Shin Ishikane

Comparison of Angiogenic, Cytoprotective, and Immunosuppressive Properties of Human Amnion- and Chorion-Derived Mesenchymal Stem Cells

Although mesenchymal stem cells (MSCs) can be obtained from the fetal membrane (FM), little information is available regarding biological differences in MSCs derived from different layers of the FM or their therapeutic potential. Isolated MSCs from both amnion and chorion layers of FM showed similar morphological appearance, multipotency, and cell-surface antigen expression. Conditioned media obtained from amnion- and chorion-derived MSCs inhibited cell death caused by serum starvation or hypoxia in endothelial cells and cardiomyocytes. Amnion and chorion MSCs secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Transplantation of human amnion or chorion MSCs significantly increased blood flow and capillary density in a murine hindlimb ischemia model. In addition, compared to human chorion MSCs, human amnion MSCs markedly reduced T-lymphocyte proliferation with the enhanced secretion of PGE2, and improved the pathological situation of a mouse model of acute graft-versus-host disease. Our results highlight that human amnion- and chorion-derived MSCs, which showed differences in their soluble factor secretion and angiogenic/immuno-suppressive function, could be ideal cell sources for regenerative medicine.

Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

Significance Postoperative cancer recurrence is a major problem following curative cancer surgery. Perioperative systemic inflammation induces the adhesion of circulating tumor cells released from the primary tumor to the vascular endothelium of distant organs, which is the first step in hematogenous metastasis. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here, we demonstrate that cancer recurrence after lung cancer surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). We show that ANP prevents cancer metastasis by suppressing the inflammatory reaction of endothelial cells, thereby inhibiting cancer cell adhesion to vascular endothelial cells. Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells.

Systemic transplantation of allogenic fetal membrane-derived mesenchymal stem cells suppresses Th1 and Th17 T cell responses in experimental autoimmune myocarditis.

We have reported that systemic administration of autologous bone marrow or allogenic fetal membrane (FM)-derived mesenchymal stem cells (MSCs) similarly attenuated myocardial injury in rats with experimental autoimmune myocarditis (EAM). Since rat EAM is a T-helper (Th) cell-mediated autoimmune disease, and recent evidence has indicated that both autologous and allogenic MSCs exert an immunosuppressive effect on Th cell activity, we focused on Th cell differentiation in allogenic FM-MSC administered EAM rats. EAM was induced in Lewis rats by injecting porcine cardiac myosin (day 0). Allogenic FM-MSCs, obtained from major histocompatibility complex mismatched ACI rats, were intravenously injected (5 × 10(5)cells/rat) on days 7, 10, or 14 (MSCd7, MSCd10, or MSCd14 groups, respectively). At day 21, echocardiography confirmed that reduced ejection fraction in the untreated EAM group (63 ± 2%) was significantly improved in the MSCd10 and MSCd14 groups (74 ± 1 and 75 ± 2%, respectively, P<0.01). CD68 immunostaining revealed that prominent macrophage infiltration in the myocardium of the EAM group (1466 ± 93 cells/mm(2)) was significantly decreased in the MSCd10 group (958 ± 139 cells/mm(2), P<0.05). To evaluate Th cell differentiation, we used flow cytometry to determine the percentage of interferon (IFN)-γ positive Th1 and interleukin (IL)-17 positive Th17 cells in peripheral CD4-positive Th cells. The percentage of Th1 cells at day 16 was significantly lower in the MSCd10 (1.3 ± 0.2%) and MSCd14 (1.6 ± 0.3%) groups compared to the EAM group (2.4 ± 0.3%, P<0.05), as was the percentage of Th17 cells in the MSCd10 group (1.9 ± 0.5%) compared to the EAM group (2.2 ± 0.9%, P<0.05). At day 21, infiltrating Th17 cells in myocardium were significantly decreased in the MSCd10 group (501 ± 132 cells/mm(2), P<0.05) compared to EAM (921 ± 109 cells/mm(2)). In addition, human CD4+ Th cells co-cultured with human FM-MSCs exhibited reduced Th1 and Th17 cell-differentiation and proliferation, with increased expression of immunosuppressive molecules including indoleamine 2,3-dioxygenase 2 and IL-6 in co-cultured FM-MSCs. These results suggest that intravenous administration of allogenic FM-MSCs ameliorates EAM via the suppression of Th1/Th17 immunity.

Allogenic fetal membrane-derived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis

Mesenchymal stem cells (MSC) have been reported to be an attractive therapeutic cell source for the treatment of renal diseases. Recently, we reported that transplantation of allogenic fetal membrane-derived MSC (FM-MSC), which are available noninvasively in large amounts, had a therapeutic effect on a hindlimb ischemia model (Ishikane S, Ohnishi S, Yamahara K, Sada M, Harada K, Mishima K, Iwasaki K, Fujiwara M, Kitamura S, Nagaya N, Ikeda T. Stem Cells 26: 2625-2633, 2008). Here, we investigated whether allogenic FM-MSC administration could ameliorate renal injury in experimental glomerulonephritis. Lewis rats with anti-Thy1 nephritis intravenously received FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (FM-MSC group) or a PBS (PBS group). Nephritic rats exhibited an increased urinary protein excretion in the PBS group, whereas the FM-MSC group rats had a significantly lower level of increase (P < 0.05 vs. PBS group). FM-MSC transplantation significantly reduced activated mesangial cell (MC) proliferation, glomerular monocyte/macrophage infiltration, mesangial matrix accumulation, as well as the glomerular expression of inflammatory or extracellular matrix-related genes including TNF-α, monocyte chemoattractant protein 1 (MCP-1), type I collagen, TGF-β, type 1 plasminogen activator inhibitor (PAI-1) (P < 0.05 vs. PBS group). In vitro, FM-MSC-derived conditioned medium significantly attenuated the expression of TNF-α and MCP-1 in rat MC through a prostaglandin E(2)-dependent mechanism. These data suggest that transplanted FM-MSC contributed to the healing process in injured kidney tissue by producing paracrine factors. Our results indicate that allogenic FM-MSC transplantation is a potent therapeutic strategy for the treatment of acute glomerulonephritis.

Allogeneic transplantation of fetal membrane-derived mesenchymal stem cell sheets increases neovascularization and improves cardiac function after myocardial infarction in rats.

Background Mesenchymal stem cell (MSC) transplantation has been pursued as a new method to repair damaged myocardium. We focused on the fetal membrane (FM) as an alternative source to bone marrow (BM)–derived MSCs. In this study, we investigated whether transplantation of allogeneic FM-MSC sheets could attenuate myocardial dysfunction in a rat chronic myocardial infarction (MI) model. Methods Sheets of allogeneic FM-MSC or autologous BM-MSC were transplanted into the scarred myocardium 4 weeks after coronary ligation. Results Four weeks after transplantation, both allogeneic FM-MSC and autologous BM-MSC sheets had significantly improved cardiac function and reduced myocardial fibrosis compared with the untreated MI group. In both MSC sheet-transplanted groups, the peri-infarct regional capillary density was increased. Some engrafted MSCs formed vascular structures and were positive for lectin I and &agr;-smooth muscle actin. The numbers of engrafted cells and differentiated cells were very low after both types of MSC sheet transplantation. CD3+ T cells did not increase in the transplantation site, but CD163+ M2 macrophages increased in the groups transplanted with allogeneic FM-MSC and autologous BM-MSC. Conclusions Transplantation of allogeneic FM-MSC or autologous BM-MSC sheets attenuated myocardial dysfunction in a rat MI model to a similar degree. The engraftment rate of transplanted cells and immune cell infiltration into the transplanted area did not differ between the two types of MSC transplants. M2 macrophage induction has possible involvement in the therapeutic effects of MSC transplantation. Allogeneic FM-MSC sheet transplantation might be a new therapeutic strategy after MI.

C/EBPβ (CCAAT/enhancer-binding protein β) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1)

The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

Transcriptional regulation of genes related to progesterone production [Review]

Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

Identification of novel steroidogenic factor 1 (SF-1)-target genes and components of the SF-1 nuclear complex.

Steroidogenic factor 1 (SF-1) is a master regulator of adrenal and reproductive development and function. Although SF-1 was identified as a transcriptional regulator for steroid metabolic enzymes, it has been shown that SF-1 also regulates other genes that are involved in various cellular processes. Previously, we showed that introduction of SF-1 into mesenchymal stem cells resulted in the differentiation of these cells to the steroidogenic lineage. By using this method of differentiation, we performed comprehensive analyses to identify the novel SF-1-target genes and components of the SF-1 nuclear complex. Genome-wide analyses with promoter tiling array and DNA microarray identified 10 genes as novel SF-1-target genes including glutathione S-transferase A family, 5-aminolevulinic acid synthase 1 and ferredoxin reductase. Using SF-1 immuno-affinity chromatography of nuclear proteins followed by MS/MS analysis, we identified 24 proteins including CCAAT/enhancer-binding protein β as components of SF-1 nuclear complex. In this review, we will describe novel roles of the newly identified genes for steroidogenesis.

Establishment and characterization of a novel orthotopic mouse model for human uterine sarcoma with different metastatic potentials

Uterine sarcomas are rare and aggressive gynecologic tumors with a poor prognosis because of recurrence and metastasis. However, the mechanisms of uterine sarcoma metastasis are largely unknown. To investigate this mechanism, we developed a novel uterine sarcoma tissue-derived orthotopic and metastatic model in KSN nude mice using a green fluorescent protein stably expressed uterine sarcoma cell line, MES-SA. Histological analysis showed that all orthotopic primary tumors were undifferentiated sarcoma. Primary tumors were characterized by high (18)F-fluorodeoxyglucose uptake with a positive correlation to the number of pulmonary metastases. In addition, we generated uterine sarcoma cell sublines with high or low metastatic potentials by serial in vivo selection. Microarray analysis between orthotopic tumors with high and low metastatic potentials revealed differential expression of genes related to cell proliferation and migration (TNNT1, COL1A2, and ZIC1). Our model would be useful to compensate for the limited clinical cases of uterine sarcoma and to investigate the molecular mechanisms of metastatic uterine sarcoma.

Injection of specific amyloid-beta oligomers (beta1-40:beta1-42 = 10:1) into rat medial septum impairs memory retention without inducing hippocampal apoptosis

Abstract Objectives: Because of the well-known neurochemical interactions between the septum and hippocampus during memory processes, we investigated the effect of amyloid-beta (A-beta) injection into the medial septum (MS) on the behavior in Wistar rats. We also assessed whether the observed effects were functional or due to apoptosis. Methods: Specific A-beta oligomers (beta1-40:beta1-42  = 10:1) were injected into MS for seven consecutive days. Behavior was assessed with the Morris water maze task. Results: Compared with the control group, rats that received A-beta oligomers exhibited significant memory retention impairment (P < 0·05) without apoptosis in the cornu ammonis (CA)1 and CA3 regions of the dorsal hippocampus. Discussion: These data indicate that septal injection of A-beta impairs memory retention, even in the absence of hippocampal apoptosis. This result might bring new insight to spatial memory-related disorders like Alzheimer’s disease (AD).