High-resolution intravital imaging for monitoring the transplanted islets in mice.

Intravital imaging techniques will be a valuable tool to monitor the post-transplantation dynamics of the cells/tissues in regenerative medicine research. Among the conventional live imaging techniques, the cranial window model has various advantages regarding resolution, longevity, and easy manipulability. We describe the use of the cranial window model to visualize the post-transplantation processes of primary pancreatic islets in the living mouse. Macroscopic or microscopic analyses were performed to evaluate the post-transplantation dynamics of primary murine islets, including the revascularization process inside the cranium. Consistent with earlier literature on clinical outcomes of islet transplantation, marked loss of transplanted islets was observed within 7 days. Intravital confocal microscope analysis revealed that functional revascularization seldom occurred in the central regions of the transplants. Our results suggest that the cranial window model offers an ideal platform for understanding cellular dynamics, through the possibility of long-term imaging studies over time scales. This platform is possibly applied not only for transplant studies of pancreatic islets, but also for other endodermal cell/tissue types in vivo.

Self-formation of vascularized hepatic tissue from human adult hepatocyte.

BACKGROUND: Recent study has demonstrated the important role of endothelial-mesenchymal interactions in 3-dimensional self-organization of immature progenitor populations with the use of mimicking of organogenesis. Here, we show that the same principle can be applicable to adult mature cells, ie, human adult hepatocytes (hAHs). METHODS: We cultivated hAHs with fluorescence-labeled human mesenchymal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in micro-well culture plates and observed them for 9 days. Fluorescence microscopy imaging analyses were performed to evaluate the internal structures of generated 3-dimensional tissues. Maintenance of in vitro protein production capacity was examined with the use of enzyme-linked immunosorbent assay (ELISA). RESULTS: hAHs started to self-organize into 3-dimensional tissue with the use of coculturing with hMSCs and HUVECs. Live imaging analyses showed that endothelial cells started sprouting inside the generated tissues after 2 days of culture. ELISA showed that human albumin production capacity was improved with the use of coculture compared with hAHs-only culture after 9 days. CONCLUSIONS: We demonstrated that 3-dimensional vascularized hepatic tissue could be generated from hAHs by reconstituting endothelial-mesenchymal interactions. Future studies are needed to evaluate the therapeutic potential of vascularized hepatic tissue transplantation, and this may pave a new way to establish a new transplantation modality as an alternative to hepatocyte transplantation.

Self-organization of human hepatic organoid by recapitulating organogenesis in vitro.

BACKGROUND: Careful orchestration among endodermal epithelial, endothelial, and mesenchymal cells initiate liver organogenesis prior to vascular function. Nonparenchymal endothelial or mesenchymal cells not only form passive conduits, but also establish an organogenic stimulus. Herein, we have evaluated the potential roles of primitive endothelial and mesenchymal cells toward hepatic organization in vitro. METHODS: To track the cellular movements and localization, we retrovirally transduced enhanced green fluorescence protein and kusabira orange into human fetal liver cells (GFP-hFLCs) and human umbilical vein endothelial cells (KO-HUVECs), respectively. GFP-hFLCs were cocultivated with KO-HUVECs and human mesenchymal stem cells (hMSCs) under conventional two-dimensional (2D) conditions. RESULTS: Even under 2D culture, fetal liver, endothelial, and mesenchymal cells self-organized into a macroscopically visible three-dimensional (3D) organoid. Time-lapse confocal imaging showed dynamic cellular organizations of GFP-hFLCs and KO-HUVECs. Endothelial cells organized into patterned clusters wrapping fetal liver cells, forming vessel-like lumens inside. Mesenchymal cells supported the generated organoid from outside. CONCLUSION: Generation of whole organ architecture remains a great challenge so far. Our preliminary results showed that recapitulation of primitive cellular interactions during organogenesis elicit the intrinsic self-organizing capacity to form hepatic organoids. Future studies to define precise conditions mimicking organogenesis may ultimately lead to the generation of a functional liver for transplantation and for other applications such as drug development.

Regenerative medicine approach as an alternative treatment to islet transplantation.

Islet transplantation is considered to be one of the most promising treatment for type I diabetes mellitus (TID). Development of the Edmonton protocol opened the possibility of insulin independence for the patients with TID. However, there is the problem of the donor shortage. Herein we have discussed recent approaches to overcome the problem. It is neccessary to develop a new cellular source for donor islets and to achieve a high engraftment rate. One advantage in TID therapy is that allogeneic islet transplantation is allowed to avoid autoimmunity. That opens broad candidates for the beta-cell source. To achieve a high engraftment rate, is several attempts have sought to develop an appropriate site for transplantation and to modify beta-cells for long-term survival. It is also important to achieve early onset of blood perfusion after transplantation by prevascularization of the islets in vitro. These multiple approaches will bring a milestone in diabetes therapy.

Generation of functional human vascular network.

BACKGROUND: One of the major obstacles in regenerating thick, complex tissues such as the liver is their need for vascularization, which is essential to maintain cell viability during tissue growth and to induce structural organization. Herein, we have described a method to engineer a functional human vascular network. METHODS: Enhanced green fluorescence protein-labeled human umbilical vein endothelial cells (GFP-HUVECs) were cocultivated with kusabira orange-labeled human mesenchymal stem cells (KO-hMSCs) inside a collagen/fibronectin matrix. Premature vascular network formation was visualized by fluorescence microscopy imaging. Furthermore, constructs prevascularized in vitro were implanted into a transparency window in immunodeficient mice. RESULTS: Following several days of cultivation, GFP-HUVECs formed vessel-like structures that were stabilized by pericytes differentiated from KO-hMSCs. After implantation in vivo, the patency of human vascular structures was proved by rhodamine dextran infusion. These functional vascular structures remained for over 2 months. DISCUSSION: Vascularization is the key challenge to organ generation. We successfully generated human vascular networks inside a matrix. Integration of parenchymal cells using our engineering technique should facilitate future efforts to reconstitute vascularized human organ systems in vitro.