Takanori Takebe

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.

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.

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.

Multilineage communication regulates human liver bud development from pluripotency

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor–ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Reconstruction of human elastic cartilage by a CD44+ CD90+ stem cell in the ear perichondrium

Despite the great demands for treating craniofacial injuries or abnormalities, effective treatments are currently lacking. One promising approach involves human elastic cartilage reconstruction using autologous stem/progenitor populations. Nevertheless, definitive evidence of the presence of stem cells in human auricular cartilage remains to be established. Here, we demonstrate that human auricular perichondrium, which can be obtained via a minimally invasive approach, harbors a unique cell population, termed as cartilage stem/progenitor cells (CSPCs). The clonogenic progeny of a single CD44+ CD90+ CSPC displays a number of features characteristic of stem cells. Highly chondrogenic CSPCs were shown to reconstruct large (>2 cm) elastic cartilage after extended expansion and differentiation. CSPC-derived cartilage was encapsulated by a perichondrium layer, which contains a CD44+ CD90+ self-renewing stem/progenitor population and was maintained without calcification or tumor formation even after 10 mo. This is a unique report demonstrating the presence of stem cells in auricular cartilage. Utilization of CSPCs will provide a promising reconstructive material for treating craniofacial defects with successful long-term tissue restoration.

Novel strategies for liver therapy using stem cells

Liver disease is an increasing clinical burden, causing over 10 000 deaths last year in the UK alone (http://www.britishlivertrust.org.uk). Liver insufficiency describes the clinical situation in which cumulative (chronic) or one-off massive (acute) insults exceed the livers normal physiological capacity to functionally regenerate. Untreated, liver insufficiency invariably leads to death. The current gold standard of care in this setting is whole organ transplantation. Due to the increasing burden of disease within the population, however, the number of patients requiring transplantation far exceeds the number of available donor organs. As a result, many patients with liver insufficiency die prematurely.1 The liver is composed of several cell types (endothelial cells, stellate cells, biliary ductal cells, Kupffer cells and natural killer cells) which together provide a supportive niche for the principle cell type—the hepatocyte.2 Treatment of liver disease by hepatocyte replacement therefore appears a logical alternative to whole organ transplant (figure 1i). Along these lines, intrahepatic hepatocyte transplantation (I-HTx) has repeatedly proven efficacious in small animal models of numerous liver diseases. Results from human applications have unfortunately proven less convincing however with the best, possibly only, positive results observed in paediatric patients suffering from hepatocyte driven inherited metabolic disorders.3 This suggests targeting the surrounding niche may be as important as replacing diseased hepatocytes themselves if one is to treat the majority of liver diseases.4 Such efforts to manipulate the complex nature of the surrounding niche will no doubt be far from trivial. So an alternative, more tangible approach that targets more specific clinical problem could be to use hepatocytes in extra-hepatic anatomical sites to ‘bridge’ patients with liver insufficiency to transplant or self repair (E-HTx).5 Some preliminary clinical experiences suggest a therapeutic effect for alginate-bead encapsulated hepatocytes delivered into the peritoneum of paediatric patients with acute liver failure …

Synergistic Engineering: Organoids Meet Organs-on-a-Chip

Organoid technology and organ-on-a-chip engineering have emerged as two distinct approaches for stem cell-derived 3D tissue preparation. Their strategic integration can address each approachs limitations and provide a path toward a superior, synergistic strategy of constructing tissues that will truly deliver on the promise of regenerative and precision medicine.

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.

Presence of Cartilage Stem/Progenitor Cells in Adult Mice Auricular Perichondrium

Background Based on evidence from several other tissues, cartilage stem/progenitor cells in the auricular cartilage presumably contribute to tissue development or homeostasis of the auricle. However, no definitive studies have identified or characterized a stem/progenitor population in mice auricle. Methodology/Principal Findings The 5-bromo-2′-deoxyuridine (BrdU) label-retaining technique was used to label dividing cells in fetal mice. Observations one year following the labeling revealed that label-retaining cells (LRCs) were present specifically in auricular perichondrium at a rate of 0.08±0.06%, but LRCs were not present in chondrium. Furthermore, LRCs were successfully isolated and cultivated from auricular cartilage. Immunocytochemical analyses showed that LRCs express CD44 and integrin-α5. These LRCs, putative stem/progenitor cells, possess clonogenicity and chondrogenic capability in vitro. Conclusions/Significance We have identified a population of putative cartilage stem/progenitor cells in the auricular perichondrium of mice. Further characterization and utilization of the cell population should improve our understanding of basic cartilage biology and lead to advances in cartilage tissue engineering and novel therapeutic strategies for patients with craniofacial defects, including long-term tissue restoration.

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.