Published online before print June 12, 2008, doi: 10.1161/CIRCRESAHA.108.178590
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Integrative Physiology |
Engineering Robust and Functional Vascular Networks In Vivo With Human Adult and Cord Blood–Derived Progenitor Cells
From the Vascular Biology Program and Department of Surgery, Children’s Hospital Boston (J.M.M.-M., M.E.D.O., S.-Y.K., Z.A.K., J.B.); and Division of Cardiology, Beth Israel Deaconess Medical Center (L.Y., P.O.), Harvard Medical School, Boston Mass.
Correspondence to Dr Joyce Bischoff, Vascular Biology Program and Department of Surgery, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115. E-mail joyce.bischoff{at}childrens.harvard.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The success of therapeutic vascularization and tissue engineering will rely on our ability to create vascular networks using human cells that can be obtained readily, can be expanded safely ex vivo, and can produce robust vasculogenic activity in vivo. Here we describe the formation of functional microvascular beds in immunodeficient mice by coimplantation of human endothelial and mesenchymal progenitor cells isolated from blood and bone marrow. Evaluation of implants after 1 week revealed an extensive network of human blood vessels containing erythrocytes, indicating the rapid formation of functional anastomoses within the host vasculature. The implanted endothelial progenitor cells were restricted to the luminal aspect of the vessels; mesenchymal progenitor cells were adjacent to lumens, confirming their role as perivascular cells. Importantly, the engineered vascular networks remained patent at 4 weeks in vivo. This rapid formation of long-lasting microvascular networks by postnatal progenitor cells obtained from noninvasive sources constitutes an important step forward in the development of clinical strategies for tissue vascularization.
Key Words: vascular networks • endothelial progenitor cells • mesenchymal stem cells • mesenchymal progenitor cells • tissue engineering • regenerative medicine • vasculogenesis • angiogenesis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Engineered tissues must have the capacity to generate a vascular network that rapidly forms anastomoses with the host vasculature to guarantee adequate nutrients, gas exchange, and elimination of waste products.1 Presently, there are no tissue-engineered (TE) constructs clinically available with an inherent microvascular bed, and therefore successes have been restricted to the replacement of relatively thin (skin) or avascular (cartilage) tissues, where postimplantation vascularization from the host is sufficient.
To overcome the problem of vascularization, strategies to promote ingrowth of microvessels by delivery of angiogenic molecules have been proposed.2–5 However, rapid and complete vascularization of thick engineered tissues is likely to require an additional process of vasculogenesis.1,6 Toward this goal, the feasibility of engineering microvascular networks in vivo has been shown using human umbilical vein endothelial cells and human microvascular endothelial cells7–9; however, such autologous tissue-derived endothelial cells (ECs) present problems for wide clinical use, because they are difficult to obtain in sufficient quantities. These limitations have instigated the search for other sources of ECs, such as those derived from embryonic and adult stem and progenitor cells.6 For instance, ECs derived from embryonic stem cells (ESCs) have been used to form blood vessels and to enhance the vascularization of engineered skeletal muscle constructs in vivo.10,11 However, the mechanisms controlling ESCs differentiation must be understood, and ethical issues surrounding their use must be resolved before their implementation in therapeutic strategies.
The identification of endothelial progenitor cells (EPCs) in blood presented an opportunity to noninvasively obtain ECs.12–14 We and other authors have shown that adult and cord blood–derived EPCs have the required vasculogenic capacity to form functional vascular networks in vivo.15–17 Importantly, these studies have also shown that to obtain stable and durable vascular networks, EPCs require coimplantation with perivascular cells. In our previous work, the role of perivascular cells was undertaken by smooth muscle cells (SMCs) isolated from human saphenous veins.15 In the work by Au et al, the mouse embryonic cell line 10T1/2 served as the perivascular component of the vascular networks.16 However, neither source is suitable for clinical utilization: harvesting SMCs from healthy vasculature would impose serious morbidity in patients and murine-derived cell lines will not be used in humans. Therefore, to exploit the full vasculogenic potential of EPCs, we set out to establish clinically viable sources of perivascular cells. The ideal perivascular cells must present several key properties: (1) isolation with minimal donor site morbidity; (2) availability in sufficient quantities; and (3) immunologic compatibility with the recipients.1 Mesenchymal stem/progenitor cells (herein referred to as MPCs)18 meet these requirements. MPCs can be isolated by minimally invasive procedures from a diversity of human tissues, including bone marrow,18 adult blood,19 umbilical cord blood,20–22 and adipose tissue.23 Furthermore, MPCs undergo self-renewal and therefore can potentially be expanded to sufficient quantities for tissue and organ regeneration.24
Here, we demonstrate that MPCs obtained from both human adult bone marrow and human cord blood can serve as perivascular cells for in vivo vasculogenesis. Subcutaneous coimplantation of EPCs and MPCs, suspended as single cells in Matrigel, into immunodeficient mice resulted in the creation of extensive microvascular beds that rapidly formed anastomoses with the host vasculature. This study constitutes a step forward in the clinical development of therapeutic vasculogenesis by showing the feasibility of using human adult and cord blood–derived progenitor cells as the basic cellular building blocks to create functional vascular networks in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In Vivo Vasculogenesis Assay
The formation of vascular networks in vivo was evaluated using a xenograft model as described.15 A total of 1.9x106 cells was resuspended in 200 µL of ice-cold Phenol Red-free Matrigel (BD Bioscience, San Jose, Calif), at ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 (EPCs:MPCs). The mixture was implanted on the back of a 6-week-old male athymic nu/nu mouse (Massachusetts General Hospital, Boston, Mass) by subcutaneous injection using a 25-gauge needle. Implants of Matrigel alone served as controls. One implant was injected per mouse. Each experimental condition was performed with 4 mice. Animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee at Children’s Hospital Boston in an AAALAC-approved facility.
An expanded Materials and Methods section, available at http://circres.ahajournals.org, describes cell isolation and expansion, flow cytometry, Western blot analysis, differentiation assays, histology and immunohistochemistry, retroviral transduction, luciferase assay, microvessel density evaluation, and statistical analysis.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Isolation of EPCs and MPCs
Cord blood–derived (cb)EPCs (Figure 1a) and adult blood (ab)EPCs were isolated from the mononuclear cells fraction of human blood samples and purified by CD31 selection as described (see supplemental Figures I and XII in online the online data supplement for morphology of cbEPCs and abEPCs, respectively).15 MPCs were isolated from the mononuclear cells fractions of human bone marrow samples (bmMPCs) and human cord blood samples (cbMPCs). bmMPCs adhered rapidly to the culture plates and proliferated until confluent, whereas cbMPCs emerged more slowly, forming mesenchymal-like colonies after one week (supplemental Figure I). cbMPC colonies were selected with cloning rings and expanded. Both bmMPCs (Figure 1b) and cbMPCs (Figure 1c) presented spindle morphology characteristic of mesenchymal cells in culture.18
|
cbEPCs and MPCs were grown in EPC medium and MPC medium, respectively, and their expansion potentials estimated by the cumulative cell numbers obtained from 25 mL of either cord blood or bone marrow samples after 25, 40, and 60 days in culture (Figure 1d). Remarkably, up to 1013 cbEPCs and 1011 bmMPCs were obtained after 40 days, consistent with previous studies.13,15 The number of cells continued to increase so that at 60 days, there were an estimated 1018 cbEPCs and 1014 bmMPCs, respectively. In the case of cbMPCs, a longer culture period was necessary to obtain such numbers. The apparent decreased number of cbMPCs was likely attributable to the smaller number of MPCs in cord blood samples (typically 1 to 2 colonies per 25 mL; data not shown) as compared with bone marrow samples, where the majority of the adherent cells contributed to the final bmMPC population (supplemental Figure I).
The phenotype of the MPCs was confirmed by 3 methods. Flow cytometry (Figure 1e) showed that bmMPCs and cbMPCs uniformly expressed the mesenchymal marker CD90 and were negative for the endothelial marker CD31 and the hematopoietic marker CD45 (see further flow cytometric evaluations in supplemental Figure II). cbEPCs served as a control. Western blot analyses (Figure 1f and 1g) confirmed the mesenchymal phenotype of bmMPCs and cbMPCs (expression of 
-smooth muscle actin [SMA] and calponin) and the endothelial phenotype of cbEPCs (expression of CD31 and VE-cadherin). These data were extended by indirect immunofluorescent staining (supplemental Figure III): bmMPCs and cbMPCs were shown to express the mesenchymal markers -SMA, calponin, and NG2 but not the EC markers CD31, VE-cadherin, and von Willebrand factor. Importantly, smooth muscle myosin heavy chain (smMHC), a specific marker of differentiated smooth muscle cells,25,26 was found in mature SMCs but not in any of the MPCs.
The ability of MPCs to differentiate into multiple mesenchymal lineages was evaluated in vitro using well-established protocols.18 Both bmMPCs and cbMPCs differentiated into osteocytes and chondrocytes, as shown by the expression of alkaline phosphatase (osteogenesis; Figure 2a and 2b) and glycosaminoglycan deposition in pellet cultures (chondrogenesis; Figure 2c and 2d), respectively (see also supplemental Figure IV). Adipogenesis was only evident with bmMPCs (Figure 2e) and not in cbMPCs (Figure 2f). This loss of adipogenic potential, reported for other mesenchymal cells in culture,27,28 was attributed to the extensive expansion that these cells required because of their lower presence in cord blood samples (the earliest cbMPCs were tested for adipogenesis was at passage 5).
|
Because we intended to test MPCs as perivascular cells to engineer microvessel networks, we evaluated the ability of MPCs to differentiate toward a smooth muscle phenotype. As already shown, MPCs and mature SMCs shared a number of cellular markers including -SMA, calponin, NG2, and platelet-derived growth factor receptor-β (supplemental Figure V). Although the definitive smooth muscle cell marker smMHC was absent in MPCs (Figure 2g and 2h), both bmMPCs and cbMPCs were induced to express smMHC when directly cocultured with cbEPCs (Figure 2i and 2j). Importantly, induction did not occur when MPCs were indirectly cocultured with cbEPCs using a Transwell culture system (supplemental Figure VI), consistent with previous reports that showed direct contact with ECs is required for mesenchymal cell differentiation into SMCs.29,30
In Vivo Formation of Human Vascular Networks
We have previously demonstrated the vasculogenic capacity of blood-derived EPCs both in vitro and in vivo.15,31 In these studies, the presence of vascular smooth muscle cells was required for formation of vascular networks. To answer the question of whether MPCs could be used instead of SMCs, we implanted different combinations of cbEPCs and MPCs (either bmMPCs or cbMPCs) into nude mice for 1 week (Figure 3). A total of 1.9x106 cells was resuspended in 200 µL of Matrigel, using ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 (percentage of cbEPCs:percentage of MPCs) and injected subcutaneously. After harvesting the Matrigel implants (Figure 3a through 3c), hematoxylin/eosin (H&E) staining revealed numerous vessels containing erythrocytes in implants containing both cbEPCs and MPCs (Figure 3e and 3g). The structures stained positive for human CD31 (Figure 3d and 3f), confirming the lumens were lined by the implanted cells (the specificity of the antihuman CD31 antibody is shown in supplemental Figure IX). Implants of Matrigel alone were devoid of vessels (supplemental Figure VII), indicating the Matrigel itself was not responsible for the presence of vascular structures. As expected,15 implants with cbEPCs alone (Figure 3h) failed to form microvessels. Implants with only MPCs (Figure 3i and 3j) presented infiltration of murine blood capillaries but no human microvessels (supplemental Figure IX). The ability of human MPCs to recruit murine vessels into Matrigel may be explained by the secretion of vascular endothelial growth factor (VEGF) from MPCs but not cbEPCs (supplemental Figure VIII).
|
4 each condition). Each bar represents the mean±SD (vessels/mm2) obtained from vascularized implants. *P<0.05 compared with implants with bmMPCs alone (n=4); 
P<0.05 compared with implants with cbMPCs alone (n=4).Microvessel density was determined by quantification of lumens containing red blood cells (Figure 3k). The extent of the engineered vascular networks was influenced by the ratio of EPCs to MPCs (Figure 3k). A progressive increase in MPCs resulted in increased microvessel density and more consistent vascularization (supplemental Table I). When the ratio of EPC:MPC was 40:60, an average density of 119±33 and 117±32 vessels/mm2 with bmMPCs or cbMPCs, respectively, was achieved in all implants. These densities were significantly higher (P<0.05) than those observed with MPCs alone, reaffirming the necessity of the endothelial component for the formation of human vessels in the implants.
Assembly of Endothelial and Mesenchymal Progenitor Cells in the Vascular Bed
In addition to the human CD31-positive lumenal structures, the engineered vessels were characterized by -SMA staining of perivascular cells (Figure 4a and 4b). With bmMPCs or cbMPCs, -SMA–positive cells were detected both in proximity and adjacent to lumenal structures, suggesting an ongoing process of perivascular cell recruitment during vessel maturation.32–34 To determine more precisely the contribution of each cell type, we implanted green fluorescent protein (GFP)-labeled cbEPCs with unlabeled MPCs. Anti-GFP staining showed cbEPCs restricted to lumenal positions in the microvessel networks, whereas anti–-SMA staining showed that the GFP-labeled vessels were covered by perivascular cells; this observation was valid with both sources of MPCs (Figure 4c and 4d). Projections of whole-mount staining showed that the GFP-expressing cells formed extensive networks throughout the implants (Figure 4e). Conversely, we implanted GFP-labeled bmMPCs with unlabeled cbEPCs to identify input MPCs without relying on anti–-SMA. Sections were stained with anti-GFP and anti-CD31 antibodies: GFP-expressing cells were detected as perivascular cells surrounding human CD31+ lumens and as individual cells dispersed throughout the Matrigel implants (Figure 4f).
|
Durability of the Vascular Bed
To test the durability of the engineered vascular beds in vivo, we evaluated implants of cbEPCs/bmMPCs (40:60) at 7, 14, 21, and 28 days after xenografting (Figure 5). H&E staining revealed the presence of lumenal structures containing erythrocytes in all implants at each time point (Figure 5a through 5d). Microvessel quantification (Figure 5e) revealed an initial reduction (statistically nonsignificant; P=0.105) in the number of patent blood vessels from 119±33 vessels/mm2 at day 7 to 83±16 vessels/mm2 at day 14. Microvessel densities remained stable thereafter (87±21 and 87±32 vessels/mm2 at days 21 and 28, respectively).
|
To further evaluate the engineered vascular bed, we used a luciferase-based imaging system to monitor perfusion of the Matrigel implants. cbEPCs were infected with lentivirus-associated vector encoding luciferase and implanted into immunodeficient mice in the presence or absence of bmMPCs. At 1 and 4 weeks, mice were given the substrate luciferin by intraperitoneal injection (Figure 5f). No bioluminescence was detected in implants with luciferase-expressing cbEPCs alone, indicating that the substrate did not diffuse into the Matrigel. In contrast, a strong bioluminescent signal was detected in xenografts in which bmMPCs were coimplanted. This result, coupled with parallel histological data, confirmed that the presence of MPCs was crucial to achieve rapid perfusion of the implants. Importantly, the luciferase-dependent signal was still detected 4 weeks after implantation, a further indication of the long-lasting nature of the engineered vessels.
The cells within the Matrigel implants appeared to undergo a process of in vivo remodeling characterized by stabilization of total cellularity (supplemental Figure X) and progressive restriction of -SMA–expressing cells to perivascular locations (Figure 5g through 5j), as expected in normal stabilized vasculature.34 Finally, after 28 days in vivo, adipocytes were identified by staining with an anti-perilipin antibody (supplemental Figure XI), suggesting a process of integration between the implants and the surrounding murine adipose tissue.35
Vascular Network Formation Using Adult Progenitor Cells
We previously showed that adult peripheral blood–derived EPCs (abEPCs) combined with mature SMCs at a ratio of 4:1 (EPCs:SMCs) are vasculogenic in vivo, yet required higher seeding densities to achieve microvessel densities similar to that obtained with cbEPCs.15 This apparently lower vasculogenic capacity of abEPCs has been suggested recently by others.16 We hypothesized that the combination of adult bmMPCs and abEPCs at an "optimized ratio" (Figure 3k) would yield a high density vascular network. Indeed, there are no previous reports on adult human bmMPCs and abEPCs in the context of in vivo vasculogenesis. To evaluate this interaction, we isolated abEPCs as described13–15 and confirmed their endothelial phenotype (supplemental Figure XII).
We implanted a total of 1.9x106 cells (40% abEPCs and 60% bmMPCs) in Matrigel by subcutaneous injection into immunodeficient mice (Figure 6). After harvesting the implants at 7 days (n=4), H&E staining consistently showed an extensive presence of blood vessels containing erythrocytes (Figure 6a and 6b). In addition, the lumenal structures stained positive for human CD31 (Figure 6d), confirming the lumens were formed by the implanted human abEPCs. Quantification of microvessel density (Figure 6c) revealed that the use of 40% abEPCs resulted in a statistically significant (P<0.05) increase in the number of blood vessels (86±26 vessels/mm2) as compared with implants with bmMPCs alone (34±25 vessels/mm2). Moreover, the difference between implants composed of abEPCs:bmMPCs and those of cbEPCs: bmMPCs (119±33 vessels/mm2) was not statistically significant (P=0.158), indicating that the presence of bmMPCs supported the vasculogenic properties of abEPCs to the same extent as was achieved with cbEPCs. These results show that a 2-cell system composed of human adult EPCs and MPCs exhibit the same robust in vivo vasculogenic activity as cbEPCs combined with adult bmMPCs, which is in contrast to conclusions drawn from experiments reported by others using a murine 10T1/2 cell line.16
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Here, we show that human postnatal EPCs and MPCs isolated from either blood or bone marrow have an inherent vasculogenic ability that can be exploited to create functional microvascular networks in vivo. Using Matrigel as a supporting scaffold,15 we have shown that coimplantation of EPCs with either bmMPCs or cbMPCs into immunodeficient mice resulted in formation of extensive vascular networks after 1 week. The presence of human EPC-lined lumens containing erythrocytes (>100 vessels/mm2) throughout the implants indicated not only a process of vasculogenesis from the 2 cell types but also the formation of functional anastomoses with the host circulatory system. In addition, MPCs were shown to reside in perivascular locations around the engineered lumens, confirming their active participation in blood vessel assembly. In vitro, MPCs were shown to differentiate into smMHC-positive cells when cocultured with EPCs, an indication that the MPCs achieved a mature smooth muscle phenotype. In a recent report, human mesenchymal stem cells combined with human umbilical vein ECs were shown to facilitate blood vessel assembly and adopt a perivascular location,36 but our study differs from this report in that we show EPCs from either adult or cord blood, combined with MPCs from adult bone marrow or cord blood, form robust vascular networks in vivo. The extent of the engineered vascular networks was highly influenced by the ratio of EPCs to MPCs, with a progressive increase in vessel density and consistency of vascularized implants achieved when the contribution of MPCs was raised to 60% (Figure 3). This was true for both abEPCs and cbEPCs (Figure 6), demonstrating that both cord blood and adult peripheral blood are excellent sources of ECs for tissue vascularization.
Previous studies suggested the possibility of using mature ECs derived from vascular tissue to create microvascular networks.7–9,37 However, the clinical use of mature ECs derived from autologous vascular tissue is limited by the difficulty of obtaining sufficient quantities of cells with minimal donor site morbidity.1 Human ESCs have unlimited expansion capacity, but the therapeutic use of ESCs-derived ECs remains years away from the clinic. Most of these hurdles would be resolved if postnatal progenitor cells with expansion and functional potential were available from individual patients or from dedicated cell banks. In this regard, the in vitro expansion of blood-derived EPCs12–15 and the recent confirmation of their ability to form vascular networks in vivo15–17 have constituted major steps forward to resolve the problem of EC sourcing for therapeutic vasculogenesis.
Importantly, these studies have also shown that to produce high density and stable vascular networks, EPCs require coimplantation with perivascular cells. This is consistent with the literature showing interactions between ECs and perivascular cells in the blood vessel wall are critical for normal vascular development.32–34 In previous attempts to create vascular networks with blood-derived EPCs, either mature SMCs15 or the mouse embryonic cell line 10T1/216 were used to serve as perivascular cells; however, neither source is suitable for clinical application. Therefore, identification of MPCs as a readily obtainable perivascular cell source to partner in vivo with EPCs constitutes a crucial step in the development of therapeutic vasculogenesis. The numbers of human MPCs we were able to obtain in this study are likely to exceed, in the case of bone marrow, and be sufficient, in the case of cord blood, what would be needed for most autologous regenerative therapies.
This study has shown that successful in vivo vascularization depends on several distinct cellular functions. Firstly, both EPCs and MPCs must be present to initiate vasculogenesis, a process that was characterized by the formation of lumenal structures composed of human EPCs surrounded by -SMA–positive mesenchymal cells. Secondly, an angiogenic response from the host vasculature is needed so that host vessels will be available to form anastomoses with the nascent vasculature. In this regard, we propose that the implanted MPCs stimulated the host angiogenic response. This is based on (1) the ability of bmMPCs alone to recruit murine vessels into the Matrigel implant and (2) the secretion of VEGF from MPCs in vitro (supplemental Figure VIII). EPCs did not secrete VEGF and did not stimulate murine vessel infiltration. Vascularization is achieved when the angiogenic and vasculogenic blood vessels meet, form anastomoses, and establish perfusion of the implants. The fact that perfusion occurred was supported by the presence of erythrocytes within the newly formed vasculature and the delivery of luciferin substrate from the peritoneal cavity to Matrigel implants containing both cbEPCs and bmMPCs. Subsequently, a process reminiscent of in vivo remodeling, characterized by a progressive restriction of -SMA–expressing cells to perivascular locations, was seen, suggesting a stabilized vasculature.34 Whether factors secreted or presented by MPCs contribute to such stabilization would be important to elucidate in a future study. Finally, our engineered vascular networks were patent for up to 4 weeks in vivo, confirming the capacity of EPC/MPC-derived vasculature to remain stable and functional.16
In summary, we have demonstrated the feasibility of engineering vascular networks in vivo with human postnatal progenitor cells that can be obtained by noninvasive procedures. In addition, we suggest that this murine model of human vasculogenesis is ideally suited for future studies on the cellular and molecular components of microvessel development and pathological neovascular responses and for the development of strategies to enhance neovascularization of engineered human tissues and organs. Further efforts are required to implement these vascularization strategies into tissue regeneration and tissue engineering applications.
|
Acknowledgments |
|---|
We thank Dr Joseph C. Wu (Department of Radiology and Molecular Imaging Program, Stanford University School of Medicine, Calif) for providing the pUb-fluc-GFP construct, Dr Masanori Aikawa (Brigham and Women’s Hospital) for providing SMCs, Elke Pravda for confocal microscopy, Sandra R. Smith for VEGF analysis, Jill Wylie-Sears for technical assistance, and Kristin Johnson for figure preparation.
Sources of Funding
This work was supported by US Army Medical Research and Material Command (W81XWH-05-1-0115).
Disclosures
None.
|
Footnotes |
|---|
Original received April 29, 2008; revision received May 29, 2008; accepted May 30, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Jain RK, Au P, Tam J, Duda DG, Fukumura D. Engineering vascularized tissue. Nat Biotechnol. 2005; 23: 821–823.[CrossRef][Medline] [Order article via Infotrieve]
2. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest. 1999; 103: 1231–1236.[Medline] [Order article via Infotrieve]
3. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet. 1996; 348: 370–374.[CrossRef][Medline] [Order article via Infotrieve]
4. Lee H, Cusick RA, Browne F, Ho Kim T, Ma PX, Utsunomiya H, Langer R, Vacanti JP. Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices. Transplantation. 2002; 73: 1589–1593.[CrossRef][Medline] [Order article via Infotrieve]
5. Li X, Tjwa M, Moons L, Fons P, Noel A, Ny A, Zhou JM, Lennartsson J, Li H, Luttun A, Ponten A, Devy L, Bouche A, Oh H, Manderveld A, Blacher S, Communi D, Savi P, Bono F, Dewerchin M, Foidart JM, Autiero M, Herbert JM, Collen D, Heldin CH, Eriksson U, Carmeliet P. Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors. J Clin Invest. 2005; 115: 118–127.[CrossRef][Medline] [Order article via Infotrieve]
6. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003; 9: 702–712.[CrossRef][Medline] [Order article via Infotrieve]
7. Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK. Tissue engineering: creation of long-lasting blood vessels. Nature. 2004; 428: 138–139.[CrossRef][Medline] [Order article via Infotrieve]
8. Nor JE, Peters MC, Christensen JB, Sutorik MM, Linn S, Khan MK, Addison CL, Mooney DJ, Polverini PJ. Engineering and characterization of functional human microvessels in immunodeficient mice. Lab Invest. 2001; 81: 453–463.[Medline] [Order article via Infotrieve]
9. Schechner JS, Nath AK, Zheng L, Kluger MS, Hughes CC, Sierra-Honigmann MR, Lorber MI, Tellides G, Kashgarian M, Bothwell AL, Pober JS. In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. Proc Natl Acad Sci U S A. 2000; 97: 9191–9196.
10. Levenberg S, Rouwkema J, Macdonald M, Garfein ES, Kohane DS, Darland DC, Marini R, van Blitterswijk CA, Mulligan RC, D'Amore PA, Langer R. Engineering vascularized skeletal muscle tissue. Nat Biotechnol. 2005; 23: 879–884.[CrossRef][Medline] [Order article via Infotrieve]
11. Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H, Arzigian M, Fukumura D, Jain RK, Scadden DT. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat Biotechnol. 2007; 25: 317–318.[CrossRef][Medline] [Order article via Infotrieve]
12. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
13. Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ, Gilley D, Yoder MC. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood. 2004; 104: 2752–2760.
14. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest. 2000; 105: 71–77.[Medline] [Order article via Infotrieve]
15. Melero-Martin JM, Khan ZA, Picard A, Wu X, Paruchuri S, Bischoff J. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood. 2007; 109: 4761–4768.
16. Au P, Daheron LM, Duda DG, Cohen KS, Tyrrell JA, Lanning RM, Fukumura D, Scadden DT, Jain RK. Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels. Blood. 2008; 111: 1302–1305.
17. Yoder MC, Mead LE, Prater D, Krier TR, Mroueh KN, Li F, Krasich R, Temm CJ, Prchal JT, Ingram DA. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 2007; 109: 1801–1809.
18. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284: 143–147.
19. Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation. 2002; 106: 1199–1204.
20. Kim JW, Kim SY, Park SY, Kim YM, Kim JM, Lee MH, Ryu HM. Mesenchymal progenitor cells in the human umbilical cord. Ann Hematol. 2004; 83: 733–738.[CrossRef][Medline] [Order article via Infotrieve]
21. Le Ricousse-Roussanne S, Barateau V, Contreres JO, Boval B, Kraus-Berthier L, Tobelem G. Ex vivo differentiated endothelial and smooth muscle cells from human cord blood progenitors home to the angiogenic tumor vasculature. Cardiovasc Res. 2004; 62: 176–184.
22. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 2004; 103: 1669–1675.
23. Traktuev D, Merfeld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, Johnstone BH, March KL. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res. 2008; 102: 77–85.
24. Marion NW, Mao JJ. Mesenchymal stem cells and tissue engineering. Methods Enzymol. 2006; 420: 339–361.[Medline] [Order article via Infotrieve]
25. Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I, Owens GK. Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence. Circ Res. 1998; 82: 908–917.
26. Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res. 1994; 75: 803–812.
27. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol. 1999; 107: 275–281.[CrossRef][Medline] [Order article via Infotrieve]
28. Wall ME, Bernacki SH, Loboa EG. Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells. Tissue Eng. 2007; 13: 1291–1298.[CrossRef][Medline] [Order article via Infotrieve]
29. Antonelli-Orlidge A, Saunders KB, Smith SR, D'Amore PA. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci U S A. 1989; 86: 4544–4548.
30. Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol. 1998; 141: 805–814.
31. Wu X, Rabkin-Aikawa E, Guleserian KJ, Perry TE, Masuda Y, Sutherland FW, Schoen FJ, Mayer JE Jr, Bischoff J. Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells. Am J Physiol Heart Circ Physiol. 2004; 287: H480–H487.
32. Darland DC, D'Amore PA. Blood vessel maturation: vascular development comes of age. J Clin Invest. 1999; 103: 157–158.[Medline] [Order article via Infotrieve]
33. Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell. 1996; 87: 1153–1155.[CrossRef][Medline] [Order article via Infotrieve]
34. Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003; 9: 685–693.[CrossRef][Medline] [Order article via Infotrieve]
35. Fukumura D, Ushiyama A, Duda DG, Xu L, Tam J, Krishna V, Chatterjee K, Garkavtsev I, Jain RK. Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ Res. 2003; 93: e88–e97.[CrossRef][Medline] [Order article via Infotrieve]
36. Au P, Tam J, Fukumura D, Jain RK. Bone marrow derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood. 2008; 111: 4551–4558.
37. Shepherd BR, Chen HY, Smith CM, Gruionu G, Williams SK, Hoying JB. Rapid perfusion and network remodeling in a microvascular construct after implantation. Arterioscler Thromb Vasc Biol. 2004; 24: 898–904.
This article has been cited by other articles:
 |
|
 |
|
  A. Reinisch, N. A. Hofmann, A. C. Obenauf, K. Kashofer, E. Rohde, K. Schallmoser, K. Flicker, G. Lanzer, W. Linkesch, M. R. Speicher, et al. Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo Blood, June 25, 2009; 113(26): 6716 - 6725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Saif, C. Heeschen, and A. Aicher Add Some Fat to Vascular Progenitor Cell Therapy Circ. Res., June 19, 2009; 104(12): 1330 - 1332. [Full Text] [PDF]
|
|
|
|
 |
|
 E. I. Chang, R. G. Bonillas, S. El-ftesi, E. I. Chang, D. J. Ceradini, I. N. Vial, D. A. Chan, J. Michaels V, and G. C. Gurtner Tissue engineering using autologous microcirculatory beds as vascularized bioscaffolds FASEB J, March 1, 2009; 23(3): 906 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Loffredo and R. T. Lee Therapeutic Vasculogenesis: It Takes Two Circ. Res., July 18, 2008; 103(2): 128 - 130. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||