Human iPSC-liver organoid transplantation reduces fibrosis through immunomodulation.

Donor organ shortages for transplantation remain a serious global concern, and alternative treatment is in high demand. Fetal cells and tissues have considerable therapeutic potential as, for example, organoid technology that uses human induced pluripotent stem cells (hiPSCs) to generate unlimited human fetal-like cells and tissues. We previously reported the in vivo vascularization of early fetal liver-like hiPSC-derived liver buds (LBs) and subsquent improved survival of recipient mice with subacute liver failure. Here, we show hiPSC-liver organoids (LOs) that recapitulate midgestational fetal liver promote de novo liver generation when grafted onto the surface of host livers in chemical fibrosis models, thereby recovering liver function. We found that fetal liver, a hematopoietic tissue, highly expressed macrophage-recruiting factors and antifibrotic M2 macrophage polarization factors compared with the adult liver, resulting in fibrosis reduction because of CD163+ M2-macrophage polarization. Next, we created midgestational fetal liver-like hiPSC-LOs by fusion of hiPSC-LBs to induce static cell-cell interactions and found that these contained complex structures such as hepatocytes, vasculature, and bile ducts after transplantation. This fusion allowed the generation of a large human tissue suitable for transplantation into immunodeficient rodent models of liver fibrosis. hiPSC-LOs showed superior liver function compared with hiPSC-LBs and improved survival and liver function upon transplantation. In addition, hiPSC-LO transplantation ameliorated chemically induced liver fibrosis, a symptom of liver cirrhosis that leads to organ dysfunction, through immunomodulatory effects, particularly on CD163+ phagocytic M2-macrophage polarization. Together, our results suggest hiPSC-LO transplantation as a promising therapeutic option for liver fibrosis.

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell-derived micro 3D spheroids.

Morphologically stable scaffold-free elastic cartilage tissue is crucial for treating external ear abnormalities. However, establishing adequate mechanical strength is challenging, owing to the difficulty of achieving chondrogenic differentiation in vitro; thus, cartilage reconstruction is a complex task. Auricular perichondrial chondroprogenitor cells exhibit high proliferation potential and can be obtained with minimal invasion. Therefore, these cells are an ideal resource for elastic cartilage reconstruction. In this study, we aimed to develop a novel in vitro scaffold-free method for elastic cartilage reconstruction, using human auricular perichondrial chondroprogenitor cells. Inducing chondrogenesis by using microscopic spheroids similar to auricular hillocks significantly increased the chondrogenic potential. The size and elasticity of the tissue were maintained after craniofacial transplantation in immunodeficient mice, suggesting that the reconstructed tissue was morphologically stable. Our novel tissue reconstruction method may facilitate the development of future treatments for external ear abnormalities.

Development of the nervous system in mouse liver.

BACKGROUND: The role of the hepatic nervous system in liver development remains unclear. We previously created functional human micro-hepatic tissue in mice by co-culturing human hepatic endodermal cells with endothelial and mesenchymal cells. However, they lacked Glisson's sheath [the portal tract (PT)]. The PT consists of branches of the hepatic artery (HA), portal vein, and intrahepatic bile duct (IHBD), collectively called the portal triad, together with autonomic nerves. AIM: To evaluate the development of the mouse hepatic nervous network in the PT using immunohistochemistry. METHODS: Liver samples from C57BL/6J mice were harvested at different developmental time periods, from embryonic day (E) 10.5 to postnatal day (P) 56. Thin sections of the surface cut through the hepatic hilus were examined using protein gene product 9.5 (PGP9.5) and cytokeratin 19 (CK19) antibodies, markers of nerve fibers (NFs), and biliary epithelial cells (BECs), respectively. The numbers of NFs and IHBDs were separately counted in a PT around the hepatic hilus (center) and the peripheral area (periphery) of the liver, comparing the average values between the center and the periphery at each developmental stage. NF-IHBD and NF-HA contacts in a PT were counted, and their relationship was quantified. SRY-related high mobility group-box gene 9 (SOX9), another BEC marker; hepatocyte nuclear factor 4α (HNF4α), a marker of hepatocytes; and Jagged-1, a Notch ligand, were also immunostained to observe the PT development. RESULTS: HNF4α was expressed in the nucleus, and Jagged-1 was diffusely positive in the primitive liver at E10.5; however, the PGP9.5 and CK19 were negative in the fetal liver. SOX9-positive cells were scattered in the periportal area in the liver at E12.5. The Jagged-1 was mainly expressed in the periportal tissue, and the number of SOX9-positive cells increased at E16.5. SOX9-positive cells constructed the ductal plate and primitive IHBDs mainly at the center, and SOX-9-positive IHBDs partly acquired CK19 positivity at the same period. PGP9.5-positive bodies were first found at E16.5 and HAs were first found at P0 in the periportal tissue of the center. Therefore, primitive PT structures were first constructed at P0 in the center. Along with remodeling of the periportal tissue, the number of CK19-positive IHBDs and PGP9.5-positive NFs gradually increased, and PTs were also formed in the periphery until P5. The numbers of NFs and IHBDs were significantly higher in the center than in the periphery from E16.5 to P5. The numbers of NFs and IHBDs reached the adult level at P28, with decreased differences between the center and periphery. NFs associated more frequently with HAs than IHBDs in PTs at the early phase after birth, after which the number of NF-IHBD contacts gradually increased. CONCLUSION: Mouse hepatic NFs first emerge at the center just before birth and extend toward the periphery. The interaction between NFs and IHBDs or HAs plays important roles in the morphogenesis of PT structure.

Dorso-ventral heterogeneity in tracheal basal stem cells.

The tracheal basal cells (BCs) function as stem cells to maintain the epithelium in steady state and repair it after injury. The airway is surrounded by cartilage ventrolaterally and smooth muscle dorsally. Lineage tracing using Krt5-CreER shows dorsal BCs produce more, larger, clones than ventral BCs. Large clones were found between cartilage and smooth muscle where subpopulation of dorsal BCs exists. Three-dimensional organoid culture of BCs demonstrated that dorsal BCs show higher colony forming efficacy to ventral BCs. Gene ontology analysis revealed that genes expressed in dorsal BCs are enriched in wound healing while ventral BCs are enriched in response to external stimulus and immune response. Significantly, ventral BCs express Myostatin, which inhibits the growth of smooth muscle cells, and HGF, which facilitates cartilage repair. The results support the hypothesis that BCs from the dorso-ventral airways have intrinsic molecular and behavioural differences relevant to their in vivo function.

A Novel Orthotopic Liver Cancer Model for Creating a Human-like Tumor Microenvironment.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. This study aims to develop a new method to generate an HCC mouse model with a human tumor, and imitates the tumor microenvironment (TME) of clinical patients. Here, we have generated functional, three-dimensional sheet-like human HCC organoids in vitro, using luciferase-expressing Huh7 cells, human iPSC-derived endothelial cells (iPSC-EC), and human iPSC-derived mesenchymal cells (iPSC-MC). The HCC organoid, capped by ultra-purified alginate gel, was implanted into the disrupted liver using an ultrasonic homogenizer in the immune-deficient mouse, which improved the survival and engraftment rate. We successfully introduced different types of controllable TME into the model and studied the roles of TME in HCC tumor growth. The results showed the role of the iPSC-EC and iPSC-MC combination, especially the iPSC-MC, in promoting HCC growth. We also demonstrated that liver fibrosis could promote HCC tumor growth. However, it is not affected by non-alcoholic fatty liver disease. Furthermore, the implantation of HCC organoids to humanized mice demonstrated that the immune response is important in slowing down tumor growth at an early stage. In conclusion, we have created an HCC model that is useful for studying HCC development and developing new treatment options in the future.

Establishment of Human Leukocyte Antigen-Mismatched Immune Responses after Transplantation of Human Liver Bud in Humanized Mouse Models.

Humanized mouse models have contributed significantly to human immunology research. In transplant immunity, human immune cell responses to donor grafts have not been reproduced in a humanized animal model. To elicit human T-cell immune responses, we generated immune-compromised nonobese diabetic/Shi-scid, IL-2RγKO Jic (NOG) with a homozygous expression of human leukocyte antigen (HLA) class I heavy chain (NOG-HLA-A2Tg) mice. After the transplantation of HLA-A2 human hematopoietic stem cells into NOG-HLA-A2Tg, we succeeded in achieving alloimmune responses after the HLA-mismatched human-induced pluripotent stem cell (hiPSC)-derived liver-like tissue transplantation. This immune response was inhibited by administering tacrolimus. In this model, we reproduced allograft rejection after the human iPSC-derived liver-like tissue transplantation. Human tissue transplantation on the humanized mouse liver surface is a good model that can predict T-cell-mediated cellular rejection that may occur when organ transplantation is performed.

The Regenerative Effect of Portal Vein Injection of Liver Organoids by Retrorsine/Partial Hepatectomy in Rats.

In this study, we reveal that liver organoid transplantation through the portal vein is a safe and effective method for the treatment of chronic liver damage. The liver organoids significantly reconstituted the hepatocytes; hence, the liver was significantly enlarged in this group, compared to the monolayer cell transplantation group in the retrorsine/partial hepatectomy (RS/PH) model. In the liver organoid transplantation group, the bile ducts were located in the donor area and connected to the recipient bile ducts. Thus, the rate of bile reconstruction in the liver was significantly higher compared to that in the monolayer group. By transplanting liver organoids, we saw a level of 70% replacement of the damaged liver. Consequently, in the transplantation group, diminished ductular reaction and a decrease of placental glutathione S-transferase (GST-p) precancerous lesions were observed. After trans-portal injection, the human induced pluripotent stem cell (hiPSC)-derived liver organoids revealed no translocation outside the liver; in contrast, the monolayer cells had spread to the lungs. The hiPSC-derived liver organoids were attached to the liver in the immunodeficient RS/PH rats. This study clearly demonstrates that liver organoid transplantation through the portal vein is a safe and effective method for the treatment of chronic liver damage in rats.

Human iPSC-Derived Posterior Gut Progenitors Are Expandable and Capable of Forming Gut and Liver Organoids.

Early endoderm progenitors naturally possess robust propagating potential to develop a majority of meter-long gastrointestinal tracts and are therefore considered as a promising source for therapy. Here, we demonstrated the reproducible generation of human CDX2+ posterior gut endoderm cells (PGECs) from five induced pluripotent stem cell clones by manipulating FGF, TGF, and WNT signaling. Transcriptome analysis suggested that putative PGECs harbored an intermediate signature profile between definitive endoderm and organ-specific endoderm. We found that combinatorial EGF, VEGF, FGF2, Chir99021, and A83-01 treatments selectively amplify storable PGECs up to 1021 cell scale without any gene transduction or feeder use. PGECs, compared with induced pluripotent stem cells, showed stable differentiation propensity into multiple endodermal lineages without teratoma formation. Furthermore, transplantation of PGEC-derived liver bud organoids showed therapeutic potential against fulminant liver failure. Together, the robustly amplified PGECs may be a promising cellular source for endoderm-derived organoids in studying human development, modeling disease, and, ultimately, therapy.

BMP signaling and cellular dynamics during regeneration of airway epithelium from basal progenitors.

The pseudostratified epithelium of the lung contains ciliated and secretory luminal cells and basal stem/progenitor cells. To identify signals controlling basal cell behavior we screened factors that alter their self-renewal and differentiation in a clonal organoid (tracheosphere) assay. This revealed that inhibitors of the canonical BMP signaling pathway promote proliferation but do not affect lineage choice, whereas exogenous Bmp4 inhibits proliferation and differentiation. We therefore followed changes in BMP pathway components in vivo in the mouse trachea during epithelial regeneration from basal cells after injury. The findings suggest that BMP signaling normally constrains proliferation at steady state and this brake is released transiently during repair by the upregulation of endogenous BMP antagonists. Early in repair, the packing of epithelial cells along the basal lamina increases, but density is later restored by active extrusion of apoptotic cells. Systemic administration of the BMP antagonist LDN-193189 during repair initially increases epithelial cell number but, following the shedding phase, normal density is restored. Taken together, these results reveal crucial roles for both BMP signaling and cell shedding in homeostasis of the respiratory epithelium.

IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells.

The pseudostratified airway epithelium of the lung contains a balanced proportion of multiciliated and secretory luminal cells that are maintained and regenerated by a population of basal stem cells. However, little is known about how these processes are modulated in vivo, and about the potential role of cytokine signaling between stem and progenitor cells and their niche. Using a clonal 3D organoid assay, we found that IL-6 stimulated, and Stat3 inhibitors reduced, the generation of ciliated vs. secretory cells from basal cells. Gain-of-function and loss-of-function studies with cultured mouse and human basal cells suggest that IL-6/Stat3 signaling promotes ciliogenesis at multiple levels, including increases in multicilin gene and forkhead box protein J1 expression and inhibition of the Notch pathway. To test the role of IL-6 in vivo genetically, we followed the regeneration of mouse tracheal epithelium after ablation of luminal cells by inhaled SO2. Stat3 is activated in basal cells and their daughters early in the repair process, correlating with an increase in Il-6 expression in platelet-derived growth factor receptor alpha(+) mesenchymal cells in the stroma. Conditional deletion in basal cells of suppressor of cytokine signaling 3, encoding a negative regulator of the Stat3 pathway, results in an increase in multiciliated cells at the expense of secretory and basal cells. By contrast, Il-6 null mice regenerate fewer ciliated cells and an increased number of secretory cells after injury. The results support a model in which IL-6, produced in the reparative niche, functions to enhance the differentiation of basal cells, and thereby acts as a "friend" to promote airway repair rather than a "foe."

Involvement of Galectin-3 with vascular cell adhesion molecule-1 in growth regulation of mouse BALB/3T3 cells.

beta-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Preferential reduction of the alpha-2-6-sialylation from cell surface N-glycans of human diploid fibroblastic cells by in vitro aging.

Human diploid fibroblastic cell line, TIG-3, has a finite life span of about 80 population doubling levels (PDL), and is used for in vitro aging studies. Young cells (PDL 23) grew to higher cell densities at a higher growth rate than aged cells (PDL 77). When the electrophoretic mobility of cells was determined, the negative surface charge of the aged cells decreased significantly when compared to that of young cells. Lectin blot analysis of membrane glycoproteins showed that the alpha-2-6-sialylation but not the alpha-2-3-sialylation of N-glycans decreases markedly in the aged cells when compared to the young cells. In support of this observation, the cDNA microarray assay and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the gene expression of the alpha-2,6-sialyltransferase I (ST6Gal I), which transfers sialic acid to galactose residues of N-glycans, decreases in the aged cells. These results indicate that the concordant decrease of the alpha-2,6-sialylation of N-glycans with the ST6Gal I gene expression is induced in TIG-3 cells by in vitro aging.