Hsin-Yang Li

Functional Improvement of Focal Cerebral Ischemia Injury by Subdural Transplantation of Induced Pluripotent Stem Cells with Fibrin Glue

Ischemic stroke is the leading cause of disability in the world. Cell transplantation has emerged in various neurological diseases as a potential therapeutic approach in the postacute stroke phase. Recently, inducible pluripotent stem (iPS) cells showed potential for multilineage differentiation and provide a resource for stem cell-based therapies. However, whether iPS transplantation could improve the function of stroke-like model is still an open question. The aim of this study is to investigate the therapeutic effects of subdural transplantation of iPS mixed with fibrin glue (iPS-FG) on cerebral ischemic rats induced by middle cerebral artery occlusion (MCAO). We demonstrated an efficient method to differentiate iPS into astroglial-like and neuron-like cells which display functional electrophysiological properties. In vivo study firstly showed that the direct injection of iPS into damaged areas of rat cortex significantly decreased the infarct size and improved the motor function in rats with MCAO. Furthermore, we found that the subdural iPS-FG can also effectively reduce the total infarct volume and greatly improve the behavior of rats with MCAO to perform rotarod and grasping tasks. Importantly, analysis of cytokine expression in iPS-FG-treated ischemic brains revealed a significant reduction of pro-inflammatory cytokines and an increase of anti-inflammatory cytokines. Taken together, these results suggest that iPS cells could improve the motor function, reduce infarct size, attenuate inflammation cytokines, and mediate neuroprotection after ischemic stroke. Subdural iPS-FG could be considered as a more safe approach because this method can avoid iatrogenic injury to brain parenchyma and enhance recovering from stoke-induced impairment.

Oestrogen‐induced epithelial–mesenchymal transition of endometrial epithelial cells contributes to the development of adenomyosis

Adenomyosis is an oestrogen‐dependent disease caused by a downward extension of the endometrium into the uterine myometrium. Epithelial–mesenchymal transition (EMT) endows cells with migratory and invasive properties and can be induced by oestrogen. We hypothesized that oestrogen‐induced EMT is critical in the pathogenesis of adenomyosis. We first investigated whether EMT occurred in adenomyotic lesions and whether it correlated with serum 17β‐oestradiol (E2) levels. Immunohistochemistry was performed on adenomyotic lesions and corresponding eutopic endometrium samples from women with adenomyosis. Endometria from women without endometrial disorders were used as a control. In the epithelial component of adenomyotic lesions, vimentin expression was up‐regulated and E‐cadherin expression was down‐regulated compared to the eutopic endometrium, suggesting that EMT occurs in adenomyosis. In adenomyosis, the serum E2 level was negatively correlated with E‐cadherin expression in the epithelial components of the eutopic endometrium and adenomyotic lesions, suggesting the involvement of oestrogen‐induced EMT in endometrial cells. In oestrogen receptor‐positive Ishikawa endometrial epithelial cells, oestrogen induced a morphological change to a fibroblast‐like phenotype, a shift from epithelial marker expression to mesenchymal marker expression, increased migration and invasion, and up‐regulation of the EMT regulator Slug. Raloxifene, a selective oestrogen receptor modulator, abrogated these effects. To determine the role of oestrogen‐induced EMT in the implantation of ectopic endometrium, we xenotransplanted eutopic endometrium or adenomyotic lesions from adenomyosis patients into ovariectomized SCID mice. The implantation of endometrium was oestrogen‐dependent and was suppressed by raloxifene. Collectively, these data highlight the crucial role of oestrogen‐induced EMT in the development of adenomyosis and suggest that raloxifene may be a potential therapeutic agent for adenomyosis patients. Copyright

Enhancement of wound healing by human multipotent stromal cell conditioned medium: the paracrine factors and p38 MAPK activation.

Wound healing can be improved by transplanting mesenchymal stem cells (MSCs). In this study, we have demonstrated the benefits of the conditioned medium derived from human MSCs (CM-MSC) in wound healing using an excisional wound model. CM-MSC accelerated wound closure with increased reepithelialization, cell infiltration, granulation formation, and angiogenesis. Notably, CM-MSC enhanced epithelial and endothelial cell migration, suggesting the contribution of increased cell migration to wound healing enhanced by CM-MSC. Cytokine array, ELISA analysis, and quantitative RT-PCR revealed high levels of IL-6 in CM-MSC. Moreover, IL-6 added to the preconditioned medium enhanced both cell migration and wound healing, and antibodies against IL-6 blocked the increase in cell motility and wound closure by CM-MSC. The IL-6 secretory pathway of MSCs was inhibited by SB203580, an inhibitor of p38 MAPK or siRNA against p38 MAPK, suggesting IL-6 secretion by MSCs is mediated through the activation of p38 MAPK. Inactivation of p38 MAPK also reduced the expression and production of IL-8 and CXCL1 by MSCs, both of which were also demonstrated to enhance cell migration and wound closure. Thus, our data suggest MSCs promote wound healing through releasing a repertoire of paracrine factors via activation of p38 MAPK, and the CM-MSC may be applied to enhance wound healing.

Resveratrol Promotes Osteogenic Differentiation and Protects Against Dexamethasone Damage in Murine Induced Pluripotent Stem Cells

Resveratrol is a natural polyphenol antioxidant that has been shown to facilitate osteogenic differentiation. A recent breakthrough has demonstrated that ectopic expression of four genes is sufficient to reprogram murine and human fibroblasts into induced pluripotent stem (iPS) cells. However, the roles of resveratrol in the differentiation and cytoprotection of iPS cells have never been studied. In this study, we showed that, in addition to cardiac cells, neuron-like cells, and adipocytes, mouse iPS cells could differentiate into osteocyte-like cells. Using atomic force microscopy that provided nanoscale resolution, we monitored mechanical properties of living iPS cells during osteogenic differentiation. The intensity of mineralization and stiffness in differentiating iPS significantly increased after 14 days of osteogenic induction. Furthermore, resveratrol was found to facilitate osteogenic differentiation in both iPS and embryonic stem cells, as shown by increased mineralization, up-regulation of osteogenic markers, and decreased elastic modulus. Dexamethasone-induced apoptosis in iPS cell-derived osteocyte-like cells was effectively prevented by pretreatment with resveratrol. Furthermore, resveratrol significantly increased manganese superoxide dismutase expression and intracellular glutathione level, thereby efficiently decreasing dexamethasone-induced reactive oxygen species (ROS) production and cytotoxicity. Transplantation experiments using iPS cell-derived osteocyte-like cells further demonstrated that oral intake of resveratrol could up-regulate osteopontin expression and inhibit teratoma formation in vivo. In sum, resveratrol can facilitate differentiation of iPS cells into osteocyte-like cells, protect these iPS cell-derived osteocyte-like cells from glucocorticoid-induced oxidative damage, and decrease tumorigenicity of iPS cells. These findings implicate roles of resveratrol and iPS cells in the stem cell therapy of orthopedic diseases.

IV Delivery of Induced Pluripotent Stem Cells Attenuates Endotoxin-Induced Acute Lung Injury in Mice

BACKGROUND Induced pluripotent stem (iPS) cells are novel stem cell populations, but the role of iPS cells in acute lung injury (ALI) is not currently known. We investigated the effect of iPS cells in modifying the pathophysiology of endotoxin-induced ALI. METHODS Male C57BL/6 8- to 12-week-old mice were enrolled in this study. Mouse iPS cells were delivered through the tail veins of mice 4 h after intratracheal instillation of endotoxin. Lung histopathologic findings, the pulmonary levels of cytokines, and functional parameters were analyzed after either 24 h or 48 h. RESULTS More iPS cells integrated into the lungs of mice with ALI than those of the control mice, as demonstrated by in vivo radionuclide imaging and in vitro Hoechst-labeled fluorescent staining. iPS cells significantly diminished the histopathologic changes of ALI and the lung injury score. There was also a significant reduction in the activity of nuclear factor-κB (NF-κB) and neutrophil accumulation in the lung, confirmed by immunostaining, electrophoretic mobility shift assays, and the decrease of myeloperoxidase activity, in the iPS-cell-treated mice with ALI. These protective effects were not replicated by the control cell therapy with fibroblasts. iPS cells mediated a downregulation of the proinflammatory response to endotoxin (reducing tumor necrosis factor-α, IL-6, and macrophage inflammatory peptide-2). In addition, iPS cells rescued the hypoxemia and pulmonary function of ALI. Treatment with a conditioned medium of iPS cells showed effects similar to those of iPS cells, which may suggest the therapeutic benefits of iPS mediated by paracrine factors. CONCLUSIONS IV delivery of iPS cells provides a beneficial effect to attenuate the severity of endotoxin-induced ALI and improve physiologic impairment, which is partly mediated by a reduction in NF-κB activity and neutrophils accumulation. The conditioned medium of iPS cells demonstrated effects equal to those of iPS cells.

Induction of Insulin-Producing Cells Derived from Endometrial Mesenchymal Stem-like Cells

Studies have demonstrated that mesenchymal stem-like cells can be isolated from endometrium. However, the potential of endometrial-derived stem cells to differentiate into insulin-positive cells and functionally secrete insulin remains undetermined. We isolated endometrial mesenchymal stem-like cells (EMSCs) from human endometrial tissue from six donors. The insulin-secreting function of EMSCs was further analyzed in vitro and in transplanted grafts in vivo. We successfully isolated EMSCs from human endometrium, and our results showed that EMSCs expressed high levels of stemness genes (Nanog, Oct-4, Nestin). Under specific induction conditions for 2 weeks, EMSCs formed three-dimensional spheroid bodies (SBs) and secreted C-peptide. The high insulin content of SB-EMSCs was confirmed by enzyme-linked immunosorbent assay, and glucose responsiveness was demonstrated by measuring glucose-dependent insulin secretion. Using cDNA microarrays, we found that the expression profiles of SB-EMSCs are related to those of islet tissues. Insulin and C-peptide production in response to glucose was significantly higher in SB-EMSCs than in undifferentiated EMSC controls. Furthermore, upon differentiation, SB-EMSCs displayed increased mRNA expression levels of NKx2.2, Glut2, insulin, glucagon, and somatostatin. Our results also showed that SB-EMSCs were more resistant to oxidative damage and oxidative damage-induced apoptosis than fibroblasts from the same patient. It is noteworthy that SB-EMSCs xenotransplanted into immunocompromised mice with streptozotocin-induced diabetes restored blood insulin levels to control values and greatly prolonged the survival of graft cells. These data suggest that EMSCs not only play a novel role in the differentiation of pancreatic progenitors, but also can functionally enhance insulin production to restore the regulation of blood glucose levels in an in vivo transplantation model.

Induction of p38 Mitogen-Activated Protein Kinase-Mediated Apoptosis Is Involved in Outgrowth of Trophoblast Cells on Endometrial Epithelial Cells in a Model of Human Trophoblast-Endometrial Interactions

Abstract During embryo implantation in species with hemochorial placentation, such as the mouse and human, trophoblast cells of the attached blastocyst penetrate the luminal epithelium of the endometrium before invasion into the endometrial stroma. Signs of apoptosis were demonstrated in luminal endometrial epithelial cells (EEC) adjacent to the trophoblast cells; however, the signaling mechanisms leading to apoptosis in EEC remain unclear. Because mitogen-activated protein kinases (MAPK) were shown to mediate apoptosis in several model systems and found to be activated in the uterus during decidualization, the possible involvement of MAPK during trophoblast-EEC interactions was studied. By coculturing BeWo human trophoblast spheroids with RL95-2 human EEC monolayers to mimic the blastocyst-endometrial interaction, we found that most spheroids rapidly attached to EEC monolayers and then progressively expanded, with marked dislodgment of EEC adjacent to the spreading trophoblast cells. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC after coculture. However, only SB203580 (a p38 MAPK inhibitor), but not PD98059 (an ERK inhibitor), inhibited trophoblast outgrowth on EEC monolayers through the suppression of p38 MAPK activation in EEC. Furthermore, trophoblast expansion caused prominent EEC apoptosis at the spheroid-EEC interface, as detected by annexin V labeling and valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (which binds activated caspases) staining, and SB203580 significantly decreased the percentage of apoptotic cells. Our results, based on a model of human trophoblast-EEC interactions, establish that trophoblast cells cause activation of p38 MAPK in EEC and, consequently, induce apoptosis and displacement of EEC, a process that may facilitate implantation.

Oestrogen‐induced angiogenesis promotes adenomyosis by activating the Slug‐VEGF axis in endometrial epithelial cells

Adenomyosis is an oestrogen‐dependent disease characterized by the invasion of endometrial epithelial cells into the myometrium of uterus, and angiogenesis is thought to be required for the implantation of endometrial glandular tissues during the adenomyotic pathogenesis. In this study, we demonstrate that compared with eutopic endometria, adenomyotic lesions exhibited increased vascularity as detected by sonography. Microscopically, the lesions also exhibited an oestrogen‐associated elevation of microvascular density and VEGF expression in endometrial epithelial cells. We previously reported that oestrogen‐induced Slug expression was critical for endometrial epithelial–mesenchymal transition and development of adenomyosis. Our present studies demonstrated that estradiol (E2) elicited a Slug‐VEGF axis in endometrial epithelial cells, and also induced pro‐angiogenic activity in vascular endothelial cells. The antagonizing agents against E2 or VEGF suppressed endothelial cells migration and tubal formation. Animal experiments furthermore confirmed that blockage of E2 or VEGF was efficient to attenuate the implantation of adenomyotic lesions. These results highlight the importance of oestrogen‐induced angiogenesis in adenomyosis development and provide a potential strategy for treating adenomyosis through intercepting the E2‐Slug‐VEGF pathway.

MafA promotes the reprogramming of placenta‐derived multipotent stem cells into pancreatic islets‐like and insulin+ cells

MafA is a pancreatic transcriptional factor that controls β‐cell‐specific transcription of the insulin gene. However, the role of MafA in the regulation of pancreatic transdifferentiation and reprogramming in human stem cells is still unclear. In this study, we investigate the role of MafA in placenta‐derived multipotent stem cells (PDMSCs) that constitutively expressed Oct‐4 and Nanog. PDMSCs were isolated and transfected with MafA using a lentivector. Our results showed that overexpression of MafA in PDMSCs significantly up‐regulated the expression of pancreatic development‐related genes (Sox17, Foxa2, Pdx1 and Ngn3). Microarray analysis suggested that the gene expression profile of MafA‐overexpressing PDMSCs was similar to that of pancreas and islet tissues. MafA increased the expression levels of the mRNAs of NKx2.2, Glut2, insulin, glucagons and somatostatin, and further facilitated the differentiation of PDMSCs into insulin+ cells. The glucose‐stimulated responses to insulin and c‐peptide production in MafA‐overexpressing PDMSCs were significantly higher than in PDMSCs with vector control. Our results indicated that MafA‐overexpressing PDMSCs were more resistant to oxidative damage and oxidative damage‐induced apoptosis than PDMSCs carrying the vector control were. Importantly, the expression of MafA in PDMSCs xenotransplanted into immunocompromised mice improved the restoration of blood insulin levels to control values and greatly prolonged the survival of graft cells in immunocompromised mice with STZ‐induced diabetes. In summary, these data suggest that MafA plays a novel role in the reprogramming of stem cells into pancreatic β‐progenitors, promotes the islet‐like characteristics of PDMSCs, as well as functionally enhances insulin production to restore the regulation of blood glucose levels in transplanted grafts.

An overview of a 30-year experience with amniocentesis in a single tertiary medical center in Taiwan.

OBJECTIVE Amniocentesis is a popular and effective prenatal diagnostic tool for chromosomal disorders. It is well-established that the risk of chromosomal abnormalities increases with maternal age; however, other related indications are seldom reported. Herein, we report our 30-year experience with amniocentesis from a single medical center, focusing on the indications and rates of abnormality. MATERIAL AND METHODS A retrospective review of 16,749 pregnant women in the mid-trimester between January 1981 and December 2010 was conducted. The medical records were analyzed. RESULTS The indications for amniocentesis were advanced maternal age (≥ 34 years old) (n=10,970, 65.5%), increasing-risk maternal triple-marker Downs screening test (≥ 1/270) (n=2090, 12.5%), history of abnormal offspring birth (n=792, 4.7%), abnormal ultrasound findings (n=484, 2.9%), parent with abnormal karyotype (n=252, 1.5%), family history of chromosomal abnormality (n=183, 1.1%), drug and radiation exposure (n=165), abnormal chorionic villus sampling (CVS) results (n=25), intrauterine fetal death (n=50), and other non-specific causes (n=1662, 9.9%). The rate of abnormality for each indication was 16% in the abnormal CVS group, 12% in the intrauterine fetal death group, 11.5% for parental chromosomal abnormality, 8.7% in the abnormal ultrasound finding group, 3.0% in the increasing-risk maternal triple-marker Downs screening test group, 2.5% in the advanced maternal age group, 1.5% for other non-specific causes, 1.4% for history of abnormal offspring birth, and 1.1% for family history of chromosomal abnormality. CONCLUSIONS Both parents with abnormal karyotype and abnormal ultrasound findings are indications for which consideration of further amniocentesis is highly recommended.