Prolonged Passaging Research Articles

Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C -Nucleoside Monophosphate Prodrug

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Trophic stimulation of articular chondrocytes by late‐passage mesenchymal stem cells in coculture

Coculture of mesenchymal stem cells (MSCs) with articular chondrocytes (ACs) increases glycosaminoglycan (GAG) accumulation compared to monoculture. MSCs might (1) differentiate into ACs (progenitor role) and/or (2) stimulate AC matrix metabolism (trophic role). MSCs lose the ability to undergo chondrogenesis after extended passaging. We hypothesized that MSCs act predominantly as progenitors, and that late-passage MSCs without chondrogenic potential would be unable to increase GAG in coculture. Early- and late-passage human MSCs (hMSCs) were grown in pellet monoculture under chondrogenic conditions and in pellet coculture with bovine ACs. Chondrogenesis was assessed with GAG quantification, safranin-O staining, and quantitative PCR (qPCR). Contributions of human and bovine cells were assessed with species-specific qPCR and human-specific immunostaining. Late-passage hMSCs did not undergo chondrogenesis in monoculture with chondrogenic stimuli or in coculture with ACs. Early-passage hMSCs underwent chondrogenesis only in response to chondrogenic stimuli. Coculture pellets in both cases accumulated as much GAG/DNA as monoculture AC pellets. Aggrecan transcription was not increased in coculture. Late-passage hMSCs that do not undergo chondrogenesis are capable of stimulating GAG accumulation in coculture with ACs. This supports a trophic effect of hMSCs on ACs. hMSCs may have therapeutic utility even after prolonged passaging.

Abstract 1569: Next generation cell line models: conditionally reprogrammed cells.

Abstract Introduction: For decades, scientists have attempted to develop models for growing and studying primary tumors outside of the human body. Even today, one of the greatest challenges in cancer research has been generation of stable cancer cell lines from primary tumors. Most primary cell cultures, regardless of the method used to generate them, suffer from limited lifespan due to cellular senescence. Cultured cells transformed with SV40 virus large T antigen or over-expression of hTERT has been limited by genomic variation and other changes that vary with the passage number. Recently, a new approach was described by the Schlegel group at Georgetown (Liu et al, AJP 2012) called “conditionally reprogrammed cells” (CRCs). They showed these cells (derived from primary tumors or normal tissue) could be grown indefinitely under defined conditions without using any extrinsic genetic immortalization technique. These cells have been shown to maintain the karyotype similar to the tissue of origin even after prolonged passaging. Here we show that we can obtain confirmatory results at a separate institution and further show that in lung cancer, the CRC lines maintain histotype specific characteristics. Method: The CRC technique requires mouse irradiated J2 cells as feeder cells and a ROCK inhibitor to rapidly grow and expand patient's epithelial cells. We have grown cultures from six tissue types including lung, breast, pancreas, kidney, colon, and salivary gland. All tissue samples were collected by Yale University Pathology Tissue Services and processed in our lab for generating CRC lines. We have characterized lung CRC lines for morphology and immunophenotyping using H&E stains and histological markers including CK7, p63 and TTF1. We have also performed gene expression profiling using Illumina bead arrays on selected lung and pancreatic samples. Results: We have successfully established CRC lines from all six tissue types. Our success rate exceeds 70% for lung and breast tumor samples while other tissues show somewhat lower rates. Even traditionally challenging cells to culture, like pancreatic and salivary gland tumors have been successfully cultured. Preliminary morphological and histological analysis on lung CRC lines show that features of lung adenocarcinoma (ADCA) and lung squamous cell carcinoma (SQCC) are maintained in CRC lines. Gene expression profiling shows that lung CRC lines from SQCC samples clustered together and are separate from ADCA. Profiles from a pancreatic CRC line recapitulated the profile of the corresponding frozen tissue. Conclusion: This novel CRC technology represents a new approach to culturing cells from tumors and normal cells, including tumors that have been historically challenging to grow. The resulting cells appear highly similar to the tumors from which they were derived. Future studies are needed to further characterize the CRC system and to test its value for assessment of personalized drug sensitivity. Citation Format: Seema Agarwal, Joanna Hu, Kelly Stanton, Kurt Schalper, Yuval Kluger, Elizabeth Zarrella, Xuefeng Liu, Richard Schlegel, David L. Rimm. Next generation cell line models: conditionally reprogrammed cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2013-1569

The effect of mesenchymal stem cell shape on the maintenance of multipotency

Human mesenchymal stem cells (MSCs) have broad therapeutic potential due to their ability to differentiate into multiple cell types. However, when cultured ex vivo MSCs will spontaneously differentiate and have been shown to lose multipotency after prolonged passaging. Cell culture conditions that promote maintenance of multipotency during in vitro expansion are a critical need to fully realize the therapeutic potential of MSCs. Here we show that by confining MSCs to small islands, we can restrict inappropriate lineage specification and enhance the expression of mesenchymal stem cell markers Stro-1 and Endoglin. Even when released from the islands and reseeded, cells previously cultured in patterns maintain higher expression of MSC markers compared to cells cultured on plastic, while maintaining their ability to differentiate into adipocytes and osteoblasts. Exposure of non-patterned cells to inhibitors of myosin and Rho-associated protein kinase (ROCK) leads to increased expression of stem cell markers. Our findings suggest that maintenance of MSC “stemness” requires a low state of actomyosin contractility. This work will prove useful in the development of culture conditions for the maintenance of multipotent MSCs in vitro and for the design of niche-mimetic biomaterials.

MDR1-associated resistance to artesunate + mefloquine does not impair blood-stage parasite fitness in a rodent malaria model

If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin combination treatment (ACT) version of artesunate+mefloquine (ATN+MF) to gain insights about their ability to compete with ATN+MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN+MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN+MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure.

Mutations in Multiple Domains of Gag Drive the Emergence of In Vitro Resistance to the Phosphonate-Containing HIV-1 Protease Inhibitor GS-8374

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.

Impact of hypoxia and long-term cultivation on the genomic stability and mitochondrial performance of ex vivo expanded human stem/stromal cells

Recent studies have described the occurrence of chromosomal abnormalities and mitochondrial dysfunction in human stem/stromal cells (SCs), particularly after extensive passaging in vitro and/or expansion under low oxygen tensions. To deepen this knowledge we investigated the influence of hypoxia (2% O2) and prolonged passaging (>P10) of human bone marrow stromal cells (BMSCs) and adipose-derived stromal cells (ASCs) on the expression of genes involved in DNA repair and cell-cycle regulation pathways, as well as on the occurrence of microsatellite instability and changes in telomere length. Our results show that hypoxic conditions induce an immediate and concerted down-regulation of genes involved in DNA repair and damage response pathways (MLH1, RAD51, BRCA1, and Ku80), concomitantly with the occurrence of microsatellite instability while maintaining telomere length. We further searched for mutations occurring in the mitochondrial genome, and monitored changes in intracellular ATP content, membrane potential and mitochondrial DNA content. Hypoxia led to a simultaneous decrease in ATP content and in the number of mitochondrial genomes, whereas the opposite effect was observed after prolonged passaging. Moreover, we show that neither hypoxia nor prolonged passaging significantly affected the integrity of the mitochondrial genome. Ultimately, we present evidence on how hypoxia selectively impacts the cellular response of BMSCs and ASCs, thus pointing for the need to optimize oxygen tension according to the cell source.

Development of five new melanoma low passage cell lines representing the clinical and genetic profile of their tumors of origin

Development of five new melanoma low passage cell lines representing the clinical and genetic profile of their tumors of origin

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×1013 fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.

Structurally Distinct LewisX Glycans Distinguish Subpopulations of Neural Stem/Progenitor Cells

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.

Abstract 502: Development of lung cancer cell lines exhibiting cancer stem cell properties through application of stem-cell culturing techniques

Abstract Introduction: Lung cancer is the most common cause of cancer death worldwide: with greater than 160,000 deaths reported in the US in 2008 and a dismal 15% 5-year survival rate, there is a clear need for improved therapeutic approaches. Considering the increasing evidence for the role of cancer stem cells (CSC) in driving tumor initiation and metastases, development of model systems to isolate and characterize such populations in lung cancer would provide important tools to drive discovery of novel therapeutic modalities. We recently reported application of selective culture conditions to generate novel cell lines from colon cancer that exhibit CSC properties (1). Here we apply a similar approach to lung cancer. Methodology: Freshly resected NSLC tissue was obtained, processed and cultured using serum-free defined culture media previously developed for fetal lung stem cells (2). Expanded cell lines were characterized for their tumor initiating properties in NSG mice upon implantation under the sub-renal capsule. Cell populations generated from lung cancer under serum-containing conditions served as controls. Tumors were analyzed by H&E and immunostaining for specific markers. Gene expression profiles were determined by U133 Affymetrix arraying, and the cell surface protein profile examined by flow cytometry using a bank of approximately ∼200 cell surface reactive mAbs. Results: Lung cancer cell lines, were successfully cultured from independent lung tumor specimens and remained genetically stable upon prolonged passaging in vitro. One such line, LUCA22, was derived from an adenosquamous carcinoma specimen and harbors a KRAS mutation. This line formed tumors in mice within 10 weeks upon implantation of as few as 500 cells; these tumors recapitulated the morphology of the patient's original tumor, with mixed features that included differentiation into both adeno- and squamous-type carcinoma. Within 16 weeks, spontaneous metastases were observed to the lung, liver and pancreas. Gene array analyses of LUCA22 cells compared to control populations revealed up-regulation of genes associated with the Wnt and TGF-beta pathways, while flow cytometry identified over-expression of several CSC-associated cell surface proteins, including CD44. Conclusion: Novel NSCL cell lines have been derived from primary tumor specimens by employing culture conditions previously established for tissue stem cells. These lines display key features ascribed to CSC including the capacity to recapitulate the original tumor morphology from where they were derived and the ability to spontaneously metastasize. Gene array analysis and mAbs library panning of the cell lines are being applied to identify lead opportunities for targeting lung cancer stem cells.

Dynamic expression patterns of imprinted genes in human embryonic stem cells following prolonged passaging and differentiation

To evaluate the overall expression patterns of imprinted genes in human embryonic stem cells following long term culture and differentiation. Expression levels of 65 imprinted genes determined by PCR array were analyzed in one human embryonic stem cell line (cHES1) following prolonged passaging and differentiation. Transcripts of 63 imprinted genes were detected in cHES1 cells. Expression levels of all but 5 imprinted genes did not correlate with passage numbers or differ in cells after passage 50 compared with those before passage 50. SLC22A2, SLC22A3, CPA, H19, COPG2IT1 and IGF2 expression were significantly increased in embryoid bodies compared with undifferentiated cells. The global expression profiles of imprinted genes are generally stable in human embryonic stem cells after prolonged passaging and differentiation.

LAT1 overexpression and function compensates downregulation of ASCT2 in an in vitro model of renal proximal tubule cell ageing

Amino acid transporters provide cells neutral amino acids indispensable for growth and proliferation-dependent protein synthesis. This study evaluates whether prolonged serial cell passaging during 6 months (over 50 passages) may induce changes in amino acid transporters properties in Opossum kidney (OK) proximal tubular cells. High passage OK cells exhibit polyploidy, but no difference in the proliferation potential was observed when compared to low passage OK cells. Increased time in culture was accompanied by an increased total, membrane and cytosol protein content. The Na(+)-insensitive [(14)C]-L-leucine uptake was promoted almost exclusively thought LAT1 (~ 90 vs 80%, high versus low passage OK cells). The increased LAT1 protein abundance in high passage OK cells correlated positively with enhanced ability to take up [(14)C]-L-leucine, despite a 4.3-fold decrease in affinity for the substrate. The Na(+)-sensitive [(14)C]-L-alanine transport was decreased by 2.5-fold in high passage OK cells. However, no differences in ASCT2 expression were observed between high and low passage OK cells. It is concluded that OK cells show functional differences in both L-leucine and L-alanine uptake as a function of passage time in culture. The increased expression and activity of LAT1 in high passage OK cells may correspond to a mechanism enabling the cell to develop the hypertrophy response to prolonged cell passaging, when the function of ASCT2 is markedly depressed.

The Phthalocyanine Prototype Derivative Alcian Blue Is the First Synthetic Agent with Selective Anti-Human Immunodeficiency Virus Activity Due to Its gp120 Glycan-Binding Potential

Alcian Blue (AB), a phthalocyanine derivative, is able to prevent infection by a wide spectrum of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains in various cell types [T cells, (co)receptor-transfected cells, and peripheral blood mononuclear cells]. With the exception of herpes simplex virus, AB is inactive against a broad variety of other (DNA and RNA) viruses. Time-of-addition studies show that AB prevents HIV-1 infection at the virus entry stage, exactly at the same time as carbohydrate-binding agents do. AB also efficiently prevents fusion between persistently HIV-1-infected HUT-78 cells and uninfected (CD4(+)) lymphocytes, DC-SIGN-directed HIV-1 capture, and subsequent transmission to uninfected (CD4(+)) T lymphocytes. Prolonged passaging of HIV-1 at dose-escalating concentrations of AB resulted in the selection of mutant virus strains in which several N-glycans of the HIV-1 gp120 envelope were deleted and in which positively charged amino acid mutations in both gp120 and gp41 appeared. A mutant virus strain in which four N-glycans were deleted showed a 10-fold decrease in sensitivity to the inhibitory effect of AB. These data suggest that AB is likely endowed with carbohydrate-binding properties and can be considered an important lead compound in the development of novel synthetic nonpeptidic antiviral drugs targeting the glycans of the envelope of HIV.

Protein cytoskeleton and overexpression of Na+,K+‐ATPase in opossum kidney cells

Previous studies have shown that over time in culture opossum kidney (OK) cells are endowed with increased Na(+),K(+)-ATPase activity and expression (Silva et al., 2006, J Membr Biol 212:163-175; Silva and Soares-da-Silva, 2007, Am J Physiol Regul Integr Comp Physiol 293:R1764-R1770). The present work evaluated the cytoskeleton reorganization in OK cells at passages 40 and 80 in culture and its possible relationship with membrane transport proteins and cell morphology. It is shown that OK cells with 80 passages in culture have increased size, internal complexity, and total protein expression. In OK cells with 80 passages in culture the use of in-cell western showed that ezrin/radixin/moesin complex was increased by 20%. The most abundant ankyrin-G isoform in OK cells with 40 passages was the approximately 200/220 kDa isoform, whereas in OK cells with 80 passages the most abundant isoform was the approximately 170 kDa isoform. The spectrin-betaII approximately 240 kDa isoform, the predominant isoform in OK cells with 40 passages, was marginally detected in OK cells with 80 passages. Besides Na(+),K(+)-ATPase, GLUT2, and NHE3 expression was also significantly increased in OK cells with 80 passages. It is concluded that the prolonged cell passaging of OK cells results in an interesting and valuable experimental model to analyze the reorganization of the renal cell cytoskeleton proteins and its relationship with transporter and signaling membrane proteins.

Establishing an indirect sandwich enzyme-linked-immunosorbent-assay (ELISA) for the detection of antibodies against Histomonas meleagridis from experimentally infected specific pathogen-free chickens and turkeys

No serological method suitable for large screening of antibodies against Histomonas meleagridis in poultry is available so far, the objective targeted in the present investigation. Consequently, an ELISA was developed as a suitable tool for this purpose. Investigating serum samples from non-infected specific pathogen-free (spf) chickens and commercial turkeys a high-background signal was noticed when ELISA plates were directly coated with purified parasitic cells. This signal was significantly reduced by coating the plates with a polyclonal rabbit antibody, raised against histomonads, prior to the addition of the antigen. Adopting this approach five antigen preparations were compared and a high reproducibility could be demonstrated reflected by a very low coefficient of variation of 5.3% and 1.7% for the chicken and turkey sera, respectively. After this initial development all further experiments were carried out with one set of plates and the same antigen preparation. Investigating chicken sera obtained from birds infected at 14 days of life, OD values above a predetermined cut-off value were observed 2 weeks post-infection and a rise of IgG antibodies was noticed until 6 weeks post-infection, when the experiment was terminated. Non-protected turkeys infected at 6 weeks of age displayed an increasing IgG response until 14 days post-infection, prior to the death of animals due to histomonosis. In comparison, the majority of turkeys vaccinated with attenuated histomonads, obtained through prolonged passaging and challenged 4 weeks later with virulent parasites, displayed a demonstrable antibody response after the challenge only. Antibody titres increased until 4 weeks post-challenge when the birds were killed and the study was terminated. Altogether, the developed indirect sandwich ELISA proved to be a quick and efficient method to detect IgG antibodies against H. meleagridis in sera of experimentally infected chickens and turkeys and will be a helpful tool to obtain more insights into the epidemiology of the parasite and the immune response of its hosts.

Nox1 Induces Differentiation Resistance in Immortalized Human Keratinocytes Generating Cells that Express Simple Epithelial Keratins

We have shown that superoxide radical-generating NADPH oxidase 1 (Nox1) is increased in intermediate human transformed cells. It was unknown whether Nox1 overexpression could accelerate early transformation steps. We demonstrated that Nox1 rendered human immortalized (GM16) keratinocytes resistant against Ca(2+)/serum-induced differentiation. Nox1-transfected cells produced fast dividing resistant cells within 7-10 days after DMEM exposure. Progenitor lines (or Nox1 lines) were reproducibly generated from Nox1-transfected cells, while no lines were obtained from control transfections. From several attempts to generate control cells, one resistant population was obtained from untransfected GM16 cells after a 6-week DMEM exposure. Prolonged passaging of the control line could induce Nox1. Compared with the control line, Nox1 lines showed greater expression of Nox1, Rac1, p47phox, p67phox, NOXO1, and NOXA1 with concomitant increased superoxide generation. All five Nox1 lines contained varying amounts of E-cadherin, involucrin, vimentin, and K8/K18, while the control line did not. Since vimentin and K8/K18 are associated with malignant progression in different types of human epithelial tumors, our data demonstrate that Nox1 accelerated neoplastic-like progression by inducing generation of progenitor cells. Our data also emphasize the importance of Nox1 in inducing resistance against differentiation-induced cell death, suggesting a contribution of Nox1 and its oxidants during early stage of cell transformation.

Adherent Neural Stem (NS) Cells from Fetal and Adult Forebrain

Stable in vitro propagation of central nervous system (CNS) stem cells would offer expanded opportunities to dissect basic molecular, cellular, and developmental processes and to model neurodegenerative disease. CNS stem cells could also provide a source of material for drug discovery assays and cell replacement therapies. We have recently reported the generation of adherent, symmetrically expandable, neural stem (NS) cell lines derived both from mouse and human embryonic stem cells and from fetal forebrain (Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A. 2005. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3(9):e283). These NS cells retain neuronal and glial differentiation potential after prolonged passaging and are transplantable. NS cells are likely to comprise the resident stem cell population within heterogeneous neurosphere cultures. Here we demonstrate that similar NS cell cultures can be established from the adult mouse brain. We also characterize the growth factor requirements for NS cell derivation and self-renewal. We discuss our current understanding of the relationship of NS cell lines to physiological progenitor cells of fetal and adult CNS.

Articular chondrocyte passage number: Influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography

The isolation and culture of articular chondrocytes is a prerequisite of their use in tissue engineering, but prolonged culture and passaging is associated with de-differentiation. In this paper we studied the influence of nanometric and micrometric grooves (85 nm to 8 microm in depth and 2 microm to 20 microm in width) on 1st and 2nd passage ovine chondrocytes since our earlier findings indicate that primary cells are not affected by such features. 1st and 2nd passage chondrocytes cultured on grooved substrata showed a polarisation of cell shape parallel to the groove long axis and F-actin condensations were evident at groove ridge boundaries. An increase in cell migration with increasing groove depth was observed. Both passages of chondrocytes maintained type II collagen expression, but to a lesser degree in 2nd. This study demonstrates that passage number alters the response of chondrocytes to micrometric and nanometric topography, and could be important in ex vivo cartilage engineering.

Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture

The in vitro culture of porcine bone marrow-derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants-rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell-cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid beta-galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP-7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long-term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging.