assay. The results are expressed as mean ± SEM. Significance was determined using the Student's t test. *, p < .05; **, p < .01. Abbreviations: bFGF, basic fibroblast growth factor; CFC, colony-forming cell; h, hours; HA, hemangioblast; HPP, high proliferative potential; No., number; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In this study, we have provided unambiguous data that the HPP-HA, a type of precursor with hemangioblastic characteristics, does exist in the P-Sp/AGM region of mouse somite-stage embryos. The developmental kinetics, including onset, amplification, and reduction of the bipotential precursor, parallel that of the hematopoietic stem/progenitor cells in the P-Sp/AGM structure. Furthermore, its inability to generate primitive erythrocyte suggests a developmental identity within the cascade of definitive hematopoiesis. An outstanding feature of the HPP-HA is the balanced bipotential, which is shown by synchronized other than biased high proliferation in both hematopoietic and endothelial lineages. Here, verification of the endothelial cells depends largely on the functional readout (DiI-Ac-LDL uptake coupled with extensive capillary-like structures in Matrigel) in addition to the immunophenotype (CD31+/vWF+/CD45–/CD11b–). Previously, an adherent bulk culture implied the existence of the hemangioblast (PCLP1+CD45– subset) in the AGM region at E10.5, exemplified by hematopoietic cluster budded from proliferating endothelial layers in the presence of bFGF, SCF, and oncostain M [36, 37]. Notwithstanding the fact that the hemangioblast has been described in E9.5 YS by Hox11 gene-mediated immortalization, the proof for an authentic and physiological hemangioblast in the somite-stage YS is lacking [38]. Recently, Ingram and Yoder et al. proposed a novel hierarchy of endothelial progenitor cells in human blood (umbilical cord and peripheral origin) and vessel walls (umbilical vein and adult arteries), as well [39–41]. Residing on the top of the hierarchy is a clonogenic endothelial precursor called high proliferative potential–endothelial colony-forming cell, which does not overlap with a monocyte ancestry (CD14+CD45+) and appears analogous in the proliferative activity to the HPP-CFC and the HPP-HA.

The exclusive localization and abundance of the HPP-HA in the AGM region further highlight that the hemangioblast serves as an important source for intraembryonic definitive hematopoiesis. Since all the HPP-HA resided within the CD45– subset of E11.5 AGM region, they may, as embryos grow, differentiate into CD45+ hematopoietic-restricted HPP-CFC with their endothelial property gradually lost. In line with this, the AGM region-derived CD45+ cells, albeit expressing a variety of endothelium-related markers (such as VE-cadherin, CD31, Tie2, Flk-1, and LDL receptor), fail to form tubule structures in vitro, reflecting their functional divergence from the vasculature compartment [42]. Upon migration into the FL, the VE-cadherin+/CD45+ HSC progressively downregulates expression of multiple endothelial-specific genes, and becomes negative for VE-cadherin by E16.5 [42, 43]. Hence, the data reinforce that the unique microenvironment cues of the AGM region are pivotal for ontogeny and self-renewal of the hemangioblast, which is rare in the postnatal and adult hematopoietic organs. Of note, inability to detect the HPP-HA in the YS, placenta, and FL may result from inappropriate culture conditions that accelerate hematopoietic differentiation at the expense of endothelial potential. For example, in vitro generation of capillary network, rather than primitive erythrocyte, by the YS mesoderm is dictated by adjacent endoderm, which cannot be substituted by exogenous VEGF [44]. At least, our findings suggest the AGM region may be a major site of hemangiopoiesis besides hemogenesis. Recently, Scott and Fleming independently reported that derivatives of a single HSC in mouse bone marrow can participate in neoangiogenesis even after serial transplantation, indicating HSC as a reservoir of endothelial progenitor cells [45–46]. Further efforts are needed to illustrate whether the hemangioblast with long-term hematopoietic reconstitution capacity develops in the dorsal aorta.

Two types of multipotential precursors are in close correlation with the HPP-HA. The first one is BL-CFC, defined as Brachuary+/Flk-1+/SCL– hemangioblast and generating blast colony with unique morphology [47]. In fact, 20% of the blast colonies derived from early embryoid bodies are able to produce macroscopic hematopoietic colonies (data not shown), implying a possible link between the HPP-HA and the BL-CFC. The HPP-HA and the BL-CFC generate bilineage colonies, depending heavily on certain cytokine cocktails. In early embryos, BL-CFC develops within a narrow window and is undetectable in a significant proportion of conceptus, further emphasizing its elusiveness and rarity [11]. The HPP-HA initially emerged in the caudal half of E8.5 embryos (data not shown), a temporal continuum of the BL-CFC in the primitive streak at E7.0–E7.5. Of particular significance, the BL-CFC system has been widely used to decipher regulatory mechanisms of hemangioblast ontogeny, including transcriptional factors (SCL, Runx1, hypoxia inducible factor, and Smad5), [35, 47–51] and cytokines (bFGF, Activin, Tpo, and bone morphogenetic protein [BMP]4) [31, 52–54]. However, it remains unknown whether BL-CFC arises in the AGM region or other hematopoietic tissues. The second one is mesoangioblast, which is identified as a common precursor for vascular and extravascular mesodermal derivatives in the dorsal aorta [55]. As compared with the HPP-HA, it emerges at a higher frequency, can easily differentiate into mesenchymal progenies including bone, fat, cartilage, and muscle, is unable to proliferate in semisolid culture, and fails to generate typical hematopoietic colonies [55–57]. As suggested, the mesoangioblast and hemangioblast may simultaneously develop from their common ancestor, the primitive angioblast, at the dorsal and ventral sides of the aorta, respectively [57].

To date, the generation, expansion, and migration of hemangioblast are poorly understood. The HPP-HA assay represents a relatively simple and sensitive way in quantitatively evaluating the hemangioblast development in the AGM region. As proposed, various cytokines may synchronize with the intrinsic transcriptional factors to dictate the ultimate behavior of multipotent precursors, as exemplified by VEGF/Flk-1/SCL [23, 58]. Our data clearly denote that bFGF is competent in promoting hemangiopoiesis, conforming to previous results that it can increase the BL-CFC number in a dose-dependent manner by elevating Flk-1 expression. In contrast, addition of VEGF does not influence the expansion of both BL-CFC and HPP-HA. However, quail/chick transplantation experiments show that VEGF is superior to bFGF in promoting hemangiopoiesis. It may reflect either the interspecies difference or distinct microenvironment cues (in vitro vs. in vivo) [59]. The suppressing effect of TGF-β1 on the HPP-HA is unexpected because the ligand can retain the stemness of HSC in vitro [60]. As the pleiotropic roles of TGF-β superfamily members and related signaling molecules, including TGF-β1, BMP4, Activin, Smad1, and Smad5, in hemangioblast specification or differentiation have been extensively reported in BL-CFC from human or mouse ES cells in vitro, gene knockout is thus required to prove if these molecules are physiologically pivotal for hemangioblast development in vivo.

Although blood-forming endothelium has been identified in human embryo and fetus [61], the hemangioblast is also found in ES cell derivatives, fetal bone marrow, and postnatal tissues [62–65]. Furthermore, clinical analysis of chronic myelogenous leukemia patients reveals the BCR/ABL fusion gene in endothelial cells as well as in Flk-1+/CD34–/CD31– precursor-derived blood and endothelial cells, suggesting the chromosome rearrangement may occur at the level of hemangioblast [66, 67]. Undoubtedly, it is of fundamental and clinical significance to illustrate whether hemangioblast is the cellular origin of human definitive hematopoiesis, which will greatly aid us in generating abundant HSC for clinical applications.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We sincerely thank Dr. Peihsien Tang and Sheng Zhou for critical review of the manuscript. This study was supported by the National Natural Science Foundation (Grant number 30300181) and the National Key Basic Research Program of China (Grant numbers 2005CB522705 and 2005CB522506). H.Y. and B.L. contributed equally to this work.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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