Recently, we established a novel in vitro differentiation system of embryonic stem (ES) cells that reproduces vascular cell differentiation and vascular formation process. F1k 1, one of the vascular endothelial growth factor receptors (VEGF-R 2), is the earliest differentiation marker for vascular system and also a marker for mesoderm. We induced Flk 1-expressing (Flk 1+) cells from undifferentiated ES cells and purified F1k 1+ cells by f lowcytometry. Purified Flk 1+cells give rise to both of vascular cell types, endothelial cells and mural cells (vascular smooth muscle cells and pericytes) by the stimulation with VEGF and PDGF-BB, respectively, and form blood vessel structures both in vitro and in vivo. These results indicate that F1k 1+cells act as common progenitors for vascular cells and suffice to form complete vessels. This system not only provides potentials to develop novel vascular regeneration therapies, but also enables us to dissect the molecular mechanisms of vascular development from a new aspect, such as global gene expression analysis during cell differentiation and in vitro functional assay of genes for vascular development. Our novel methodological approach should offer new possibilities to ES cell research.

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Conditions that destroy the limbal area of the peripheral cornea, such as Stevens-Johnson syndrome, ocular pemphigoid, and chemical and thermal injuries, can deplete stem cells of the corneal epithelium. The result is scarring and opacification of the normally clear cornea. Standard corneal transplantation cannot treat this form of functional blindness.
Transplantation of corneal epithelial stem cells can restore useful vision in some patients with severe ocularsurface disorders.
Corneal epithelial stem-cell transplantation permits sustained reconstruction of the corneal epithelium in many eyes with severe primary disorders of the ocular surface. The control of persistent epithelial defects, ocular hypertension, dry eye, and graft rejection may further increase the efficacy of this method of transplantation.

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Liver development requires a number of growth factors (GFs) to lead differentiation of liver parenchymal cells. However, because the liver contains cells of many types, it is difficult to examine their functional effects on a rare cell population, such as a hepatic stem cell population. Prospective isolation and clonal assays for hepatic stem cells enable to investigate direct effects of GFs on their differentiation. Using previously purified cells that fulfill the criteria for hepatic stem cells, we examined how GFs regulate differentiation in the developing liver. These experiments would allow us to describe sequential liver cell lineages derived from hepatic stem cells precisely. Furthermore, consecutive production of stem cell-derived hepatocytes may provide a useful cell source for developing therapeutic strategies, such as cell transplantation, gene therapy, and artificial liver organ to treat various liver disorders.

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Identification and isolation of pancreatic stem cells (PSCs) responsible for both endocrine and exocrine tissue formation have excited much interest due not only to their developmental importance but also to therapeutic potential. In a previous report, we have established an in vitro clonal colony forming assay system for pancreatic stem/progenitor cells, in which they could form relative large colonies. Our data showed that clonal colonies contained cells expressing several markers for endocrine and exocrine lineage cells. These results suggest that cells initiating colony formation are defined as pancreatic stem cells or common progenitors for endocrine and exocrine cells. To distinguish PSC from other cell types, prospective identification is critically required. The prospective study gives us much information about specific characteristics of stem cells and allows us to selectively separate them from differentiated cells. By combining colony-forming assay with fluorescence activated cell sorting (FACS), we can analyze cells sorted from distinct fractions and attempt to purify pancreatic stem cells.

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One of the major risk factors triggering or worsening human inflammatory bowel disease (IBD) is the administration of non-steroidal anti-inflammatory drugs (NSAIDs) . Since NSAIDs share inhibition of the enzyme-cyclooxygenase (COX) that catalyzes production of prostanoids, some prostanoids are formed and negatively modulate the extension of IBD. We used mice deficient in prostaglandin (PG) E₂ receptor EP1, EP2, EP3, and EP4, and examined the roles of prostaglandin E₂ in DSS-induced colitis model. Among the PGE₂ receptor-deficient mice, only EP4-deficient mice developed severe colitis with 3% DSS treatment, which induced only marginal colitis in wild-type mice. Conversely, administration of an EP 4-selective agonist (AE 1-734) to wild type mice ameliorated severe colitis normally induced with 7% DSS, while that of an EP 4 agonist, AE 3-208, suppressed recovery from colitis and induced significant proliferation of CD 4⁺ T cells. In vitro AE 3-208 enhanced and AE 1-734 suppressed the proliferation and Th 1 cytokine production of lamina propria mononuclear cells from the colon. DNA microarray analysis revealed elevated expression of genes associated with immune response and reduced expression of genes with mucosal repair and remodeling in the colon of EP 4-deficient mice. We conclude that EP 4 maintains intestinal homeostasis by keeping mucosal integrity and downregulating immune response.

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Embryonic stem cells can be applicable for neural cell transplantation. Here we differentiated mouse embryonic stem cells into neuroepithelial stem cells with tertiary structures in floating cell aggregates. We first cultured ES cells with 0.5μM retinoic acid to induce neural differentiation. RT-PCR and immunostainning studies showed most of the RA treated cells at day 8 differentiated into Nestin expressing neural stem cells without tertiary structures. About 80% of the treated cells become panNCAM+ neural cells by FACS analysis. The findings indicated the RA treatment successfully induced neural stem cells from ES cells. The RA treated cells at day 8 were recovered and were cultured in FCS depleted medium consisted of DMEM/F 12, N 2 supplement and fibronectin on nontreated dishes for additional 6-8 days. The cell aggregates contained a cluster of pseudostratified Nestin+ neuroepithelial stem cells surrounded by the Masson staining+ basement membrane, whereas mature neural cells located outside of the structures. Thus, we have generated neuroepithelial stem cells from ES cells. These findings may get access to induce purposive and transplantable neurons for several diseases and get a cue to resolve inaccessible problems of neurogenesis.

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Polysaccharide 70% deacetylated chitin (DAC-70) possessing some immunopotential is a derivative of beta-poly-N-acetyl D-glucosamine chitin. In this study, examination was made of the effects of systemic DAC-70 over a period of four months on knee joints of MRL/lpr mice, the sites of SLE and RA-like arthritis. In 54% of the non treated mice (n=13), severe pannus formation and cartilage destruction were evident and in 38% of these animals, synovial hyperplasia and/or proliferation with inflammatory infiltration and distinct cartilage erosion could be seen. 56% of DAC-70 treated mice (n=9) showed virtually no synovial inflammatory change; the cartilage was nearly intact and in 33% of the animals, there was only mild cartilage erosion, with only one mouse (11%) showing pannus formation and cartilage destruction. All the mice (n=9) on the low nutrition diet had intact joints. This agent is thus shown effective for lessening RA joint destruction and possibly may thus also be used for safely treating human RA patients.

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We report a case of myositis ossificans progressiva in Japan. The patient was 7 yearsold who had swelling and pain of his back and arms. He had surgically treated for halluces valgus of his left foot when he was 2 years old. Laboratory examination on admission of our hospital showed elevated serum phosphate and calcium. On plain roentogenogram, ectopic ossification of bilateral biceps muscles was noted. Chest computed tomography showed severe deformity and constriction of his thorax, and the ectopic ossification was confirmed. Magnetic resonance imaging of his back and neck revealed massive swelling of his muscles including left sternocleidomastoid muscle. Muscle biopsy has not been performed, because his diagnosis of having myositis ossificans progressiva is definitive, and histological examination of the specimen has been reported to have difficulty on differential diagnosis of myositis ossif icans progressiva from other types of malignancies of muscle origin including soft tissue sarcoma and inflammatory myositis. In addition, muscle injury caused by the biposy procedure accelerates progression of the disease including inflammation and ossification. There is no clear cut explanation of the pathophysiology of nor effective treatment of the disease. We need to accumulate and experience more patients with the same disease to elucidate their pathophysiology and to carry out the genetic linkage study of this disease in Japan.

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