Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.     

15-Deoxy-Δ¹²,¹⁴ prostaglandin J₂ reduces the formation of atherosclerotic lesions in apolipoprotein E knockout mice

Aim

15-Deoxy-Δ^12,14 Prostaglandin J₂ (15d-PGJ₂) is a ligand of peroxisome proliferator-activated receptor γ (PPARγ) having diverse effects such as the differentiation of adipocytes and atherosclerotic lesion formation. 15d-PGJ₂ can also regulate the expression of inflammatory mediators on immune cells independent of PPARγ. We investigated the antiatherogenic effect of 15d-PGJ₂.

Methods

We fed apolipoprotein (apo) E-deficient female mice a Western-type diet from 8 to 16 wk of age and administered 1 mg/kg/day 15d-PGJ₂ intraperitoneally. We measured atherosclerotic lesions at the aortic root, and examined the expression of macrophage and inflammatory atherosclerotic molecules by immunohistochemical and real-time PCR in the lesion.

Results

Atherosclerotic lesion formation was reduced in apo E-null mice treated with 15d-PGJ₂, as compared to in the controls. Immunohistochemical and real-time PCR analyses showed that the expression of MCP-1, TNF-α, and MMP-9 in atherosclerotic lesions was significantly decreased in 15d-PGJ₂ treated mice. The 15d-PGJ₂ also reduced the expression of macrophages and RelA mRNA in atherosclerotic lesions.

Conclusion

This is the first report 15d-PGJ₂, a natural PPARγ agonist, can improve atherosclerotic lesions in vivo. 15d-PGJ₂ may be a beneficial therapeutic agent for atherosclerosis.     

Neural induction: Historical views and application to pluripotent stem cells

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state‐of‐the‐art stem cell research.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Biological activities of homologous loop regions in the laminin alpha chain G domains

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.     

Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

CrkL directs ASAP1 to peripheral focal adhesions

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.     

Specific inhibitor of puromycin-sensitive aminopeptidase with a homophthalimide skeleton: identification of the target molecule and a structure-activity relationship study

2-(2,6-Diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3-dione (2: PIQ-22) was found to be a potent and specific inhibitor of puromycin-sensitive aminopeptidase (PSA). Lineweaver-Burk plot analysis showed that PSA is inhibited by PIQ-22 in a non-competitive manner. Structure -activity relationship studies indicated that tautomerism of the imidobenzoylketone group in the cyclic imide moiety of the PIQ-22 skeleton is important for the inhibitory activity.