Endothelial Network Research Articles

Serine 26 in Early Growth Response-1 Is Critical for Endothelial Proliferation, Migration, and Network Formation.

BackgroundVascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type.Methods and ResultsA CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr ‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis.ConclusionsThese findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells

PurposeLimbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. MethodsHuman corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. ResultsLMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. ConclusionThese findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

The Adipose-Derived Stem Cell and Endothelial Cell Coculture System-Role of Growth Factors?

Adequate vascularization is a fundamental prerequisite for bone regeneration, formation and tissue engineering applications. Endothelialization of scaffold materials is a promising strategy to support neovascularization and bone tissue formation. Besides oxygen and nutrition supply, the endothelial network plays an important role concerning osteogenic differentiation of osteoprogenitor cells and consecutive bone formation. In this study we aimed to enhance the growth stimulating, proangiogenic and osteogenic features of the ADSC and HUVEC coculture system by means of VEGFA165 and BMP2 application. We were able to show that sprouting phenomena and osteogenic differentiation were enhanced in the ADSC/HUVEC coculture. Furthermore, apoptosis was unidirectionally decreased in HUVECs, but these effects were not further enhanced upon VEGFA165 or BMP2 application. In summary, the ADSC/HUVEC coculture system per se is a powerful tool for bone tissue engineering applications.

UTP is a regulator of in vitro and in vivo angiogenic properties of cardiac adipose-derived stem cells.

The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3days after ligation. We observed a significant increase of capillary density 14 and 30days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.

Interleukin-17A Triggers the Release of Platelet-Derived Factors Driving Vascular Endothelial Cells toward a Pro-Angiogenic State.

Platelets comprise a highly interactive immune cell subset of the circulatory system traditionally known for their unique haemostatic properties. Although platelets are considered as a vault of growth factors, cytokines and chemokines with pivotal role in vascular regeneration and angiogenesis, the exact mechanisms by which they influence vascular endothelial cells (ECs) function remain underappreciated. In the present study, we examined the role of human IL-17A/IL-17RA axis in platelet-mediated pro-angiogenic responses. We reveal that IL-17A receptor (IL-17RA) mRNA is present in platelets transcriptome and a profound increase is documented on the surface of activated platelets. By quantifying the protein levels of several factors, involved in angiogenesis, we identified that IL-17A/IL17RA axis selectively induces the release of vascular endothelial growth factor, interleukin -2 and -4, as well as monocyte chemoattractant protein -1 from treated platelets. However, IL-17A exerted no effect on the release of IL-10, an anti-inflammatory factor with potentially anti-angiogenic properties, from platelets. Treatment of human endothelial cell two-dimensional tubule networks or three-dimensional spheroid and mouse aortic ring structures with IL-17A-induced platelet releasate evoked pro-angiogenic responses of ECs. Our findings suggest that IL-17A may critically affect platelet release of pro-angiogenic factors driving ECs towards a pro-angiogenic state.

An efficient method to generate kidney organoids at the air-liquid interface.

The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

Effect of Pig-Adipose-Derived Stem Cells' Conditioned Media on Skin Wound-Healing Characteristics In Vitro.

The primary mechanism by which adipose-derived stem cells (ASCs) exert their reparative or regenerative potential relies predominantly on paracrine action. Secretory abilities of ASCs have been found to be amplified by hypoxia pre-conditioning. This study investigates the impact of hypoxia (1% O2) on the secretome composition of pig ASCs (pASCs) and explores the effect of pASCs’ conditioned media (CM) on skin cell functions in vitro and the expression of markers attributed to wound healing. Exposure of pASCs to hypoxia increased levels of vascular endothelial growth factor (VEGF) in CM-Hyp compared to CM collected from the cells cultured in normoxia (CM-Nor). CM-Hyp promoted the migratory ability of pig keratinocytes (pKERs) and delayed migration of pig dermal fibroblasts (pDFs). Exposure of pKERs to either CM-Nor or CM-Hyp decreased the levels of pro-fibrotic indicators WNT10A and WNT11. Furthermore, CM-Hyp enhanced the expression of KRT14, the marker of the basal epidermis layer. In contrast, CM-Nor showed a stronger effect on pDFs manifested by increases in TGFB1, COL1A1, COL3A1, and FN1 mRNA expression. The formation of three-dimensional endothelial cell networks was improved in the presence of CM-Hyp. Overall, our results demonstrate that the paracrine activity of pASCs affects skin cells, and this property might be used to modulate wound healing.

Conversion of stem cells from apical papilla into endothelial cells by small molecules and growth factors

ObjectivesRecently, a new strategy has been developed to directly reprogram one cell type towards another targeted cell type using small molecule compounds. Human fibroblasts have been chemically reprogrammed into neuronal cells, Schwann cells and cardiomyocyte-like cells by different small molecule combinations. This study aimed to explore whether stem cells from apical papilla (SCAP) could be reprogrammed into endothelial cells (ECs) using the same strategy.Materials and methodsThe expression level of endothelial-specific genes and proteins after chemical induction of SCAP was assessed by RT-PCR, western blotting, flow cytometry and immunofluorescence. The in vitro functions of SCAP-derived chemical-induced endothelial cells (SCAP-ECs) were evaluated by tube-like structure formation assay, acetylated low-density lipoprotein (ac-LDL) uptake and NO secretion detection. The proliferation and the migration ability of SCAP-ECs were evaluated by CCK-8 and Transwell assay. LPS stimulation was used to mimic the inflammatory environment in demonstrating the ability of SCAP-ECs to express adhesion molecules. The in vivo Matrigel plug angiogenesis assay was performed to assess the function of SCAP-ECs in generating vascular structures using the immune-deficient mouse model.ResultsSCAP-ECs expressed upregulated endothelial-specific genes and proteins; displayed endothelial transcriptional networks; exhibited the ability to form functional tubular-like structures, uptake ac-LDL and secrete NO in vitro; and contributed to generate blood vessels in vivo. The SCAP-ECs could also express adhesion molecules in the pro-inflammatory environment and have a similar migration and proliferation ability as HUVECs.ConclusionsOur study demonstrates that the set of small molecules and growth factors could significantly promote endothelial transdifferentiation of SCAP, which provides a promising candidate cell source for vascular engineering and treatment of ischemic diseases.

Perfusable host microfluidic endothelial networks for quantitative assessment of the effects of interstitial flows, macrophage polarization and monoclonal‐based immunotherapy on immune cell infiltration into a tumor spheroid

Monoclonal antibody-based immunotherapy is a promising treatment for several diseases such as cancer and virus infection. However, due to its highly selective target, drug testing using animal models or classical 2D human cell cultures are not sufficient to predict the drug effects on patients. New 3D organotypic models that employ 3D cell culture of human cells within a microfluidic device allow detailed characterization of critical biological interactions. In this project, we describe a new 3D cell culture model that allows the creation of in-vitro human perfusable vasculature containing hollow spaces for the insertion of tumor spheroids. A polydimethylsiloxane device with several vertical holes on top of the gel channel is used to create the host microvascular endothelial (HOME) network (Fig.1A). Endothelial cells and fibroblasts create perfusable vascular networks (Fig. 1B). Tumor spheroids are formed by coculturing MDA-MB-468 tumor cells (T), fibroblasts (F), and non-polarized (M0) or M2 macrophages, and denoted by TFM0 and TFM2 respectively (Fig. 1C). One day after inserting a tumor spheroid in collagen into the HOME networks through the hole, freshly-isolated monocytes are perfused into the vascular networks by luminal flows. We observe that after 2 days, the majority of monocytes remain either within the vasculature or extravasated but stay inside the fibrin gel outside of the hole containing the tumor spheroid (Fig. 1Di). On day 2, interstitial flows from the tumor spheroid cause monocyte migration from the vascular network side into the hole containing the tumor spheroid (figure 1Dii). When a TFM2 spheroid is inserted into the HOME networks, it recruits more monocytes than the TFM0 spheroid (Fig. Ei and Eii). Recruitment % is the percentage of monocytes that leave the blood vessel and migrate into the hole among the total number of monocytes in 3mmx3mm region of interest around the hole. Monocytes are recruited better by the TFM2 spheroid than the TFM0 spheroid (Fig. 1Gi). These results showed that interstitial flows from the tumor spheroid to the vascular networks and macrophage polarization are essential for monocyte recruitment. Multi-specific antibody UniTI102 developed by Elstar Therapeutics (MA, USA) neutralizes chemokine CCL2 to block monocyte recruitment. To characterize the response to the drug UniTI102, we compared devices having a TFM0 spheroid that are untreated (Fig. 1D) or treated with UniTI102 (Fig. 1F) and observed that less monocytes has migrated into the hole in the presence of UniTI102 at 100nM. Quantitative data also showed that less monocytes are recruited when devices are treated with 100nM UniTI102 than 100nM IgG (Fig. 1Gii).

Biofabrication of endothelial cell, dermal fibroblast, and multilayered keratinocyte layers for skin tissue engineering

The skin serves a substantial number of physiological purposes and is exposed to numerous biological and chemical agents owing to its large surface area and accessibility. Yet, current skin models are limited in emulating the multifaceted functions of skin tissues due to a lack of effort on the optimization of biomaterials and techniques at different skin layers for building skin frameworks. Here, we use biomaterial-based approaches and bioengineered techniques to develop a 3D skin model with layers of endothelial cell networks, dermal fibroblasts, and multilayered keratinocytes. Analysis of mechanical properties of gelatin methacryloyl (GelMA)-based bioinks mixed with different portions of alginate revealed bioprinted endothelium could be better modeled to optimize endothelial cell viability with a mixture of 7.5% GelMA and 2% alginate. Matrix stiffness plays a crucial role in modulating produced levels of Pro-Collagen I alpha-1 and matrix metalloproteinase-1 in human dermal fibroblasts and affecting their viability, proliferation, and spreading. Moreover, seeding human keratinocytes with gelatin-coating multiple times proved to be helpful in reducing culture time to create multiple layers of keratinocytes while maintaining their viability. The ability to fabricate selected biomaterials for each layer of skin tissues has implications in the biofabrication of skin systems for regenerative medicine and disease modeling.

Exogenous FGF-2 prolongs endothelial connection in multilayered human skeletal muscle cell sheet

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking invivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design invitro pre-vascularized tissue and graft integration prospects.

Coculture of Endothelial and Stromal Cells to Promote Concurrent Osteogenesis and Vasculogenesis.

A key challenge in the treatment of large bone defects is the need to provide an adequate and stable vascular supply as new tissue develops. Bone tissue engineering applies selected biomaterials and cell types to create an environment that promotes tissue formation, maturation, and remodeling. Mesenchymal stromal cells (MSCs) have been widely used in these strategies because of their established effects on bone formation, and their ability to act as stabilizing pericytes that support vascular regeneration by endothelial cells (ECs). However, the creation of vascularized bone tissue in vitro requires coupling of osteogenesis and vasculogenesis in a three-dimensional (3D) biomaterial environment. In the present study, 3D fibrin hydrogels containing MSCs and ECs were prevascularized in vitro for 7 days to create an endothelial network in the matrix, and were subsequently cultured for a further 14 days under either continued vasculogenic stimulus, a combination of vasculogenic and osteogenic (hybrid) stimulus, or only osteogenic stimulus. It was found that ECs produced robust vessel networks in 3D fibrin matrices over 7 days of culture, and these networks continued to expand over the 14-day treatment period under vasculogenic conditions. Culture in hybrid medium resulted in maintenance of vessel networks for 14 days, while osteogenic culture abrogated vessel formation. These trends were mirrored in data representing overall cell viability and cell number in the 3D fibrin constructs. MSCs were found to colocalize with EC networks under vasculogenic and hybrid conditions, suggesting pericyte-like function. The bone marker alkaline phosphatase increased over time in hybrid and osteogenic media, but mineral deposition was evident only under purely osteogenic conditions. These results suggest that hybrid media compositions can support some aspects of multiphase tissue formation, but that alternative strategies are needed to obtain robust, concomitant vascularization, and osteogenesis in engineered tissues in vitro. Impact statement The combined use of mesenchymal stromal cells (MSCs) and endothelial cells to concomitantly produce mature bone and a nourishing vasculature is a promising tissue engineering approach to treating large bone defects. However, it is challenging to create and maintain vascular networks in the presence of osteogenic cues. This study used a 3D fibrin matrix to demonstrate that prevascularization of the construct can lead to maintenance of vessel structures over time, but that osteogenesis is compromised under these conditions. This work illuminates the capacity of MSCs to serve as both supportive pericytes and as osteoprogenitor cells, and motivates new strategies for coupling osteogenesis and vasculogenesis in engineered bone tissues.

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 µM genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 µM genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

Transforming growth factor-β1 disrupts angiogenesis during the follicular-luteal transition through the Smad-serpin family E member 1 (SERPINE1)/serpin family B member 5 (SERPINB5) signalling pathway in the cow.

Intense angiogenesis is critical for the development of the corpus luteum and is tightly regulated by numerous factors. However, the exact role transforming growth factor-β1 (TGFB1) plays during this follicular-luteal transition remains unclear. This study hypothesised that TGFB1, acting through TGFB receptor 1 (TGFBR1) and Smad2/3 signalling, would suppress angiogenesis during the follicular-luteal transition. Using a serum-free luteinising follicular angiogenesis culture system, TGFB1 (1 and 10ngmL-1 ) markedly disrupted the formation of capillary-like structures, reducing the endothelial cell network area and the number of branch points (P <0.001 compared with control). Furthermore, TGFB1 activated canonical Smad signalling and inhibited endothelial nitric oxide synthase (NOS3 ) mRNA expression, but upregulated latent TGFB-binding protein and TGFBR1 , serpin family E member 1 (SERPINE1 ) and serpin family B member 5 (SERPINB5 ) mRNA expression. SB431542, a TGFBR1 inhibitor, reversed the TGFB1-induced upregulation of SERPINE1 and SERPINB5 . In addition, TGFB1 reduced progesterone synthesis by decreasing the expression of steroidogenic acute regulatory protein (STAR ), cytochrome P450 family 11 subfamily A member 1 (CYP11A1 ) and 3β-hydroxysteroid dehydrogenase (HSD3B1 ) expression. These results show that TGFB1 regulates NOS3 , SERPINE1 and SERPINB5 expression via TGFBR1 and Smad2/3 signalling and this could be the mechanism by which TGFB1 suppresses endothelial networks. Thereby, TGFB1 may provide critical homeostatic control of angiogenesis during the follicular-luteal transition. The findings of this study reveal the molecular mechanisms underlying the actions of TGFB1 in early luteinisation, which may lead to novel therapeutic strategies to reverse luteal inadequacy.

Hepatocytes and Endothelial Networks in a Fluid-Based In Vitro Model of Liver Drug Metabolism.

Drug-induced liver toxicity remains a major cause of drug withdrawal from animal testing and human clinical trials. A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. Previously reported fluid-based systems have some advantages for hepatocyte culture, but have insufficient liver-specific functions because they simply involve moving conventional hepatocyte cultures from a dish into a fluid-based system. Importantly, these cultures have no liver tissue-specific structures that construct liver-specific cellular polarities, such as apical, basolateral, and basal faces. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions. Impact statement A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions.

(Invited) Cell Analysis Using Integrated Electrochemical Devices

For the fields of tissue engineering and organs-on-a-chip, three-dimensional (3D) cultured cells are widely used. These applications require a method that can easily measure cell activities of 3D cultured cells without any cell damages. For the purpose, electrochemical approaches have been proposed [1-3]. By using these electrochemical approaches, several kinds of cell activities such as respiratory activities can be measured.My group has developed several kinds of integrated electrochemical devices including chip and probe devices. The chip devices contained many electrodes based on complementary metal oxide semiconductor (CMOS)-based devices [3-9]. The detection system is useful for real-time imaging, because it rapidly obtains 2D electrochemical images consisting redox current signals at target positions. By using the CMOS-based electrochemical devices, respiration activity and cell differentiation status were clearly visualized. Also, the device provided electrochemical images of released molecules such as dopamine from 3D cultured cells.In addition to CMOS-based devices, bipolar electrode (BPE)-based electrode arrays was developed [10]. In BPE system, anodic and cathodic reactions are induced at both ends of the BPE with only a pair of driving electrodes. Therefore, many sensing elements can be incorporated into a small chip without complex wiring. In my BPE device, oxygen reduction currents at the sensor near 3D cultured cells converted electrochemiluminescence (ECL) to detect respiratory activity of 3D cultured cells.In addition to chip devices, scanning electrochemical microscopy was applied for electrochemical measurement of respiration activity of fibroblast spheroids containing endothelial cell networks [11]. This study reported the effects of endothelial cell networks on cell activity of 3D cultured cells.Thus, these integrated electrochemical devices were applied for cell analysis. They will be utilized for tissue engineering and organs on a chip. In this presentation, I will summarize these devices.

Abstract IA-06: Heterotypic 3D bioprinted tissues to study pancreatic cancer

Abstract Crosstalk between tumor cells and the tumor microenvironment contributes critically to tumor initiation, tumor progression, and therapeutic response. The tumor microenvironment (TME) is composed of multiple cell types (e.g. fibroblast, vascular, and immune cells) that are temporally and spatially dynamic. Stromal cells in the TME respond not only to the growing tumor cells, but are also affected by concurrent changes in the accessibility and composition of the extracellular matrix (ECM), signaling molecules, nutrients and oxygen. Together, the interactions of tumor cells and stromal cells regulates the tumor phenotype and, ultimately, affects patient outcomes. As more is understood about the dynamic relationship of multiple cell types in the tumor microenvironment, it has become clear that better models of human cancer are needed. In order to understand the impact of heterotypic crosstalk on tumor behaviors such as initiation, progression, and response to therapies, we recently developed heterotypic, scaffold-free tissue models of advanced cancer using an extrusion-based bioprinter system. Multiple cell types including cancer cells, fibroblasts, endothelial cells, mesenchymal stem cells, and immune cells can be incorporated into bioprinted tissues with defined spatial architecture, and the system is compatible with patient-derived cells. Within these structures, cells exhibit a tissue-like cellular density, deposit ECM, and self-organize to form complex structures, as illustrated by formation of nascent endothelial networks. Cell intrinsic, extrinsic, and spatial phenotypes, including cell survival, cell proliferation, differentiation state, ECM deposition, and cellular migration, can be assessed within these tissues following exposure to extrinsic signals or therapies. Our current work is focused in three main areas. First, we aim to understand the signaling between tumor and stromal cell types in early cancer development that contributes to malignant progression. Second, we are working to understand the influence of fibroblast heterogeneity on tumor cell phenotypes, using genetic or non-genetic perturbagens to alter fibroblast activation. Finally, we are building tissues comprising patient-derived cells to understand how the microenvironment influences therapeutic response. Together, our work demonstrates that bioprinted tumor tissues recapitulate many aspects of in vivo neoplastic tissues, and provides a manipulable model system to interrogate molecular mechanisms of tumor development, progression, and treatment response. Citation Format: Ellen M Langer, Brittany L Allen-Petersen, Isabel A English, Jason M Link, Rosalie C Sears. Heterotypic 3D bioprinted tissues to study pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-06.

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks