Antitumor effect of chondroitin sulfate-coated ternary granulocyte macrophage-colony-stimulating factor plasmid complex for ovarian cancer.

BACKGROUND: Although replication-competent viruses have been developed for treating cancers, their cytotoxic effects are insufficient as a result of infection inhibited by the generation of neutralizing antibodies, and systemic administration is difficult as a result of the life-threatening serious side-effects of virus-induced cytokine surge. To overcome these critical problems, we devised a plasmid/polycation/polyanion complex and assessed the potential of ternary plasmid complexes coated with chondroitin sulfate in gene therapy for ovarian cancer. The antitumor effects of chondroitin sulfate-coated complex as an anionic component were compared with those of hyaluronic acid on ovarian cancer. METHODS: Plasmid harboring the gene of murine granulocyte macrophage-colony-stimulating factor (mGM-CSF) was complexed with polyethyleneimine (PEI) and hyaluronic acid or chondroitin sulfate. Murine ovarian cancer cells were injected into (C57BL/6 × C3H/He) F(1) mice to prepare a subcutaneous or intraperitoneal tumor model. RESULTS: DNA/PEI was charged positively and DNA/PEI/chondroitin sulfate or DNA/PEI/hyaluronic acid was charged negatively. Plasmid-green fluorescent protein (GFP)/PEI coated with 10-kilodalton (kDa) chondroitin sulfate increased transfection efficiency compared to coating with chondroitin sulfate of higher-molecular-weight or hyaluronic acid. The transfection efficiency of GFP/PEI/10-kDa chondroitin sulfate in ovarian cancer cells was six-fold higher than that in normal cells. Intraperitoneal injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate prolonged survival compared to that coated with hyaluronic acid. Intratumoral injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate achieved mouse survival rates of 100%, although that with hyaluronic acid did not. CONCLUSIONS: These findings suggest that GM-CSF/PEI coated with 10-kDa chondroitin sulfate has the potential for use in gene therapy of ovarian cancer.

Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

Immunological characteristics of amniotic epithelium.

We review recent experimental evidence of the immunosuppressive and immunogenic potential of amniotic epithelial cells. Since cryopreserved amniotic membrane (AM) has been used in clinical applications, much research has focused on the beneficial effects of amniotic stromal matrix rather than on the function of viable amniotic cells. However, viable human amniotic epithelial cells (HAECs) have been shown to elicit beneficial effects on secretion of anti-inflammatory factors. Topical application of culture supernatant from HAECs leads to profound suppression of suture-induced neovascularization in cornea and fewer major histocompatibility complex (MHC) class II antigen-presenting cells (APCs) in inflamed cornea after thermal cautery. Furthermore, expression of interleukin (IL)-1beta mRNA was suppressed in cauterized cornea. These results suggest that HAECs are a source of soluble anti-inflammatory factors that suppress corneal inflammation. However, viable amniotic epithelial cells display antigenicity and immunogenicity as allografts. Fresh allogeneic amniotic epithelium (AE) expresses MHC class I antigens and sensitizes recipients when placed in the eye, although long-term memory of allo-specific delayed hypersensitivity (DH) was not acquired. Allogeneic AE was clearly vulnerable to acute immune rejection in specifically sensitized recipients and recipients of repeated AE transplantation. We therefore suggest that immunogenicity of AE should not be ignored, and use of AM from different donor placentas should be emphasized when repeated AM transplantation is required in patients clinically.

Transplantation of amniotic epithelial cells into fetal rat liver by in utero manipulation.

It has been hoped that amniotic epithelial cells would be a gene carrier to neural and hepatic tissue, because of 1) the presence of neural and hepatic stem-like cells, 2) the ability to cryopreserve them, 3) long-term survival in the transplanted site, and 4) few ethical problems concerning procurement. But transplantation of a sufficient number of cells to adult tissue needs large-scale cell supply and may lead to vascular embolism. We attempted transplantation of amniotic epithelial cells into fetal liver, because 1) the fetal liver is at the proliferative stage, 2) the number of cells required is small, and 3) the fetal stage is advantageous for the induction of immunological tolerance. Amniotic epithelial cells from day 18.5-20.5 fetuses were transfected with adenoviral AdlacZ and harvested to inject into fetal rat liver of the syngeneic strain (day 18.5-20.5). The efficacy of cell transplantation into the liver increased in the order: intraplacental < intraumbilical vein < intrahepatic route. LacZ-transfected amniotic cells (1-8 x 10(5) cells), hepatocytes (5 x 10(5) cells), or AdlacZ vector solution (1.7 x 10(7) pfu) were injected through the uterine membrane into the liver. Transplanted cells formed a cellular mass and survived for up to 14 days after birth, whereas lacZ-transfected cells were rapidly decreased after the injection of AdlacZ vector or rat hepatocytes as a gene carrier so that the use of amniotic epithelial cells as a gene carrier will result in long-term expression of exogenous genes in the liver.

Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions.

We recently found that human amniotic epithelial (HAE) cells secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine (DA) neurons. The present study explored whether implantation of HAE cells can be a possible means to deliver trophic factors into the brain to prevent the death of DA neurons in a rat model of Parkinson's disease. We first investigated the ability of HAE cells to produce factors capable of promoting DA cell survival in vitro, and then tested whether HAE cell grafts survive and prevent the death of nigral DA neurons in rats with 6-hydroxydopamine lesions. A treatment with conditioned medium derived from HAE cell cultures enhanced the survival of tyrosine hydroxylase (TH)-immunopositive DA cells in serum-free cultures. The conditioned medium also protected the morphological integrity of TH-positive neurons against toxic insult with 6-hydroxydopamine. HAE cells were grafted into the midbrain of immunosuppressed rats. The rats were then subjected to a unilateral nigrostriatal lesion induced by intrastriatal infusions of 6-hydroxydopamine. HAE cell transplants were found to survive without evidence for overgrowth 2 weeks postgrafting. The number of nigral DA cells, detected with either TH-immunohistochemistry or retrograde labelling with fluorogold, was significantly increased in rats given the grafts as compared to that in control animals without the grafts. The results indicate that HAE cells produce diffusible molecules that can enhance the survival of DA neurons. Although the factors that contribute to the currently observed effects remain to be fully determined, implantation of HAE cells could be a viable strategy to counteract the loss of DA neurons in Parkinson's disease.

Uptake of dopamine by cultured monkey amniotic epithelial cells.

In this study, the ability of monkey amniotic epithelial (MAE) cells to take up dopamine was tested by incubating the cells in buffer containing unlabeled dopamine under different experimental conditions followed by assaying dopamine content using high performance liquid chromatography with electrochemical detection. Results showed the capability of MAE cells to take up dopamine in a time- and concentration-dependent fashion, and also this uptake is sodium-dependent. Further, selective dopamine transporter blockers inhibited dopamine uptake with rank order of potency that is consistent with the pharmacology of the dopamine transporter. These results suggest that MAE cells may be potential model to study dopamine uptake and release, and to explore new drugs affecting these processes.

Characterization of the dopamine transporter gene expression and binding sites in cultured human amniotic epithelial cells.

In this study we sought to investigate whether the dopamine transporter, DAT, and its binding sites are expressed in the human amniotic epithelial cells (HAEC) using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding studies, respectively. The RT-PCR findings showed that HAEC expressed DAT mRNA with 100% homology to the human brain DAT. Saturation binding studies using [3H]mazindol showed a high affinity DAT binding site with K(D) and B(max) values of 12.32+/-1.67 nM and 82.7+/-9.74 fmol/mg protein, respectively. Competition experiments showed that selective DAT blockers are potent displacers of [3H]mazindol binding. The rank order of potency of the competing drugs is consistent with the pharmacology of the DAT. The present results provide compelling evidence that HAEC natively express the DAT mRNA and binding sites. More importantly, these results may suggest that HAEC is an appropriate human cell model for studying dopamine release and uptake processes and potential ligands at these sites.

Evidence of dopamine D1 receptor mRNA and binding sites in cultured human amniotic epithelial cells.

In this study, we employed RT-PCR and radioligand binding studies to evaluate the gene expression and binding characteristics, respectively, of dopamine D(1) receptors in human amniotic epithelial cells (HAEC). The results showed that HAEC natively expressed D(1) receptor mRNA, as measured by RT-PCR, which was identical to that of human brain. Saturation binding studies using [(3)H]SCH 23390 demonstrated the presence of a high affinity D(1) site in HAEC with K(D) and B(max) values of 2.01+/-0.25 nM and 32.5+/-3.7 fmol/mg protein, respectively. Competition studies showed that selective D(1) antagonists were potent displacers of [(3)H]SCH 23390 binding with a potency order consistent with D(1) receptor characteristics. The current results present compelling evidence that HAEC natively express D(1) receptor mRNA and binding sites. The results also establish a primate cell model that can possibly be used for studying D(1) receptor signal transduction and molecular mechanisms and exploring newly developed drugs acting at these receptors.

Factors secreted by human amniotic epithelial cells promote the survival of rat retinal ganglion cells.

We evaluated whether factors secreted by human amniotic epithelial cells (HAECs) have the neuroprotective effect on rat retinal ganglion cells (RGCs) purified by immunopanning. After culture in B27 complete medium containing B27 supplement, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and forskolin, the medium was changed to: (1). medium containing N2 supplement and forskolin (N2 basal medium); (2). medium conditioned by HAECs containing N2 supplement and forskolin (HAEC-CM); and (3). N2 basal medium containing several neurotrophic factors. HAEC-CM promoted the RGC survival compared to N2 basal medium. The effect of HAEC-CM was significantly higher than that of BDNF, neurotrophin-3 and CNTF. These results suggest that HAECs may produce unknown neuroprotective factors, suggesting its potential for the treatment of RGC degenerative diseases.

Acetylcholine increase in amniotic fluid of experimental rats for intrauterine growth retardation.

Previous reports from this laboratory have demonstrated evidence for synthesis and release of acetylcholine (ACh) and catecholamines (CAs) by human amniotic epithelial cells (HAEC) and the presence of ACh and CAs in amniotic fluid. To study the physiological role of amniotic ACh, we used an experimental pregnant rat model for intrauterine growth retardation. Prior to this experiment, we confirmed the presence of choline acetyltransferase in the HAEC by immunocytochemical staining. Amniotic fluid was collected at 48 and 72 h after a transient ligation of the uterine vessels near the lower and upper ends of the right horn of the pregnant rat. The ACh concentration in the amniotic fluid from rats received intrauterine ischemia increased with time to a greater degree compared with the control rat, although the increase was not statistically significant. These results suggest that intrauterine hypoxic conditions cause a tendency to increase ACh concentrations in the amniotic fluid.

New GAA mutations in Japanese patients with GSDII (Pompe disease).

Glycogen storage disease type II (Pompe disease) is inherited by autosomal recessive transmission and caused by a deficiency of acid alpha-glucosidase (GAA), resulting in impaired degradation and lysosomal accumulation of glycogen. The GAA gene, responsible for this disease, has been mapped to chromosome 17q25.2-25.3. To date, more than 70 disease-causing mutations have been identified. In this study, we present four mutations found in three Japanese patients with the juvenile form of glycogen storage disease type II; three of these mutations were new (R224W, S619R, and R660H). The pathogenicity of these new mutations was verified by the loss of function of the mutant enzymes expressed in COS cells.

Progesterone, but not 17beta-estradiol, up-regulates erythropoietin (EPO) production in human amniotic epithelial cells.

Human amniotic epithelial (HAE) cells have great potential for successful use in cell therapy, since they do not cause acute rejection upon allotransplantation. However, to date, HAE cells have not well been studied. We previously reported that HAE cells produce erythropoietin (EPO), which is known to be a regulator of hematopoiesis, and that the induction mechanism of HAE cells is unknown, although EPO production from HAE cells is not increased by hypoxia which induces several cell types to produce EPO. In this study, we determined whether female sex hormones, including progesterone and 17beta-estradiol, affect the EPO production of HAE cells. Bioactive measurement of EPO activity in the culture supernatants of HAE-SV40 cells, which were immortalized by transfection with a simian virus 40 large T antigen, revealed that EPO bioactivity was significantly increased by treatment with progesterone, but not 17beta-estradiol. Treatment of HAE-SV40 cells with progesterone transiently increased the EPO mRNA level by fivefold, while there was no change in response to 17beta-estradiol. Furthermore, the progesterone receptor (PR)-B was detected in both HAE cells and HAE-SV40 cells by Western blotting. These results suggest that EPO synthesis in HAE-SV40 cells is stimulated by progesterone, but not by 17beta-estradiol, and thus it is highly likely that the EPO synthesis of HAE cells is also regulated by progesterone.

[Possible use of amniotic cells for regenerative medicine].

Human amniotic epithelial cells are able to express differentiation markers of at least two cell lineages, neuronal cells and hepatic parenchymal cells in vitro(Neurosci Lett 209: 9, 1996, J Hum Genet 45: 171, 2000). The former was characterized by the expression of neurofilament and glial fibrillary acidic protein, and the latter, by albumin and alpha-fetoprotein. Since amnion is formed in the early stage of embryogenesis, immature undifferentiated cells may be reserved. In addition to biological features, amniotic epithelial cells are obtained from the placenta during Cesarean section without obvious ethical problems. Amniotic membrane may be one novel cell source in regenerative medicine for neuronal and hepatic disease, including an ex vivo gene carrier.

Hypoxia enhances the induction of human amniotic mesenchymal side population cells into vascular endothelial lineage.

Human amniotic mesenchymal side population (hAM-SP) cells have pluripotency and weak immunogenicity, and have promising roles in the field GAPDH of regenerative medicine. The aim of the present study was to determine whether hypoxic conditions induce the differentiation of hAM-SP cells into the vascular endothelial lineage. Mesenchymal cells were isolated from enzyme-treated amniotic membranes and stained with Hoechst 33342. The hAM-SP cells were negatively sorted by FACS and cultured in induction medium containing vascular endothelial growth factor (VEGF) under normoxic (20% O2) or hypoxic (1% O2) conditions for 1 or 2 weeks. The expression of endothelial markers such as kinase domain region (KDR), fms-like tyrosine kinase (Flt)-1, von Willebrand factor (vWF), vascular endothelial (VE)-cadherin and human vascular cell adhesion molecule (VCAM) at the gene and protein level was evaluated by real-time PCR and fluorescent immunostaining, respectively. The gene expression of KDR, Flt-1, VE-cadherin and vWF peaked after 2 weeks of culture. The protein expression of KDR and VE-cadherin was also enhanced after 2 weeks of culture under hypoxic conditions. To confirm the involvement of hypoxia-inducible factor (HIF) in the induction under hypoxic conditions, the expression of genes which are known to be upregulated by HIF was analyzed by DNA microarray. The expression of these genes increased under hypoxic conditions. hAM-SP cells cultured under hypoxic conditions differentiated into the vascular endothelial lineage, probably due to upregulation of the gene expression associated with angiogenesis through activation of the HIF system.

Encapsulation cell therapy for mucopolysaccharidosis type VII using genetically engineered immortalized human amniotic epithelial cells.

Mucopolysaccharidosis type VII (MPSVII) is a lysosomal storage disease resulted from a deficiency of the enzyme beta-glucuronidase (GUSB), which is necessary for degradation of glycosaminoglycans (GAGs). The deficiency of GUSB causes progressive accumulation of GAGs and subsequent lysosomal distension in multiple tissues, including the central nervous system (CNS). In murine experiments, bone marrow transplant, enzyme replacement, viral vectors, and genetically modified cells were successfully used for correction of the visceral accumulation of GAGs, but little improvement was seen in the brain, because these therapeutic agents cannot cross the blood-brain barrier (BBB). Although direct intracerebral injection of GUSB-encoding viral vectors has been developed to bypass the BBB, the possibility of tumor formation and the toxicity of over-expressed GUSB have been reported. In this study, we generated immortalized human amniotic epithelial (IHAE) cells to maintain the effect of implantation, and encapsulated these cells to prevent harmful immunological response and tumor formation and to regulate the level of GUSB expression within the host. Moreover, we generated IHAE cells that over-express and secrete human GUSB following transduction with an adenoviral vector encoding human GUSB. Therapeutic efficacy for MPSVII was evaluated in and ex vivo experiments using these encapsulated genetically engineered GUSB-encoding IHAE cells. We confirmed that encapsulated genetically engineered IHAE cells could secrete significant amounts of GUSB outside the capsule in vitro and into the cerebral parenchyma of C3H mice seven days after the capsule implantation. Thus, encapsulation cell therapy using genetically engineered IHAE cells is an effective armamentarium for the treatment of MPSVII.

Topical application of culture supernatant from human amniotic epithelial cells suppresses inflammatory reactions in cornea.

Human amniotic epithelial cells (HAEC) may be a source of soluble anti-inflammatory factors. The purpose of this study is to determine the effect of topically applied HAEC culture supernatant on corneal inflammatory reactions. HAEC were obtained from a placenta and cultured for 48 hr, and the supernatant was collected. The conditioned medium from HAEC contained small amounts of human interleukin-1 receptor antagonist (IL-1ra). Intrastromal sutures were placed in the cornea of BALB/c mice to induce corneal neovascularisation. Superficial cauterisation was applied to induce recruitment or activation of antigen presenting cells (APCs) in the cornea without neovascularisation. HAEC conditioned medium, placebo, or recombinant human IL-1ra was topically applied three times daily for 2 weeks. Suture-induced corneal neovascularisation was evaluated microscopically for 8 weeks. The cauterised corneas were harvested at 2 weeks, and the MHC class II(+) APCs were quantified by immunofluorescent staining and confocal microscopy. Inflammatory cytokine gene expression in the cauterised corneas was analyzed by a multiprobe ribonuclease protection assay. Conditioned medium from HAEC led to a profound suppression of corneal neovascularisation and fewer MHC class II(+) APCs in the epithelium. In contrast, human IL-1ra was only slightly effective in suppressing corneal inflammatory reactions. mRNA expression of murine IL-1ra and IL-1beta in the cauterised corneas was markedly suppressed after application of the conditioned medium. These results suggest that HAEC are a source of soluble anti-inflammatory factors and that conditioned medium from HAEC contains factors other than IL-1ra that suppress corneal inflammation.

Synthesis and release of activin and noggin by cultured human amniotic epithelial cells.

Recent studies suggest that extra-embryonic tissues may be essential sources of early organizing signals for the mouse embryo. In vitro studies of human amniotic epithelial cells (HAEC) have shown that the amnion can produce various biologically active substances. In this study, the synthesis and release of activin A and noggin, and the activin signaling pathway, was investigated in HAEC. Conditioned medium from cultured HAEC contained activin A which was functionally active in Xenopus laevis animal cap assays. Immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction confirmed that HAEC also synthesize and release noggin. Noggin transcripts were induced by the addition of recombinant activin A, and activin A was inhibited by activin antibody except in the presence of cycloheximide (CHX). These data demonstrate that noggin mRNA expression is induced directly by activin A without new protein synthesis, indicating that noggin is a primary response gene. The results suggest that there is an activin signaling pathway in HAEC, and that the human amnion might therefore be involved in neural formation during early development.

Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells.

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin-positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66-82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT-PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti-Tuj1 and NF-M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein-positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC-derived stem cells may be a favorable cell vehicle in regenerative medicine.