Cells In Serum-containing Medium Research Articles

Isolation, culture and identification of brain tumor stem cells from glioma

Objectives: This study aims to provide a feasible method for culturing tumor stem cells to facilitate further research on the occurrence and development of brain tumors, making it applicable for in vitro studies of brain tumor stem cells. Methods: Samples of glioma were collected and subsequently isolated and cultured, then immunocytochemical staining, including CD133, Nestin, and GFAP staining, was performed on the cultured cells. Results: Some of the primary cultured tumor stem cells grew in the shape of sphere and formed into clusters. With the extension of culture time, the cell mass gradually increased, with strong proliferation and self-renewal ability. Meanwhile, the tumor derived spherical cells could differentiate into different types of tumor cells in serum-containing medium since most of them were positive to CD133, Nestin, and GFAP on the 7 days. Conclusion: The tumor stem cell can differentiate into different cell types, carrying significant implications for tumor research and treatment. More importantly, the methods introduced in this study are simple and feasible to isolate and culture tumor stem cell, contributing to cell source for future studies.

Targeted Anticancer Agent with Original Mode of Action Prepared by Supramolecular Assembly of Antibody Oligonucleotide Conjugates and Cationic Nanoparticles.

Despite their clinical success, Antibody-Drug Conjugates (ADCs) are still limited to the delivery of a handful of cytotoxic small-molecule payloads. Adaptation of this successful format to the delivery of alternative types of cytotoxic payloads is of high interest in the search for novel anticancer treatments. Herein, we considered that the inherent toxicity of cationic nanoparticles (cNP), which limits their use as oligonucleotide delivery systems, could be turned into an opportunity to access a new family of toxic payloads. We complexed anti-HER2 antibody-oligonucleotide conjugates (AOC) with cytotoxic cationic polydiacetylenic micelles to obtain Antibody-Toxic-Nanoparticles Conjugates (ATNPs) and studied their physicochemical properties, as well as their bioactivity in both in vitro and in vivo HER2 models. After optimising their AOC/cNP ratio, the small (73 nm) HER2-targeting ATNPs were found to selectively kill antigen-positive SKBR-2 cells over antigen-negative MDA-MB-231 cells in serum-containing medium. Further in vivo anti-cancer activity was demonstrated in an SKBR-3 tumour xenograft model in BALB/c mice in which stable 60% tumour regression could be observed just after two injections of 45 pmol of ATNP. These results open interesting prospects in the use of such cationic nanoparticles as payloads for ADC-like strategies.

Effective RNAi in leukemia cells is enhanced by spermine-modified pullulan combined with desloratadine

RNA interference is a very useful tool for clinical treatment as well as mechanistic studies of leukemia. However, leukemia cells are known as hard-to-transfected by non-virus carriers. The low capacity of endocytosis and lysosomal escape of synthetic carriers are two obstacles for successful RNAi in leukemia cells. In this work, we established a universal powerful strategy for mediating effective RNAi in different types of leukemia cells by sequentially using spermine-modified pullulan (PS) as siRNA carrier and desloratadine (DL) to promote the lysosomal escape. It was shown that the complex of PS and siRNA could be largely internalized by human T-cell acute lymphoblastic leukemia cells, acute myeloid leukemia cells and chronic myeloid leukemia cells in serum-containing media, and the internalized complex was able to escape from the lysosomes by the aid of DL, resulting in effective RNAi against the key genes for the different leukemia cells.

Derivation of neural stem cells from human teratomas.

Human teratoma is a germ cell tumor that contains normal tissues (e.g., hair, skin or cartilage) differentiated from embryonal germ layers. Because of the feature of this tumor, we hypothesized that human teratomas contain multipotent stem cells that can develop into various non-cancerous normal tissues. In this study, we cultured neurospheres originally derived from a human infantile teratoma tissue, and the sphere cells were found to possess the characteristics of neural stem cells. Tumor tissues were obtained from an infantile immature teratoma at the time of surgical resection. In the primary cell culture, colonies were formed in two weeks and were individually cultured in serum-free conditioned neural stem cell medium (NSC medium). Colonies changed into spheres and grew in smooth round forms, or attached to the bottom of the dishes and extended processes and filaments around. Sphere cells were dissociated into single cells, and new spheres (secondary spheres) were formed in NSC medium. Cell differentiation was induced by culturing cells in serum-containing medium (differentiation medium), as cells spread and attached to the bottom of dishes and changed form. The expression of Nestin, Sox2, CXCR4, and (stem cell markers), β3-tubulin (a neural marker) GFAP (a glial marker) CNPase, SOX10 (oligodendrocyte markers) and NF-L in cells was analyzed by immunofluorescence and a Q-PCR. Nestin, SOX2, CXCR4 were abundant in both primary and secondary spheres. Neural and glial markers (β3-tubulin and GFAP, respectively) were increased in cells cultured in differentiation medium while stem cell markers were diminished. The oligodendrocyte markers SOX10 and CNPase were also found in both spheres and differentiated cells. In conclusion, spheres with the characteristics of neural stem cells were obtained from the primary culture of a human infantile teratoma. These spheres are considered to have the potential to undergo a natural course of neural development in humans.

Resolving Single-Nanoconstruct Dynamics during Targeting and Nontargeting Live-Cell Membrane Interactions.

This paper describes how differences in the dynamics of targeting and nontargeting constructs can provide information on nanoparticle (NP)-cell interactions. We probed translational and rotational dynamics of functionalized Au nanostar (AuNS) nanoconstructs interacting with cells in serum-containing medium. We found that AuNS with targeting ligands had a larger dynamical footprint and faster rotational speed on cell membranes expressing human epidermal growth factor receptor 2 (HER-2) receptors compared to that of AuNS with nontargeting ligands. Targeting and nontargeting nanoconstructs displayed distinct membrane dynamics despite their similar protein adsorption profiles, which suggests that targeted interactions are preserved even in the presence of a protein corona. The high sensitivity of single-NP dynamics can be used to compare different nanoconstruct properties (such as NP size, shape, and surface chemistry) to improve their design as delivery vehicles.

Isolation and identification of chemotherapy-enriched sphere-forming cells from a patient with gastric cancer.

Gastric cancer (GC) is the third and fifth cause of cancer-associated mortality for men and women throughout the world, respectively. Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Cancer stem cells (CSCs) due to their pivotal role in tumor initiation, growth, progression, invasion, distant metastasis, recurrence and resistance to anticancer drugs are very appealing targets for cancer therapies. Here, we isolated and identified CSCs from a chemotherapy-treated patient. Small subpopulation of dissociated cells after tissue digestion formed spheroid colonies in serum-free media under the non-adherent condition. These spheroid colonies differentiated into epithelial like cells in serum-containing medium. Few sphere-forming cells carried CD44 and CD54 markers overexpressed DLL4 that is responsible for tumor growth and angiogenesis. Subcutaneous injections of sphere-forming cells in different passages conferred tumorigenicity in nude mice. Sphere-forming cells upregulated CD44 polymorphisms CD44v3, -v6, and -v8 -10, stemness factors OCT4, SOX2, SALL4 and Cripto-1, self-renewal molecules IHh, Wnt, β-catenin and BMI1, and epithelial mesenchymal transition (EMT) markers Twist1 and Snail1 in vitro and in vivo. Moreover, these cells similar to sphere-forming cells isolated from a chemotherapy-free patient expressed Oct-4 and β-catenin proteins. However, the Twist1 protein was only expressed by sphere-forming cells derived from the chemotherapy-treated patient. Thus, these cells have all the characteristics of stationary and migratory CSCs, including tumorigenicity, self-renewal, pluripotency, invasion and metastasis. Taken together, targeting chemotherapy-enriched CSCs as chemo-resistance cells observed in GC patients can provide more effective therapeutic strategies compared to untreated patients.

Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality

BackgroundSuspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease.MethodsChanges to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation.ResultsCapsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A24-2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media.ConclusionThe selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.

Abstract 5834: Serum induces differentiation in aggressive MYCN-amplified neuroblastoma patient-derived xenograft cells

Abstract Background: Cancer cell lines have traditionally been established in serum-containing medium though it has been questioned whether serum-grown cell lines faithfully represent the tumors they are derived from. Potential serum-induced differentiation of in vitro cultured cells could partly explain why xenografted cell lines often lack invasive growth and robust metastasis. Direct implantation of patient tumor material into mice, i.e. patient derived xenografts (PDXs), has shown to more closely mimic the original tumor growth pattern. By isolating cells from neuroblastoma PDXs and culturing the PDX cells under serum-free conditions, we expect to preserve a less differentiated phenotype, enabling us to use PDX cells as a source for identifying potential neuroblastoma stem-like cells. Moreover, to facilitate future drug screening, we investigated conditions that would allow us to grow neuroblastoma PDX cells adherently, without compromising their tumorigenic or metastatic capacity. Materials & Methods: PDX cells were cultured in stem cell medium or serum-containing medium for up to 7 days. IHC, RT-qPCR and WB were used to investigate the expression of sympathetic neuronal differentiation markers. For monolayer culture, cells were grown on laminin. Results: PDX cells can routinely be established as neurospheres in serum-free medium with retained tumorigenic and metastatic capacity. Addition of serum induces loss of sphere formation, adherent growth and a robust increase in sympathetic differentiation markers, both at mRNA and protein level. Serum-cultured cells also show a decreased cell proliferation (without increased apoptotic rate, as determined by sub-G0/G1 analyses). The serum-induced differentiation was not irreversible since transferring serum-grown cells back to serum-free medium resulted in a phenotypic switch, with recovered proliferation and decreased expression of differentiation markers. Growing cells on laminin achieved adherent culture of PDX cells. This resulted only in slight morphological differentiation but did not affect growth rate or the cells tumorigenic and metastatic capacity. Conclusion: Culturing neuroblastoma PDX cells in serum-containing medium results in a more differentiated phenotype. Thus, avoiding serum appears to be a key strategy in order to preserve the phenotypic origin of these cells. The phenotypic effects of serum on tumor cells have largely been overlooked and might be a general effect on tumor cells explaining why serum-established cell lines do not metastasize and grow invasively. Citation Format: Camilla U. Persson, Kristoffer von Stedingk, Daniel Bexell, My Merselius, David Gisselsson, Siv Beckman, Sofie Mohlin, Sven Påhlman, Caroline Wigerup. Serum induces differentiation in aggressive MYCN-amplified neuroblastoma patient-derived xenograft cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5834. doi:10.1158/1538-7445.AM2017-5834

Unveiling gene trait relationship by cross-platform meta-analysis on Chinese hamster ovary cell transcriptome.

In the past few years, transcriptome analysis has been increasingly employed to better understand the physiology of Chinese hamster ovary (CHO) cells at a global level. As more transcriptome data accumulated, meta-analysis on data sets collected from various sources can potentially provide better insights on common properties of those cells. Here, we performed meta-analysis on transcriptome data of different CHO cell lines obtained using NimbleGen or Affymetrix microarray platforms. Hierarchical clustering, non-negative matrix factorization (NMF) analysis, and principal component analysis (PCA) accordantly showed the samples were clustered into two groups: one consists of adherent cells in serum-containing medium, and the other suspension cells in serum-free medium. Genes that were differentially expressed between the two clusters were enriched in a few functional classes by Database for Annotation, Visualization, and Integrated Discovery (DAVID) of which many were common with the enriched gene sets identified by Gene Set Enrichment Analysis (GSEA), including extracellular matrix (ECM) receptor interaction, cell adhesion molecules (CAMs), and lipid related metabolism pathways. Despite the heterogeneous sources of the cell samples, the adherent and suspension growth characteristics and serum-supplementation appear to be a dominant feature in the transcriptome. The results demonstrated that meta-analysis of transcriptome could uncover features in combined data sets that individual data set might not reveal. As transcriptome data sets accumulate over time, meta-analysis will become even more revealing. Biotechnol. Bioeng. 2017;114: 1583-1592. © 2017 Wiley Periodicals, Inc.

Laboratory Scale Production and Purification of a Therapeutic Antibody.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

Lipophilic 2'-O-Acetal Ester RNAs: Synthesis, Thermal Duplex Stability, Nuclease Resistance, Cellular Uptake, and siRNA Activity after Spontaneous Naked Delivery.

The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi-based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2'-O-phenylisobutyryloxymethyl (PiBuOM)-modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM-modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)-modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM-modified siRNA and of substantial target suppression in human cells in serum-containing medium.

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines.

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

Serum-induced differentiation of human meibomian gland epithelial cells.

We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland.

Single Unpurified Breast Tumor-Initiating Cells from Multiple Mouse Models Efficiently Elicit Tumors in Immune-Competent Hosts

The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.

A Serum‐Tolerant Hydroxyl‐Modified Polyethylenimine as Versatile Carriers of pDNA/siRNA

The polyethylenimine (PEI) derivatives (PTn) are prepared by treating PEI25k with Tris(hydroxymethyl) acrylamidomethane via the Michael addition. These PTns can effectively condense nucleic acids into nanosized particles with positive surface charges. The PTns show lower cytotoxicity and better serum-resistant capacity than PEI25k. Specially, the transfection efficiency of PT26/DNA is 29-fold higher than that of PEI25k in HeLa cells in serum-containing medium. The PTn/siRNA complexes show superior knockdown effect in CT26 cells in serum-containing medium. In addition, flow cytometry analysis shows that the PTns can efficiently mediate the entry of nucleic acids into the cell. Thus, PTns are potentially applicable as non-viral carriers of nucleic acids and warrant further development for use in gene therapy.

The protective effect of GD1a ganglioside and inhibitors of nitric oxide synthase after the application of bacterial lipopolysaccharide to PC12 cells

Lipopolysaccharide from Escherichia coli, serotype 0111: B4 (LPS), at concentrations of 0.125 and 0.25 mg/mL exhibited toxic effects in cells of the neuronal PC12 line in DMEM medium. Higher concentrations of LPS are necessary to induce the death of PC12 cells in serum-containing medium. The nonspecific inhibitor of nitric oxide synthase (NOS) and the inhibitor of inducible NOS (iNOS) increased the survival of the PC12 cells, whereas an inhibitor of cyclooxygenase 1/2 (COX 1/2) enhanced cell death. We found that GD1a ganglioside at concentrations of 50 and 100 μM increased the survival of PC12 cells in DMEM medium after LPS application and decreased the LPS-induced accumulation of reactive oxygen species. Our data suggest that the activation of NOS and, specifically, iNOS, is involved in the toxic LPS effect in PC12 cells, whereas the activation of COX 1/2 weakens this effect. The protective and antioxidant effects of GD1a ganglioside in PC12 cells after LPS application probably depends on the capability of gangliosides to modify raft structure after their incorporation into membranes and to prevent the activation of TLR receptors in cell membranes.

Cardiac Cell Generation from Encapsulated Embryonic Stem Cells in Static and Scalable Culture Systems

Heart diseases are major causes of morbidity and mortality linked to extensive loss of cardiac cells. Embryonic stem cells (ESCs) give rise to cardiomyocyte-like cells, which may be used in heart cell replacement therapies. Most cardiogenic differentiation protocols involve the culture of ESCs as embryoid bodies (EBs). Stirred-suspension bioreactor cultures of ESC aggregates may be employed for scaling up the production of cardiomyocyte progeny but the wide range of EB sizes and the unknown effects of the hydrodynamic environment on differentiating EBs are some of the major challenges in tightly controlling the differentiation outcome. Here, we explored the cardiogenic potential of mouse ESCs (mESCs) and human ESCs (hESCs) encapsulated in poly-L-lysine (pLL)-coated alginate capsules. Liquefaction of the capsule core led to the formation of single ESC aggregates within each bead and their average size depended on the concentration of seeded ESCs. Encapsulated mESCs were directed along cardiomyogenic lineages in dishes under serum-free conditions with the addition of bone morphogenetic protein 4 (BMP4). Human ESCs in pLL-layered liquid core (LC) alginate beads were also differentiated towards heart cells in serum-containing media. Besides the robust cell proliferation, higher fractions of cells expressing cardiac markers were detected in ESCs encapsulated in LC than in solid beads. Furthermore, we demonstrated for the first time that ESCs encapsulated in pLL-layered LC alginate beads can be coaxed towards heart cells in stirred-suspension bioreactors. Encapsulated ESCs yielded higher fractions of Nkx2.5- and GATA4-positive cells in the bioreactor compared to dish cultures. Differentiated cells formed beating foci that responded to chronotropic agents in an organotypic manner. Our findings warrant further development and implementation of microencapsulation technologies in conjunction with bioreactor cultivation to enable the production of stem cell-derived cardiac cells appropriate for clinical therapies and applications.

Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs

Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum-free media (K-M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum-containing media (FBS-M) with cells cultured in four different serum-free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS-M. Additional proliferation assays were performed using pre-coated fibronectin (FN) tissue culture plates and of the four serum-free medium, only K-M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS-M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K-M or FBS-M, and, additionally, cells retained their multipotency in K-M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K-M or FBS-M. Taken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency.

Characterization of Human Pancreatic Progenitor Cells

β-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for β-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed "cobblestone" morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed "fibroblast-like" morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6-10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.

Abstract 3336: Upregulated inflammatory cytokines in glioma stem cells: Targets for glioma therapies

Abstract Gliomas are the most common primary brain tumors in adults. With the highly invasive growth pattern, gliomas often escape current therapeutic modalities including combinations of surgery, radiotherapy and chemotherapy. Recent studies implicate that a minority population of glioma stem cells (GSCs) are responsible for glioma maintenance and recurrence. Interleukin (IL)-1 beta, IL-6 and IL-8 are pleiotropic proinflammatory cytokines and their excessive production in the tumor microenvironment is associated with poor disease outcome in many cancer types, including glioma. These cytokines promote tumor growth via induction of proteins associated with tumor invasion and angiogenesis and anti-apoptosis. We hypothesize that GSCs acquire their chemo-resistance and invasion by excessive cytokine production. To test the hypothesis, we compared the cytokine gene expression of a human GSC-enriched population in serum-free medium (SFM) in the presence of epidermal growth factor and fibroblast growth factor, to that of glioma cells in serum-containing medium (SCM). SFM is the neural stem/progenitor cell (NPC) medium in which NPCs proliferate in neurospheres and this medium also has been used to enrich GSCs. We found that IL-1 beta and IL-6 were upregulated in GSC-enriched population along with increased expression of Nestin, a marker of neural stem cells. More interestingly, Smad interacting protein 1 (SIP1), which is thought to be involved in glioma cell migration and invasion, was also increased in the GSC-enriched population. The paralleled upregulation of IL-1 beta and IL-6 with Nestin and SIP1 in GSC-enriched population suggest that there is a possible correlation between them. The correlation is now under investigation. Since IL-1 beta is elevated in GSC enriched population, we further determined whether IL-1 beta plays a critical role in glioma growth. Glioma cells were co-cultured with mouse NPCs expressing IL-1 receptor antagonist (IL-1ra), which can bind to IL-1 receptors without transmitting activation signals and represents a competitive inhibitor of IL-1. Proliferation of glioma cells, which were modified to express Renilla luciferase, was measured using bioluminescence imaging. Our results showed that proliferation of glioma cells was inhibited in the co-culture with NPCs expressing IL-1ra, but not in the co-culture with NPCs without expression of IL-1ra, compared to glioma cells cultured alone. The results suggest that IL-1 has an important role in glioma cell growth and NPCs engineered to express IL-1ra have therapeutic potential for gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3336.