Vascular-like Network Research Articles

Laser Printing of Three-Dimensional Multicellular Arrays for Studies of Cell–Cell and Cell–Environment Interactions

Utilization of living cells for therapies in regenerative medicine requires a fundamental understanding of the interactions between different cells and their environment. Moreover, common models based on adherent two-dimensional cultures are not appropriate to simulate the complex interactions that occur in a three-dimensional (3D) cell-microenvironment in vivo. In this study, we present a computer-aided method for the printing of multiple cell types in a 3D array using laser-assisted bioprinting. By printing spots of human adipose-derived stem cells (ASCs) and endothelial colony-forming cells (ECFCs), we demonstrate that (i) these cell spots can be arranged layer-by-layer in a 3D array; (ii) any cell-cell ratio, cell quantity, cell-type combination, and spot spacing can be realized within this array; and (iii) the height of the 3D array is freely scalable. As a proof of concept, we printed separate spots of ASCs and ECFCs within a 3D array and observed cell-cell interactions in vascular endothelial growth factor-free medium. It has been demonstrated that direct cell-cell contacts trigger the development of stable vascular-like networks. This method can be applied to study complex and dynamic relationships between cells and their local environment.

Oxygen Sensing of Mesenchymal Stem and Progenitor Cells Facilitates Neo-Vasculogenesis In Vivo

AbstractAbstract 4313 Background:Vascular homeostasis and regeneration are maintained by proliferating vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs). Despite promising experimental data, regenerative stem cell therapy approaches employing ECFCs have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. We and others have recently shown that ECFC function in vivo requires a stringent interaction with mesenchymal stem and progenitor cells (MSPCs) * [Blood 2009; 113 (26):6716-25]. Co-transplantation of ECFCs and MSPCs is considered to be an advantageous strategy for vascular regenerative medicine.Hypoxia in ischemic tissue is considered to be a key factor influencing pro- and anti-angiogenic treatment by driving the revascularization machinery. In vivo most cells exist under an O2 pressure considerably below air oxygen. In vitro cells are usually expanded under air oxygen and suddenly encounter reduced O2 conditions when re-injected for therapy. Preliminary data suggests that low oxygen conditions differentially regulate stem cell function. We hypothesized that MSPCs act as hypoxia sensors and drive ECFCs to form functioning vessels in vivo. Methods:Adult human ECFCs were isolated and propagated directly from whole venous blood using a novel recovery strategy **[J Vis Exp. 2009;(32) pii: 1524]. MSPCs were isolated from human bone marrow aspirates. During cell culture, pooled human platelet lysate (pHPL) entirely replaced fetal bovine serum. Throughout this study we designated the oxygen level present in vivo in the venous environment as euoxia (41.5±3.4 mmHg). Oxygen levels below euoxia are defined as hypoxia (27.4±7.3 mmHg). Air-oxygen commonly used in standard laboratory practice is above euoxia and is therefore referred to as hyperoxia (139.8±2.9 mmHg). Progenitor cell phenotype, hierarchy, long-term proliferation, wound repair as well as migratory and vasculogenic functions were monitored under euoxia as compared to hypoxic or hyperoxic conditions. Molecular regulation of cellular responses to different oxygen levels was assessed by flow cytometry, immune cytochemistry and proteomic profiling. ECFC and MSPC interactions in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after sub-cutaneous co-implantation in matrigel plugs. Immune histochemistry and TUNEL assays were performed on plugs at day 1, 7 and 14 after transplantation. Results:Compared to hyperoxic standard laboratory conditions in vitro, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced under euoxia, and even more under hypoxic conditions. Hyperoxic conditioning resulted in a shift in progenitor hierarchy with an augmented number of ECFC high proliferative potential (HPP) colonies (60±18% of total colonies) as compared to euoxia (9±6%) and a complete loss of HPP colonies under hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair in scratch assays and matrigel vascular-like network formation in vitro were improved with escalating oxygen supply. The reoxygenation of hypoxic and euoxic ECFCs led to enhanced proliferation and function. Furthermore, MSPCs stabilized hypoxia inducible factor-1α (HIF-1α) under hypoxic as well as euoxic conditions, whereas ECFCs only stabilized HIF-1α when confronted with hypoxia in vitro. In a mouse model, subcutaneously injected ECFCs in matrigel underwent apoptosis after 1 day and attracted mouse leucocytes which infiltrated the matrigel plug. Co-implantation of ECFCs and MSPCs in these matrigel plugs resulted in reduced apoptosis and formation of perfused human vessels as soon as 7 days after transplantation. In this in vivo setting, perivascular cells but not endothelial cells were positive for HIF-1α in immune histochemistry. Background:These data indicate that oxygen levels differentially regulate ECFC and MSPC function during vascular homeostasis and regeneration. While hypoxic ECFCs alone are not able to function in vitro and form patent vessels in vivo, MSPCs react to the low oxygen environment and support ECFCs to perform vessel formation in vivo at least in part by rescuing ECFCs from hypoxia-induced apoptosis. This suggests that oxygen appears to be a key factor in stem cell transplantation and regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

449 Inhibition of vascular-like network formation of highly aggressive melanoma

449 Inhibition of vascular-like network formation of highly aggressive melanoma

Cell-responsive hydrogel for encapsulation of vascular cells

The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive poly(ethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin β4 (Tβ4), was examined. We show that the physical incorporation of Tβ4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tβ4 increased the survival of HUVEC compared to gels without Tβ4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of Tβ4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled Tβ4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tβ4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues.

Direct Fabrication of a Hybrid Cell/Hydrogel Construct by a Double-nozzle Assembling Technology

Assembling multitype cells and biomimic extracellular matrix materials in a single construct is a promising method to regenerate complex tissues/organs in vitro. A new double-nozzle assembling technique was used to fabricate a hybrid cell/hydrogel construct with a vascular-like network to perform as a mass exchange conduit. Using a digital model to mimic an anatomical liver structure, adipose-derived stromal cells (ADSC) were combined within a gelatin/alginate/fibrinogen hydrogel to form a vascular-like network and hepatocytes combined gelatin/alginate/chitosan were placed around it. The construct was stabilized in a thrombin/CaCl2/Na5P3O10 solution after assembly. The ADSC were induced to differentiate into endothelial-like cells with endothelial growth factor. The albumin secretion level of the embedded hepatocytes increased during the 2 weeks culture, while the levels of urea and alanine transaminase were decreased after an increasing profile. These preliminary results indicate that this double-nozzle assembling technique could be a powerful tool for fabricating complex constructs with special intrinsic/ extrinsic structures. This technology has the potential to be widely used in medical regeneration and drug screening.

Microgenomics profile the endogenous angiogenic phenotype in subpopulations of aggressive Melanoma

Beyond the elemental role of blood vessels in tumor growth, fluid conducting networks lacking endothelium (termed vasculogenic mimicry) were identified previously in metastatic melanoma and other cancer types. The etiology remains unclear, though it appears to involve dysregulation of the tumor-specific phenotype and transdifferentiation. Instigating the molecular deciphering of this phenomenon, we established a novel technique for microdissecting the spontaneously formed vascular-like networks and the randomly arranged cells (nests) from living 3D cultures of melanoma and performed microgenomics analysis. For the first time we show that despite the shared genotype, transcription was differentially regulated among the phenotypically distinct melanoma structures in vasculogenic mimicry. Several angiogenesis-specific genes were differentially expressed in higher levels in network cells of both uveal and cutaneous melanoma with intriguing representation of the ephrin family of angiogenesis factors, which was confirmed with immunocytochemistry. Interestingly, the adjacent nest-cells over-expressed ECM-related genes. Moreover, expression of angiogenesis-specific genes in melanoma resembled that of normal microvascular cells and was enhanced in melanoma disseminating hematogenously. The findings suggest that melanoma plasticity could enable autopoiesis of vascular-mimicking elements within the tumor infrastructure with significant clinical implications, such as response to anti-angiogenic treatments. Identifying factors regulating tumor plasticity and heterogeneity at the molecular level is essential in designing effective anti-cancer therapies.

Time‐lapse analysis and microdissection of living 3D melanoma cell cultures for genomics and proteomics

A novel technique is presented for the monitoring and morphological characterization of 3D cell cultures targeted for laser capture microdissection (LCM). A custom-made chamber enables time-lapse topography and pre-selection of cell targets in order to minimize microdissection time, optimizing the quality of biomolecules for downstream analyses. The method complements the recently presented novel application of LCM in living 3D cultures, whose compatibility with standard genomics and proteomics assays such as microarrays, real-time PCR, and 2D gel electrophoresis is further corroborated here. Specifically, the above techniques are employed in tandem to study, as a proof of principle, the dynamics of in vitro vasculogenic mimicry. It was shown previously that aggressive melanoma cells spontaneously differentiate on collagen gels into vascular-like networks with strong endogenous angiogenic potential. Here the evolution of vasculogenic mimicry was quantified by three time-dependent variables: the distribution of the vascular-like network lengths, widths, and area coverage. Based on these morphological descriptors the networks were locally classified over time as "early" or "mature" stage. LCM of networks and randomly oriented cells followed by real-time PCR for select genes revealed that differential expression was time-dependent and increased with network maturity. The method is widely applicable for microgenomics and microproteomics analyses in phenotypically evolving 3D cultures (i.e., of stem cells), under spontaneous or directed differentiation. Therefore beyond enabling future rigorous analyses on the mechanistics of vasculogenic mimicry, it provides a practical discovery engine for a range of developmental studies and tissue regenerative engineering applications.

Humanized System to Propagate Cord Blood-Derived Multipotent Mesenchymal Stromal Cells for Clinical Application

Umbilical cord blood (UCB) is an easily accessible alternative source for multipotent mesenchymal stromal cells (MSCs) and is generally believed to provide MSCs with a higher proliferative potential compared with adult bone marrow. Limitations in cell number and strict dependence of expansion procedures from selected lots of fetal bovine serum have hampered the progress of clinical applications with UCB-derived MSCs. We analyzed the isolation and proliferative potential of human UCB MSCs compared with bone marrow MSCs under optimized ex vivo culture conditions. We further investigated human platelet lysate as an alternative to replace fetal bovine serum for clinical-scale MSC expansion. Clonogenicity was determined in colony-forming units-fibroblast assays. MSC functions were tested in hematopoiesis support, vascular-like network formation and immune modulation potency assays. MSCs could be propagated from UCB with and without fetal bovine serum. MSC propagation was effective in 46% of UCB samples. Once established, the proliferation kinetics of UCB MSCs did not differ significantly from that of bone marrow MSCs under optimized culture conditions, resulting in more than 50 population doublings after 15 weeks. A clinical quantity of 100 million MSCs with retained differentiation potential could be obtained from UCB MSCs within approximately 7 weeks. Ex vivo expansion of hematopoietic UCB-derived CD34+ cells as well as immune inhibition and vascular-like network formation could be shown for UCB MSCs propagated under both culture conditions. We demonstrate for the first time that human MSCs can be obtained and propagated to a clinical quantity from UCB in a completely bovine serum-free system. Surprisingly, our data argue against a generally superior proliferative potential of UCB MSCs. Functional data indicate the applicability of clinical-grade UCB MSCs propagated with human platelet lysate-conditioned medium for hematopoiesis support, immune regulation and vascular regeneration.

Human Embryonic Stem Cells Demonstrate That Mpl Expression Initially Appears on a Population of CD31+ Endothelial Cells with Hemogenic Potential.

Mammalian hematopoiesis occurs adjacent to endothelial cells in the yolk sac and in the AGM region during fetal development, and in vivo cell labeling with Ac-LDL in fetal birds and mice indicates that hematopoietic cells can arise from endothelial cells. Blood forming potential has also been reported in endothelial cells isolated from human fetal liver and fetal bone marrow. However, the role of endothelial cells in the generation of hematopoietic cells remains poorly understood. While thrombopoietin (TPO) and its receptor, Mpl, are known for their critical role in megakaryocytopoiesis, Mpl−/ − mice and patients with congenital amegakaryocytic thrombocytopenia demonstrate that Mpl signaling is also important for the establishment and/or maintenance of hematopoietic stem cells. However, the nature of that role and the relevant Mpl-expressing cells remain unknown. To determine if Mpl signaling in early hematopoiesis involves hemogenic endothelium, we used human embryonic stem cells (hESCs) co-cultured with OP9 stromal cells, a robust model system for generating human hematopoietic cells. We found that CD34+ cells appeared by day 3–4 of co-culture, followed one day later by the appearance of CD31+ cells, nearly all of which appeared within the CD34+ population. The CD34+ cells increased to 20–40% of the total cell population by day 10, with nearly half co-expressing CD31+. Mpl+ cells first appeared on day 6–7, almost exclusively within the CD31+ population, increasing to 5–10% of total cells by day 10. The hESC:OP9 system generated a vascular-like network lined with von Willebrand Factor-expressing cells, which combined with FACS data showing the onset of Mpl expression on CD31+ cells, suggested that Mpl may first be expressed on vascular lining cells. Interestingly, Mpl expression precedes the detection of CD45+ cells, which are first observed on day 8–9. Moreover, round CD45+ cells appeared within the vascular networks, raising the possibility that vascular lining cells may give rise to hematopoietic cells. To begin to characterize the role of TPO/Mpl signaling in the CD31+ cell population, FACS-sorted CD31+ cells from day-9 and day-10 cultures were plated onto a fibronectin (FN)-coated surface, and non-adherent cells were removed after 1 hour. The adherent CD31+ cells uptake Ac-LDL and express vWF, characteristic of endothelial cells. When cultured in media supplemented with endothelial growth factors, including VEGF, EGF, bFGF, IGF-1, and heparin, the FN-adherent cells generated areas of cobblestone-like cell clusters and CD45+/CD34+ cells. When cultured in the same conditions plus TPO, the number of cobblestone-like clusters increased, and the number of CD45+/CD34+ hematopoietic cells generated increased 3–4 fold. Our data demonstrate that Mpl is expressed on a CD31+, vWF-expressing cell population with hemogenic potential, and that TPO/Mpl signaling increases the yield of hematopoietic cells generated from these cells. Ongoing sorting experiments, including the isolation and characterization of CD31+/Mpl+ cells, will further our understanding of where and how Mpl signaling affects early human hematopoietic development. HESCs provide a novel system for defining the role of TPO/Mpl signaling in early human hematopoietic development, which may lead to improved treatment of hematopoietic disorders.

In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent.

For patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split-thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular-like network inside tissue-engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel-Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue-engineered skin in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary-like network with the patient's own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.