| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Embryo |
Biotechnology Interdisciplinary Unit and Rappaport Faculty of Medicine,3 Technion–Israel Institute of Technology, Haifa 31906, Israel
Department of Obstetrics and Gynecology,4 Rambam Medical Center, Haifa 31906, Israel
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, early development, embryo, embryonic development, endothelial cells, human embryonic stem cells, smooth muscle cells, vasculogenesis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In humans, by the end of Week 5 two jugular lymph sacs develop and start collecting fluids from the lymphatics of the upper limbs, upper trunk, head, and neck. By Week 6, four additional lymph sacs develop and start collecting lymph from the trunk and lower extremities [3, 9]. The first anlagen of lymphatics, like those of developing blood vessels, are composed solely of endothelial cells, although the origin of lymphatic endothelial cells (LECs) remains unclear, mainly owing to lack of specific markers.
In vitro isolation and propagation of the inner cell mass of the blastocyst stage can result in the generation of a human embryonic stem cell (hESC) line [10, 11]. Human ESCs are able to remain in an undifferentiated state in specific culture conditions for prolonged periods [10, 11]. Removal of hESCs from such culture conditions, which support their pluripotency and self-renewal, causes their spontaneous differentiation [10–12]. The hESC differentiation process follows a reproducible temporal pattern of development that for some extent recapitulates early embryogenesis. When implanted into severe combined immunodeficiency (SCID) mice, hESCs develop teratomas containing complex structures comprising differentiated cell types representing derivatives of all three major embryonic lineages [10, 11]. In particular, hESCs have been shown by means of specific markers and features to differentiate spontaneously in vitro into ECs [13, 14] and SMCs [14]. These characteristics permit the use of hESCs in models to improve our basic understanding of vasculogenesis and angiogenesis in normal and abnormal development of the embryo and placenta.
Accordingly, we aimed to follow vasculogenesis during the early stages of developing human embryos and to examine whether teratomas generated from hESCs in SCID mice may provide a useful in vivo platform to study human neovasculogenesis. To this end, human vascular markers for v-SMC, ECs, and LECs were explored in 4- to 8-week-old human embryos (6–10 weeks of gestation) and also in teratomas generated in SCID mice from hESCs.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fourteen human embryos ranging from 4- to 8-wk-old (estimated time of postfertilization = 6–10 weeks of pregnancy) were obtained following elective abortions induced by RU486 and Misoprostol or by dilatation and curettage. Embryonic developmental age was determined based on the date of the last menstrual period, ultrasound measurement of crown-rump length, and postabortion measurements of limb size and shape. Whole embryos aged 4 wk (n = 4), 7 wk (n = 2), and 8 wk (n = 2) were immediately frozen in liquid nitrogen for RNA extraction. Embryos aged 4 wk (n = 2), 7 wk (n = 2), and 8 wk (n = 2) were fixed and sectioned for histological and immunohistochemical analyses (see below). The study was approved by the local ethics committee at Rambam Medical Center (protocol approval 1646), and patients gave informed consent.
Human Embryonic Stem Cell (hESC) Culture
Nondifferentiating hESC lines H9.2, H13, and I6 were grown as previously described [14]. In brief, the cells were grown on mouse embryonic fibroblasts and passaged every 4–6 days using 1 mg/ml type IV collagenase (Gibco Invitrogen Co., San Diego, CA).
Teratoma Formation
Teratoma formation by injection of hESCs in SCID mice was induced as previously described [15]. Teratomas could be detected 4 wk following injection of hESCs and were removed for histological and immunohistochemical examination at no less than 8 wk after the injection.
Histology and Immunohistochemistry
For histological analyses the aborted embryos and teratomas were fixed in 10% neutral-buffered formalin, dehydrated in graduated alcohols (70%– 100%), and embedded in paraffin for routine histology. Six to eight micrometer thick sections were stained with hematoxylin/eosin. Immunostaining was performed (with at least five repetitions) using a Dako LSAB+ staining kit with specific anti-human HLA-a, -b, -c, and VE-Cad (both from Chemicon Int., Temecula, CA); anti-human Tal1 and VE-Cad (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-human KDR (R&D Systems, Minneapolis, MN); and anti-human CD31, CD34, glycophorin A, smooth muscle actin (SMA), calponin, von Willebrand factor (vWF), and smooth muscle myosin heavy chain (SM-MHC; all from DAKO, Denmark). All markers were human specific and showed no cross-reactivity with mice. Mouse IgG isotype-matching (R&D Systems) or secondary antibody alone (from DAKO LSAB+ staining kit) served as negative controls. For positive controls, paraffin sections of human breast or rat heart were used. All positive controls were fixed, embedded, and stained using the same procedures. For quantification, 10 fields of teratomas stained with HLA-a, -b, and -c (n = 5) were viewed at x100 total magnification. Blood vessels were scored for positive and negative staining and calculated as percentage out of total blood vessels. The results were expressed as mean ± SD.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted using TriZol (Gibco Invitrogen Co., San Diego, CA) according to manufacturer's instructions. To ensure no DNA contamination, RNA samples were treated with a DNA-free kit (Ambion Inc., Austin, TX) and examined for DNA contamination before performing reverse transcription (RT) reaction. Total RNA was quantified by a UV spectrophotometer and 1 µg was used for each RT sample. RNA was reverse transcribed with M-MLV (Promega Co., Madison, WI) and oligo (dT) primers (Promega) according to manufacturer's instructions. PCRs were performed with BIOTAQ DNA Polymerase (Bioline Ltd., London, UK) using 1 µl RT product per reaction according to manufacturer's instructions. In some cases MgCl2 concentration (normally 1.5 mmol) was calibrated (indicated in Table 1). To ensure semiquantitative results of the RT–polymerase chain reaction (RT-PCR) assays, the number of PCR cycles for each set of primers was verified to be in the linear range of the amplification. In addition, all RNA samples were adjusted to yield equal amplification of GAPDH as an internal standard. RT reaction mix was used for negative controls. PCR conditions consisted of 5 min at 94°C (hot start), and 30–40 cycles (actual number noted in Table 1) of 94°C for 30 sec, annealing temperature (Ta, noted in Table 1) for 30 sec, and 72°C for 30 sec. A final 7-min extension at 72°C was performed. The amplified products were separated on 2% agarose gels containing ethidium bromide. Each RT experiment was done in triplicate and repeated at least three times. Human-specific PCR primers were used, and PCR reaction conditions are described in Table 1.
|
Real-time Quantitative RT-PCR
Two-step RT-PCR was performed on teratomas and on 4-and 8-wk-old human embryos. First strand cDNA was synthesized as described above. TaqMan Universal PCR Master Mix and Assays-on-Demand Gene Expression Probes (Applied Biosystems, Foster City, CA) for Ang2, Tie2, VEGFC, VEGFR3, and ß-actin were used according to manufacturer's instructions. Each real-time PCR measurement was done in duplicate and repeated three times. The relative standard curve method (Applied Biosystems) was used to calculate the amplification differences between 4-wk-old embryos and 8-wk-old embryos or teratomas for each primer set. A Student t-test was performed for comparison of cycle threshold (Ct) values between 4-wk-old embryos and 8-wk-old embryos or teratomas.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Vascular development was studied using specific markers for ECs and v-SMCs. Four- to 8-wk-old embryos stained with specific endothelial markers (CD34, CD31, KDR, and vWF) revealed the presence of blood vessels in a range of developing embryonic tissues and organs, including hindlimbs, nervous tissues, heart, liver, kidney, and vertebral column (Fig. 1). The 4-wk human embryonic heart is surrounded by relatively few blood vessels (Fig. 1D). Developing blood vessels of the 4-wk human embryos contained predominantly erythrocytes (Fig. 1H). Expression of VE-cad was detected in 8-wk-old embryos, whereas the expression of Tal1 could not be detected at this time.
|
|
Immunolocalization was used to identify expression of v-SMCs during the development of human embryonic vasculature. Only smooth muscle 
-actin (SMA) positive cells were detectable in the 4-wk embryos (Fig. 2A). Intensive SMA staining was prominent in the developing heart and in regions of connective tissue (Fig. 2, B and C). Vascular SMCs in 7-wk embryos were clearly observed in blood vessels of the major arteries (Fig. 2D), as well as in various peripheral blood vessels such as those of the perichondrium of developing cartilage (Fig. 2, E and F). However, at this embryonic stage neither calponin nor SM-MHC could be detected by immunolabeling.
|
Immunolabeling of Human Vasculature in Teratomas
When maintained in strict culture conditions, hESCs remain in an undifferentiated state. When injected into the hindlimb muscles of SCID mice, they differentiate spontaneously to form teratomas [10]. In the current study, three different hESC lines—H9.2 [15], H13 [10], and I6 [16]— were examined to follow neovasculogenesis during teratoma development. Various small blood vessels, including arterioles, venules, and capillaries, were observed within the developing teratomas. These vessels were found to spread and invade the various developing tissues of human origin [17]. As MHC class I is expressed in teratomas generated from hESCs [18], anti-HLA-a, -b, and -c was used to detect tissues and blood vessels of human origin. Most of the blood vessels within the teratomas (93.15% ± 7.07%) were HLA–. HLA+ blood vessels (6.84% ± 0.71%) were detected mainly in the more central regions of the teratomas near the tissues of human origin (e.g., in connective tissue of mouse origin and in perichondrium of cartilage of human origin; Fig. 3, A–C). These were mostly small (5–30 µm diameter) blood vessels. We were unable to detect blood vessels staining with anti-human EC–specific markers CD34 or CD31 (data not shown).
|
Investigation of the participation of v-SMCs during this de novo human vasculogenesis revealed SMA+ cells in small blood vessels near various tissues of human origin, such as cartilage, connective tissue, and epithelial tubes (Fig. 3, D and E). Human SM-MHC was rarely detected in the teratomas derived from the hESCs, and no expression of calponin could be detected.
Molecular Profiles of Human Neovasculogenesis
To analyze the maturation kinetics of the blood vessels, a molecular profiling of 4- to 8-wk embryos and teratomas generated from hESCs was performed. Figure 4A demonstrates RT-PCR showing the expression pattern of specific genes of human ECs and v-SMCs. Expression levels of specific v-SMC markers other than SMA, such as caldesmon and calponin, but not SM-MHC, were detectable already by Week 4 of development, whereas SM-MHC was observed by Week 8. Human embryos after Week 4 of development were found to express progenitor markers of ECs such as vascular endothelial growth factor receptor 2 (VEGFR2)/KDR, Tal-1, and CD133, as well as other EC markers such as CD31, CD34, and vWF. Production of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), and the endothelial receptor tyrosine kinase Tie2 were already detected by Week 4 of development. However, expression of sprouting and remodeling genes such as vascular endothelial cadherin (VE-Cad) and vascular cell adhesion molecule-1 (VCAM-1) were detectable only by Week 8 of development. Real-time RT-PCR analysis revealed substantial upregulation of Ang2 by Week 8 of development with no significant change in the expression of Tie2 (Fig. 4B).
|
Vascular gene profiling during teratoma formation from hESCs revealed expression of human CD133 and the production of human VEGF and Ang2 (Fig. 4A). Real-time RT-PCR analysis further verified the minor expression of human Ang2 mRNA during teratoma formation and the absence of Tie2 expression within developing teratomas (Fig. 4B).
Furthermore, detectable levels of expression of the specific v-SMC marker SM-MHC and caldesmon were also observed in the developing teratomas (Fig. 4A). The use of mouse-specific primers clearly showed expression of blood vessel–related genes in the developing teratomas, including CD31, CD34, FLK1, VE-cad, and SMA (data not shown).
Molecular Profiles of Human Lymphangiogenesis
To analyze lymphangiogenesis during these developmental stages, molecular profiling of 4- to 8-wk embryos was determined. Figure 5A demonstrates RT-PCR and the expression pattern of specific genes of human LECs. Four-week-old human embryos were found to express LYVE1, a marker of LECs. However, no significant increase in the expression of VEGFR3 and VEGFC could be detected by Week 8 of development (Fig. 5B). In teratomas, markers of human LECs, such as LYVE1 and 5' nucleotidase, were observed (Fig. 5A). Furthermore, real-time RT-PCR analysis revealed a minor expression of human VEGFC mRNA during teratoma formation and the absence of VEGFR3 expression within developing teratomas (Fig. 5B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Using immunolabeling and RT-PCR, expression of specific endothelial markers CD34, KDR, and CD31 was detected in the early stages of human vasculature development. This agrees with similar observations recently reported by Oberlin et al. [19]. RT-PCR of 4-wk-old embryos further revealed expression of specific progenitor markers of ECs, such as Tal-1 and CD133 [20–22]. At the same time, the presence of specific angiogenic cytokines VEGF, Ang1, and Ang2 was observed by RT-PCR. VEGF mRNA species encoding VEGF isoforms of 121, 145, 165, 189, and 206 are produced by alternative splicing of the VEGF mRNA [23]. The three shorter forms, VEGF165, VEGF145, and VEGF121, are all capable of inducing angiogenesis, vascular permeability, and proliferation of ECs [24]. Using RT-PCR, it was observed that in 4-wk-old embryos four isomers of VEGF are expressed, whereas by Week 8 the expression of the angiogenic isoform VEGF121 is further upregulated. This corresponds to the expression of the growth factors Ang1 and Ang2, which are involved in determining the arrangement and remodeling of the vascular system [25]. The endothelial receptor tyrosine kinase Tie2 was shown to be expressed predominantly on ECs, hematopoietic cells, or their embryonic precursors and to have an apparent important role in angiogenesis, particularly for vascular network formation by endothelial cells [26]. Moreover, Tie2 was shown to be stimulated by angiopoietin ligand Ang1 (see review in [27]). It appears that during embryonic development, VEGF and Ang1 play coordinated and complementary roles, with VEGF required early in development and Ang1 required for vascular remodeling and sprouting [28, 29]. Our results (using immunolabeling and RT-PCR) indicate that by Week 4 of human development, ECs are present (expressing CD31, CD34, and vWF), but with a primary vascular arrangement and remodeling as indicated by low expression of structural proteins. However, by Week 8 of development expression of sprouting and remodeling genes such as VE-Cad (RT-PCR and immunolabeling) and VCAM-1 (RT-PCR) reveals an intensive remodeling mode. It has been proposed that Ang2 may play a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis [30] as well as postnatal angiogenesis remodeling [31]. Significant upregulation of Ang2 at Week 8 of human embryonic development provides further indication of intensive angiogenesis and permeability of ECs.
Vascular Markers and Gene Profiling in Developing Human Embryos: Vascular Smooth Muscle Cells
Although the molecular basis for phenotypic modulation of v-SMCs is poorly understood, specific variants of cytoskeletal and contractile proteins such as SMA, calponin, caldesmon, and SM-MHC have been found to characterize v-SMC differentiation [32, 33]. Similar to events occurring in other species [34, 35], the dominant early marker of v-SMCs was found to be SMA. Vasculature arrangement of SMA+ cells could already be detected in 4-wk-old embryos. By Week 7, SMA+ cells were present in many arteries. Expression levels of other specific v-SMCs markers such as caldesmon and calponin were already detectable by Week 4 of development, with upregulation of SM-MHC by Week 8. Ang1 appears to be involved in mediating the interactions between ECs and surrounding supporting cells such as v-SMCs [36]. As some differentiation of SMCs takes place during the first 4 wk of human embryonic development, it seems that these SMCs produce the Ang1 that acts paracrinely on ECs expressing its receptor, Tie2 (shown in vitro by [36]).
Vascular Markers and Gene Profiling in Developing Human Embryos: Lymphatic Endothelial Cells
The lymphatic vessels appear later than blood vessels of the arterial and venous system [37]. In humans, lymphatic vessels do not begin to appear before the end of Week 5, and functional lymph sacs have been found in 6- to 7-wk-old embryos [9]. We demonstrated that the specific marker for LECs, LYVE1, is already expressed during Week 4 of development. A recent study showed that VEGFC activation of VEGFR3 is required for the sprouting of lymphatic vessels [38]. During Weeks 4–8 of human development, we could not demonstrate any significant increase of the lymphangiogenesis activating growth factor VEGFC or its receptor VEGFR3. Further studies of more advanced stages of human embryonic development need to be done to shed further light on the mechanisms and timing of human lymphangiogenesis.
Vasculogenesis Within Teratomas Generated From Human Embryonic Stem Cells
The potential of teratomas generated in vivo from hESCs to use as a useful model to study early events of human vasculature development was explored and compared with early developing human vasculature. Small-diameter vessels of human origin could be detected mainly in the center of the teratomas. Vascular gene profiling revealed expression of CD133, a progenitor marker for ECs and VEGF production. Surprisingly, KDR, a known early mesodermal-vascular marker, was not expressed in the teratomas. Furthermore, none of the genes involved in the remodeling and sprouting of the vascular system could be detected. In addition to the expression of SMA, some levels of expression of caldesmon and SM-MHC were detected in the teratomas. This differentiation of human v-SMCs during teratoma formation might explain the moderate production of human Ang2 in the teratomas. These results suggest that spontaneous human in vivo vasculogenesis from hESCs does occur but is immature and still lacks mature endothelial markers or indicators associated with sprouting remodeling processes. It seems that simultaneously with mouse angiogenesis in the developing tumor, some primitive human vasculogenesis processes also occur. Human lymphangiogenesis processes appear to be present in the developing teratomas (as evidenced by the expression of LYVE1 and 5' nucleotidase), although the low expression levels of human VEGFR3 and VEGFC in the teratomas indicate that this process of lymphangiogenesis is very restricted. Overall, the developing teratomas, which typically reach dimensions of 2 cm or more, are vascularized mainly by invasion from the murine host and to a much lesser degree by de novo–generated blood and lymphatic vessels of human origin. We conclude that the teratoma model has limited value in studying early events of human vascular development and that other in vitro models for the spontaneous and guided differentiation of hESCs may prove more useful.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Joseph Itskovitz-Eldor, Department of Obstetrics and Gynecology, Rambam Medical Center, P.O. Box 9602, Haifa 31096, Israel. FAX: 972 4 854 2503; Itskovitz{at}rambam.health.gov.il
Received: 25 May 2004.
First decision: 15 June 2004.
Accepted: 3 August 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Levenberg, J. Zoldan, Y. Basevitch, and R. Langer Endothelial potential of human embryonic stem cells Blood, August 1, 2007; 110(3): 806 - 814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gerecht, J. A. Burdick, L. S. Ferreira, S. A. Townsend, R. Langer, and G. Vunjak-Novakovic Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells PNAS, July 3, 2007; 104(27): 11298 - 11303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tzukerman, T. Rosenberg, I. Reiter, S. Ben-Eliezer, G. Denkberg, R. Coleman, Y. Reiter, and K. Skorecki The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts. Cancer Res., April 1, 2006; 66(7): 3792 - 3801. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
