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Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies.
Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via co-culture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that co-express multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development.
Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.
Vasculogenesis, the process of de novo endothelial cell differentiation and blood vessel formation, initiates in mammals shortly after gastrulation. During gastrulation, epiblast cells migrate through the primitive streak forming a mesodermal layer which lies adjacent to the visceral endoderm within the extraembryonic yolk sac1. Mesodermal progenitors are thought to receive cues from the visceral endoderm to direct their differentiation into primordial endothelial and hematopoietic cells, which constitute blood islands. Later stages of vasculogenesis include formation of vascular channels and capillary plexus which then remodels into a circulatory network via the process of angiogenesis.
Current knowledge of mammalian regulation of endothelial cell differentiation has been largely derived from studies of mouse embryonic development. This model system suggests that during vasculogenesis, endoderm-derived soluble factors, such as Indian Hedgehog (Ihh)2, 3, vascular endothelial growth factor (VEGF)4-6, and basic fibroblast growth factor (bFGF)7-9 promote endothelial cell formation within the mesoderm where their receptors, Patched (Ptc), VEGFR2/Flk1 and FGFR2, respectively, are localized2, 6, 10-16. Other molecules proposed to be downstream Ihh signaling, such as bone morphogenic protein 4 (BMP4)2, 17, are similarly localized within the mesoderm. While these factors have been shown, individually, to be important for regulating murine blood vessel formation, the signaling hierarchy among them has not been delineated.
Whether similar signals, in a hierarchy, regulate human endothelial cell commitment and differentiation has not been investigated. It is known that vascular differentiation kinetics differ between mouse and human ES cells18, 19; therefore, the molecular regulation of this process may also differ. A better understanding of the regulation of human endothelial cell development is needed to gain insights applicable to promoting human vascular regeneration and optimizing human clinical therapies.
These studies investigated the molecular regulation of human endothelial cell development using a hES-OP9 co-culture system20, wherein we could generate, isolate and culture CD31 and VE-cadherin co-expressing endothelial cells. Since bFGF and VEGF did not promote endothelial cell differentiation from hES cells (please see http://atvb.ahajournals.org; Supplemental Fig. I), we investigated the role of Ihh, which is also expressed in the yolk sac visceral endoderm as early as 6.5dpc3, 21. Although the specific cellular role for Ihh in murine vascular development is not defined, Ihh-null mutants are embryonic lethal, exhibit impaired yolk sac vasculogenesis and vascular remodeling and the yolk sacs have fewer endothelial cells22. Similar expression patterns and function were also seen in mouse embryoid bodies lacking Ihh21.
We found that exogenous Ihh increased expression of vascular inductive genes BMP4, VEGF and VEGFR2/Flk1, as well as generation of differentiated endothelial and hematopoietic cells. Conversely, inhibition of hedgehog signaling using cyclopamine suppressed formation of endothelial cells, as well as hematopoietic cells19, 23, 24. Furthermore, inhibition of BMP signaling using the pan inhibitor Noggin, or BMP4, specifically, using neutralizing antibodies, abolished the Ihh-mediated effects, indicating that BMP4 signals downstream of Ihh to modulate human endothelial cell development. Consistent with this idea, addition of rhBMP4 to hES cells treated with the hedgehog inhibitor, cyclopamine, rescued endothelial cell formation to control levels.
Collectively, our studies demonstrate that Ihh signals via the BMP pathway, and specifically through BMP4, to promote endothelial cell differentiation from pluripotent hES cells. These novel insights into the molecular regulation of human endothelial cell development should aid in optimizing human vascular regeneration and clinical therapies for prevalent pathologies, as well as provide insights into the signaling hierarchy that may regulate other human stem cells such as induced pluripotent stem (iPS) cells.
Undifferentiated H9 and H1 hES cells were obtained from WiCell (with Institutional approval) and maintained on irradiated CF-1 MEFs and passaged manually weekly, as described25, 26. The OP9 bone marrow stromal cells were obtained from ATCC (CRL-2749) and plated at a density of 50,000 cells/ml on 0.2% gelatinized 6 well multi-well plates in media containing α-MEM (Invitrogen, Carlsbad, CA, USA) with 20% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). Cells were grown for nine days, with half media changes on days 4, 6 and 8. Differentiation of hES cells on OP9 cells was performed as described 20. In brief, hES cells were treated with collagenase type IV (Invitrogen, Carlsbad, CA, USA) and transferred onto day 9 growing OP9 cells and maintained in differentiation media consisting of α-MEM, 10% FBS, and 100μM monothioglycerol (MTG; Sigma Aldrich, St. Louis, MO, USA).
For hedgehog inhibition experiments, cells were treated with 30μM KAAD-cyclopamine (Toronto Research Chemicals, Inc., Toronto, CA). The Ihh over-expression experiments were carried out either with 100ng/ml Ihh (R&D Systems, Minneapolis, MN, USA) alone or in conjunction with 200ng/ml anti-BMP4 (R&D Systems, Minneapolis, MN, USA) or 200ng/ml Noggin (R&D Systems, Minneapolis, MN, USA). For the rescue experiments, cells were initially treated with cyclopamine and then with 20ng/ml rhBMP4 (R&D Systems, Minneapolis, MN, USA) beginning at day 4 of differentiation. Initial differentiation experiments were conducted using both H1 and H9 hES cell lines to demonstrate that the differentiation process was not specific to one hES cell line,(please see http://atvb.ahajournals.org; Supplemental Fig. II); all subsequent experiments were performed using H9 hES cells that exhibited more robust endothelial cell differentiation.
Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA), and subsequently DNase-treated (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed using random primers and SuperScriptII RNaseH− Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative PCR (qPCR) was performed using the TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA). The samples were amplified for 40 cycles on the ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA). Target gene transcripts were amplified, as well as β-actin, for a housekeeping control gene. All reactions were performed in triplicate in each of at least three experiments. The ΔΔCt 27 method was used to compare and quantify fold induction between undifferentiated hES cells and all other differentiated sample time points, normalizing to β-actin.
Cells were fixed in 4% paraformaldehyde (Electron Microscopy Services, Hatfield, PA, USA) for 20 minutes at RT, then washed 3 times for 5 minutes each with PBS, and incubated with PBST buffer consisting of 0.5% BSA (Sigma Aldrich, St. Louis, MO, USA), 0.1% Triton-X 100 (Sigma Aldrich) and PBS, for 10 minutes. Cells were then placed into blocking buffer (PBST + 10% Donkey Serum, Sigma Aldrich) for 10 minutes. Primary antibodies were diluted in blocking buffer as follows: VE-cadherin 1:50 and PECAM-1 (CD31) 1:50 (R&D Systems, Minneapolis, MN, USA) and incubated on samples for 2 hours at RT. Cells were washed 3 times for 5 minutes each with PBS. The appropriate secondary antibodies (R&D Systems, Minneapolis, MN, USA) were diluted 1:500 in blocking buffer and incubated on the samples for 30 minutes at room temperature, in the dark. Cells were then washed with PBS 3 times for 5 minutes each, mounted with VectaShield mounting media with DAPI (Vector Laboratories H-1200, Burlingham, CA, USA), and visualized and photographed with the Zeiss Axiovert 200M equipped with digital camera.
Human ES cells were subjected to the endothelial differentiation protocol for 14 days, as described, then manually dissected from cultures and embedded in O.C.T. compound (Sakura Finetek, Torrance, CA, USA), wherein they were flash frozen in liquid nitrogen. Frozen blocks were then sectioned at a thickness of 10 microns and fixed onto slides. Slides were subjected to immunocytochemistry with aforementioned endothelial cell markers and visualized with the Zeiss Axiovert 200M microscope.
CD31 positive cells were cultured for 7 days and then washed twice with PBS. Cells were then placed in serum free media and incubated with DiI-Ac-LDL (Invitrogen, Carlsbad, CA, USA) at a concentration of 10μg/ml for 30 minutes. Cells were then washed three times with Hank's Balanced Salt Solution and visualized and photographed with a Zeiss Axiovert 200M and digital camera.
For mesodermal cell production, cells were differentiated for 3 days in media containing α-MEM (Invitrogen, Carlsbad, CA, USA) and 20% FBS (Invitrogen, Carlsbad, CA, USA). Cells were treated with trypsin, triturated to a single cell suspension, and then fixed, permeabilized and incubated with T-Brachyury antibodies (1:100) (Abcam, Cambridge, MA, USA). For endothelial and hematopoietic assessment, cells were differentiated and isolated at either 14 or 21 days. Once in a single cell suspension, cells were labeled with CD31-PE (1:100) and/or CD45-PE (1:100) monoclonal antibodies (R&D Systems, Minneapolis, MN, USA). Samples were then analyzed on the FACScan (BD Bioscience, San Jose, CA, USA) with CellQuest software (BDIS). All antibodies were initially tested for cross-reactivity with OP9 stromal cells that showed no reactivity and were subsequently used as a negative control. All samples were normalized to an isotype control.
The culture of hES cells on OP9 stromal cells was shown to promote hematopoietic cell development20. Since endothelial and hematopoietic cells are proposed to arise from common mesodermal progenitors, we investigated whether the culture of hES cells on OP9 cells could be used as a model system to reproducibly generate endothelial cells. hES cells were plated onto OP9 cells for 3, 7, 10, 14 and 21 days and subsequently analyzed via immunohistochemistry for the co-expression of CD31 and VE-cadherin proteins, which confirmed the generation of differentiated endothelial cells (Fig. 1A). Cells differentiated for 14 and 21 days were analyzed via fluorescence-activated cell sorting (FACS) for the expression of CD31 and CD45 proteins to quantify endothelial and hematopoietic cell production, respectively. Endothelial cell generation was maximal at day 14, at which time endothelial cells constituted 10−15% of the cells in culture; 1−2% were hematopoietic cells (data shown in Fig. 4). CD31 positive cells were then isolated using FACS at day 14 and cultured on collagen IV-coated plates for seven days. The cultured cells exhibited expected endothelial cell morphology (Fig. 1B) and function, as evidenced by uptake of DiI-Ac-LDL, and plexus and lumen formation (Figs. 1C and 1D). Thus, we could consistently achieve robust generation of differentiated endothelial cells in this culture system.
We next determined via qPCR the time course of expression of the mesoderm-specific T-Brachyury gene (maximal expression at day 3 of differentiation; data not shown) and putative vascular-inductive genes including Ihh, BMP4, VEGF and VEGFR2 (KDR/Flk1) in this co-culture system. As expected, all genes were expressed after mesodermal induction and prior to the detection of differentiated endothelial cells. Ihh expression was highest at days 3 and 7 of differentiation, whereas the expression of BMP4 peaked at day 7 (Fig. 2). The expression of Flk1 and VEGF peaked later, at day 14. Expression of genes indicative of differentiated endothelial cells, CD31 and VE-cadherin, was also highest at day 14 (Fig. 2). These data correlated with the FACS analysis, which revealed that the day 14 cultures contained the highest proportion of cells expressing CD31 (Fig. 4). Expression of CD45, which marks cells committed to blood cell lineages, was induced by day 14 and continued to increase at day 21 (Figs. 2 and and4).4). This reproducible time course provides a baseline of gene expression levels, and allows for the dissection of molecular signals that regulate the process endothelial cell differentiation from pluripotent human stem cells.
To elucidate the molecular regulators of human endothelial cell development, we first focused on investigating the role of the hedgehog signaling pathway. We treated hES cells, in co-culture with OP9 cells, with the pan hedgehog inhibitor, cyclopamine, at day 1 of culture and performed qPCR and FACS analysis to measure the expression of vascular-inductive, endothelial and hematopoietic genes and proteins, respectively, at time points up to 21 days, as described above. We found that inhibition of hedgehog signaling significantly decreased the expression of BMP4 mRNA starting at day 3 and VEGF starting at day 10 (please see http://atvb.ahajournals.org; Supplemental Fig. III). Furthermore, in the absence of hedgehog signaling, there was suppressed induction of endothelial gene expression at day 10 (CD31) and day 14 (CD31 and VE-cadherin) when they are usually at their peak. There was also a lack of induction of hematopoietic genes, such as CD45, in cyclopamine treated cells at days 14 and 21 (Fig. 3). FACS analysis of the same samples revealed suppressed generation of cells expressing CD31 and CD45 (Fig. 4). These data indicate that hedgehog signaling is required for endothelial, as well as hematopoietic, cell development from hES cells.
In subsequent experiments, we examined the specific role of Ihh, the hedgehog family member implicated in the regulation of vasculogenesis, in promoting endothelial cell differentiation from hES cells. We added exogenous Ihh to the culture medium throughout the differentiation process and performed qPCR and FACS analyses of cells isolated after 0, 3, 7, 10, 14 and 21 days of culture. We found that Ihh significantly increased the expression of BMP4, above OP9 co-culture conditions, starting at day 7 and maximally (3-fold) at day 14 (please see http://atvb.ahajournals.org; Supplemental Fig. III). Flk1 gene expression was upregulated above OP9 co-culture conditions by day 10, and maximally (2-fold) at day 14, as was VEGF expression (elevated 4-fold at day 14). Consistent with these findings was the observation that CD31 and VE-cadherin mRNA expression was maximally upregulated (4- to 5-fold) by day 14 in response to Ihh (Fig. 3). Generation of CD31-expressing cells, as determined via FACS, correlated with the qPCR results; maximal indcution (1.5-fold) was observed at day 14 (Fig. 4). CD45 mRNA expression was maximally upregulated (2.5-fold) by Ihh treatment at 21 days, which is consistent with maximal generation of CD45-expressing cells at the same time point. These data indicate the Ihh promotes the production of endothelial and hematopoietic cells from hES cells.
It has been speculated that Ihh and BMP act together in a signaling pathway2, 28, 29, but has not been definitively shown in either the mouse or human system. Thus, we aimed to determine whether BMP4 was the downstream mediator of the Ihh-induced endothelial cell differentiation that we observed. Toward this end, hES cells were differentiated with Ihh as described above, but at day 4 of differentiation they were treated with either BMP4 neutralizing antibodies or the pan BMP inhibitor, Noggin. This time point for inhibition (day 4) was chosen because BMP4 gene expression, and mesodermal progenitor cell formation (as evidenced by T-Brachury-expression), is not induced in the OP9 co-culture system until day 3. At days 3−21, RNA was collected and real time qPCR analysis was performed. The results demonstrated that in the absence of BMP4 signaling, genes associated with vascular induction (BMP4, Flk1 and VEGF; please see http://atvb.ahajournals.org; Supplemental Fig. IV)) were significantly down-regulated, as were endothelial cell-specific genes (CD31 and VE-cadherin; Fig. 5). This effect was even more pronounced when cells were treated with Noggin, which inhibits both BMP2 and BMP4 (Fig. 5). Therefore, BMP signaling is needed for endothelial cell differentiation and may act downstream of Ihh.
Interestingly, when the BMP signaling pathway was inhibited with Noggin, CD45 expression was increased ~9-fold (Fig. 5). These results suggest that Ihh does not signal via the BMP pathway to promote hematopoietic differentiation of hES cells. Thus, human endothelial and hematopoietic cell differentiation are likely mediated via distinct molecular pathways.
To more specifically test whether BMP4 signals downstream of Ihh, we performed a rescue experiment wherein we suppressed endothelial cell differentiation from hES cells via treatment with the hedgehog inhibitor, cyclopamine, and then added rhBMP4 to the cultures at day 4 of differentiation. RNA was isolated from hES cells over the time course of differentiation (0−21 days), and subjected to qPCR analysis. As previously discussed, cyclopamine significantly suppressed endothelial cell-specific gene expression. The addition of rhBMP4 to cyclopamine-treated cells restored endothelial cell formation (Fig. 6A). These data indicate that BMP4 signals downstream of the hedgehog pathway to promote endothelial cell differentiation from hES cells.
Ihh, and downstream BMP, signaling may promote endothelial cell formation either by increasing the generation of mesodermal progenitors (endothelial cell precursors) from hES cells or by increasing the differentiation of mesodermal progenitors toward an endothelial cell lineage. To determine whether Ihh promoted the overall production of mesodermal progenitors from hES cells, we measured the number of T-Brachyury positive cells at day 3 of differentiation, when we found T-Brachyury to be maximally expressed in hES cells co-cultured with OP9 cells. We found that treatment of cultures with either Ihh or the pan hedgehog inhibitor, cyclopamine, had no effect on the number of T-Brachyury positive cells generated from hES cells, as compared with untreated co-cultures (please see http://atvb.ahajournals.org; Supplemental Fig. V). Therefore, Ihh signaling does not appear to induce the formation of mesodermal progenitors from pluripotent human stem cells, but rather their differentiation toward an endothelial lineage.
The molecular regulation of human endothelial cell differentiation was previously undefined. Herein, we delineate the signaling hierarchy that mediates the differentiation of pluripotent human stem cells toward human endothelial cells and their immediate precursors. We utilized a hES-OP9 cell co-culture system to reproducibly and robustly generate endothelial cells that co-express endothelial-specific genes/proteins, take up DiI-AcLDL and form a plexus of tubes with lumens in three-dimensional culture, in addition to generating hematopoietic cells19.
We found that blocking hedgehog signaling inhibited endothelial cell formation from hES cells, and exogenous Ihh, increased production of cells co-expressing endothelial-specific genes and proteins. Our finding that Ihh signaling plays a role in mediating human endothelial cell formation is consistent with recent observations that Ihh is essential for mouse vasculogenesis30. Since during murine vasculogenesis in vivo, such endoderm-derived factors promote endothelial cell development in adjacent mesoderm, we aimed to elucidate downstream, mesoderm-derived mediator(s) of this signaling pathway.
Mesoderm-generated BMP factors have been previously proposed to signal downstream of hedgehog in the development of other organ systems31-33; therefore, we investigated whether BMP factors mediated the observed Ihh induction of endothelial cell differentiation from hES cells. In initial experiments, we found that inhibition of BMP4 signaling led to suppressed endothelial cell formation, but increased hematopoietic cell production, suggesting that BMP signaling may act to balance the differentiation of mesodermal progenitors to potential hemato-vascular cell fates. Subsequent experiments demonstrated that BMP2/4 rescued the effects of hedgehog inhibition on endothelial cell differentiation. Thus, BMP signaling appears to function downstream of hedgehog signaling to control human endothelial cell differentiation from pluripotent stem cells. These results are consistent with recent studies suggesting that BMP can signal downstream of hedgehog to regulate mouse vasculogenesis30. Although there may be multiple roles for BMP signaling during embryogenesis, in vascular development, specifically, it appears to mediate hedgehog induction of endothelial cell differentiation.
In summary, we have made the novel discovery that hedgehog signaling drives human endothelial cell development from pluripotent human stem cells, and its effects are mediated via BMP signaling (Fig. 6B). In addition, other factors that regulate the development of murine endothelium, such as bFGF and VEGF, do not directly promote human endothelial cell differentiation (please see http://atvb.ahajournals.org; Supplemental Fig. I). Thus, further investigation is needed to define similarities and differences among human and murine vascular development, as well as to determine the mechanism by which BMP factors promote human endothelial cell formation. Understanding the molecular pathways that regulate cell type specific differentiation of hES cells will allow for more efficient generation of cells that can be utilized for human clinical therapies in the future. Furthermore, insights gained from these studies will be applicable to directing the differentiation of other pluripotent human stem cells, such as recently generated induced pluripotent stem (iPS) cells.
This work was supported by NIH grants EB-005173, EB-007076 R01 and DK-075355 to KKH. MAK was supported by NIH grant T32 DK-064717.