Background & Aims: Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. Methods: Functional analyses of polycomb-group protein Bmi1 and Wnt/β-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit⁻CD29⁺CD49f^+/lowCD45⁻Ter-119⁻ hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. Results: Forced expression of Bmi1 and constitutively active β-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or β-catenin–transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. Conclusions: These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/β-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.

Therapies against hepatitis B virus (HBV) have improved in recent decades; however, the development of individualized treatments has been limited by the lack of individualized infection models. In this study, we used human induced pluripotent stem cell (hiPSC) to generate a functional liver organoid (LO) that inherited the genetic background of the donor, and evaluated its application in modeling HBV infection and exploring virus-host interactions. To establish a functional hiPSC-LO, we cultured hiPSC-derived endodermal, mesenchymal, and endothelial cells with a chemically defined medium in a three-dimensional microwell culture system. Based on cell-cell interactions, these cells could organize themselves and gradually differentiate into a functional organoid, which exhibited stronger hepatic functions than hiPSC derived hepatic like cell (HLC). Moreover, the functional LO demonstrated more susceptibility to HBV infection than hiPSC-HLC, and could maintain HBV propagation and produce infectious virus for a prolonged duration. Furthermore, we found that virus infection could cause hepatic dysfunction of hiPSC-LOs, with down-regulation of hepatic gene expression, induced release of early acute liver failure markers, and altered hepatic ultrastructure. Therefore, our study demonstrated that HBV infection in hiPSC-LOs could recapitulate virus life cycle and virus induced hepatic dysfunction, suggesting that hiPSC-LOs may provide a promising individualized infection model for the development of individualized treatment for hepatitis.

The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self-renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell-based analysis to examine precisely bilineage differentiation potential and self-renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133+ cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo . Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self-renewing cell divisions. The clonogenic progeny of an LC-forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC-forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell-like capacity of isolated and clonally cultured CD133+ LC-forming cells. Conclusion: Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis. (Hepatology 2008;48:1964-1978.)

Glucagon-like peptide (GLP) 1 is produced through posttranslational processing of proglucagon and acts as a regulator of various homeostatic events. Among its analogs, however, the function of GLP-1-(1–37), synthesized in small amounts in the pancreas, has been unclear. Here, we find that GLP-1-(1–37) induces insulin production in developing and, to a lesser extent, adult intestinal epithelial cells in vitro and in vivo , a process mediated by up-regulation of the Notch-related gene ngn3 and its downstream targets, which are involved in pancreatic endocrine differentiation. These cells became responsive to glucose challenge in vitro and reverse insulin-dependent diabetes after implantation into diabetic mice. Our findings suggest that efficient induction of insulin production in intestinal epithelial cells by GLP-1-(1–37) could represent a new therapeutic approach to diabetes mellitus.

Acute liver failure (ALF) is a life-threatening disease with a high mortality rate. However, there are limited treatments or devices available for ALF therapy. Here, we aimed to develop a new strategy for ALF treatment by transplanting functional liver organoids (LOs) generated from single donor-derived human induced pluripotent stem cell (hiPSC) endoderm, endothelial cells (ECs), and mesenchymal cells (MCs). First, we isolated ECs and MCs from a single donor umbilical cord (UC) through enzyme digestion and characterized the UC-ECs and UC-MCs by flow cytometry. Second, using a nonviral reprogramming method, we generated same donor-derived hiPSCs from the UC-ECs and investigated their hepatic differentiation abilities. Finally, we simultaneously plated EC-hiPSC endoderm, UC-ECs, and UC-MCs in a three-dimensional (3D) microwell culture system, and generated single donor cell-derived differentiated LOs for ALF mouse treatment. We obtained ECs and MCs from a single donor UC with high purity, and these cells provided a multicellular microenvironment that promoted LO differentiation. hiPSCs from the same donor were generated from UC-ECs, and the resultant EC-hiPSCs could be differentiated efficiently into pure definitive endoderm and further into hepatic lineages. Simultaneous plating of EC-hiPSC endoderm, UC-ECs, and UC-MCs in the 3D microwell system generated single donor cell-derived LOs (SDC-LOs) that could be differentiated into functional LOs with enhanced hepatic capacity as compared to that of EC-hiPSC-derived hepatic-like cells. When these functional SDC-LOs were transplanted into the renal subcapsules of ALF mice, they rapidly assumed hepatic functions and improved the survival rate of ALF mice. These results demonstrate that functional LOs generated from single donor cells can improve the condition of ALF mice. Functional SDC-LO transplantation provides a promising novel approach for ALF therapy.

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.