Passage In Cell Culture Research Articles

Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation

The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with mitochondrial DNA (mtDNA) mutation. Skin biopsies were obtained from two diabetic patients with mtDNA A3243G mutation. The fibroblasts thus obtained were infected with retroviruses encoding OCT4 (also known as POU5F1), SOX2, c-MYC (also known as MYC) and KLF4. The stem cell characteristics were investigated and the mtDNA mutation frequencies evaluated by Invader assay. From the two diabetic patients we isolated four and ten putative mitochondrial disease-specific iPS (Mt-iPS) clones, respectively. Mt-iPS cells were cytogenetically normal and positive for alkaline phosphatase activity, with the pluripotent stem cell markers being detectable by immunocytochemistry. The cytosine guanine dinucleotide islands in the promoter regions of OCT4 and NANOG were highly unmethylated, indicating epigenetic reprogramming to pluripotency. Mt-iPS clones were able to differentiate into derivatives of all three germ layers in vitro and in vivo. The Mt-iPS cells exhibited a bimodal degree of mutation heteroplasmy. The mutation frequencies decreased to an undetectable level in six of 14 clones, while the others showed several-fold increases in mutation frequencies (51-87%) compared with those in the original fibroblasts (18-24%). During serial cell culture passage and after differentiation, no recurrence of the mutation or no significant changes in the levels of heteroplasmy were seen. iPS cells were successfully generated from patients with the mtDNA A3243G mutation. Mutation-rich, stable Mt-iPS cells may be a suitable source of cells for human mitochondrial disease modelling in vitro. Mutation-free iPS cells could provide an unlimited, disease-free supply of cells for autologous transplantation therapy.

Frequency and patterns of protease gene resistance mutations in HIV-infected patients treated with lopinavir/ritonavir as their first protease inhibitor.

Selection of protease mutations on antiretroviral therapy (ART) including a ritonavir-boosted protease inhibitor (PI) has been reported infrequently. Scarce data exist from long-term cohorts on resistance incidence or mutational patterns emerging to different PIs. We studied UK patients receiving lopinavir/ritonavir as their first PI, either while naive to ART or having previously received non-PI-based ART. Virological failure was defined as viral load ≥ 400 copies/mL after previous suppression <400 copies/mL, or failure to achieve <400 copies/mL during the first 6 months. pol sequences whilst failing lopinavir or within 30 days after stopping were analysed. Major and minor mutations (IAS-USA 2008-after exclusion of polymorphisms) were considered. Predicted susceptibility was determined using the Stanford HIVdb algorithm. Three thousand and fifty-six patients were followed for a median (IQR) of 14 (6-30) months, of whom 811 (27%) experienced virological failure. Of these, resistance test results were available on 291 (36%). One or more protease mutations were detected in 32 (11%) patients; the most frequent were I54V (n = 12), M46I (n = 11), V82A (n = 7) and L76V (n = 3). No association with viral subtype was evident. Many patients retained virus predicted to be susceptible to lopinavir (14, 44%), tipranavir (26, 81%) and darunavir (27, 84%). This study reflects the experience of patients in routine care. Selection of protease gene mutations by lopinavir/ritonavir occurred at a much higher rate than in clinical trials. The mutations observed showed only partial overlap with those previously identified by structural chemistry models, serial cell culture passage and genotype-phenotype analyses. There remained a low degree of predicted cross-resistance to other widely used PIs.

Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis

We aimed to evaluate the stability of the Chlamydia trachomatis multi locus VNTR analysis (MLVA-ompA) and multi sequence typing (MST) systems through multiple passages in tissue culture. Firstly, we analyzed the stability of these markers through adaptation of C. trachomatis to tissue culture and secondly, we examined the stability of a four-locus MLVA-ompA and a five-locus MST system after multiple passages in tissue culture. Marker sequences were monitored through successive chlamydial developmental cycles to evaluate the stability of the individual DNA markers through many bacterial divisions and this, in turn, informed us of the usefulness of using such typing systems for short and long-term molecular epidemiology. Southampton genitourinary medicine (GUM) clinic isolates from endocervical swabs collected from C. trachomatis positive women were passaged through tissue culture. MLVA-ompA typing was applied to primary swab samples and to the same samples after C. trachomatis had been passaged through cell culture (eight passages). Sequence data from time-zero and passage-eight isolates were aligned with reference sequences to determine the stability of the markers. The Swedish new variant (nvCT) underwent 72 passages in cell culture and the markers of the two schemes were similarly analyzed. Analysis of genetic markers of the MLVA-ompA typing system before and after the isolates were introduced to tissue culture showed no change in the dominant sequence. The nvCT that had been passaged 72 times over the duration of a year also showed no variation in the dominant sequence for both the genotyping schemes. MLVA-ompA and MST markers are stable upon adaptation of C. trachomatis to tissue culture following isolation of strains from primary endocervical swab samples. These markers remain stable throughout multiple rounds of cell-division in tissue culture, concomitant with the incubation period and appearance of symptoms normally associated with host-infection. Both genotyping schemes are, therefore, suitable for epidemiology of C. trachomatis.

Development and laboratory evaluation of two lateral flow devices for the detection of vesicular stomatitis virus in clinical samples

Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3 of subtype 3 [VSV-IND-3]) and 189 positive for VSV-NJ (162 vesicular epithelial suspensions and 27 cell culture antigens) from suspected cases of vesicular disease in cattle and horses collected from 11 countries between 1937 and 2008 or else were derived from experimental infection and 777 samples that were either shown to be positive or negative for foot-and-mouth disease (FMD) virus (FMDV) and swine vesicular disease virus (SVDV) or else collected from healthy cattle or pigs and collected from 68 countries between 1965 and 2011. The diagnostic sensitivity of the VSV-IND (for reaction with VSV-IND-1) and VSV-NJ LFDs was either similar or identical at 94.6% (VSV-IND) and 97.4% (VSV-NJ) compared to 92.5% and 97.4% obtained by the reference method of antigen ELISA. The VSV-IND LFD failed to react with viruses of VSV-IND-2 and 3, while the VSV-NJ device recognized all VSV-NJ virus strains. The diagnostic specificities of the VSV-IND and VSV-NJ LFDs were 99.1% and 100, respectively, compared to 99.6% and 99.8% for the ELISA. Reactions with FMDV which can produce indistinguishable syndromes clinically in cattle, pigs and sheep and SVDV (vesicular disease in pigs) did not occur. These data illustrate the potential for the LFDs to be used next to the animal for providing rapid and objective support to veterinarians in their clinical judgment of vesicular disease and for the subtype (VSV-IND-1) and type-specific (VSV-NJ) pen-side diagnosis of VS and differential diagnosis from FMD.

Genomic RNA Editing and Its Impact on Ebola Virus Adaptation During Serial Passages in Cell Culture and Infection of Guinea Pigs

Synthesis of the structural, surface glycoprotein (GP) of Ebola virus (EBOV) is dependent on transcriptional RNA editing phenomenon. Editing results in the insertion of an extra adenosine by viral polymerase at the editing site (7 consecutive template uridines) during transcription of GP gene of the wild-type virus (EBOV/7U). In this study, we demonstrate that passage of EBOV/7U in Vero E6 cells results in the appearance and rapid accumulation of a variant (EBOV/8U) containing an additional uridine at the editing site in the viral genome. EBOV/8U outgrows and eventually replaces the wild-type EBOV during 4-5 passages. On the contrary, infection of guinea pigs with EBOV/8U leads to the appearance and rapid predominance by EBOV/7U. These rapid conversions suggest that editing of the genomic RNA occurs at a higher frequency than previously thought. In addition, it indicates that the EBOV/7U phenotype has a selective advantage that is linked to controlled expression of GP and/or expression of secreted sGP, the primary gene product for wild-type EBOV. This study demonstrates the potential for insertion and deletion of uridines in the editing site of the EBOV genomic RNA, depending on environmental constraints.

SIRT1 is required for long-term growth of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Compensatory mutations rescue the virus replicative capacity of VIRIP-resistant HIV-1

VIRIP has been identified as a highly specific natural inhibitor of HIV-1 that blocks HIV-1 gp41-dependent fusion by interacting with the gp41 fusion peptide. Two analogues of VIRIP (VIR-353 and VIR-576) with a few amino acid changes increase its antiretroviral potency by two orders of magnitude in cell culture. VIR-576 has been shown effective in a phase I/II clinical trial. Resistance to VIRIP and its analogue VIR-353 were generated after long-term passage in cell culture suggesting a high genetic barrier to resistance. Mutations conferring resistance to VIRIP and VIR-353 significantly reduced virus fitness. However, accumulation of additional mutations rescued the replication capacity of the virus while retaining resistance to VIR-353 and full sensitivity to T20. Combinations of VIR-353 and T20 had an additive effect on the inhibition of wild type HIV-1 replication, but only a single agent was active when combinations were tested against T20-resistant HIV-1, suggesting that both gp41 peptides do not interfere with each other in their binding to gp41. Our results provide additional support to the development of a new class of antiretroviral agents targeting gp41-dependent fusion.

β-Hairpin Peptide That Targets Vascular Endothelial Growth Factor (VEGF) Receptors

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.

Forced Activation of β-Catenin Signaling Supports the Transformation of hTERT-Immortalized Human Fetal Hepatocytes

Hepatocarcinogenesis is a multistep process driving the progressive transformation of normal liver cells into highly malignant derivatives. Unlimited proliferation and telomere maintenance have been recognized as prerequisites for the development of liver cancer. Moreover, recent studies identified illegitimate β-catenin signaling as relevant hit in a considerable subset of patients. To further investigate the currently not well-understood malignant evolution driven by telomerase and β-catenin, we monitored cytogenetic and phenotypic alterations in untransformed telomerase-immortalized human fetal hepatocytes following forced activation of β-catenin signaling. As expected, constitutive activation of β-catenin signaling significantly enhanced proliferation with decreasing serum dependence. Previously intact contact inhibition was almost completely eliminated. Interestingly, after several passages in cell culture, immortalized clones with dominant-positive β-catenin signaling acquired additional chromosomal aberrations, in particular translocations, anchorage-independent growth capabilities, and formed tumors in athymic nude mice. In further support for the driving role of β-catenin during hepatocarcinogenesis, improved colony growth in soft agar and accelerated tumor formation was also confirmed in Huh7 cells following stable expression of the constitutively active S33Y β-catenin mutant. Telomerase inhibition showed that short-term expansion of transformed clones was not telomerase dependent. Finally, cancer pathway profiling in derived tumors revealed upregulation of characteristic genes associated with invasion and angiogenesis. In conclusion, illegitimate activation of β-catenin signaling enhances the transformation from immortalization to malignant growth in human fetal hepatocytes. Our data functionally confirm a permissive role for β-catenin signaling in the initial phase of hepatocarcinogenesis.

Analysis of deletion within the reindeer pseudocowpoxvirus genome

Cases of contagious pustular stomatitis have been reported in Finnish reindeer for many years. Two species of the genus Parapoxvirus of the family Poxviridae have been identified as the causative agent of the disease; orf virus (ORFV) was found during the 1992–1993 epidemic and pseudocowpoxvirus (PCPV) was connected to the 1999–2000 epidemic. The genome of reindeer parapoxvirus from the latter outbreak, isolate F00.120R, was recently sequenced and confirmed as PCPV. The six gene deletion of the right terminus of the F00.120R genome, in comparison to ORFV, was investigated in an attempt to use it in differentiating viruses causing pustular stomatitis in reindeer. The present study describes discovery and analysis of genes 116–121 in reindeer PCPV and in an Italian field isolate of bovine PCPV. The results show that a 5431bp sequence containing genes 116–121 was likely to have been deleted from the F00.120R genome between the 6th and 7th passage in cell culture, and that these genes are present in other isolates of reindeer and bovine PCPV isolated in Finland during the years 2005–2010. The data presented here extends our knowledge of the PCPV genome, confirming that it contains homologues of all known ORFV genes and further reinforces their close genetic relationship. The similarity between the EEV envelope and GM-CSF inhibitory factor genes from reindeer PCPV and ORFV isolates, Finnish sheep ORFV and cattle PCPV isolates indicate that these viruses have been circulating among Finnish reindeer, cattle and sheep over a long period of time.

Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny.

Defective Interfering Viral Particles in Acute Dengue Infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3′ and 5′ ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6–36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

K Influx Parameters in Rat Aortic Vascular Smooth Muscle Cells (RASMCs)

Background RASMCs serve as major experimental tool in cardiovascular research. Here we studied further the effect of cell culture passage on K influxes with Rb as congener. Methods Rb uptake was measured by atomic absorption spectrophotometry in RASMCs ± inhibitors of the Na/K pump (ouabain, O), Na-K-2Cl cotransport (NKCC, bumetanide, B), and K-Cl cotransport (KCC, Cl replacement by sulfamate). Rb influx was calculated in nmol/mg protein × time, with O inhibition maximized for NKCC and KCC measurements. RASMC identity was verified immunologically. Results Positive IF anti-αactin staining. Rb uptake nonlinear with time in the absence (total, T) and presence of 1 mM O, and both O and 10 μM B. Rb flux by NKCC {O-insensitive (OIS) – [O+B-insensitive, OBIS]) = > 30% than that by the Na/K pump (T- OIS). Presence of O during the flux alone or flux + preincubation inhibited the Na/K pump, IC50 = 0.14 or 0.17 mM and 45% and 60% of T at 2 mM O, respectively. At 2 mM O, B inhibited 80% of OIS Rb influx with an IC50 = 0.1 μM. OBIS KCC activity (53%) saturated with increasing Cl mol fractions, remainder in 100% sulfamate due to channel Rb influx. KCC Vmax = ~ 1 and 2 nmol/(mg protein × min), and Km = 89 and 59 mM [Cl]o, with Hill n=1, in old vs young RASMCs passages, respectively. Conclusions Na/K pump, NKCC and KCC measurement required inhibitor presence in both preincubation + flux. Apparent Na/K pump O affinity was consistent with the low rodent α1Na/K ATPase isoform O affinity and NKCC B affinity s close to that for renal NKCC2. KCC parameters, except Hill n, were age-dependent, consistent with KCC1, 3 & 4 isoforms present in RASMCs.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

Dentinogenic potential of human adult dental pulp cells during the extended primary culture

Despite the frequent use of primary dental pulp cells in dental regenerative research, few systematic studies of stemness for osteogenic and dentinogenic differentiation of human adult pulp cells have been reported. To investigate the stemness of human adult dental pulp cells, pulp tissues were obtained from extracted third molars and used as a source of pulp cells. In FACS analysis and immunophenotyping, the general mesenchymal stem cell markers CD44, CD90, and CD146 were highly expressed in early passages of the pulp cell culture. The stem cell population was dramatically decreased in an expansion culture of human dental pulp cells. When pulp cells were treated with additives such as β-glycerophosphate, ascorbic acid, and dexamethasone, nodule formation was facilitated and mineralization occurred within 2 weeks. Expression of osteogenic markers such as alkaline phosphatase, osteocalcin, and osteonectin was relatively low in undifferentiated cells, but increased significantly under differentiation conditions in whole passages. Dentinogenic markers such as dentin sialophosphoprotein and dentin matrix protein-1 appeared to decrease in their expression with increasing passage number; however, peak levels of expression occurred at around passage 5. These data suggested that stem cells with differentiation potential might exist in the dental pulp primary culture, and that their phenotypes were changed during expansion culture over 8-9 passages. Under these conditions, a dentinogenic population of pulp cells occurred in limited early passages, whereas osteogenic cells occurred throughout the whole passage range.

Toxoplasma gondii RH Ankara: Production of evolving tachyzoites using a novel cell culture method

Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem.The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain tachyzoites to harvest abundant tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12–0.5×106/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3×106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12×106/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55×106/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2–99.4% and 0.31–0.37×106/ml, respectively.ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays’ antigen from local evolving strains.

Enzyme-passage free culture of mouse embryonic stem cells on thermo-responsive polymer surfaces

Cell based therapies offer potentially revolutionary treatments for a number of diseases, but are dependent on the culture and supply of defined cell types in appropriate numbers. In turn, this supply requires changes to current cell culture passaging methods, which commonly use enzymatic digestion. Here we demonstrate, for the first time, the use of thermoresponsive poly(methoxyethoxyethylmethacrylate-co-oligoethyleneglycolmethacrylate) (poly(MEO2MA-co-OEGMA)) as a responsive surface on which to culture embryonic stem cells (mouse; used as a model embryonic stem cell type). Thermo-responsive copolymer brushes composed of MEO2MA and OEGMA were grafted from hydroxyl plasma-polymer functionalised glass slides using atom transfer radical polymerisation (ATRP) and their thermo-responsive behaviour characterised. Feeder-free mouse embryonic stem cells (mESCs) were seeded on these surfaces following adsorption of fibronectin (to encourage cell attachment) and cultured for three days. Subsequent passaging experiments were performed over 10 passage cycles and the cells analysed for population doubling times and retention of the stem cell geno/phenotype. These thermoresponsive polymer/fibronectin surfaces were found to be suitable for mESC culture without promoting differentiation during the early stages of culture. This indicated that these materials have promise as a new generation of culture materials for enzyme free cell culture and passage suitable for generating cell populations for clinical applications.

Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture

BackgroundHaematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.Methods and FindingsUCB-derived mononuclear cells (MNC) or selected CD34+ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34+ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin+, CD133+ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.ConclusionsThis novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.

Effects of antibiotics on Chlamydia trachomatis viability as determined by real-time quantitative PCR

The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA and the groEL1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones, L2-OFXR, obtained by in vitro selection. Using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at time zero. This method allowed the easy determination of MICs by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed the16S rRNA and groEL1 genes, even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell-culture passaging.

Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy

Overproduction of collagen and its abnormal assembly are hallmarks of keloid scars. Type I/III collagen ratios are altered in keloids compared with normal skin. Fibroblasts from different sites in keloid tissue, perilesional compared with intralesional and extralesional sites, show differential apoptosis and contraction. Additionally, early vs. later cell culture passages display differential collagen expression. We therefore hypothesize that keloid fibroblasts from the growing margin of the keloid express higher levels of collagen type I and III, and that collagen production is altered by extended cell culture passage. (i) To measure collagen I and III at mRNA and protein levels quantitatively in keloid fibroblasts, growth media and tissue sections; and (ii) to perform tissue staining for collagen I and III expression in different lesional sites. Keloid fibroblast cultures from intralesional, perilesional and extralesional sites (n = 8 separate keloid cases, yielding 64 biopsy samples) were established from passage 0 to passage 3. Collagen I and III mRNA was quantified using quantitative reverse transcription-polymerase chain reaction. We also measured the protein levels quantitatively by developing a highly specific and sensitive capture sandwich enzyme-linked immunosorbent assay. A novel in-cell Western blotting was carried out in addition to haematoxylin and eosin and Herovici staining on keloid tissue sections for collagen I and III. Collagen types I and III were significantly higher (P < 0·03) in fibroblasts from the growing margin (perilesional site) compared with extralesional and intralesional keloid biopsy sites. As the passage number increased, the amount of collagen I significantly (P < 0·05) decreased and the collagen I/III ratio also decreased. Our data show that cells from the growing margin of keloid scars have a higher production of collagen I and III compared with other lesional sites. Additionally, temporal extension of cell passage affects collagen production. Clinically these findings may influence selection and interpretation of extended cell passage and provide future direction for lesional site-specific therapy in keloid scars.