Inja Lim

Human Astrocytes: Secretome Profiles of Cytokines and Chemokines

Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction.

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Human neural stem cells overexpressing choline acetyltransferase restore cognitive function of kainic acid-induced learning and memory deficit animals.

Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.

Habitual physical exercise has beneficial effects on telomere length in postmenopausal women

Objective It has been reported that women benefit from the maintenance of telomere length by estrogen. Exercise may favorably influence telomere length, although results are inconsistent regarding the duration and type of exercise and the cell type used to measure telomere length. The purpose of this study was to investigate the relationship between habitual physical exercise and telomere length in peripheral blood mononuclear cells (PBMCs) in postmenopausal women. Postmenopausal women were chosen as study participants because they are typically estrogen deficient. Methods This experimental-control, cross-sectional study included 44 healthy, nondiabetic, nonsmoking, postmenopausal women. Habitual exercisers and sedentary participants were matched for age and body mass index. Body weight, height, blood pressure, and waist and hip circumference were measured. Mitochondrial DNA copy number and telomere length in PBMCs were determined, and biochemical tests were performed. Habitual physical exercise was defined as combined aerobic and resistance exercise performed for at least 60 minutes per session more than three times a week for more than 12 months. Results The mean age of all participants was 58.11 ± 6.84 years, and participants in the habitual exercise group had been exercising more than three times per week for an average of 19.23 ± 5.15 months. Serum triglyceride levels (P = 0.01), fasting insulin concentrations (P < 0.01), and homeostasis model assessment of insulin resistance (P < 0.01) were significantly lower and high-density lipoprotein cholesterol levels (P < 0.01), circulating adiponectin (P < 0.01), mitochondrial DNA copy number (P < 0.01), and telomere length (P < 0.01) were significantly higher in the habitual exercise group than in the sedentary group. In a stepwise multiple regression analysis, habitual exercise (&bgr; = 0.522, P < 0.01) and adiponectin levels (&bgr; = 0.139, P = 0.03) were the independent factors associated with the telomere length of PBMCs in postmenopausal women. Conclusions Habitual physical exercise is associated with greater telomere length in postmenopausal women. This finding suggests that habitual physical exercise in postmenopausal women may reduce telomere attrition.

Mesenchymal stem cells overexpressing hepatocyte growth factor (HGF) inhibit collagen deposit and improve bladder function in rat model of bladder outlet obstruction.

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly male. This study was performed to examine the capability of human mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF) to inhibit collagen deposition in rat model of bladder outlet obstruction (BOO). HGF is known for its antifibrotic effect and the most promising agent for treating bladder fibrosis. BM3.B10 stable immortalized human MSC line (B10) was transduced to encode human HGF with a retroviral vector was prepared (B10.HGF). Two weeks after the onset of BOO, B10, and B10.HGF cells were injected into the rats bladder wall. After 4 weeks, bladder tissues were harvested and Massons trichrome staining was performed. Transgene expression in HGF-expressing B10 cells was demonstrated by reverse transcriptase polymerase chain reaction and immunohistochemical staining, and the high levels of HGF secreted by B10. HGF cells was confirmed by ELISA. The mean bladder weight in BOO rats was 5.8 times of the normal controls, while in animals grafted with B10.HGF cells, the weight was down to four times of the control [90.2 ± 1.6 (control), 89.9 ± 2.8 (sham), 527.9 ± 150.9 (BOO), 447.7 ± 41.0 (BOO + B10), and 362.7 ± 113.2 (BOO + B10. HGF)]. The mean percentage of collagen area increased in BOO rats, while in the animals transplanted with B10.HGF cells, the collagen area decreased to the normal control level [12.2 ± 1.3, (control), 12.8 ± 1.1 (sham), 26.6 ± 2.7 (BOO), 19.9 ± 6.0 (BOO + B10), and 13.3 ± 2.1 (BOO + B10.HGF)]. The expression of collagen and TGF-β protein increased after BOO, while the expression of HGF and c-met protein increased in the group with B10.HGF transplantation after BOO. Intercontraction interval decreased after BOO, but it recovered after B10.HGF transplantation. Maximal voiding pressure (MVP) increased after BOO, and it recovered to levels of the normal control after transplantation of B10.HGF cells. Residual urine volume (RU) increased after BOO, but the RU increase was not reversed by transplantation of B10.HGF cells. Human MSCs overexpressing HGF inhibited collagen deposition and improved cystometric parameters in bladder outlet obstruction of rats. The present study indicates that transplantation of MSCs modified to overexpress HGF could serve as a novel therapeutic strategy against bladder fibrosis in patients with bladder outlet obstruction.

Transplantation of Human Adipose Tissue-Derived Stem Cells Delays Clinical Onset and Prolongs Life Span in ALS Mouse Model:

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons in the cortex, brain stem, and spinal cord. The precise pathogenic mechanism remains unknown, and there is currently no effective therapy. We evaluated the therapeutic effects of human adipose tissue-derived stem cells (ASCs) in an animal model of ALS. Human abdominal subcutaneous fat tissues were obtained by simple liposuction from donors, and ASCs were isolated from the fat stromal vascular fraction. ASCs were found to differentiate into adipocytes, chondrocytes, osteocytes, and neurons. SOD1G93A ALS mice were divided into three groups: sham, intravenous (IV), and intracerebroventricular (ICV) groups. Human ASCs were transplanted in the ALS mice at 70 postnatal days before the appearance of clinical symptoms. Behavior of transplanted animals was assessed by rotarod test, paw grip endurance (PaGE), and reflex index. Mice in every group were sacrificed after 4 weeks posttransplantation. Transplanted ASCs were identified in the lumbar spinal cords with an antihuman mitochondria antibody and cell type-specific markers for neurons or astrocytes. Delayed onset of clinical symptoms (26 days) and extended survival of animals (24 days) were observed in ALS mice transplanted with ASCs via ICV route. ASCs were found to secrete high levels of neurotrophic factors such as NGF, BDNF, IGF-1, and VEGF. Reduction of apoptotic cell death by these factors was confirmed in cultured CNS cells and in the ALS spinal cord. These results indicate that transplantation of ASCs in ALS mice provides neuroprotective effects by production of cytokines/growth factors, delays disease progression, and prolongs the life span of ALS mice.

Inhibition of Collagen Deposit in Obstructed Rat Bladder Outlet by Transplantation of Superparamagnetic Iron Oxide-Labeled Human Mesenchymal Stem Cells as Monitored by Molecular Magnetic Resonance Imaging (MRI)

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly males. The present study is to investigate if human mesenchymal stem cells (MSCs) are capable of inhibiting collagen deposition and improve cystometric parameters in bladder outlet obstruction in rats. Human MSCs were labeled with nanoparticles containing superparamagnetic iron oxide (SPION), and transplanted in rat BOO lesion site. Forty 6-week-old female Sprague-Dawley rats were divided into four groups (group 1: control, group 2: sham operation, group 3: BOO, and group 4: BOO rats receiving SPION-hMSCs). Two weeks after the onset of BOO, 1 × 106 SPION-hMSCs were injected into the bladder wall. Serial T2-weighted MR images were taken immediately after transplantation of SPION-labeled human MSCs and at 4 weeks posttransplantation. T2-weighted MR images showed a clear hypointense signal induced by the SPION-labeled MSCs. While the expression of collagen and TGF-β protein increased after BOO, the expression of both returned to the original levels after MSC transplantation. Expression of HGF and c-met protein also increased in the group with MSC transplantation. Maximal voiding pressure and residual urine volume increased after BOO but they recovered after MSC transplantation. Human MSCs transplanted in rat BOO models inhibited the bladder fibrosis and mediated recovery of bladder dysfunction. Transplantation of MSC-based cell therapy could be a novel therapeutic strategy against bladder fibrosis in patients with bladder outlet obstruction.

Human Motor Neurons Generated from Neural Stem Cells Delay Clinical Onset and Prolong Life in ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neuron disease. The etiology and pathogenic mechanisms of the disease remain unknown, and there is no effective treatment. Here we show that intrathecal transplantation of human motor neurons derived from neural stem cells (NSCs) in spinal cord of the SOD1G93A mouse ALS model delayed disease onset and extended life span of the animals. When HB1.F3.Olig2 (F3.Olig2) cells, stable immortalized human NSCs encoding the human Olig2 gene, were treated with sonic hedgehog (Shh) protein for 5–7 days, the cells expressed motor neuron cell type-specific phenotypes Hb9, Isl-1 and choline acetyltransferase (ChAT). These F3.Olig2-Shh human motor neurons were transplanted intrathecally in L5–L6 spinal cord of SOD1G93A mice, and at 4 weeks post-transplantation, transplanted F3.Olig2-Shh motor neurons expressing the neuronal phenotype markers NF, MAP2, Hb9, and ChAT were found in the ventral horn of the spinal cord. Onset of clinical signs in ALS mice with F3.Olig2-Shh motor neuron implants was delayed for 7 days and life span of animals was significantly extended by 20 days. Our results indicate that this treatment modality of intrathecal transplantation of human motor neurons derived from NSCs might be of value in the treatment of ALS patients without significant adverse effects.

Expression profiling of mitochondrial voltage-dependent anion channel-1 associated genes predicts recurrence-free survival in human carcinomas.

Background Mitochondrial voltage-dependent anion channels (VDACs) play a key role in mitochondria-mediated apoptosis. Both in vivo and in vitro evidences indicate that VDACs are actively involved in tumor progression. Specifically, VDAC-1, one member of the VDAC family, was thought to be a potential anti-cancer therapeutic target. Our previous study demonstrated that the human gene VDAC1 (encoding the VDAC-1 isoform) was significantly up-regulated in lung tumor tissue compared with normal tissue. Also, we found a significant positive correlation between the gene expression of VDAC1 and histological grade in breast cancer. However, the prognostic power of VDAC1 and its associated genes in human cancers is largely unknown. Methods We systematically analyzed the expression pattern of VDAC1 and its interacting genes in breast, colon, liver, lung, pancreatic, and thyroid cancers. The genes differentially expressed between normal and tumor tissues in human carcinomas were identified. Results The expression level of VDAC1 was uniformly up-regulated in tumor tissue compared with normal tissue in breast, colon, liver, lung, pancreatic, and thyroid cancers. Forty-four VDAC1 interacting genes were identified as being commonly differentially expressed between normal and tumor tissues in human carcinomas. We designated VDAC1 and the 44 dysregulated interacting genes as the VDAC1 associated gene signature (VAG). We demonstrate that the VAG signature is a robust prognostic biomarker to predict recurrence-free survival in breast, colon, and lung cancers, and is independent of standard clinical and pathological prognostic factors. Conclusions VAG represents a promising prognostic biomarker in human cancers, which may enhance prediction accuracy in identifying patients at higher risk for recurrence. Future therapies aimed specifically at VDAC1 associated genes may lead to novel agents in the treatment of cancer.

Long-term survival and differentiation of human neural stem cells in nonhuman primate brain with no immunosuppression.

Cellular fate of human neural stem cells (hNSCs) transplanted in the brain of nonhuman primates (NHPs) with no immunosuppression was determined at 22 and 24 months posttransplantation (PTx) regarding survival, differentiation, and tumorigenesis. Survival of hNSCs labeled with magnetic nanoparticles was successfully detected around injection sites in the brain at 22 months PTx by MRI. Histological examination of brain sections with H&E and Prussian blue staining at 24 months revealed that most of the grafted hNSCs were found located along the injection tract. Grafted hNSCs were found to differentiate into neurons at 24 months PTx. In addition, none of the grafted hNSCs were bromodeoxyuridine positive in the monkey brain, indicating that hNSCs did not replicate in the NHP brain and did not cause tumor formation. This study serves as a proof of principle and provides evidence that hNSCs transplanted in NHP brain could survive and differentiate into neurons in the absence of immunosuppression. It also serves as a preliminary study in our scheduled preclinical studies of hNSC transplantation in NHP stroke models.