Generation of Cortical Brain Organoid with Vascularization by Assembling with Vascular Spheroid.

BACKGROUND AND OBJECTIVES: Brain organoids have the potential to improve our understanding of brain development and neurological disease. Despite the importance of brain organoids, the effect of vascularization on brain organoids is largely unknown. The objective of this study is to develop vascularized organoids by assembling vascular spheroids with cerebral organoids. METHODS AND RESULTS: In this study, vascularized spheroids were generated from non-adherent microwell culture system of human umbilical vein endothelial cells, human dermal fibroblasts and human umbilical cord blood derived mesenchymal stem cells. These vascular spheroids were used for fusion with iPSCs induced cerebral organoids. Immunostaining studies of vascularized organoids demonstrated well organized vascular structures and reduced apoptosis. We showed that the vascularization in cerebral organoids up-regulated the Wnt/ß-catenin signaling. CONCLUSIONS: We developed vascularized cerebral organoids through assembly of brain organoids with vascular spheroids. This method could not only provide a model to study human cortical development but also represent an opportunity to explore neurological disease.

Establishing a 3D In Vitro Hepatic Model Mimicking Physiologically Relevant to In Vivo State.

Three-dimensional (3D) bioprinting is a promising technology to establish a 3D in vitro hepatic model that holds great potential in toxicological evaluation. However, in current hepatic models, the central area suffers from hypoxic conditions, resulting in slow and weak metabolism of drugs and toxins. It remains challenging to predict accurate drug effects in current bioprinted hepatic models. Here, we constructed a hexagonal bioprinted hepatic construct and incorporated a spinning condition with continuous media stimuli. Under spinning conditions, HepG2 cells in the bioprinted hepatic construct exhibited enhanced proliferation capacity and functionality compared to those under static conditions. Additionally, the number of spheroids that play a role in boosting drug-induced signals and responses increased in the bioprinted hepatic constructs cultured under spinning conditions. Moreover, HepG2 cells under spinning conditions exhibited intensive TGFß-induced epithelial-to-mesenchymal transition (EMT) and increased susceptibility to acetaminophen (APAP)-induced hepatotoxicity as well as hepatotoxicity prevention by administration of N-acetylcysteine (NAC). Taken together, the results of our study demonstrate that the spinning condition employed during the generation of bioprinted hepatic constructs enables the recapitulation of liver injury and repair phenomena in particular. This simple but effective culture strategy facilitates bioprinted hepatic constructs to improve in vitro modeling for drug effect evaluation.

Modeling of Hypoxic Brain Injury through 3D Human Neural Organoids.

Brain organoids have emerged as a novel model system for neural development, neurodegenerative diseases, and human-based drug screening. However, the heterogeneous nature and immature neuronal development of brain organoids generated from pluripotent stem cells pose challenges. Moreover, there are no previous reports of a three-dimensional (3D) hypoxic brain injury model generated from neural stem cells. Here, we generated self-organized 3D human neural organoids from adult dermal fibroblast-derived neural stem cells. Radial glial cells in these human neural organoids exhibited characteristics of the human cerebral cortex trend, including an inner (ventricular zone) and an outer layer (early and late cortical plate zones). These data suggest that neural organoids reflect the distinctive radial organization of the human cerebral cortex and allow for the study of neuronal proliferation and maturation. To utilize this 3D model, we subjected our neural organoids to hypoxic injury. We investigated neuronal damage and regeneration after hypoxic injury and reoxygenation. Interestingly, after hypoxic injury, reoxygenation restored neuronal cell proliferation but not neuronal maturation. This study suggests that human neural organoids generated from neural stem cells provide new opportunities for the development of drug screening platforms and personalized modeling of neurodegenerative diseases, including hypoxic brain injury.

Development of highly functional bioengineered human liver with perfusable vasculature.

Liver tissue engineering offers a promising strategy for liver failure patients. Since transplantation rejection resulting in vessel thrombosis is regarded as a major hurdle, vascular reconstruction is one of indispensable requirements of whole organ engineering. Here we demonstrated a novel strategy for reconstruction of a vascularized bioengineered human liver (VBHL) using decellularized liver scaffolds in an efficient manner. First we achieved fully functional endothelial coverage of scaffolds by adopting the anti-CD31 aptamer as a potent coating agent for re-endothelialization. Through an ex vivo human blood perfusion that recapitulates the blood coagulation response in humans, we demonstrated significantly reduced platelet aggregation in anti-CD31 aptamer coated scaffolds. We then produced VBHL constructs using liver parenchymal cells and nonparenchymal cells, properly organized into liver-like structures with an aligned vasculature. Interestingly, VBHL constructs displayed prominently enhanced long-term liver-specific functions that are affected by vascular functionality. The VBHL constructs formed perfusable vessel networks in vivo as evidenced by the direct vascular connection between the VBHL constructs and the renal circulation. Furthermore, heterotopic transplantation of VBHL constructs supported liver functions in a rat model of liver fibrosis. Overall, we proposed a new strategy to generate transplantable bioengineered livers characterized by highly functional vascular reconstruction.

Repeated intramuscular transplantations of hUCB-MSCs improves motor function and survival in the SOD1 G93A mice through activation of AMPK.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by loss of motor neurons and degeneration of neuromuscular junctions. To improve disease progression, previous studies have suggested many options that have shown beneficial effects in diseases, especially stem cell therapy. In this study, we used repeated intramuscular transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and observed positive effects on muscle atrophy and oxidative stress. In an in vivo study, motor function, body weight and survival rate were assessed, and skeletal muscle tissues were analyzed by western blotting and immunohistochemistry. After intramuscular transplantation, the hUCB-MSCs survived within the skeletal muscle for at least 1 week. Transplantation ameliorated muscle atrophy and the rate of neuromuscular degeneration in skeletal muscle through reductions in intracellular ROS levels. Both expression of skeletal muscle atrophy markers, muscle atrophy F-box (MAFbx)/atrogin1 and muscle RING finger 1 (MuRF1), were also reduced; however, the reductions were not significant. Moreover, transplantation of hUCB-MSCs improved protein synthesis and inhibited the iNOS/NO signaling pathway through AMPK activation. Our results suggest that repeated intramuscular transplantation of hUCB-MSCs can be a practical option for stem cell therapy for ALS.

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.