- Email: [email protected]
Therapeutic effect of maternal molecular hydrogen administration in a rat model of preeclampsia
A6
Abstracts / Placenta 35 (2014) A1eA23
hyalinized material. Surgical procedures, such as dilatation and curettage and cesarean section, might increase the risk of PSN/P. It has been suggested that PSN/P might be consequence of disturbances to the implantation, resulting in abnormal involution of the placental site. The subjective symptom is menorrhagia or intermenstral bleeding, the effect on fertility is unknown.
O-001. A PREGNANCY CASE REPORT OF ALAGILLE SYNDROME; EXPRESSION OF NOTCH SIGNALING IN THE PLACENTA Kazue Togashi a, Megumi Sato a, Akira Sato a, Yurina Kameta b, Reiko Takayama a, Hiroshi Miura c, Yukihiro Terada a. a Akita University Graduate School of Medicine, Department of Obstetrics and Gynecology, Japan; b Akita City Hospital, Japan; c Kazuno Kosei Hospital, Japan Background: Pregnancy in patients with Alagille syndrome (AS) is rare. It is characterized by chronic cholestasis resulting from intrahepatic bile duct paucity. 114 Japanese AS patients were reported in 2009. Case report: A 22 year old female with history of a miscarriage 7months previously was seen at a hospital for edema and amenorrhea. She was diagnosed at 27 weeks and 4 days of gestation by fetal biparietal diameter and transferred to our maternity unit. Her physical examination was remarkable for low platelets, hypertension, proteinuria and ascites. Her medical record from pediatrics in our hospital revealed that she had AS; hepatic, cardiac, facial and skeletal anomalies. Anesthetic difficulty was predicted. Her thrombocytopenia was caused by liver cirrhosis and hypersplenism. The patient had no esophageal varices and no intracranial vessel abnormalities. Due to the deterioration of her renal function a cesarean section under general anesthesia was performed at 29 weeks. Atonic bleeding was treated with an intrauterine balloon. Management of acute pulmonary edematous, severe proteinuria and hypertension required several days of observation and treatment. After a couple of months her blood pressure and proteinuria had returned to normal levels; meaning that she had been preeclamptic. Conclusion: An awareness of possible organ involvement and a multidisciplinary approach is required in the management of pregnancy with AS. This fascinating case might hopefully provide perspectives for both future research into treatment of Alagille syndrome and human placentation of pregnancy complicated by preeclampsia.
O-002. IDENTIFICATION AND CHARACTERIZATION OF HTR-8/SVNEO SP CELLS. Tetsunori Inagaki a, Kiyoko Kato b, Soshi Kusunoki a, Koichi Tabu c, Hitomi Okabe a, Izumi Yamada a, Tetsuya Taga c, Akemi Matsumoto a, Toshiaki Takezawa d, Shintaro Makino a, Satoru Takeda a. a Jyuntendo Hospital, Japan; b Kyushu University, Japan; c Tokyo medical and dental University, Japan; d National Institute of Agrobiological Sciences, Japan Goals: We investigated the side population cells in HTR-8/SVneo to identify the trophoblast stem cell marker, and characterize trophoblast stem cells. Method: We performed microarray expression analysis to search for upregulated genes in trophoblast stem cell using a set of HTR-8/SVneo side population and non-side population cells. The expression of several candidate trophoblast stem cell markers were investigated by real time PCR, and performed knockdown of up-regulated genes expression using siRNA to analyze the association of up-regulated genes expression with trophoblast phenotype. Finally three dimensional cell culture system was established to analyze the association with epithelial-mesenchymal transition. Result: LY6D gene was highly up-regulated in side population cells. LY6D gene reduced its expression in the course of seven days’ cultivation in differentiation medium. Side population cells tended to reduce its fraction by treatment of LY6D siRNA. On ontology analysis, two Gene GO pathways of epithelial-mesenchymal transition were involved. Epithelial-
mesenchymal transition was induced by the co-existence of endometrial cells and side population cells. Conclusion: This is the first report to have identified the expression of LY6D and demonstrated induction of epithelial-mesenchymal transition in HTR-8/SVneo side population cells.
O-004. CLINICAL APPLICATION OF HUMAN AMNION-DERIVED MESENCHYMAL STEM CELLS FOR THE TREATMENT OF ACUTE GVHD Kenichi Yamahara a, Akihiko Taguchi b, Toshihiro Soma c, Hiroyasu Ogawa c, Tomoaki Ikeda d, Jun Yoshimatsu e. a Department of Regenerative Medicine, National Cerebral and Cardiovascular Center, Japan; b Department of Regenerative Medicine Research, Foundation for Biomedical Research and Innovation, Japan; c Division of Hematology, Department of Internal Medicine, Hyogo College of Medicine, Japan; d Department of Obstetrics & Gynecology, Mie University Faculty of Medicine, Japan; e Division of Maternal Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Purpose: We have demonstrated that, similar to bone marrow-derived mesenchymal stem cells (BM-MSCs), fetal appendage-derived MSCs possess the differentiation, angiogenic and immunosuppressive property. Fetal appendage, normally discarded as medical waste, is a rich source of MSCs, which secret significant amounts of cytokines/growth factors, indicating a suitable cell source to perform a cell therapy at low-cost, faster, and with less MSCs. Among fetal appendage, amnion is anatomically easy to separate MSCs by enzymatic digestion, and we started a multicenter clinical trial to treat acute GVHD using amnion-derived MSCs. Methods and Results: For the clinical study of cell therapy using amnion MSCs, we established a multicenter clinical team as follows: National Cerebral and Cardiovascular Center (amnion MSC manufacturing), Foundation for Biomedical Research and Innovation (quality control of amnion MSCs), and Hyogo College of Medicine (administration of amnion MSCs in patients with acute GVHD). We also established a method to isolate amnion MSCs with ease and high efficiency (Japanese patent application No. 2013-170008). After a consultation with the Pharmaceuticals and Medical Devices Agency (PMDA), we started to manufacture amnion MSCs adhering to GMP, and are planning to perform a phase I clinical trial within this year to assess the safety of amnion MSCs in patients with acute GVHD. Conclusion: Outside our country, BM-MSCs have already received approval for the treatment of children with acute GVHD. However, an invasive procedure and a long-term culturing is required to obtain an adequate number of BM-MSCs. Since a large number of MSCs could be obtained easily and non-invasively, amnion MSCs would be an ideal cell source for regenerative medicine.
O-006. THERAPEUTIC EFFECT OF MATERNAL MOLECULAR ADMINISTRATION IN A RAT MODEL OF PREECLAMPSIA
HYDROGEN
Takafumi Ushida, Yukio Mano, Hiroyuki Tsuda, Seiji Sumigama, Tomomi Kotani, Fumitaka Kikkawa. Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Japan Objective: Preeclampsia is a pregnancy-specific condition characterized by new-onset hypertension and proteinuria. It is also occasionally related to fetal growth restriction. Some studies suggest that placental oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) is reported to prevent a variety of diseases associated with oxidative stress in model systems and in human. We studied the efficacy of H2 using a rat model of preeclampsia. Methods: We used the well-established reduced utero-placental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in rat that mimics features of preeclampsia. RUPP was performed on day 14 of pregnancy, clips were placed around the aorta below the renal arteries and on both uterine arcade at the ovarian artery. The sham group underwent
Abstracts / Placenta 35 (2014) A1eA23
laparotomy on day 14 without RUPP procedure. Hydrogen-saturated water (HW) was orally administered ad libitum from day 12 to 19. 24 hours urinary protein was measured on day 18. Mean arterial pressure (MAP) was measured via carotid catheters, and fetus and placenta were collected by cesarean section on day 19. And we assessed placenta histologically by HE staining. Results: MAP was significantly increased in the RUPP group compared with the sham group. Hydrogen water treatment improved MAP and attenuated urinary protein (sham 102.5mmHg, 4.73mg/dL, RUPP 118.1mmHg, 8.14mg/dL and RUPP+HW 107.7mmHg, 5.88mg/dL). RUPP fetuses and placentas were significantly smaller than those of sham group. In the RUPP+HW group, fetal and placental weights were improved (sham 2.52g, 0.433g, RUPP 1.91g 0.379g and RUPP+HW 2.13g, 0.441g). Spongiotrophoblast cells layer of placenta was reduced in RUPP group compared with sham group. This layer was increased by Hydrogen water. Conclusion: The administration of HW significantly attenuated features of preeclampsia. These results suggest that maternal administration of HW could have a potential benefit for the prevention of preeclampsia as a novel intra-uterine therapy.
O-008. CLINICOPATHOGICAL INTERVILLOSITIS
FEATURES
OF
CHRONIC
HISTIOCYTIC
Yuichiro Sato a, Sayaka Moriguchi a, Atsushi Yamashita b, Yujiro Asada b, Hiroshi Sameshima c. a Departments of Diagnostic Pathology, University Miyazaki Hospital, Faculty of Medicine, University of Miyazaki, Japan; b Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan; c Departments of Obstetrics & Gynecology, Faculty of Medicine, University of Miyazaki, Japan Context: Chronic histiocytic intervillositis (CHI) is a rare entity, defined by inflammatory placental lesions prevailing in the intervillous space. CHI is associated with poor perinatal prognosis by placental insufficiency. Objective: To evaluate the clinicopathological of CHI, we reviewed the CHI cases and study the relation between clinical features and histological findings. Design: We studied the placentas with CHI in the University Miyazaki Hospital. Perinatal outcome was evaluated according to the clinical records. Results: Sixteen pregnancies complicated by CHI were included (10 patients). Four patients had a recurrence of CHI. Mean gestational week was 26 weeks (8-37weeks). Intrauterine growth restriction was seen in 8 cases, and spontaneous abortion was noted in 5 cases. Histological examination showed histiocytic infiltration and fibrin deposition in the intervillous spaces. Conclusions: CHI has a poor perinatal outcome and a high rate of recurrence. There is a relation between clinical expression and histological lesions.
O-009. EFFECTS OF A RHO-ASSOCIATED KINASE INHIBITOR ON CELLULAR FUNCTIONS OF TERM HUMAN PLACENTA-DERIVED PRIMARY CYTOTROPHOBLASTS Kenichro Motomura a, b, Naoko Okada a, Akio Matsuda a, Haruhiko Sago b, Hirohisa Saito a, Kenji Matsumoto a. a Department of Allergy and Immunology, National Research Institute for Child Health and Development, Japan; b Center of Maternal-Fetal, Neonatal and Reproductive Medicine, National Center for Child Health and Development, Japan Objectives: Term human placenta-derived primary cytotrophoblasts (primary human trophoblasts, PHT), which naturally fuse and differentiate into syncytiotrophoblast-like cells in vitro, are a useful tool for analyzing placental functions in vitro. However, in vitro culture of PHT is not easy because very few cells adhere to culture plates, and the cells merely proliferate in vitro. Rho-associated kinases (ROCKs) reportedly regulate various cell functions, including cell adhesion and survival. Specific
A7
inhibitors of ROCK enhance various cells adhesion to culture plates. In the present study, we investigated the effects of a ROCK inhibitor, Y-27632, on several functions of term human placenta-derived primary cytotrophoblasts in vitro. Methods: PHT were isolated from term, uncomplicated placentas by trypsin-DNase I-Dispase II treatment and percoll density gradient centrifugation, followed by negative-selection using anti-HLA-A, -B and -C mAbcoated immunomagnetic beads. Purified PHT were suspended in 10% FBS, 10 ng/ml EGF-containing medium with various concentrations of Y-27632 and cultured for up to 96 h. Adhesion was assessed by counting the number of cell nuclei on the culture plates and measuring the area of cytoplasm using immunofluorescent dyes. Mitochondrial activity was assessed by WST-8 assay. Syncytium formation and trophoblast maturation were assessed by qPCR assay of syncytialization markers (syncytin 1 and syncytin 2) and ELISA of b-HCG, respectively. Results: Y-27632 treatment induced significant PHT adhesion to culture plates in a concentration-dependent manner. The mitochondrial activity of PHT was also significantly enhanced. Y-27632 treatment up-regulated syncytin 1 and syncytin 2 mRNA expression and b-HCG secretion. Conclusion: ROCK is likely to regulate several different functions of PHT. A ROCK inhibitor, Y-27632, is capable of inducing PHT adhesion, syncytium formation and b-HCG production in vitro.
O-011. TRANSPLANTATION OF HUMAN AMNIOTIC MEMBRANE-DERIVED MESENCHYMAL STEM CELLS IMPROVES RADIATION ENTERITIS Minori Honda a, Shunsuke Ohnishi b, Masayoshi Ono b, Kenichi Yamahara c, Jun Yoshimatsu d, Koji Nakagawa a, Hiroshi Takeda a, Naoya Sakamoto b. a Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan; b Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Japan; c Department of Regenerative Medicine, National Cerebral and Cardiovascular Center, Japan; d Division of Maternal and Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Aims: Mesenchymal stem cells (MSCs) have been reported to be a cell source in cell therapy, and several studies have shown that MSCs can be easily isolated from amnionic membrane (AM), and a large amount of cells can be obtained. Therapeutic irradiations, which are performed for the treatment of intrapelvic cancers, often cause radiation enteritis; however, there is no effective treatment at present. Therefore, we examined the effects of transplantation of human AM-derived MSCs (hAM-MSCs) in rats with radiation enteritis. Methods: The Medical Ethical Committee approved this examination and all pregnant women gave written informed consent. AM was obtained at Cesarean delivery, and hAM-MSCs were isolated by collagenase treatment, and expanded with culture medium containing fetal bovine serum. Sprague-Dawley rats were exposed to X-ray (5 Gy/day) from day 1 to day 5. At day 5, hAM-MSCs (1X106 cells) were transplanted intravenously. Rats were sacrificed on day 8. Histological analyses were performed, and mRNA expressions of inflammatory mediators were measured by quantitative RTPCR. In vitro, after rat intestinal epithelial cells (IEC-6) were irradiated with X-ray (4 Gy), the cells were cultured with MSC-conditioned medium (CM). The effect of CM was evaluated by caspase 3/7 assay and quantitative RTPCR. Results: Transplantation of hAM-MSCs tended to reduce mRNA expression of inflammatory cytokines such as CXCL1 and CCL2. In addition, the number of goblet cells was significantly recovered in the rectum of hAMMSC-treated rats. Furthermore, infiltration of monocytes/macrophages was suppressed to the level of non-irradiated group. In vitro experiments demonstrated that the apoptosis of IEC-6 cells by radiation tended to be inhibited by incubation with CM. The mRNA expression of p21, one of the target genes of p53, was significantly decreased. Conclusion: Transplantation of hAM-MSCs is likely to improve radiation enteritis, and would be considered as a new treatment of radiation enteritis.
Abstracts / Placenta 35 (2014) A1eA23
hyalinized material. Surgical procedures, such as dilatation and curettage and cesarean section, might increase the risk of PSN/P. It has been suggested that PSN/P might be consequence of disturbances to the implantation, resulting in abnormal involution of the placental site. The subjective symptom is menorrhagia or intermenstral bleeding, the effect on fertility is unknown.
O-001. A PREGNANCY CASE REPORT OF ALAGILLE SYNDROME; EXPRESSION OF NOTCH SIGNALING IN THE PLACENTA Kazue Togashi a, Megumi Sato a, Akira Sato a, Yurina Kameta b, Reiko Takayama a, Hiroshi Miura c, Yukihiro Terada a. a Akita University Graduate School of Medicine, Department of Obstetrics and Gynecology, Japan; b Akita City Hospital, Japan; c Kazuno Kosei Hospital, Japan Background: Pregnancy in patients with Alagille syndrome (AS) is rare. It is characterized by chronic cholestasis resulting from intrahepatic bile duct paucity. 114 Japanese AS patients were reported in 2009. Case report: A 22 year old female with history of a miscarriage 7months previously was seen at a hospital for edema and amenorrhea. She was diagnosed at 27 weeks and 4 days of gestation by fetal biparietal diameter and transferred to our maternity unit. Her physical examination was remarkable for low platelets, hypertension, proteinuria and ascites. Her medical record from pediatrics in our hospital revealed that she had AS; hepatic, cardiac, facial and skeletal anomalies. Anesthetic difficulty was predicted. Her thrombocytopenia was caused by liver cirrhosis and hypersplenism. The patient had no esophageal varices and no intracranial vessel abnormalities. Due to the deterioration of her renal function a cesarean section under general anesthesia was performed at 29 weeks. Atonic bleeding was treated with an intrauterine balloon. Management of acute pulmonary edematous, severe proteinuria and hypertension required several days of observation and treatment. After a couple of months her blood pressure and proteinuria had returned to normal levels; meaning that she had been preeclamptic. Conclusion: An awareness of possible organ involvement and a multidisciplinary approach is required in the management of pregnancy with AS. This fascinating case might hopefully provide perspectives for both future research into treatment of Alagille syndrome and human placentation of pregnancy complicated by preeclampsia.
O-002. IDENTIFICATION AND CHARACTERIZATION OF HTR-8/SVNEO SP CELLS. Tetsunori Inagaki a, Kiyoko Kato b, Soshi Kusunoki a, Koichi Tabu c, Hitomi Okabe a, Izumi Yamada a, Tetsuya Taga c, Akemi Matsumoto a, Toshiaki Takezawa d, Shintaro Makino a, Satoru Takeda a. a Jyuntendo Hospital, Japan; b Kyushu University, Japan; c Tokyo medical and dental University, Japan; d National Institute of Agrobiological Sciences, Japan Goals: We investigated the side population cells in HTR-8/SVneo to identify the trophoblast stem cell marker, and characterize trophoblast stem cells. Method: We performed microarray expression analysis to search for upregulated genes in trophoblast stem cell using a set of HTR-8/SVneo side population and non-side population cells. The expression of several candidate trophoblast stem cell markers were investigated by real time PCR, and performed knockdown of up-regulated genes expression using siRNA to analyze the association of up-regulated genes expression with trophoblast phenotype. Finally three dimensional cell culture system was established to analyze the association with epithelial-mesenchymal transition. Result: LY6D gene was highly up-regulated in side population cells. LY6D gene reduced its expression in the course of seven days’ cultivation in differentiation medium. Side population cells tended to reduce its fraction by treatment of LY6D siRNA. On ontology analysis, two Gene GO pathways of epithelial-mesenchymal transition were involved. Epithelial-
mesenchymal transition was induced by the co-existence of endometrial cells and side population cells. Conclusion: This is the first report to have identified the expression of LY6D and demonstrated induction of epithelial-mesenchymal transition in HTR-8/SVneo side population cells.
O-004. CLINICAL APPLICATION OF HUMAN AMNION-DERIVED MESENCHYMAL STEM CELLS FOR THE TREATMENT OF ACUTE GVHD Kenichi Yamahara a, Akihiko Taguchi b, Toshihiro Soma c, Hiroyasu Ogawa c, Tomoaki Ikeda d, Jun Yoshimatsu e. a Department of Regenerative Medicine, National Cerebral and Cardiovascular Center, Japan; b Department of Regenerative Medicine Research, Foundation for Biomedical Research and Innovation, Japan; c Division of Hematology, Department of Internal Medicine, Hyogo College of Medicine, Japan; d Department of Obstetrics & Gynecology, Mie University Faculty of Medicine, Japan; e Division of Maternal Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Purpose: We have demonstrated that, similar to bone marrow-derived mesenchymal stem cells (BM-MSCs), fetal appendage-derived MSCs possess the differentiation, angiogenic and immunosuppressive property. Fetal appendage, normally discarded as medical waste, is a rich source of MSCs, which secret significant amounts of cytokines/growth factors, indicating a suitable cell source to perform a cell therapy at low-cost, faster, and with less MSCs. Among fetal appendage, amnion is anatomically easy to separate MSCs by enzymatic digestion, and we started a multicenter clinical trial to treat acute GVHD using amnion-derived MSCs. Methods and Results: For the clinical study of cell therapy using amnion MSCs, we established a multicenter clinical team as follows: National Cerebral and Cardiovascular Center (amnion MSC manufacturing), Foundation for Biomedical Research and Innovation (quality control of amnion MSCs), and Hyogo College of Medicine (administration of amnion MSCs in patients with acute GVHD). We also established a method to isolate amnion MSCs with ease and high efficiency (Japanese patent application No. 2013-170008). After a consultation with the Pharmaceuticals and Medical Devices Agency (PMDA), we started to manufacture amnion MSCs adhering to GMP, and are planning to perform a phase I clinical trial within this year to assess the safety of amnion MSCs in patients with acute GVHD. Conclusion: Outside our country, BM-MSCs have already received approval for the treatment of children with acute GVHD. However, an invasive procedure and a long-term culturing is required to obtain an adequate number of BM-MSCs. Since a large number of MSCs could be obtained easily and non-invasively, amnion MSCs would be an ideal cell source for regenerative medicine.
O-006. THERAPEUTIC EFFECT OF MATERNAL MOLECULAR ADMINISTRATION IN A RAT MODEL OF PREECLAMPSIA
HYDROGEN
Takafumi Ushida, Yukio Mano, Hiroyuki Tsuda, Seiji Sumigama, Tomomi Kotani, Fumitaka Kikkawa. Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Japan Objective: Preeclampsia is a pregnancy-specific condition characterized by new-onset hypertension and proteinuria. It is also occasionally related to fetal growth restriction. Some studies suggest that placental oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) is reported to prevent a variety of diseases associated with oxidative stress in model systems and in human. We studied the efficacy of H2 using a rat model of preeclampsia. Methods: We used the well-established reduced utero-placental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in rat that mimics features of preeclampsia. RUPP was performed on day 14 of pregnancy, clips were placed around the aorta below the renal arteries and on both uterine arcade at the ovarian artery. The sham group underwent
Abstracts / Placenta 35 (2014) A1eA23
laparotomy on day 14 without RUPP procedure. Hydrogen-saturated water (HW) was orally administered ad libitum from day 12 to 19. 24 hours urinary protein was measured on day 18. Mean arterial pressure (MAP) was measured via carotid catheters, and fetus and placenta were collected by cesarean section on day 19. And we assessed placenta histologically by HE staining. Results: MAP was significantly increased in the RUPP group compared with the sham group. Hydrogen water treatment improved MAP and attenuated urinary protein (sham 102.5mmHg, 4.73mg/dL, RUPP 118.1mmHg, 8.14mg/dL and RUPP+HW 107.7mmHg, 5.88mg/dL). RUPP fetuses and placentas were significantly smaller than those of sham group. In the RUPP+HW group, fetal and placental weights were improved (sham 2.52g, 0.433g, RUPP 1.91g 0.379g and RUPP+HW 2.13g, 0.441g). Spongiotrophoblast cells layer of placenta was reduced in RUPP group compared with sham group. This layer was increased by Hydrogen water. Conclusion: The administration of HW significantly attenuated features of preeclampsia. These results suggest that maternal administration of HW could have a potential benefit for the prevention of preeclampsia as a novel intra-uterine therapy.
O-008. CLINICOPATHOGICAL INTERVILLOSITIS
FEATURES
OF
CHRONIC
HISTIOCYTIC
Yuichiro Sato a, Sayaka Moriguchi a, Atsushi Yamashita b, Yujiro Asada b, Hiroshi Sameshima c. a Departments of Diagnostic Pathology, University Miyazaki Hospital, Faculty of Medicine, University of Miyazaki, Japan; b Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan; c Departments of Obstetrics & Gynecology, Faculty of Medicine, University of Miyazaki, Japan Context: Chronic histiocytic intervillositis (CHI) is a rare entity, defined by inflammatory placental lesions prevailing in the intervillous space. CHI is associated with poor perinatal prognosis by placental insufficiency. Objective: To evaluate the clinicopathological of CHI, we reviewed the CHI cases and study the relation between clinical features and histological findings. Design: We studied the placentas with CHI in the University Miyazaki Hospital. Perinatal outcome was evaluated according to the clinical records. Results: Sixteen pregnancies complicated by CHI were included (10 patients). Four patients had a recurrence of CHI. Mean gestational week was 26 weeks (8-37weeks). Intrauterine growth restriction was seen in 8 cases, and spontaneous abortion was noted in 5 cases. Histological examination showed histiocytic infiltration and fibrin deposition in the intervillous spaces. Conclusions: CHI has a poor perinatal outcome and a high rate of recurrence. There is a relation between clinical expression and histological lesions.
O-009. EFFECTS OF A RHO-ASSOCIATED KINASE INHIBITOR ON CELLULAR FUNCTIONS OF TERM HUMAN PLACENTA-DERIVED PRIMARY CYTOTROPHOBLASTS Kenichro Motomura a, b, Naoko Okada a, Akio Matsuda a, Haruhiko Sago b, Hirohisa Saito a, Kenji Matsumoto a. a Department of Allergy and Immunology, National Research Institute for Child Health and Development, Japan; b Center of Maternal-Fetal, Neonatal and Reproductive Medicine, National Center for Child Health and Development, Japan Objectives: Term human placenta-derived primary cytotrophoblasts (primary human trophoblasts, PHT), which naturally fuse and differentiate into syncytiotrophoblast-like cells in vitro, are a useful tool for analyzing placental functions in vitro. However, in vitro culture of PHT is not easy because very few cells adhere to culture plates, and the cells merely proliferate in vitro. Rho-associated kinases (ROCKs) reportedly regulate various cell functions, including cell adhesion and survival. Specific
A7
inhibitors of ROCK enhance various cells adhesion to culture plates. In the present study, we investigated the effects of a ROCK inhibitor, Y-27632, on several functions of term human placenta-derived primary cytotrophoblasts in vitro. Methods: PHT were isolated from term, uncomplicated placentas by trypsin-DNase I-Dispase II treatment and percoll density gradient centrifugation, followed by negative-selection using anti-HLA-A, -B and -C mAbcoated immunomagnetic beads. Purified PHT were suspended in 10% FBS, 10 ng/ml EGF-containing medium with various concentrations of Y-27632 and cultured for up to 96 h. Adhesion was assessed by counting the number of cell nuclei on the culture plates and measuring the area of cytoplasm using immunofluorescent dyes. Mitochondrial activity was assessed by WST-8 assay. Syncytium formation and trophoblast maturation were assessed by qPCR assay of syncytialization markers (syncytin 1 and syncytin 2) and ELISA of b-HCG, respectively. Results: Y-27632 treatment induced significant PHT adhesion to culture plates in a concentration-dependent manner. The mitochondrial activity of PHT was also significantly enhanced. Y-27632 treatment up-regulated syncytin 1 and syncytin 2 mRNA expression and b-HCG secretion. Conclusion: ROCK is likely to regulate several different functions of PHT. A ROCK inhibitor, Y-27632, is capable of inducing PHT adhesion, syncytium formation and b-HCG production in vitro.
O-011. TRANSPLANTATION OF HUMAN AMNIOTIC MEMBRANE-DERIVED MESENCHYMAL STEM CELLS IMPROVES RADIATION ENTERITIS Minori Honda a, Shunsuke Ohnishi b, Masayoshi Ono b, Kenichi Yamahara c, Jun Yoshimatsu d, Koji Nakagawa a, Hiroshi Takeda a, Naoya Sakamoto b. a Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan; b Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Japan; c Department of Regenerative Medicine, National Cerebral and Cardiovascular Center, Japan; d Division of Maternal and Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Aims: Mesenchymal stem cells (MSCs) have been reported to be a cell source in cell therapy, and several studies have shown that MSCs can be easily isolated from amnionic membrane (AM), and a large amount of cells can be obtained. Therapeutic irradiations, which are performed for the treatment of intrapelvic cancers, often cause radiation enteritis; however, there is no effective treatment at present. Therefore, we examined the effects of transplantation of human AM-derived MSCs (hAM-MSCs) in rats with radiation enteritis. Methods: The Medical Ethical Committee approved this examination and all pregnant women gave written informed consent. AM was obtained at Cesarean delivery, and hAM-MSCs were isolated by collagenase treatment, and expanded with culture medium containing fetal bovine serum. Sprague-Dawley rats were exposed to X-ray (5 Gy/day) from day 1 to day 5. At day 5, hAM-MSCs (1X106 cells) were transplanted intravenously. Rats were sacrificed on day 8. Histological analyses were performed, and mRNA expressions of inflammatory mediators were measured by quantitative RTPCR. In vitro, after rat intestinal epithelial cells (IEC-6) were irradiated with X-ray (4 Gy), the cells were cultured with MSC-conditioned medium (CM). The effect of CM was evaluated by caspase 3/7 assay and quantitative RTPCR. Results: Transplantation of hAM-MSCs tended to reduce mRNA expression of inflammatory cytokines such as CXCL1 and CCL2. In addition, the number of goblet cells was significantly recovered in the rectum of hAMMSC-treated rats. Furthermore, infiltration of monocytes/macrophages was suppressed to the level of non-irradiated group. In vitro experiments demonstrated that the apoptosis of IEC-6 cells by radiation tended to be inhibited by incubation with CM. The mRNA expression of p21, one of the target genes of p53, was significantly decreased. Conclusion: Transplantation of hAM-MSCs is likely to improve radiation enteritis, and would be considered as a new treatment of radiation enteritis.