Lumen-like Structures Research Articles

Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

Collagen plays a critical role in regulating breast cancer progression and therapeutic resistance. An improved understanding of both the features and drivers of tumor-permissive and -restrictive collagen matrices are critical to improve prognostication and develop more effective therapeutic strategies. In this study, using a combination of in vitro, in vivo and bioinformatic experiments, we show that type III collagen (Col3) plays a tumor-restrictive role in human breast cancer. We demonstrate that Col3-deficient, human fibroblasts produce tumor-permissive collagen matrices that drive cell proliferation and suppress apoptosis in non-invasive and invasive breast cancer cell lines. In human triple-negative breast cancer biopsy samples, we demonstrate elevated deposition of Col3 relative to type I collagen (Col1) in non-invasive compared to invasive regions. Similarly, bioinformatics analysis of over 1000 breast cancer patient biopsies from The Cancer Genome Atlas BRCA cohort revealed that patients with higher Col3:Col1 bulk tumor expression had improved overall, disease-free, and progression-free survival relative to those with higher Col1:Col3 expression. Using an established 3D culture model, we show that Col3 increases spheroid formation and induces the formation of lumen-like structures that resemble non-neoplastic mammary acini. Finally, our in vivo study shows co-injection of murine breast cancer cells (4T1) with rhCol3-supplemented hydrogels limits tumor growth and decreases pulmonary metastatic burden compared to controls. Taken together, these data collectively support a tumor-suppressive role for Col3 in human breast cancer and suggest that strategies that increase Col3 may provide a safe and effective therapeutic modality to limit recurrence in breast cancer patients.

Prox1a promotes liver growth and differentiation by repressing cdx1b expression and intestinal fate transition in zebrafish

The liver is a key endoderm-derived multifunctional organ within the digestive system. Prospero homeobox 1 (Prox1) is an essential transcription factor for liver development, but its specific function is not well understood. Here, we show that hepatic development, including the formation of intrahepatic biliary and vascular networks, is severely disrupted in prox1a mutant zebrafish. We find that Prox1a is essential for liver growth and proper differentiation but not required for early hepatic cell fate specification. Intriguingly, prox1a depletion leads to ectopic initiation of a Cdx1b-mediated intestinal program and the formation of intestinal lumen-like structures within the liver. Morpholino knockdown of cdx1b alleviates liver defects in the prox1a mutant zebrafish. Finally, chromatin immunoprecipitation analysis reveals that Prox1a binds directly to the promoter region of cdx1b, thereby repressing its expression. Overall, our findings indicate that Prox1a is required to promote and protect hepatic development by repression of Cdx1b-mediated intestinal cell fate in zebrafish.

Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography.

The vital role of bile canaliculus (BC) in liver function is closely related to its morphology. Electron microscopy has contributed to understanding BC morphology; however, its invasiveness limits its use in living specimens. Here, we report non-invasive characterization of BC formation using refractive index (RI) tomography. First, we investigated and characterized the RI distribution of BCs in two-dimensional (2D) cultured HepG2 cells. BCs were identified based on their distinct morphology and functionality, as confirmed using a fluorescence-labeled bile acid analog. The RI distribution of BCs exhibited three common features: (1) luminal spaces with a low RI between adjacent hepatocytes; (2) luminal spaces surrounded by a membranous structure with a high RI; and (3) multiple microvillus structures with a high RI within the lumen. Second, we demonstrated the characterization of BC structures in a three-dimensional (3D) culture model, which is more relevant to the in vivo environment but more difficult to evaluate than 2D cultures. Various BC structures were identified inside HepG2 spheroids with the three features of RI distribution. Third, we conducted comparative analyses and found that the BC lumina of spheroids had higher circularity and lower RI standard deviation than 2D cultures. We also addressed comparison of BC and intracellular lumen-like structures within a HepG2 spheroid, and found that the BC lumina had higher RI and longer perimeter than intracellular lumen-like structures. Our demonstration of the non-destructive, label-free visualization and quantitative characterization of living BC structures will be a basis for various hepatological and pharmaceutical applications.

Investigation of the biological activity of hydroxyapatite on vascular endothelial cells

ObjectiveThis study aims to explore the impact of nano-hydroxyapatite (na-HA) and micron-hydroxyapatite (mi-HA) on human umbilical vein endothelial cells (HUVEC) using in vitro experiments, assessing their influence on cellular biological activity. These findings offer crucial experimental data for informing the development of more vascularized tissue-engineered bone constructs. MethodsWe employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of various concentrations of both HA extracts on HUVEC metabolic activity post 48, 72, and 96 h of treatment. Transwell experiments were conducted to evaluate the influence of HA extract on HUVEC migratory capabilities. The cell proliferation activity was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, elucidating the impact of varying concentrations of both HA extracts on cell proliferation. Lumen formation experiments were conducted to assess the capacity of HA-treated HUVECs to form lumen-like structures. The Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the impact of HA extract on vascular endothelial growth factor (VEGF) secretion by HUVECs. Western blotting (WB) was utilized to analyze alterations in the expression levels of PI3K/Akt signaling pathway-related proteins following HA extract treatment of cells. ResultsAt extract concentrations of 100 g/L and 12.5 g/L, both the mi-HA and na-HA groups demonstrated suppression of cell metabolic activity, migration, and proliferation. Conversely, at 25 g/L, increased cell metabolic activity and proliferative activity were observed. Lumen formation experiments demonstrated that both HA extracts at 100 g/L concentration facilitated lumen formation, with the na-HA group at 25 g/L concentration displaying a more pronounced impact on lumen formation. The ELISA results indicated a notable reduction in VEGF secretion within the mi-HA group at a concentration of 100 g/L. WB experiments revealed that within the na-HA group, treatment of HUVECs with 25 g/L and 12.5 g/L extract concentrations led to upregulation of PI3K and Akt protein expression, while at 100 g/L concentration, Akt protein expression decreased. In the mi-HA group, intracellular expression of both PI3K and Akt proteins exhibited reduction. ConclusionHydroxyapatite extract at both high and low concentrations impacts the biological activity of vascular endothelial cells, with the potential mechanism of action involving the PI3K/Akt signaling pathway.

Vascularization ability of glioma stem cells in different three-dimensional microenvironments.

Glioblastoma (GBM) is among the most common and aggressive adult central nervous system tumors. One prominent characteristic of GBM is the presence of abnormal microvessels. A significant correlation between angiogenesis and prognosis has been observed. Accurately reconstructing this neovascularization and tumor microenvironment through personalized in vitro disease models presents a significant challenge. However, it is crucial to develop new anti-angiogenic therapies for GBM. In this study, 3D bioprinted glioma stem cell (GSC)-laden hydrogel scaffolds, hybrid GSC hydrogels and cell-free hydrogel scaffolds were manufactured to investigate the vascularization ability of GSCs in varying 3D microenvironments. Our results demonstrated that the bioactivity of GSCs in the 3D bioprinted GSC-laden hydrogel scaffold was preferable and stable, and the amounts of vascular endothelial growth factor A and basic fibroblast growth factor were the highest in the microenvironment. When the three different models were co-cultured with human umbilical vein endothelial cells, the expression of angiogenesis-related markers was the most abundant in the bioprinted GSC-laden hydrogel scaffold. Additionally, xenograft tumors formed by bioprinted GSC-laden hydrogel scaffolds more closely resembled human gliomas regarding color, texture and vascularization. Notably, in xenograft tumors derived from 3D bioprinted GSC-laden hydrogel scaffolds, the number of human CD105+ cells was significantly higher, and human endothelial vascular lumen-like structures were observed. This indicates that the 3D bioprinted GSC-laden hydrogel scaffold is a suitable model for mimicking the glioma microenvironment and studying tumor angiogenesis.

Nestin identifies a subpopulation of rat ventricular fibroblasts and participates in cell migration.

Fibroblast progenitor cells migrate to the endocardial region during cardiogenesis, and the migration of ventricular fibroblasts to the ischemically damaged region of the infarcted adult heart is a seminal event of reparative fibrosis. The intermediate filament protein nestin is implicated in cell migration and expression identified in a subpopulation of scar-derived myofibroblasts. The present study tested the hypothesis that fibroblast progenitor cells express nestin, and the intermediate filament protein drives the migratory phenotype of ventricular fibroblasts. Transcription factor 21 (Tcf21)- and Wilms tumor 1 (WT1)-fibroblast progenitor cells identified in the epicardial/endocardial regions of the E12.5- to E13.5-day embryonic mouse heart predominantly expressed nestin. Nuclear Tcf21/WT1 staining was identified in neonatal rat ventricular fibroblasts (NNVFbs), and a subpopulation coexpressed nestin. Nuclear Tcf21/WT1 expression persisted in adult rat ventricular fibroblasts, whereas nestin protein levels were downregulated. Nestin-expressing NNVFbs exhibited a unique phenotype as the subpopulation was refractory to cell cycle reentry in response to selective stimuli. Nestin(-)- and nestin(+)-scar-derived rat myofibroblasts plated in Matrigel unmasked a migratory phenotype characterized by the de novo formation of lumen-like structures. The elongated membrane projections emanating from scar myofibroblasts delineating the boundary of lumen-like structures expressed nestin. Lentiviral short-hairpin RNA (shRNA)-mediated nestin depletion inhibited the in vitro migratory response of NNVFbs as the wound radius was significantly larger compared with NNVFbs infected with the empty lentivirus. Thus, nestin represents a marker of embryonic Tcf21/WT1(+)-fibroblast progenitor cells. The neonatal rat heart contains a distinct subpopulation of nestin-immunoreactive Tcf21/WT1(+) fibroblasts refractory to cell cycle reentry, and the intermediate filament protein may preferentially facilitate ventricular fibroblast migration during physiological/pathological remodeling.NEW & NOTEWORTHY Tcf21/WT1(+)-fibroblast progenitor cells of the embryonic mouse heart predominantly express the intermediate filament protein nestin. A subpopulation of Tcf21/WT1(+)-neonatal rat ventricular fibroblasts express nestin and are refractory to selective stimuli influencing cell cycle reentry. Scar-derived myofibroblasts plated in Matrigel elicit the formation of lumen-like structures characterized by the appearance of nestin(+)-membrane projections. Lentiviral shRNA-mediated nestin depletion in a subpopulation of neonatal rat ventricular fibroblasts suppressed the migratory response following the in vitro scratch assay.

Vascularization of a Bone Organoid Using Dental Pulp Stem Cells.

Bone organoids offer a novel path for the reconstruction and repair of bone defects. We previously fabricated scaffold-free bone organoids using cell constructs comprising only bone marrow-derived mesenchymal stem cells (BMSCs). However, the cells in the millimetre-scale constructs were likely to undergo necrosis because of difficult oxygen diffusion and nutrient delivery. Dental pulp stem cells (DPSCs) are capable of differentiating into vascular endothelial lineages and have great vasculogenic potential under endothelial induction. Therefore, we hypothesized that DPSCs can serve as a vascular source to improve the survival of the BMSCs within the bone organoid. In this study, the DPSCs had greater sprouting ability, and the proangiogenic marker expressions were significantly greater than those of BMSCs. DPSCs were incorporated into the BMSC constructs at various ratios (5%-20%), and their internal structures and vasculogenic and osteogenic characteristics were investigated after endothelial differentiation. As a result, the DPSCs are differentiated into the CD31-positive endothelial lineage in the cell constructs. The incorporation of DPSCs significantly suppressed cell necrosis and improved the viability of the cell constructs. In addition, lumen-like structures were visualized by fluorescently labelled nanoparticles in the DPSC-incorporated cell constructs. The vascularized BMSC constructs were successfully fabricated using the vasculogenic ability of the DPSCs. Next, osteogenic induction was initiated in the vascularized BMSC/DPSC constructs. Compared with only BMSCs, constructs with DPSCs had increased mineralized deposition and a hollow structure. Overall, this study demonstrated that vascularized scaffold-free bone organoids were successfully fabricated by incorporating DPSCs into BMSC constructs, and the biomimetic biomaterial is promising for bone regenerative medicine and drug development.

Biliary epithelial cell differentiation of bipotent human liver-derived organoids by 2D and 3D culture

Organoid culture is a technology for creating three-dimensional (3D) tissue-like structures in vitro, and is expected to be used in various fields. It was reported that human adult bile duct cells derived from human biopsy can be expanded as organoids in vitro that exhibit stem cell-like properties including high proliferative ability and differentiation ability toward both hepatocytes and biliary epithelial cells (BECs). Although many studies have achieved the efficient differentiation of bipotent human liver-derived organoids (hLOs) toward mature hepatocytes, the differentiation potency toward mature BECs remains unclear. In this study, we attempted to evaluate the differentiation potency of bipotent hLOs, which were generated from primary (cryopreserved) human hepatocytes (PHHs), toward BECs by sequential treatment with epidermal growth factor (EGF), Interleukin-6 (IL-6), and sodium taurocholate hydrate. Along with the differentiation toward bipotent hLOs-derived BECs (Org-BECs), increases in the gene expression levels of BEC markers and formation of the lumen-like structures typical of BECs were observed. In addition, Org-BECs exhibited P-glycoprotein-mediated drug transport capacity. Finally, in order to expand the applicability of Org-BECs, we succeeded in the differentiation of bipotent hLOs toward BECs in a two-dimensional (2D) culture system. Our findings demonstrated that bipotent hLOs can indeed differentiate into mature BECs, meaning that they possess a capacity for differentiation toward both hepatocytes and BECs.

Role of Heparan Sulfate in Vasculogenesis of Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell–extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, EXT1-silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of EXT1-silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with EXT1-silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein–transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with EXT1-silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.

P-023 Exogenous glial cell-derived neurotrophic factor contributes hepatocyte growth factor-induced in vitro organization of Sertoli cells on matrigel-based three-dimensional culture

Abstract Study question Does glial cell-derived neurotrophic factor (GDNF), a crucial factor for spermatogonial stem cell (SSC) self-renewal, have a role in in vitro tubule-formation capacity of Sertoli cells? Summary answer Exogenous GDNF treatment together with hepatocyte growth factor (HGF) significantly increased the in vitro tubule-forming capacity of Sertoli cells on matrigel-based three-dimensional (3D) culture system. What is known already GDNF increases the tubule-forming capacity of HGF in vitro by regulating c-Met expression during renal tubulogenesis. In vitro testicular organization of Sertoli cells consists of two stages: testicular cord-like structures and tubule-like structures. HGF secreted from Sertoli cells plays a direct role in forming seminiferous tubules in vivo and in vitro by activating c-Met. GDNF maintains SSC numbers by activating GFRα1 and Ret receptors in SSCs and can also directly phosphorylate c-Met in GFRα1-positive cells. Study design, size, duration Four groups were established using the 15P-1 Sertoli cell line: Control, HGF-administrated, GDNF-administrated and, HGF and GDNF-administrated. All groups were pursued on a matrigel-based 3D culture system for seven days. Experiments were repeated three times in all groups. Kit ligand (KitL, Sertoli cell marker; positive control) and c-kit (germ cell marker; negative control) were used as the control for Sertoli cell characterization. This work was supported by TUBITAK (SBAG-120S554) and Akdeniz University Research Foundation (TYL-2020-5481). Participants/materials, setting, methods The cord-forming and tubule-forming capacity of Sertoli cells on matrigel-based 3D culture was evaluated under an inverted microscope, and c-Met and p-c-Met expression levels were analyzed using western blot. In addition to KitL and c-kit protein expression, zonula occludens-1 (ZO-1) protein expression for polarization of Sertoli cells was evaluated by immunofluorescence staining. Data were analyzed using one-way ANOVA and Tukey post hoc test for multiple comparisons. p < 0.05 was considered statistically significant. Main results and the role of chance In the first 24 hours, interaction and aggregation between Sertoli cells began. The highest cellular interaction was found in the group treated with HGF and GDNF compared to the other groups (p < 0.05). At 48 and 72 hours, the formation of cord-like structures was observed by forming connections between the clusters formed in all groups in the first 24 hours. The number of cord-like structures was higher in the groups treated with only HGF and HGF and GDNF at 48th and 72nd hours (p < 0.05). At 96th and 120th hours, structures forming spaces were observed, and these structures were determined to be lumen-like structures. The capacity of 15P-1 Sertoli cells to form tubule-like structures was higher in the HGF and GDNF treated groups than in the other groups (p < 0.05). c-Met and p-c-Met protein expression levels were high in HGF, GDNF, HGF, and GDNF groups compared to control (P < 0.05). Sertoli cells retained their identity until the end of the culture and formed tight junctions in the HGF and GDNF-administrated group as evaluated by ZO-1 expression. Limitations, reasons for caution The cord- and tubule-forming capacity of Sertoli cells was evaluated in vitro utilizing exogenous GDNF treatment but the effect of testicular endothelial cell-derived GDNF should be also evaluated. Wider implications of the findings Exogenous GDNF treatment enhances HGF-induced in vitro tubule-formation capacity of Sertoli cells by directly phosphorylating c-Met in Sertoli cells. Our findings highlight the importance of designing studies related to GDNF-induced cell signaling pathway for in vitro tubulogenesis of Sertoli cells to support ex vivo spermatogenesis. Trial registration number None

Soluble ECM promotes organotypic formation in lung alveolar model

Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

Pancreatic extracellular matrix and platelet-rich plasma constructing injectable hydrogel for pancreas tissue engineering.

The development of pancreatic extracellular matrices enriched with insulin-secreting β-cells is a promising tissue engineering approach to treat type 1 diabetes. However, its long-term therapeutic efficacy is restricted by the defensive mechanism of host immune response and the lack of developed vascularization as appropriate after transplantation. Platelet-rich plasma (PRP), as an autologous platelet concentrate, contains a large number of active factors that are essential for the cell viability, vascularization, and immune regulation. In this study, we have incorporated pancreatic extracellular matrix (PEM) with PRP to develop a three-dimensional (3D) injectable PEM-PRP hydrogel to coculture and transplant rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVECs). Results from this study demonstrated that PEM-PRP hydrogel mimicked the biochemical compositions of native extracellular matrices, and possessed the enhanced elastic modulus and resistance to enzymatic degradation that enabled biomaterials to maintain its volume and slowly degrade. Additionally, PEM-PRP hydrogel could release growth factors in a sustained manner. In vitro, PEM-PRP hydrogel significantly promoted the viability, insulin-secreting function, and insulin gene expression of gel encapsulated INS-1 cells. Moreover, HUVECs encapsulated in PEM-PRP hydrogel were found to constitute many lumen-like structures and exhibited high expression of proangiogenic genes. In vivo transplantation of PEM-PRP hydrogel encapsulated with INS-1 cells and HUVECs improved the viability of INS-1 cells, promoted vascularization, inhibited the host inflammatory response, and reversed hyperglycemia of diabetic rats. Our study suggests that the PEM-PRP hydrogel offers excellent biocompatibility and proangiogenic property, and may serve as an effective biomaterial platform to maintain the long-term survival and function of insulin-secreting β cells.

Bioprinting of Complex Vascularized Tissues.

Functional vasculature is crucial for the maintenance of living tissues via the transport of oxygen, nutrients, and metabolic waste products. As a result, insufficient vascularization in thick engineered tissues will lead to cell death and necrosis due to mass transport and diffusional constraints. To circumvent these limitations, we describe the development of a microscale continuous optical bioprinting (μCOB) platform for 3D printing complex vascularized tissues with superior resolution and speed. By using the μCOB system, endothelial cells and other supportive cells can be printed directly into hydrogels with precisely controlled distribution and subsequent formation of lumen-like structures in vitro.

Coculture with noncardiac cells promoted maturation of human stem cell-derived cardiomyocyte microtissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CM) provided a promising cell source for cell therapy, drug screening, and disease modeling. However, hPSC-CM are immature and phenotypically more similar to fetal rather than adult cardiomyocytes in vitro. We explored the impact of coculture of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) and endothelial cells (ECs) with human embryonic stem cells-derived cardiac progenitor cells (hESC-CPC) on the gene expression and electrophysiological properties of hESC-CPC in 3D culture (microtissue spheroid). In this regard, hESC-CPC were cultured either alone (CM microtissue) or in coculture with EC and hESC-MSC (CMEM microtissue) on agar-coated 96-well round-bottomed plates for 1 week. Lumen-like structures were formed in CMEM but not in CM microtissue. Cardiac progenitor markers (TBX5, GATA4) were downregulated and cardiac sarcomeric transcripts (MLC2v and β-MHC) were upregulated in CMEM compared with CM microtissue. Furthermore, beating frequencies, beating cycles, and field potential durations of CMEM resided in the range of adult cardiomyocytes rather than fetal like phenotypes observed in CM microtissue. These findings demonstrated that CPC spheroids in coculture with EC and hESC-MSC may undergo greater maturation toward an adult-like cardiomyocyte.

SiRNA-mediated silencing of PAI-1 gene acts as a promoter over the recanalization of endothelial progenitor cells in rats with venous thrombosis.

With the changing lifestyle, venous thrombosis (VT) is becoming increasingly prevalent and poses a burden on the health economy. Endothelial progenitor cells (EPCs) are recruited into resolving VT. We aimed to investigate the effect of plasminogen activator inhibitor 1 (PAI-1) silencing on the recanalization of VT in rat EPCs. EPCs and VT rat models were cultured and treated with negative control-siRNA vector and PAI-1-siRNA vector, respectively. 4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound-healing test, and Matrigel-induced tubular experiment were performed to detect the ability of cell proliferation, migration, and EPCs lumen formation. Immunohistochemistry was used to observe the recanalization of thrombus. The messenger RNA (mRNA) and protein expression of PAI-1 and vascular endothelial growth factor (VEGF) were determined by reverse transcription quantitative polymerase chain reaction and Western blot analysis. PAI-1-siRNA enhances the luminal formation ability of EPCs and significantly promotes EPCs homing. In response to PAI-1 gene silencing, tissues from inferior vena cava displayed reduced mRNA and protein expression of PAI-1, increased VEGF expression as well as promoted lumen-like structures. PAI-1 gene silencing can promote the recanalization of VT by enhancement of the luminal formation ability of rats' EPCs.

A microfluidic device for study of the effect of tumor vascular structures on the flow field and HepG2 cellular flow behaviors

To build a microfluidic device with various morphological features of the tumor vasculature for study of the effects of tumor vascular structures on the flow field and tumor cellular flow behaviors. The designed microfluidic device was able to approximatively simulate the in vivo structures of tumor vessels and the flow within it. In this models, the influences of the angle of bifurcation, the number of branches, and the narrow channels on the flow field and the influence of vorticity on the retention of HepG2 cells were significant. Additionally, shear stress below physiological conditions of blood circulation has considerable effect on the formation of the lumen-like structures (LLSs) of HepG2 cells. These results can provide some data and reference in the understanding of the interaction between hemorheological properties and tumor vascular structures in solid tumors.

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

Direct 3D bioprinting of prevascularized tissue constructs with complex microarchitecture.

Living tissues rely heavily on vascular networks to transport nutrients, oxygen and metabolic waste. However, there still remains a need for a simple and efficient approach to engineer vascularized tissues. Here, we created prevascularized tissues with complex three-dimensional (3D) microarchitectures using a rapid bioprinting method - microscale continuous optical bioprinting (μCOB). Multiple cell types mimicking the native vascular cell composition were encapsulated directly into hydrogels with precisely controlled distribution without the need of sacrificial materials or perfusion. With regionally controlled biomaterial properties the endothelial cells formed lumen-like structures spontaneously invitro. Invivo implantation demonstrated the survival and progressive formation of the endothelial network in the prevascularized tissue. Anastomosis between the bioprinted endothelial network and host circulation was observed with functional blood vessels featuring red blood cells. With the superior bioprinting speed, flexibility and scalability, this new prevascularization approach can be broadly applicable to the engineering and translation of various functional tissues.

A novel 3-D bio-microfluidic system mimicking in vivo heterogeneous tumour microstructures reveals complex tumour-stroma interactions.

A 3-D microfluidic system consisting of microchamber arrays embedded in a collagen hydrogel with tuneable biochemical gradients that mimics the tumour microenvironment of mammary glands was constructed for the investigation on the interactions between invasive breast cancer cells and stromal cells. The hollow microchambers in collagen provide a very similar 3-D environment to that in vivo that regulates collective cellular dynamics and behaviour, while the microfluidic channels surrounding the collagen microchamber arrays allow one to impose complex concentration gradients of specific biological molecules or drugs. We found that breast epithelial cells (MCF-10A) seeded in the microchambers formed lumen-like structures similar to those in epithelial layers. When MCF-10A cells were co-cultured with invasive breast cancer cells (MDA-MB-231), the formation of lumen-like structures in the microchambers was inhibited, indicating the capability of cancer cells to disrupt the structures formed by surrounding cells for further invasion and metastasis. Subsequent mechanism studies showed that down regulation of E-cad expression due to MMPs produced by the cancer cells plays a dominant role in determining the cellular behaviour. Our microfluidic system offers a robust platform for high throughput studies that aim to understand combinatorial effects of multiple biochemical and microenvironmental factors.