3D Brain Vascular Niche Model Captures Invasive Behavior and Gene Signatures of Glioblastoma.

Glioblastoma (GBM) is a lethal brain cancer with no effective treatment; understanding how GBM cells respond to tumor microenvironment remains challenging as conventional cell cultures lack proper cytoarchitecture while in vivo animal models present complexity all at once. Developing a culture system to bridge the gap is thus crucial. Here, we employed a multicellular approach using human glia and vascular cells to optimize a 3-dimensional (3D) brain vascular niche model that enabled not only long-term culture of patient derived GBM cells but also recapitulation of key features of GBM heterogeneity, in particular invasion behavior and vascular association. Comparative transcriptomics of identical patient derived GBM cells in 3D and in vivo xenotransplants models revealed that glia-vascular contact induced genes concerning neural/glia development, synaptic regulation, as well as immune suppression. This gene signature displayed region specific enrichment in the leading edge and microvascular proliferation zones in human GBM and predicted poor prognosis. Gene variance analysis also uncovered histone demethylation and xylosyltransferase activity as main themes for gene adaption of GBM cells in vivo . Furthermore, our 3D model also demonstrated the capacity to provide a quiescence and a protective niche against chemotherapy. In summary, an advanced 3D brain vascular model can bridge the gap between 2D cultures and in vivo models in capturing key features of GBM heterogeneity and unveil previously unrecognized influence of glia-vascular contact for transcriptional adaption in GBM cells featuring neural/synaptic interaction and immunosuppression.

SOX17/ETV2 improves the direct reprogramming of adult fibroblasts to endothelial cells.

An autologous source of vascular endothelial cells (ECs) is valuable for vascular regeneration and tissue engineering without the concern of immune rejection. The transcription factor ETS variant 2 (ETV2) has been shown to directly convert patient fibroblasts into vascular EC-like cells. However, reprogramming efficiency is low and there are limitations in EC functions, such as eNOS expression. In this study, we directly reprogram adult human dermal fibroblasts into reprogrammed ECs (rECs) by overexpressing SOX17 in conjunction with ETV2. We find several advantages to rEC generation using this approach, including improved reprogramming efficiency, increased enrichment of EC genes, formation of large blood vessels carrying blood from the host, and, most importantly, expression of eNOS in vivo. From these results, we present an improved method to reprogram adult fibroblasts into functional ECs and posit ideas for the future that could potentially further improve the reprogramming process.

3D Printing of Interpenetrating Network Flexible Hydrogels with Enhancement of Adhesiveness.

3D printing of hydrogels has been widely explored for the rapid fabrication of complex soft structures and devices. However, using 3D printing to customize hydrogels with both adequate adhesiveness and toughness remains a fundamental challenge. Here, we demonstrate mussel-inspired (polydopamine) PDA hydrogel through the incorporation of a classical double network (2-acrylamido-2-methylpropanesulfonic acid) PAMPS/(polyacrylamide) PAAm to achieve simultaneously tailored adhesiveness, toughness, and biocompatibility and validate the 3D printability of such a hydrogel into customized architectures. The strategy of combining PDA with PAMPS/PAAm hydrogels leads to favorable adhesion on either hydrophilic or hydrophobic surfaces. The hydrogel also shows excellent flexibility, which is attributed to the reversible cross-linking of PDA and PAMPS, together with the long-chain PAAm cross-linking network. Among them, the reversible cross-linking of PDA and PAMPS is capable of dissipating mechanical energy under deformation. Meanwhile, the long-chain PAAm network contributes to maintaining a high deformation capability. We establish a theoretical framework to quantify the contribution of the interpenetrating networks to the overall toughness of the hydrogel, which also provides guidance for the rational design of materials with the desired properties. Our work manifests a new paradigm of printing adhesive, tough, and biocompatible interpenetrating network hydrogels to meet the requirements of broad potential applications in biomedical engineering, soft robotics, and intelligent and superabsorbent devices.

Direct differentiation of human pluripotent stem cells into vascular network along with supporting mural cells.

During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.

Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis, and maturation.

Neural progenitor cells (NPCs) have the capability to self-renew and differentiate into neurons and glial cells. In the adult brain, NPCs are found near brain microvascular networks (BMVNs) in specialized microenvironments called the neurovascular niche (NVN). Although several in vitro NVN models have been previously reported, most do not properly recapitulate the intimate cellular interactions between NPCs and perfused brain microvessels. Here, we developed perfused BMVNs composed of primary human brain endothelial cells, pericytes, and astrocytes within microfluidic devices. When induced pluripotent stem cell-derived NPCs were introduced into BMVNs, we found that NPC survival, neurogenesis, and maturation were enhanced. The application of flow during BMVN coculture was also beneficial for neuron differentiation. Collectively, our work highlighted the important role of BMVNs and flow in NPC self-renewal and neurogenesis, as well as demonstrated our model's potential to study the biological and physical interactions of human NVN in vitro.

Sox17 mediates adult arterial endothelial cell adaptation to hemodynamics.

Sox17 is a critical regulator of arterial identity during early embryonic vascular development. However, its role in adult endothelial cells (ECs) are not fully understood. Sox17 is highly expressed in arterial ECs but not in venous ECs throughout embryonic development to adulthood suggesting that it may play a functional role in adult arteries. Here, we investigated Sox17 mediated phenotypical changes in adult ECs. To precisely control the temporal expression level of Sox17, we designed a tetracycline-inducible lentiviral gene expression system to express Sox17 selectively in cultured venous ECs. We confirmed that Sox17-induced ECs exhibit a gene profile favoring arterial and tip cell identity. Furthermore, in comparison to control ECs, Sox17-activated ECs under shear leads to greater expression of arterial markers and suppression of venous identity. These data suggest that Sox17 enables greater hemodynamic adaptability of ECs in response to fluid shear stress. Here, we also demonstrate key morphogenic behaviors of Sox17-mediated ECs. In both vasculogenic and angiogenic 3D fibrin gel studies, Sox17-mediated ECs prefer to form cohesive vessels with one another while interfering the vessel formation of the control ECs. Sox17-mediated ECs elicit hyper-sprouting behavior in the presence of pericytes but not fibroblasts, suggesting Sox17 mediated sprouting frequency is dependent on supporting cell type. Using a microfluidic chip, we also show that Sox17-mediated ECs maintain thinner diameter vessels that do not widen under interstitial flow like the control ECs. Taken together, these data showed that Sox17 mediated EC gene expression and phenotypical changes are highly modulated in the context of biomechanical stimuli, suggesting Sox17 plays a role in regulating the arterial ECs adaptability under arterial hemodynamics as well as tip cells behavior during angiogenesis and vasculogenesis. The results from this study may be valuable in improving vein graft adaptation to arterial hemodynamics and bioengineering microvasculature for tissue engineering applications.

3D direct printing of mechanical and biocompatible hydrogel meta-structures.

Direct Ink Writing (DIW) has demonstrated great potential as a versatile method to 3D print multifunctional structures. In this work, we report the implementation of hydrogel meta-structures using DIW at room temperature, which seamlessly integrate large specific surface areas, interconnected porous characteristics, mechanical toughness, biocompatibility, and water absorption and retention capabilities. Robust but hydrophobic polymers and weakly crosslinked nature-origin hydrogels form a balance in the self-supporting ink, allowing us to directly print complex meta-structures without sacrificial materials and heating extrusion. Mechanically, the mixed bending or stretching of symmetrical re-entrant cellular lattices and the unique curvature patterns are combined to provide little lateral expansion and large compressive energy absorbance when external forces are applied on the printed meta-structures. In addition, we have successfully demonstrated ear, aortic valve conduits and hierarchical architectures. We anticipate that the reported 3D meta-structured hydrogel would offer a new strategy to develop functional biomaterials for tissue engineering applications in the future.

Differentiating Human Pluripotent Stem Cells to Vascular Endothelial Cells for Regenerative Medicine, Tissue Engineering, and Disease Modeling.

Vasculature plays a vital role in human biology as blood vessels transport nutrients and oxygen throughout the body. Endothelial cells (ECs), specifically, are key as they maintain barrier functions between the circulating blood and the surrounding tissues. ECs derived from human pluripotent stem cells (hPSCs) are utilized to study vascular development and disease mechanisms within in vitro models. Additionally, ECs derived from induced pluripotent stem cells (iPSCs) hold great promise for advancing personalized medicine, cell therapies, and tissue-engineered constructs by creating patient-specific cell populations. Here, we describe a xeno-free, serum-free differentiation protocol for deriving ECs from hPSCs. In brief, mesoderm progenitor cells are derived via WNT pathway activation. Following this, EC maturation is achieved with exogenous vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor 2 (bFGF2). We have characterized these cells as expressing mature EC markers and have illustrated their functionality in vitro.

Interstitial flow enhances the formation, connectivity, and function of 3D brain microvascular networks generated within a microfluidic device.

The bulk flow of interstitial fluid through tissue is an important factor in human biology, including the development of brain microvascular networks (MVNs) with the blood-brain barrier (BBB). Bioengineering perfused, functional brain MVNs has great potential for modeling neurovascular diseases and drug delivery. However, most in vitro models of brain MVNs do not implement interstitial flow during the generation of microvessels. Using a microfluidic device (MFD), we cultured primary human brain endothelial cells (BECs), pericytes, and astrocytes within a 3D fibrin matrix with (flow) and without (static) interstitial flow. We found that the bulk flow of interstitial fluid was beneficial for both BEC angiogenesis and vasculogenesis. Brain MVNs cultured under flow conditions achieved anastomosis and were perfusable, whereas those under static conditions lacked connectivity and the ability to be perfused. Compared to static culture, microvessels developed in flow culture exhibited an enhanced vessel area, branch length and diameter, connectivity, and longevity. Although there was no change in pericyte coverage of microvessels, a slight increase in astrocyte coverage was observed under flow conditions. In addition, the immunofluorescence intensity of basal lamina proteins, collagen IV and laminin, was nearly doubled in flow culture. Lastly, the barrier function of brain microvessels was enhanced under flow conditions, as demonstrated by decreased dextran permeability. Taken together, these results highlighted the importance of interstitial flow in the in vitro generation of perfused brain MVNs with characteristics similar to those of the human BBB.

Recent Advances in Cellular and Molecular Bioengineering for Building and Translation of Biological Systems.

In January of 2020, the Biomedical Engineering Society (BMES)- Cellular and Molecular Bioengineering (CMBE) conference was held in Puerto Rico and themed "Vision 2020: Emerging Technologies to Elucidate the Rule of Life." The annual BME-CMBE conference gathered worldwide leaders and discussed successes and challenges in engineering biological systems and their translation. The goal of this report is to present the research frontiers in this field and provide perspectives on successful engineering and translation towards the clinic. We hope that this report serves as a constructive guide in shaping the future of research and translation of engineered biological systems.

Bioengineering the neurovascular niche to study the interaction of neural stem cells and endothelial cells.

The ability of mammalian neural stem cells (NSCs) to self-renew and differentiate throughout adulthood has made them ideal to study neurogenesis and attractive candidates for neurodegenerative disease therapies. In the adult mammalian brain, NSCs are maintained in the neurovascular niche (NVN) where they are found near the specialized blood vessels, suggesting that brain endothelial cells (BECs) are prominent orchestrators of NSC fate. However, most of the current knowledge of the mammalian NVN has been deduced from nonhuman studies. To circumvent the challenges of in vivo studies, in vitro models have been developed to better understand the reciprocal cellular mechanisms of human NSCs and BECs. This review will cover the current understanding of mammalian NVN biology, the effects of endothelial cell-derived signals on NSC fate, and the in vitro models developed to study the interactions between NSCs and BECs.

Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics.

Infiltrative growth is a major cause of high lethality of malignant brain tumors such as glioblastoma (GBM). We show here that GBM cells upregulate guidance receptor Plexin-B2 to gain invasiveness. Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies establish Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness.

Reconfigurable Microphysiological Systems for Modeling Innervation and Multitissue Interactions.

Tissue-engineered models continue to experience challenges in delivering structural specificity, nutrient delivery, and heterogenous cellular components, especially for organ-systems that require functional inputs/outputs and have high metabolic requirements, such as the heart. While soft lithography has provided a means to recapitulate complex architectures in the dish, it is plagued with a number of prohibitive shortcomings. Here, concepts from microfluidics, tissue engineering, and layer-by-layer fabrication are applied to develop reconfigurable, inexpensive microphysiological systems that facilitate discrete, 3D cell compartmentalization, and improved nutrient transport. This fabrication technique includes the use of the meniscus pinning effect, photocrosslinkable hydrogels, and a commercially available laser engraver to cut flow paths. The approach is low cost and robust in capabilities to design complex, multilayered systems with the inclusion of instrumentation for real-time manipulation or measures of cell function. In a demonstration of the technology, the hierarchal 3D microenvironment of the cardiac sympathetic nervous system is replicated. Beat rate and neurite ingrowth are assessed on-chip and quantification demonstrates that sympathetic-cardiac coculture increases spontaneous beat rate, while drug-induced increases in beating lead to greater sympathetic innervation. Importantly, these methods may be applied to other organ-systems and have promise for future applications in drug screening, discovery, and personal medicine.

CEREBRAL ORGANOIDS AS A MODEL FOR GLIOBLASTOMA MULTIFORME.

Glioblastoma multiforme (GBM) is a highly lethal and elusive cancer. While many in vitro and in vivo models have been developed to recapitulate the factors that contribute to its invasive behavior, they suffer from drawbacks related to genetic variability, expense and scope. Technologies utilizing human pluripotent stem cells can now generate organoids which can recapitulate the relative complexity the cytoarchitecture and microenvironment of human brain tissue. In conjunction with protocols which effectively induce GBM tumors within these "cerebral organoids", such approaches represent an unprecedented model to investigate GBM invasion and its effect on the brain ECM. This review focuses on methods of brain organoid development, protocols for inducing GBM, the relevant findings on invasion and microenvironmental changes, and discusses their limitations and potential future direction.

High-resolution tomographic analysis of in vitro 3D glioblastoma tumor model under long-term drug treatment.

Glioblastoma multiforme (GBM) is a lethal type of brain tumor that often develop therapeutic resistance over months of chemotherapy cycles. Recently, 3D GBM models were developed to facilitate evaluation of drug treatment before undergoing expensive animal studies. However, for long-term evaluation of therapeutic efficacy, novel approaches for GBM tissue construction are still needed. Moreover, there is still a need to develop fast and sensitive imaging methods for the noninvasive assessment of this 3D constructs and their response to drug treatment. Here, we report on the development of an integrated platform that enable generating (i) an in vitro 3D GBM model with perfused vascular channels that allows long-term culture and drug delivery and (ii) a 3D imaging modality that enables researchers to noninvasively assess longitudinal fluorescent signals over the whole in vitro model.

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells.

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.

Engineering transferrable microvascular meshes for subcutaneous islet transplantation.

The success of engineered cell or tissue implants is dependent on vascular regeneration to meet adequate metabolic requirements. However, development of a broadly applicable strategy for stable and functional vascularization has remained challenging. We report here highly organized and resilient microvascular meshes fabricated through a controllable anchored self-assembly method. The microvascular meshes are scalable to centimeters, almost free of defects and transferrable to diverse substrates, ready for transplantation. They promote formation of functional blood vessels, with a density as high as ~220 vessels mm-2, in the poorly vascularized subcutaneous space of SCID-Beige mice. We further demonstrate the feasibility of fabricating microvascular meshes from human induced pluripotent stem cell-derived endothelial cells, opening a way to engineer patient-specific microvasculature. As a proof-of-concept for type 1 diabetes treatment, we combine microvascular meshes and subcutaneously transplanted rat islets and achieve correction of chemically induced diabetes in SCID-Beige mice for 3 months.

Recent advances in high-strength and elastic hydrogels for 3D printing in biomedical applications.

Three-dimensional (3D) printing enables the production of personalized tissue-engineered products with high tunability and complexity. It is thus an attractive and promising technology in the pharmaceutical and medical fields. Printable and biocompatible hydrogels are attractive materials for 3D printing applications because they offer favorable biomimetic environments for live cells, such as high water content, porous structure, bioactive molecule incorporation, and tunable mechanical properties and degradation rates. However, most conventional hydrogel materials are brittle and mechanically weak and hence cannot meet the mechanical needs for handling and soft and elastic tissue use. Thus, the development of printable, high-strength, and elastic hydrogel materials for 3D printing in tissue repair and regeneration is critical and interesting. In this review, we summarized the recent reports on high-strength and elastic hydrogels for printing use and categorized them into three groups, namely double-network hydrogels, nanocomposite hydrogels, and single-network hydrogels. The reinforcing mechanisms of these high-strength hydrogels and the strategies to improve their printability and biocompatibility were further discussed. These high-strength and elastic hydrogels may offer opportunities to accelerate the development of 3D printing technology and provide new insights for 3D-printed product design in biomedicine. STATEMENT OF SIGNIFICANCE: Biocompatible and biodegradable hydrogels are highly attractive in 3D printing because of their desirable printability and friendly environment for loading bioactive molecules and living cells. The development of high-strength and elastic hydrogels changes the conventional impression of weak and brittle hydrogels and provides new opportunities and inspirations for 3D printing and biomedical applications. In this review, we analyzed the hydrogel reinforcement mechanisms, summarized recent progresses in developing high-strength and elastic hydrogels for 3D printing, and discussed the strategies to improve the printability and biocompatibility of the hydrogel inks.

3D bioprinting of vascular conduits for pediatric congenital heart repairs.

In children with congenital heart defects, surgical correction often involves the use of valves, patches or vascular conduits to establish anatomic continuity. Due to the differences between the pediatric and adult populations, tissue reconstruction in pediatric patients requires a substantially different approach from those in adults. Cardiovascular anatomy of children with congenital heart defect vary, which requires tailored surgical operations for each patient. Since grafts used in these palliative surgeries are sensitive to the local hemodynamic environments, their geometries need to be precisely designed to ensure long-term performance. Tissue engineered vascular grafts (TEVGs) have made tremendous progress over the past decade, but it remains difficult to fabricate patient- and operation-specific vascular grafts. This review summarizes historical milestones of TEVG development for repairing pediatric congenital defects and current clinical outcomes. We also highlight ongoing works on 3D bioprinting of TEVGs with complex geometries and address the current limitations of each technique. Although 3D bioprinted vascular grafts with appropriate functions are yet to be developed, some of the current researches are promising to create better patient specific tissue engineered vascular grafts in the future.