Differentiation of embryonic stem cells into lung-like cells using lung-derived matrix sheets.

Various extracellular matrix (ECM) in the lungs regulate tissue development and homeostasis, as well as provide support for cell structures. However, few studies regarding the effects of lung cell differentiation using lung-derived ECM (LM) alone have been reported. The present study investigated the capability of lung-derived matrix sheets (LMSs) to induce lung cell differentiation using mouse embryonic stem (ES) cells. Expressions of lung-related cell markers were significantly upregulated in ES-derived embryoid bodies (EBs) cultured on an LMS for two weeks. Moreover, immunohistochemical analysis of EBs grown on LMSs revealed differentiation of various lung-related cells. These results suggest that an LMS can be used to promote differentiation of stem cells into lung cells.

Isolation and culture of pleural mesothelial cells.

Purpose: Although the isolation of rat and mouse mesothelial cells has previously been reported, most mesothelial cells used for experimental studies are obtained from peritoneal cells. Here, we describe an optimized method for the isolation and in vitro propagation of rodent pleural mesothelial cells without the requirement for specialized surgical techniques. Materials and Methods: To harvest pleural mesothelial cells, the pleural space of 8-9-week-old rats or older mice was filled with 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) buffer for 20 min at 37 °C. Cells were then harvested, and incubated at 37 °C in a humidified atmosphere with 5% CO2. Immunofluorescence analysis of plated pleural mesothelial cells was performed using Alexa 546 (calretinin). To investigate optimal proliferation conditions, medium enriched with various concentrations of fetal calf serum (FCS) was used for pleural mesothelial cell proliferation. Results: By day 10, confluent cell cultures were established, and the cells displayed an obvious cobblestone morphology. Immunofluorescence analysis of the cells demonstrated that all stained positive for Alexa 546 (calretinin) expression. Mesothelial cells grew better in medium containing 20% FCS than with 10% FCS. Conclusions: This is a simple procedure for the efficient collection of primary pleural mesothelial cells, which were obtained in defined culture conditions from the euthanized rodent thoracic cavity using trypsin-EDTA treatment. The ability to easily culture and maintain identifiable pleural mesothelial cells from rodents will be helpful for future experiments using these cells.

Antitumor effects of minodronate, a third-generation nitrogen-containing bisphosphonate, in synergy with γδT cells in human glioblastoma in vitro and in vivo.

Nitrogen-containing bisphosphonates (N-BPs), which prevent bone resorption, exert direct and γδT cell (GDT)-mediated antitumor effects against several tumor cell types, including glioblastoma (GBM). However, limited information is available regarding the antitumor effects of N-BPs in GBM. Specifically, the antitumor effects of minodronate (MDA), a third-generation N-BP, in GBM are yet unclear. This study aimed to investigate the antitumor effects of MDA in GBM in vitro and in vivo. We performed growth inhibition and apoptosis detection assays using the GBM cell lines U87MG and U138MG. Apoptosis inhibition assays were also conducted. In vivo xenograft assays were performed in highly immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Sug)/Jic mice subcutaneously implanted with U87MG and U138MG cells. Growth inhibition and apoptosis detection assays demonstrated that MDA inhibited GBM cell growth via apoptosis, which was markedly enhanced by ex vivo expanded GDT. A pan-caspase inhibitor, z-VAD-fmk, inhibited MDA-induced U138MG apoptosis and MDA/GDT-induced U87MG and U138MG apoptosis. But z-VAD-fmk increased MDA-induced U87MG apoptosis. MDA/GDT-mediated apoptosis was blocked by the anti-T cell receptor (TCR) Vγ9, mevalonate pathway inhibitor, granzyme B inhibitor, and antitumor necrosis factor (TNF)-α. In vivo xenograft assays showed that combined intraperitoneal administration of MDA/GDT induced antitumor effects on unestablished U87MG-derived subcutaneous tumors. MDA exerted direct and GDT-mediated anti-GBM apoptotic effects in a caspase-dependent manner. GDT recognized MDA-exposed GBM cells via TCRVγ9 and induced apoptosis via granzyme B and TNF-α release. Because MDA elicited anti-GBM effects in synergy with GDT in vivo, a combination of MDA and ex vivo-generated GDT could be an effective treatment in patients with GBM.

Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells.

In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-ß-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage.

Cytotoxic human peripheral blood-derived γδT cells kill glioblastoma cell lines: implications for cell-based immunotherapy for patients with glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell-cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αßT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32% U87MG, 15% U138MG, 1% A172, and 50% K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.

Canonical Wnts, specifically Wnt-10b, show ability to maintain dermal papilla cells.

Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cell proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.

Culture of organoids with vestibular cell-derived factors promotes differentiation of embryonic stem cells into inner ear vestibular hair cells.

Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs.

Expression and distribution of generated neurons and endogenous precursors in rat cerebral cortical venous ischemia.

Neurogenesis in the subventricular zone (SVZ), subgranular zone (SGZ), and cerebral cortex is now a familiar event to confirm by cerebral arterial ischemia in rat models. However, it remains unclear whether cerebral venous ischemia (CVI) alone causes neurogenesis, and where that neurogenesis occurs. After creating CVI rat models via a two-vein occlusion (2-VO) method, neurogenesis was immunohistochemically evaluated by double-labeling 5-bromo-2'-deoxyuridine (BrdU)-positive cells with neuronal nuclei (NeuN) or doublecortin (DCX) antibody. Fifty Wistar rats were divided into two major groups (BrdU-NeuN and BrdU-DCX) and then separated into two subgroups (2-VO or sham). The total number of double-positive cells expressed inside a predefined region of interest (ROI) covering the ischemic area was compared between the two subgroups. Then, we divided the ROI into six sections to evaluate and compare the distribution of double-positive cells generated in each section between the two subgroups. The 2-VO subgroup presented more double-positive cells than the sham group in both BrdU-NeuN and BrdU-DCX groups, while the BrdU-DCX+2-VO group showed a characteristic distribution of double-positive cells in ROI 2 and ROI 3, suggesting areas of the ischemic core and penumbra, with a significant difference compared to the BrdU-DCX+sham group. This study demonstrates that CVI has the potential to induce endogenous neurogenesis, with significant numbers of both newly generated neurons and precursors observed in the ischemic area. The distribution of these cells suggests that the cortex could be the main origin of neurogenesis after cortical CVI.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Local administration of shikonin improved the overall survival in orthotopic murine glioblastoma models with temozolomide resistance.

BACKGROUND: Glioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin cannot easily cross the blood-brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice. MATERIALS AND METHODS: The cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells. RESULTS: In flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively). CONCLUSIONS: The direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.

Impaired differentiation potential of CD34-positive cells derived from mouse hair follicles after long-term culture.

Hair follicle epithelial stem cells (HFSCs), which exist in the bulge region, have important functions for homeostasis of skin as well as hair follicle morphogenesis. Although several methods for isolation of HFSCs using a variety of stem cell markers have been reported, few investigations regarding culture methods or techniques to yield long-term maintenance of HFSCs in vitro have been conducted. In the present study, we screened different types of commercially available culture medium for culturing HFSCs. Among those tested, one type was shown capable of supporting the expression of stem cell markers in cultured HFSCs. However, both the differentiation potential and in vivo hair follicle-inducing ability of HFSCs serially passaged using that optimal medium were found to be impaired, probably because of altered responsiveness to Wnt signaling. The changes noted in HFSCs subjected to a long-term culture suggested that the Wnt signaling-related environment must be finely controlled for maintenance of the cells.

Schistosoma mansoni larvae in vitro cultures using Biomphalaria glabrata extracts.

Schistosomiasis is one of the most prevalent waterborne parasitic diseases affecting humans. In natural conditions, snails are necessary for maintenance of its lifecycle and also required as intermediate hosts to maintain the lifecycle in laboratory settings. In the present study, the location of S. mansoni larvae in Biomphalaria glabrata snails after infection (inoculation of miracidia) was investigated. Larvae were found located in the head-foot (HF) area of B. glabrata snails at 10 days post-infection (DPI), then their location was predominantly changed to the hepatopancreas and ovotestis (HPOT) area by 56 DPI. Next, the effects of extracts from various organs of B. glabrata snails including HF and HPOT for in vitro culturing of S. mansoni larvae were investigated. The HF extract enabled prolonged culturing of S. mansoni larvae. Furthermore, sequential use of that followed by the HPOT extract supported larval development or reproduction of daughter sporocysts. These results may provide important information for identifying essential factors and molecules for culturing Schistosoma larvae in vitro.

Role of syndecan-1 (CD138) in cell survival of human urothelial carcinoma.

Heparan sulfate proteoglycan syndecan-1, CD138, is well known to be associated with cell proliferation, adhesion, and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human urothelial carcinoma of the urinary bladder. Silencing of syndecan-1 by siRNA transfection down-regulated transcriptional factor junB and the long isoform of FLICE-inhibitory protein (FLIP long), resulting in the induction of apoptosis in the urothelial carcinoma cell lines UMUC2 and UMUC3. Knockdown of junB and FLIP long as well as syndecan-1 silencing mediated apoptosis that was inhibited by pan-caspase inhibitors. Transurethral injection of syndecan-1 siRNA into the urinary bladder significantly reduced syndecan-1 gene expression and growth of red fluorescent-labeled KU-7/RFP bladder cancer cells in the mouse orthotopic bladder cancer model. Immunohistochemical examination showed high syndecan-1 protein expression in high-grade, superficial, and deep invasive carcinomas (pT1 and >or=pT2) as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes (pTa). In addition, the percentage of cancer cells positive for syndecan-1 at initial diagnosis was statistically associated with the frequency of bladder cancer recurrence after transurethral resection. In conclusion, syndecan-1 might contribute to urothelial carcinoma cell survival and progression; therefore, this molecule could be a new therapeutic target in human urinary bladder cancer.

Syndecan-1, a new target molecule involved in progression of androgen-independent prostate cancer.

Heparan sulfate proteoglycan syndecan-1 (CD138) is well known to be associated with cell proliferation, adhesion and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human prostate cancer. Immunohistochemical analysis revealed either no or rare expression of syndecan-1 in normal secretory glands and prostate cancer cells at hormone naïve status, whereas the expression was significantly increased in viable cancer cells following neo-adjuvant hormonal therapy. Syndecan-1 expression was much higher in the androgen independent prostate cancer cell lines DU145 and PC3, rather than the androgen-dependent LNCaP, but the level in LNCaP was up-regulated in response to long-term culture under androgen deprivation. Silencing of syndecan-1 by siRNA transfection reduced endogenous production of reactive oxygen species through down-regulating NADPH oxidase 2 and induced apoptosis in DU145 and PC3 cells. Consistently, NADPH oxidase 2 knockdown induced apoptosis to a similar extent. Subcutaneous inoculation of PC3 cells in nude mice demonstrated the reduction of tumor size by localized injection of syndecan-1 siRNA in the presence of atelocollagen. Moreover, the mouse model and chorioallantoic membrane assay demonstrated significant inhibition of vascular endothelial growth factor and tumor angiogenesis by silencing of syndecan-1. In conclusion, syndecan-1 might participate in the process of androgen-dependent to -independent conversion, and be a new target molecule for hormone resistant prostate cancer therapy.

Cotransplantation of mouse embryonic stem cells and bone marrow stromal cells following spinal cord injury suppresses tumor development.

Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 x 10(-9) M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.

Treatment of Parkinson's disease model mice with allogeneic embryonic stem cells: necessity of immunosuppressive treatment for sustained improvement.

OBJECTIVE: The purpose of the present study was to examine the efficacy of transplantation of mouse embryonic stem (ES) into Parkinson's disease (PD) model mice as well as the necessity of immunosuppression in allogeneic donor-host combinations. MATERIALS AND METHODS: ES cells, derived from SvJ129 strain mice, were differentiated into tyrosine hydroxylase (TH)-positive neurons in vitro by an embryoid body (EB)-based multistep differentiation method and used as graft cells for PD mice, which were prepared by injection of 6-hydroxydopamine (OHDA) into C57BL/6, BALB/c and C3H/HeN strains. Mice from each strain were divided into Groups 1-3. Four weeks after the 6-OHDA injection, Group 1 received phosphate-buffered saline in the striatum wounds, while Group 2 received 2 x 10(4) graft cells, and Group 3 mice received 2 x 10(4) graft cells and were also treated with cyclosporine A. RESULTS: Apomorphine-induced rotational behavior was improved in Groups 2 and 3, but not in Group 1. However, the behavioral improvement ceased later in Group 2, whereas sustained improvement was observed in Group 3 throughout the 8 week observation period after transplantation. ES-derived TH(+) cells were found at the grafted sites at the end of the experiment in Groups 2 and 3, and tended to be more abundant in Group 3. CONCLUSION: Intra-striatum transplantation of ES-derived dopaminergic neurons was effective in treating PD mice, even in allogeneic donor-host combinations. Immunosuppressive treatment did not have an effect on initial behavioral restoration after transplantation; however, it was necessary for sustained improvement over a prolonged period.

Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium.

Vestibular hair cells (V-HCs) in the inner ear have important roles and various functions. When V-HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V-HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V-HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V-HCs.

Effect of covering with cross-linked gelatin glue on tissue regeneration in a rat lung injury model.

OBJECTIVES: Thoracic reintervention is a common treatment; however, preventing adhesion of the lung to the thoracic cavity wall remains a problem. This study aimed to investigate the effect on pleural adhesion of covering the postoperative pleural injury site with cross-linked gelatin glue (gelatin plus glutaraldehyde, hereafter 'gelatin glue') and to evaluate the proliferation of healing cells on gelatin glue. METHODS: We created a rat incisional lung-wound model and compared the effects of sealing the wound with gelatin glue (group A, n = 5), fibrin glue (group B, n = 5) or fibrin glue with a polyglycolic acid sheet (group C, n = 5). Adhesions were assessed 28 days postoperatively and compared among the groups using the Karacam's scoring method. Lung-wound healing was studied histologically at day 7 postoperatively. Mesothelial cell proliferation was investigated on gelatin and fibrin glues in vitro. RESULTS: There were no or few adhesions of the chest wall in group A. The adhesion scores (mean ± standard deviation) were 1.2 ± 0.4, 2.6 ± 1.4 and 3.2 ± 1.2 in groups A, B and C, respectively (A vs C, P = 0.0496). During the healing process, the gelatin glue surface was covered by mesothelial-like cells. Proliferation of cultured mesothelial cells was promoted on the gelatin glue compared with the fibrin glue. CONCLUSIONS: Covering lung wounds with the gelatin glue reduced adhesions and promoted the growth of healing cells compared with the fibrin glue. These findings suggest that the gelatin glue may help prevent adhesions and thus be a therapeutically effective biomaterial in lung surgery.

Wnt-10b, uniquely among Wnts, promotes epithelial differentiation and shaft growth.

Although Wnts are expressed in hair follicles throughout life from embryo to adult, and considered to be critical for their development and maturation, their roles remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, Wnt-5a, Wnt-10b, and Wnt-11) on epithelial cell differentiation using adult mouse-derived primary skin epithelial cell (MPSEC) cultures and hair growth using hair follicle organ cultures. Only Wnt-10b showed evident promotion of epithelial cell differentiation and hair shaft growth, in contrast to Wnt-3a, 5a, and 11. Our results suggest that Wnt-10b is unique and plays an important role in differentiation of epithelial cells in the hair follicle.

Embryonic stem cells reduce liver fibrosis in CCl4-treated mice.

We transplanted undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl(4))-treated mice to determine their effects on liver fibrosis. Carbon tetrachloride at 0.5 ml/kg of body weight was injected intraperitoneally into C57BL/6 mice twice weekly for up to 20 weeks. Four weeks after the first injection, the mice were divided into two groups and those in group 1 received 1 x 10(5) ES cells genetically labelled with enhanced green fluorescent protein (GFP) in the spleens, while group 2 mice received 0.1 ml of phosphate-buffered saline. In group 1, GFP-immunopositive cells were retained and found in areas of fibrosis in the liver, and reduced liver fibrosis was observed as compared with group 2. Secondary transplantation of ES cells at 12 weeks after the initial transplantation enhanced the reduction in liver fibrosis. No teratoma formation or uncontrolled growth of ES cells in organs, including the liver and spleen, was observed in any of the mice. In the livers of group 1 mice, metalloproteinase 9-immunopositive cells derived from ES cells as well as those from the recipient were observed. These cells were also found to be immunopositive for the hepatoblast marker Delta-like (DlK-1), a member of the DlK-1 family of transmembrane proteins. These results suggest that ES-based cell therapy is potentially useful for liver fibrosis treatment and that reduction in CCl(4)-induced liver fibrosis by transplantation of ES cells may be related closely to the emergence of metalloproteinase-producing hepatoblast-like cells.