Optimizing cell migration assays: Critical roles of fluorescent labeling and chemoattractant gradients.

Cell migration assays, also known as chemotaxis assays, are widely used to measure the migratory capacities of cancer cells, leukocytes, macrophages, and other motile cell types. In these assays, fluorescently labeled cells are seeded onto cell culture inserts with microporous membranes that block light transmission from 490 to 700 nm. The migrated cells are then observed and quantified from the bottom of the microporous membrane using a fluorescence microscope.

Generation of human iPSC-derived 3D bile duct within liver organoid by incorporating human iPSC-derived blood vessel.

In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs.

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.

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.

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)].

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).

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.

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.

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.

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.

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.

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.

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.