Human Stem Cell Lines Research Articles

ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy.

We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.

Identification of RNA-Binding Protein LARP4B as a Tumor Suppressor in Glioma.

Transposon-based insertional mutagenesis is a valuable method for conducting unbiased forward genetic screens to identify cancer genes in mice. We used this system to elucidate factors involved in the malignant transformation of neural stem cells into glioma-initiating cells. We identified an RNA-binding protein, La-related protein 4b (LARP4B), as a candidate tumor-suppressor gene in glioma. LARP4B expression was consistently decreased in human glioma stem cells and cell lines compared with normal neural stem cells. Moreover, heterozygous deletion of LARP4B was detected in nearly 80% of glioblastomas in The Cancer Genome Atlas database. LARP4B loss was also associated with low expression and poor patient survival. Overexpression of LARP4B in glioma cell lines strongly inhibited proliferation by inducing mitotic arrest and apoptosis in four of six lines as well as in two patient-derived glioma stem cell populations. The expression levels of CDKN1A and BAX were also upregulated upon LARP4B overexpression, and the growth-inhibitory effects were partially dependent on p53 (TP53) activity in cells expressing wild-type, but not mutant, p53. We further found that the La module, which is responsible for the RNA chaperone activity of LARP4B, was important for the growth-suppressive effect and was associated with BAX mRNA. Finally, LARP4B depletion in p53 and Nf1-deficient mouse primary astrocytes promoted cell proliferation and led to increased tumor size and invasiveness in xenograft and orthotopic models. These data provide strong evidence that LARP4B serves as a tumor-suppressor gene in glioma, encouraging further exploration of the RNA targets potentially involved in LARP4B-mediatd growth inhibition. Cancer Res; 76(8); 2254-64. ©2016 AACR.

Establishment and applications of male germ cell and Sertoli cell lines.

Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine.

Establishment and Characterization of Primary Human Ovarian Cancer Stem Cell Line (CD44+ve)

Ovarian cancer is ranked as the 7th most lethal cancer worldwide with 239,000 new cases annually. The mortality rate is high because most ovarian tumors are diagnosed at advanced stages and are resistant to chemotherapy and thus incurable due to the lack of effective early detection of ovarian tumors. There is a small sub-population of ovarian tumor cells capable of self-renewal and differentiation into different cancer cell types, called cancer stem cells (CSCs), which might be responsible for cancer relapse. The CD44+ phenotype in ovarian tumor cells elucidates cancer initiating cell-like properties of promoting differentiation, metastasis, and chemotherapy-resistance. Increased expression of genes previously associated with CSCs promotes regenerative capacity by promoting stem cell function that can drive cancer relapse and metastasis. In this study we present a method to isolate the primary epithelial ovarian cancer cells from human solid tumor and establish CD44+ve primary ovarian cancer stem cell (OCSCCD44+ve) line using magnetic microbeads. Also we evaluated the expression of stemness genes Nanog, Sox2, Oct4, and Nestin by real-time qPCR analysis. Thequantitative analysis by real-time qPCRshows that OCSCCD44+ve overexpressed the embryonic stem cell marker genes Nanog, Oct4, Sox2, and Nestin when compared with ovarian cancer cells OCCCD44-ve as positive control and ovarian cells as negative control. We demonstrate that CD44 in malignant ovarian tumors is a critical molecule that exhibits cancer stem cell properties that enhance tumorigenicity and cancer metastasis. Our results provide a better understanding of ovarian CSCs, which is important for future in vivo studies with subsequent target therapy for preclinical studies.

PD35-09 DIFFERENTIAL ACTIONS OF ERα AND ERβ VIA NON-GENOMIC SIGNALING IN HUMAN PROSTATE STEM-PROGENITOR CELLS

You have accessJournal of UrologyStem Cell Research1 Apr 2016PD35-09 DIFFERENTIAL ACTIONS OF ERα AND ERβ VIA NON-GENOMIC SIGNALING IN HUMAN PROSTATE STEM-PROGENITOR CELLS Shyama Majumdar, Neha Malhotra, Susan Kasper, Lishi Xie, Timothy Gauntner, Wen-yang Hu, and Gail Prins Shyama MajumdarShyama Majumdar More articles by this author , Neha MalhotraNeha Malhotra More articles by this author , Susan KasperSusan Kasper More articles by this author , Lishi XieLishi Xie More articles by this author , Timothy GauntnerTimothy Gauntner More articles by this author , Wen-yang HuWen-yang Hu More articles by this author , and Gail PrinsGail Prins More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1081AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Estrogens have been implicated in prostate development and cancer; however, specific roles of estrogen receptor-α and estrogen receptor-β are not well established. Using human prostate stem-progenitor cells we have previously shown that non-genomic pathways are involved in estrogen actions. Our present study sought to elucidate specific roles for ERα and ERβ via membrane initiated signaling in stem-progenitor cells. METHODS Human prostate stem-progenitor cells were enriched from primary prostate epithelial cell culture of young, disease-free donors using a 3D prostasphere (PS) model as previously described. Cells were labeled using ERα or ERβ antibodies and prostate stem cell markers CD49f and TROP2 followed by triple channel FACS to quantify ERα+/ERβ+ cell numbers. To explore ERα, the benign human prostate stem cell line WPE-stem, with extremely low levels of ERα and ERβ, was stably transfected with a lentiviral-ERα expression vector. The human prostate cancer stem-like cell line HuSLC (ERβ++, ERα-) was used to interrogate ERβ actions. Cells were exposed to 10 nM estradiol (E2) over a 15 to 60 minute time course +/- ICI 182,870 (ICI), an ERα/β antagonist. Western Blotting was used to quantify phosphorylation of target proteins. RESULTS FACS analysis of day 7 PS cells labeled for ERα or ERβ revealed 66% of day 7 PS cells as ERα+ and 40% as ERβ+. Among ERα or ERβ positive PS cells, 4% were Trop2+/CD49fhigh (stem-like cells) and 10-12% were Trop2+/CD49fmedium (early stage progenitor cells). PS exposed to 10 nM E2 showed sequential phosphorylation of Src, Erk1/2, p38, Akt and NFκB (p65) over 60 minutes. ICI attenuated Akt and Erk1/2 phosphorylation, confirming membrane bound ERs are involved in downstream signaling. E2 treatment of HuSLCs showed phosphorylation of Erk1/2 but not Akt, indicating that ERβ signals exclusively through the MAPK pathway in these cells. Conversely, E2 treatment of WPE-stem cells overexpressing ERα resulted in robust phosphorylation of Akt but lower levels of Erk1/2 phosphorylation suggesting that Akt activation may be more reliant on ERα signaling. CONCLUSIONS ERs trigger cellular responses via activation of signaling cascades that originate at the membrane (non-genomic signaling) in addition to nuclear signaling. The present findings reveal that human prostate stem-progenitor cells express both ERα and ERβ, which differentially activate membrane signaling cascades. This may lead to unique downstream actions that influence prostate stem-progenitor cell proliferation as well as lineage commitment decisions. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e847 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Shyama Majumdar More articles by this author Neha Malhotra More articles by this author Susan Kasper More articles by this author Lishi Xie More articles by this author Timothy Gauntner More articles by this author Wen-yang Hu More articles by this author Gail Prins More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Novel MET/TIE2/VEGFR2 inhibitor altiratinib inhibits tumor growth and invasiveness in bevacizumab-resistant glioblastoma mouse models.

Glioblastoma highly expresses the proto-oncogene MET in the setting of resistance to bevacizumab. MET engagement by hepatocyte growth factor (HGF) results in receptor dimerization and autophosphorylation mediating tumor growth, invasion, and metastasis. Evasive revascularization and the recruitment of TIE2-expressing macrophages (TEMs) are also triggered by anti-VEGF therapy. We investigated the activity of altiratinib (a novel balanced inhibitor of MET/TIE2/VEGFR2) against human glioblastoma stem cell lines in vitro and in vivo using xenograft mouse models. The biological activity of altiratinib was assessed in vitro by testing the expression of HGF-stimulated MET phosphorylation as well as cell viability after altiratinib treatment. Tumor volume, stem cell and mesenchymal marker levels, microvessel density, and TIE2-expressing monocyte infiltration were evaluated in vivo following treatment with a control, bevacizumab alone, bevacizumab combined with altiratinib, or altiratinib alone. In vitro, HGF-stimulated MET phosphorylation was completely suppressed by altiratinib in GSC17 and GSC267, and altiratinib markedly inhibited cell viability in several glioblastoma stem cell lines. More importantly, in multiple xenograft mouse models, altiratinib combined with bevacizumab dramatically reduced tumor volume, invasiveness, mesenchymal marker expression, microvessel density, and TIE2-expressing monocyte infiltration compared with bevacizumab alone. Furthermore, in the GSC17 xenograft model, altiratinib combined with bevacizumab significantly prolonged survival compared with bevacizumab alone. Together, these data suggest that altiratinib may suppress tumor growth, invasiveness, angiogenesis, and myeloid cell infiltration in glioblastoma. Thus, altiratinib administered alone or in combination with bevacizumab may overcome resistance to bevacizumab and prolong survival in patients with glioblastoma.

Establishment of a Drug-Induced, Bile Acid–Dependent Hepatotoxicity Model Using HepaRG Cells

Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 μM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [3H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool.

RNA-Seq Reveals the Angiogenesis Diversity between the Fetal and Adults Bone Mesenchyme Stem Cell.

In this research, we used RNA sequencing (RNA-seq) to analyze 23 single cell samples and 2 bulk cells sample from human adult bone mesenchyme stem cell line and human fetal bone mesenchyme stem cell line. The results from the research demonstrated that there were big differences between two cell lines. Adult bone mesenchyme stem cell lines showed a strong trend on the blood vessel differentiation and cell motion, 48/49 vascular related differential expressed genes showed higher expression in adult bone mesenchyme stem cell lines (Abmsc) than fetal bone mesenchyme stem cell lines (Fbmsc). 96/106 cell motion related genes showed the same tendency. Further analysis showed that genes like ANGPT1, VEGFA, FGF2, PDGFB and PDGFRA showed higher expression in Abmsc. This work showed cell heterogeneity between human adult bone mesenchyme stem cell line and human fetal bone mesenchyme stem cell line. Also the work may give an indication that Abmsc had a better potency than Fbmsc in the future vascular related application.

Abstract A34: Decline in Arylsulfatase B activates Wnt signaling by effects on DKK-3 methylation

Abstract Immunostaining and activity of the enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) were reduced in prostatic malignancies, and decline in ARSB was associated with higher Gleason score and biochemical recurrence in two prostate cancer tissue microarrays. Since the only known function of ARSB is to remove 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate, a mechanism for this association of ARSB with more aggressive prostate cancer was unknown. Subsequent studies showed transcriptional effects of ARSB, mediated by reduced binding of galectin-3 to the more highly sulfated C4S present when ARSB was reduced. Decline in ARSB induced increased expression of the extracellular matrix proteoglycan versican, which has chondroitin 4-sulfate attachments and has been shown to be increased in more aggressive prostate cancers. The increase in versican was mediated by increased nuclear galectin-3 and AP-1 effects on the versican promoter. Other experiments following decline in ARSB showed increased expression of Wnt9A, mediated by increased nuclear galectin-3 and Sp1. These findings suggested that ARSB acted as a tumor suppressor gene and decline in ARSB enhanced tumor progression. Experiments were performed to address transcriptional mechanisms by which decline in ARSB and the accompanying increase in chondroitin 4-sulfation could activate Wnt signaling and lead to more aggressive prostate cancers. The impact of ARSB on Wnt signaling was evaluated in prostate tissue from the ARSB-null mouse, and human prostate stromal (WPMY-1, ATCC), epithelial (RWPE-1, ATCC), and stem (WPE-stem, ATCC) cell lines. The effects of ARSB on Wnt signaling were assessed by ELISA, Western blot, silencing by siRNA, ARSB overexpression, DNA methyltransferase (DNMT) activity assays, QRT-PCR, and measurements of promoter methylation. Decline in ARSB increased Wnt signaling, manifested by increases in nuclear beta-catenin, DNA-bound TCF-LEF, nuclear c-myc and cyclin D1. To determine if increased Wnt signaling was attributable to reduction of the inhibitory effect of Dickkopf (DKK), an established negative regulator of Wnt signaling and tumor suppressor, the expression of DKK 1-4 was determined by QRT-PCR. DKK-3 expression in the prostate cells following ARSB silencing and in the ARSB-null tissue was significantly reduced. ARSB overexpression increased the DKK-3 expression. DNA methyltransferase (DNMT) activity was significantly increased when ARSB was silenced, and reduced when ARSB was overexpressed. Expression of DNMT 1 and 3a was increased in the ARSB-null mouse prostate tissue and prostate cells, consistent with the decline in DKK-3 expression. Determinations of DKK-3 promoter methylation showed increased methylation following ARSB silencing and in the malignant prostate tissue. Hence, decline in ARSB activated Wnt signaling by increased methylation of DKK-3, leading to disinhibition of Wnt signaling, attributable to reduced binding of DKK-3 with the Frizzled - LRP5/6 receptor complex. These findings demonstrate an epigenetic mechanism whereby decline in ARSB increases Wnt signaling in prostate tissue and can lead to more aggressive prostate malignancy. Also, the role of ARSB as a tumor suppressor gene is supported by this demonstration of inverse effects of decline and overexpression of ARSB on Wnt signaling and on DNMT expression and activity. Experiments are ongoing to clarify the intermediates in the pathway by which ARSB affects DNMT expression. Note: This abstract was not presented at the conference. Citation Format: Sumit Bhattacharyya, Leo Feferman, Joanne Kramer Tobacman. Decline in Arylsulfatase B activates Wnt signaling by effects on DKK-3 methylation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A34.

Establishment and characterization of a novel human endometrial mesenchymal stem cell line from a patient with adenomyosis

Adenomyosis, previously termed “endometriosis interna,” is a widespread disease affecting the female reproductive system and frequently resulting in infertility in women. The aim of this work was to examine the properties of endometrial mesenchymal stem cells (eMSCs) from a patient with adenomyosis. We established the cell line from a patient with adenomyosis and compared the properties of these cells with cells derived from a healthy donor. It was found that patient-derived eMSCs and eMSCs from healthy donors had a fibroblast-like morphology and did not differ in expression of surface markers and adipogenic potential. Karyotype analysis of G-banded metaphase chromosomes was performed in cells of both lines at the six or seventh passages. Cells from the healthy donor mostly had normal karyotype. Karyotype of eMSCs from the patient with adenomyosis usually had chromosomal abnormalities. The abnormalities concerned aneuploidy and nonrandom chromosomes breaks more often involving chromosomes 7 and 11. Although karyotype instability may be a sign of cell transformation, the patient-derived eMSCs stopped cycling after about 26 passages and entered into replicative senescence. This shows that the karyotypic abnormalities that we observed in adenomyosis-derived eMSCs are not relevant to the cell transformation and immortalization in vitro.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

Inhibition of PERK and NFKB pathway can reduce death of stressed dopaminergic neurons differentiated from human stem cell line

Inhibition of PERK and NFKB pathway can reduce death of stressed dopaminergic neurons differentiated from human stem cell line

Inhibition of PERK and NFKB pathway can reduce death of stressed dopaminergIC neurons differentiated from human stem cell line

Inhibition of PERK and NFKB pathway can reduce death of stressed dopaminergIC neurons differentiated from human stem cell line

Use of Human Pluripotent Stem Cells to Model the Hematopoietic Defect and Repair of Human Telomerase Deficiency

Telomeres are repetitive DNA protein structures that cap the ends of chromosomes, protect chromosome ends from degradation and fusion, and are essential for maintenance of genomic integrity. Telomere length has been shown to gradually shorten over time as cells divide. When telomeres become critically short, the cells enter a state of senescence. As such, telomere shortening has been implicated in accelerated aging. Defects in telomerase function have been associated with the development of bone marrow failure. Patients with inherited mutations in telomerase components have significantly shortened telomere lengths and reductions in telomere length have been associated with a worse prognosis in myelodysplastic syndrome (MDS) and Aplastic anemia (AA). We have isolated fibroblasts from patients with a novel mutation in the telomerase RNA component (TERC), a 6 nucleotide in frame duplication at position +334, which results in bone marrow failure. We have derived several lines of human induced pluripotent stem cells (iPSCs) from these patients. We have demonstrated that these telomerase-deficient iPSCs appropriately express markers of pluripotency: Oct4, Sox2, SSEA4, and Nanog. Using quantitative real time PCR, we were able to measure the average telomere length as a T/S ratio of kilobases of telomere length per genome. We have determined that reprogramming results in significant increase in telomere length of control fibroblasts. However despite the typical induction of endogenous TERT expression during reprograming in our telomerase-deficient iPSCs, these TERC-mutant iPSCs did not demonstrate significant telomere elongation. We found telomerase-deficient fibroblasts have telomere lengths of 130-150kb/diploid genome compared to normal human fibroblasts with telomeres of ~250kb/diploid genome. After reprogramming, the iPSCs generated from wild type fibroblasts can have markedly increased telomere lengths to as high as 2000kb/diploid genome. The telomere lengths in TERC-mutant iPSCs from two different patients are less than 300kb/diploid genome (replicates=3). Expression of telomerase components TERT and TERC, DKC was compared to the mRNA level by qRT-PCR in the TERC-mutant iPSCs. We found significant variation in mRNA expression levels of telomerase components the different lines of telomerase deficient iPSCs, and even variation among different clones of the same telomerase deficient line. We have also found that mutations in TERC results in defective hematopoietic differentiation from these iPSCs in in vitro assays. In the TERC-mutant iPSCs, the proportion of CD34+CD45+ hematopoietic cells was reduced compared to wild type controls. Wild type iPSCs produce 30% CD34+ cells compared to 15-20% in TERC-mutant iPSCs (n=3). Additionally, wild type iPSC controls produce 9-10% CD34+CD45+ hematopoietic progenitor cells compared to 1-2% of TERC mutant cells. Interestingly, approximately equal proportions (8-9%) of wild type and TERC-mutant cells differentiate into CD34+CD31+ endothelial cells, suggesting this pathway is less affected by the TERC mutation. The TERC-mutant iPSCs demonstrate reduced development of hematopoietic progenitor cells in standard hematopoietic colony forming cell assays: 100 CFCs per 50,000 differentiated wild type iPSC-derived cells compared to 65 CFCs per 50,000 differentiated TERC mutant iPSC-derived cells. In order to rescue the hematopoietic defect in telomerase deficient TERC-mutant iPSCs we have used the Sleeping Beauty transposon system to over express these telomerase components TERC and TERT in the TERC mutant iPSCs. We are currently characterizing the effect of overexpression of these telomerase components on hematopoietic differentiation to determine if this provides a strategy to enable use of gene-corrected iPSCs to provide a future therapy for patients with bone marrow failure due to defined telomerase deficiencies. DisclosuresNo relevant conflicts of interest to declare.

Abstract B23: Characterization of canine glioma cancer stem cells for human glioblastoma models

Abstract Glioblastoma (GBM) is the most aggressive and common form of glioma in humans with an average survival period of twelve to fourteen months. Due to its highly invasive and malignant nature, current treatment options have not been able to improve the survival rate over the past several decades. Several studies claim that cancer stem cells (CSC), self-renewalable and differentiable into neuron- and glia-like cells, may play a key role in initiating tumor formation and cancer reoccurrence. Cancer stem cells are known to have tumorigenic ability and resistance to current cancer treatment options. Similar to humans, naturally occurring glioma in canines is highly common and devastating. Cancer in canines and humans shares strong anatomical and physiological similarities, including histological appearance, tumor genetics, molecular targets, biological behavior, and response to therapies. The goal of this study is to characterize and test the presence of canine glioma cancer stem cells (CSC) in order to utilize the spontaneously occurring cancer models to deepen understanding of human and canine GBM cancer stem cells and investigate better cancer model systems utilizing naturally occurring canine glioma since artificial rodent models have not been able to translate into human GBM studies effectively. Two different canine glioma cells lines, Roxanne and Ike, were harvested and provided from the College of Veterinary Medicine Small Animal Hospital at Purdue University. Canine cell line Roxanne, Grade II Oligodendroglioma, was harvested from a six-year-old female mixed breed by post-mortem examination. The second canine cell line Ike, Grade II Oligodendroglioma neoplastic cells with negative GFAP expression, was harvested from a six-year-old male Boxer by surgery. In order to compare these canine cell lines with the human GBM cancer stem cells GBAM1 human GBM stem cell line (known to be 98% CD133 positive) was obtained from Moffitt Cancer Center. These three cell lines were investigated by immunofluorescence with CD133, sex determining region Y-box 2 (Sox2), Notch1, glial acidic fibrillary protein (GFAP), and O6-alkylguanine DNA alkyltransferase (MGMT) protein markers. Cells were fixed and permeabilized prior to antibody attachment. Confocal images for detection of CD133, Sox2, Notch, GFAP, and MGMT were taken with a Nikon A1R-MP confocal microscope equipped with an objective Plan Fluor 20X-oil/water (0.75 NA). Roxanne and GBAM1 were tested for immunofluorescence since Ike was not able to grow confluent in in vitro environment. GBAM1 expressed all protein markers, CD133, Sox2, Notch, and MGMT, except for GFAP from the immunofluorescent detection. However, canine Roxanne only expressed Sox2 proteins, which is a transcription factor for undifferentiated stem cell. Other immunofluorescent signals were either weak or not expressed. Since the cancer stem cell population is very low in heterogeneous glioma tumor environment, it was difficult to conclude the presence of these protein markers in canine samples. In order to validate and further explore these results, western blot will be performed in the three cell lines. Characterization of both human and canine cancer stem cells has a potential to provide a better understanding of both human and canine glioma cancer stem cells, improve an animal cancer model system, and develop more effective treatments for glioblastoma. Citation Format: Soo Jung Ha, Marisol Herrera-Perez, Jiri Adamec, R. Timothy Bentley, Jenna L. Rickus, Kari L. Clase. Characterization of canine glioma cancer stem cells for human glioblastoma models. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B23.

Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare, and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry, and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years, and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

MiR-218-5p inhibits the stem cell properties and invasive ability of the A2B5⁺CD133⁻ subgroup of human glioma stem cells.

MicroRNAs (miRs) act as oncogenes or tumor-suppressor genes, and regulate the proliferation, apoptosis, invasion, differentiation, angiogenesis and behavior of glioma stem cells, which are important in glioma development and recurrence. The present study was performed to investigate the impact of miR-218-5p on stem cell properties and invasive ability of the A2B5+CD133- human glioma stem cell subgroup. qRT-PCR was used to detect miR-218-5p expression in non-cancerous brain and human glioma tissues, human glioma cell lines and human glioma stem cell lines. Lentivirus vectors encoding miR-218-5p and anti-miR-218-5p were constructed and stably transfected into A2B5+CD133- SHG-139s cells. Neurosphere formation Cell Counting Kit-8 (CCK-8) and Transwell assays, immunofluorescence and qRT-PCR analyses were used to explore the role of miR-218-5p in SHG-139s cells. qRT-PCR analysis showed that miR-218-5p expression was lower in human glioma tissues and cells than in non-cancerous brain tissues and normal human astrocyte cells, and lower in A2B5+CD133- (SHG-139s) cells than in CD133+ (SU2 and U87s) cells. The CCK-8 assay demonstrated that the growth curve was significantly inhibited in the miR-218-5p-SHG-139s cells compared to the miR-control, blank and anti-miR‑218-5p groups. The neurosphere formation assay indicated that upregulation of miR‑218-5p expression inhibited SHG-139s neurosphere formation. Immunofluorescence staining and qRT-PCR showed that miR-218-5p reduced stem cell marker (A2B5, nestin, PLAGL2, ALDH1 and Sox2) expression compared with the controls; however, immunofluorescence staining analysis showed that upregulation of miR-218-5p expression led to no difference in CD133 expression. miR‑218-5p reduced SHG-139s cell invasiveness in the Transwell assay and reduced MMP9 expression as detected in qRT-PCR and immunofluorescence analyses. All differences were statistically significant. miR-218-5p expression was lower in human glioma tissues, cells and the A2B5+CD133- human glioma stem cell subgroup. miR-218-5p may be a tumor-suppressor gene in glioma that functions by upregulating miR-218-5p expression, which inhibits the stem cell properties and invasive properties of SHG-139s cells.

Parthenogenesis and Human Assisted Reproduction.

Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself, somatic cell, and nuclear transfer experiments and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. Different activating methods have been tested on human and nonhuman oocytes, with varying degrees of success in terms of parthenote generation rates, embryo development stem cell derivation rates. Success in achieving a standardized artificial activation methodology for human oocytes and the subsequent potential therapeutic gain obtained from these embryos depends mainly on the availability of gametes donated from infertility treatments. This review will focus on the creation of parthenotes from clinically unusable oocytes for derivation and establishment of human parthenogenetic stem cell lines and their potential applications in regenerative medicine.

Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds.

Grafted synthetic polypeptides hold appeal for extending the range of biophysical properties achievable in synthetic extracellular matrix (ECM) hydrogels. Here, N-carboxyanhydride polypeptide, poly(γ-propargyl-l-glutamate) (PPLG) macromers were generated by fully grafting the "clickable" side chains with mixtures of short polyethylene glycol (PEG) chains terminated with inert (-OH) or reactive (maleimide and/or norbornene) groups, then reacting a fraction of these groups with an RGD cell attachment motif. A panel of synthetic hydrogels was then created by cross-linking the PPLG macromers with a 4-arm PEG star molecule. Compared to well-established PEG-only hydrogels, gels containing PPLG exhibited dramatically less dependence on swelling as a function of cross-link density. Further, PPLG-containing gels, which retain an α-helical chain conformation, were more effective than standard PEG gels in fostering attachment of a human mesenchymal stem cell (hMSC) line for a given concentration of RGD in the gel. These favorable properties of PPLG-containing PEG hydrogels suggest they may find broad use in synthetic ECM.

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo.

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1–2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo´s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.