Development of a Method for Scaffold-Free Elastic Cartilage Creation.

Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.

Vascularized and Complex Organ Buds from Diverse Tissues via Mesenchymal Cell-Driven Condensation.

Transplantation of in-vitro-generated organ buds is a promising approach toward regenerating functional and vascularized organs. Though it has been recently shown in the context of liver models, demonstrating the applicability of this approach to other systems by delineating the molecular mechanisms guiding organ bud formation is critical. Here, we demonstrate a generalized method for organ bud formation from diverse tissues by combining pluripotent stem cell-derived tissue-specific progenitors or relevant tissue samples with endothelial cells and mesenchymal stem cells (MSCs). The MSCs initiated condensation within these heterotypic cell mixtures, which was dependent upon substrate matrix stiffness. Defining optimal mechanical properties promoted formation of 3D, transplantable organ buds from tissues including kidney, pancreas, intestine, heart, lung, and brain. Transplanted pancreatic and renal buds were rapidly vascularized and self-organized into functional, tissue-specific structures. These findings provide a general platform for harnessing mechanical properties to generate vascularized, complex organ buds with broad applications for regenerative medicine.

Efficient hepatic differentiation of human induced pluripotent stem cells in a three-dimensional microscale culture.

Human induced pluripotent stem cells (iPSCs) represent a novel source of hepatocytes for drug development, disease modeling studies, and regenerative therapy for the treatment of liver diseases. A number of protocols for generating functional hepatocytes have been reported worldwide; however, reproducible and efficient differentiation remained challenging under conventional two-dimensional (2D) culture. In this study, we describe an efficient differentiation protocol for generating functional hepatocyte-like cells from human iPSC-derived homogenous hepatic endoderm cells combined with three-dimensional (3D) microscale culture system. First, hepatic endoderm cells (iPSC-HEs) were directly differentiated using two-step approaches, and then cultured in the 3D micropattern plate. Human iPSC-HEs quickly reaggregated and formed hundreds of round-shaped spheroids at day 4 of cell plating. The size distribution of iPSC-HEs derived spheroids was relatively uniform around 100-200 µm in diameter. After 14 days, iPSC-HEs efficiently differentiated into hepatocyte-like cells in terms of hepatic maker gene expression compared with conventional 2D approach. We conclude that our scalable and three-dimensional culture system would be one promising approach to generate a huge number of hepatocyte-like cells from human iPSCs aiming at future industrial and clinical applications.

Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant.

Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCs) will facilitate our understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from human iPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSC-derived specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1-2 d. To facilitate additional maturation, 4-d-old iPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple hepatic functions in a chronological manner in vivo.

Vascularized and functional human liver from an iPSC-derived organ bud transplant.

A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.