Special Growth Medium Research Articles

Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder-Free Conditions.

The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful; however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.

Evaluation of the ELITe InGenius PCR Platform for Detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia in persons of all ages. Due to the fastidious nature of this bacterium and the necessary specialized growth media, nucleic acid amplification testing is currently the most reliable means for patient diagnostics. Analytical sensitivity, specificity, reproducibility, and clinical performance of the ELITe InGenius automated PCR platform with its MGB Alert M. pneumoniae real-time PCR research use only reagents (ELITechGroup, Inc., Bothell, WA) were compared with those of a laboratory-developed real-time PCR assay targeting repMp1 for detection of M. pneumoniae The ELITe InGenius PCR assay successfully detected 31 distinct M. pneumoniae clinical isolates and reference strains, and there was no cross-reactivity with other mollicutes, Gram-positive bacteria, or Gram-negative bacteria. In testing 223 clinical samples, the ELITe InGenius PCR showed 95.79% and 99.22% positive and negative agreement with the repMp1 assay, respectively. Additionally, the ELITech platform showed 98.91% positive and 96.95% negative predictive values, and there was no significant difference detected between the two assays (McNemar's test, P = 0.375). The ELITe InGenius PCR assay limit of detection was 0.16 CFU/PCR test or 4.16 genome copies (GCs)/test. Accuracy, instrument ease-of-use, and decreased hands-on time make the ELITe InGenius platform suitable for detection of M. pneumoniae directly from clinical specimens.

Isolation of Anaerobic Bacteria During Quality Analysis of Medicinal Products

Important features of the isolation of anaerobic microorganisms were examined. The detection limit and frequency and time for isolating several anaerobic bacteria by direct inoculation on thioglycollate medium and by a BacT/ALERT 3D automated system were established. Isolation of individual cells of anaerobic microorganisms with different tolerances to oxygen was shown to be possible by using thioglycollate growth medium to analyze the sterility of medicinal products. The minimum number of cells in a sample could be reliably determined in less time by using a BacT/ALERT 3D automated system and special growth medium.

Abstract 3017: A spontaneous developmental lineage plasticity that underlies the response of prostate cancer cells to androgen deprivation

Abstract Androgen-dependent prostate cancer (PCa) cells can acquire features of neuroendocrine- (NE-) cells under acute androgen deprivation (AD). Gene expression studies show that other neuroectodermal/mesenchymal (NE/M) cell phenotypes develop under chronic AD. We propose that a transient PCa stem-like cell mediates these transdifferentiations and we sought to reveal and maintain this stem-like state using a special growth medium that is compatible with stem cells that could give rise to these complex lineages. LNCaP or CWR22r cells were plated at low density on coated plates then switched to an androgen-free “stem-transition medium” compatible with NE/M-derived stem cells. Eight days later, cells were transferred in the same medium and were characterized for morphology and 3-dimensional (3D) growth properties. Growth in the medium converted these cells, en masse, to small, rounded proliferating cells that grew as 3D rosettes then to spheroids. Gene expression and cell surface CD antigen profiling of converted LNCaP cells were most consistent with a neural/neural crest stem-like (N/NCSL) identity. N/NCSL PCa cells fail to express androgen receptor (AR) or PSA protein and they are significantly more resistant to hypoxia and enzalutamide than parental LNCaPs. LNCaP-N/NCSL cells have significantly increased tumor-initiating capability and formed tumors in nude mice from only 100 xenografted cells vs 1 X 106 parental LNCaP cells needed to form tumors. When LNCaP N/NCSL cells were placed into differentiation mediums without androgen, they acquired morphological features and expressed biomarkers of neuron-, glia-, oligodendrocyte or osteocyte-like cells. When they were redifferentiated in androgen-containing medium, they reverted to an epithelial cell that resembles parental LNCaPs but they overexpressed AR and retained expression of nestin, a neural stem marker. Our outcomes show that differentiated (AR+) PCa cells have a previously unsuspected developmental plasticity that allows them to return back to a neural/neural crest stem-like state with drastically increased tumor initiating capability under certain microenvironmental conditions. Our new model cell culture system allows us to maintain them in this state or to differentiate them to a variety of NE/M-derived cell lineages. Reactivation of androgen signaling, however, returns them to a modified prostate epithelial-like state that overexpresses AR and retains expression of some stem genes. We believe that our model culture system reveals a novel pathway associated with progression of PCa to therapy-resistance. *Authors contributed equally Citation Format: Josselin Caradec*, Amy Lubik*, Mannan Nouri, Na Li, Jennifer Bishop, Martin Gleave, Ralph Buttyan. A spontaneous developmental lineage plasticity that underlies the response of prostate cancer cells to androgen deprivation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3017. doi:10.1158/1538-7445.AM2014-3017

The effects of stress hormones on growth of selected periodontitis related bacteria

The focus of this study was to examine in vitro the effects of stress hormones (catecholamines: epinephrine, norepinephrine, dopamine and hydrocortisone: cortisol) on the growth of four anaerobic species of periodontitis-related bacteria (Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia) and one facultative anaerobic species (Eikenella corrodens).Bacterial growth was determined by two different methods: fluorescence in situ hybridization (FISH), and the viable count by culture method. To simulate stress, each single strain was grown in a special growth medium with three different concentrations of each hormone, using an anaerobic chamber at 37 °C.Growth of F. nucleatum increased in the presence of all stress hormones. Growth of P. gingivalis was not significantly influenced by any hormone. Growth of P. intermedia and E. corrodens was inhibited by almost all stress hormones tested. Both methods of analysis revealed that the highest concentrations of norepinephrine and cortisol increased the growth of T. forsythia. Different hormones have a different effect on the growth of periodontitis-related bacteria in vitro. It appears that bacterial viability is more strongly influenced than is bacterial metabolic activity. The growth of F. nucleatum particularly and partially of T. forsythia is increased by several stress hormones and may have an additional negative impact on periodontal disease.

In vitro development and characterization of canine epidermis on a porcine acellular dermal matrix

The aim of this study was to develop and to characterize a canine skin epidermal model able to form a proper epidermis on a porcine acellular dermal matrix (PADM). In addition, the role of fibroblasts in skin barrier formation was studied by incorporating or omitting canine dermal fibroblasts in the PADM. Canine epidermal composites were developed by seeding keratinocytes onto the surface of PADM that were previously seeded or non-seeded with dermal fibroblasts. After 14 days of culture under air-exposed conditions and in a special growth medium, skin composites were histologically processed and immunohistochemically characterized to determine the expression of cytokeratins and of vimentin and the presence of basement membrane. In all composites, keratinocytes underwent differentiation to a multilayer epidermis with 5-7 viable cell layers. The stratum basalis, stratum spinosum, stratum granulosum and stratum corneum were identified. The expression of cytokeratins was similar to that described in healthy canine epidermis. Laminin and collagen IV immunostaining revealed a homogeneous layer in the epidermal-dermal junction only when the matrix had been seeded by canine dermal fibroblasts. The model may become a simple, useful and cost-effective tool to investigate the biology and pathology of canine epidermis and could partially replace animal testing in several areas of dermatological research.

Fast investigations from biological matrices using CE – Test of a blood–brain barrier model

In order to adopt a general workflow for complex biological matrices with respect to a new blood-brain barrier (BBB) model, a micellar electrokinetic chromatography method has been developed. The cells forming the BBB have been cultivated in a special cell growth medium in which six drugs (acetaminophen, caffeine, carbamazepine, cimetidine, indometacin and propranolol) have been dissolved and tested for their penetration properties. The results showed good to very good accordance to the reference values. Samples were directly injected onto the capillary without any pretreatment (fused silica capillary, id: 50 μm, L: 48 cm, l: 40 cm). After method development, separations were carried out using a 60 mM borate buffer containing 200 mM of SDS at 30 kV, leading to an analysis time of less than 10 min. Between two runs the capillary was rinsed with a mixture of equal parts of running buffer and isopropanol (70% v/v), which proved to be very effective to remove matrix compounds. An appropriate choice of the detection wavelength (220 or 254 nm) could avoid major interferences between analytes and matrix. The typical RSD% for migration times was approximately 2%, for peak areas, it ranged from 2 to 6%, which was very well acceptable for the generic method used in this study and the low concentrations investigated. The LODs ranged from 10 to 30 ng/mL.

Zebrafish stromal cells have endothelial properties and support hematopoietic cells

The goal of this study was to determine if we could establish a mesenchymal stromal line from zebrafish that would support hematopoietic cells. Such a coculture system would be a great benefit to study of the hematopoietic cell-stromal cell interaction in both invitro and invivo environments. Zebrafish stromal cells (ZStrC) were isolated from the "mesenchymal" tissue of the caudal tail and expanded in a specialized growth media. ZStrC were evaluated for phenotype, gene expression, and ability to maintain zebrafish marrow cells in coculture experiments. ZStrC showed mesenchymal and endothelial gene expression. Although ZStrC lacked the ability to differentiate into classic mesenchymal stromal cell lineages (i.e., osteocytes, adipocytes, chondrocytes), they did have the capacity for endotube formation on Matrigel and low-density lipoprotein uptake. ZStrC supported marrow cells for >2 weeks invitro. Importantly, marrow cells were shown to retain homing ability in adoptive transfer experiments. ZStrC were also shown to improve hematopoietic recovery after sublethal irradiation after adoptive transfer. As the zebrafish model grows in popularity and importance in the study of hematopoiesis, new tools to aid in our understanding of the hematopoietic cell-stromal cell interaction are required. ZStrC represent an additional tool in the study of hematopoiesis and will be useful in understanding the factors that mediate the stromal cell-hematopoietic cell interactions that are important in hematopoietic cell maintenance.

Apophysomyces variabilisInfections in Humans

To the Editor: The fungus Apophysomyces elegans (order Mucorales) is a thermotolerant species that causes severe infections among humans. In contrast to other fungi that cause zygomycosis, which have a worldwide distribution and are rarely found in immunocompetent hosts, A. elegans has been reported mainly in areas with warm climates as an emerging pathogen that causes mostly cutaneous infections after injury to the skin (1). This fungus was discovered in 1979 (2) and until recently was considered the only species in the genus. A polyphasic study of clinical and environmental strains of A. elegans, including analysis of several genes, showed that the genus contained 4 well-characterized species (3). Of 16 isolates tested in this study, only 2 from soil in India were A. elegans. Most of the isolates were A. variabilis. The incidence of A. variabilis in humans is unknown and difficult to ascertain because most cases had isolates that were not properly preserved. These fungi usually cause necrotizing fasciitis, but rhino-orbito-cerebral or renal infections have also been reported (1). Whether these infections are produced by different Apophysomyces spp., have different responses to antifungal drugs, or have differences in virulence is unknown. To assess incidence of Apophysomyces spp. in a tertiary hospital (Government Medical College Hospital, Chandigarh, India), which usually receives patients with zygomycosis, a retrospective study was conducted during November 2001–April 2009. Nine patients were identified as having primary cutaneous zygomycosis. For 4 patients, fungal isolates were morphologically identified as A. elegans. A description of clinical findings, their management, and outcomes for these 9 patients has been reported (4). The 4 isolates were sent to the Universitat Rovira i Virgili (Reus, Spain) for molecular analysis. The internal transcribed spacer region of these isolates was sequenced and compared with those of type strains of Apophysomyces spp. Fungi were identified by morphologic (Figure, panel A) and molecular analysis as A. variabilis (99.6%–99.7% sequence identity with sequence of type strain CBS 658.93 [{type:entrez-nucleotide,attrs:{text:FN556436,term_id:306480963}}FN556436]). GenBank accession nos. of the 4 isolates are {type:entrez-nucleotide,attrs:{text:FN813491,term_id:307133352}}FN813491, {type:entrez-nucleotide,attrs:{text:FN813490,term_id:307133351}}FN813490, {type:entrez-nucleotide,attrs:{text:FN556442,term_id:306480968}}FN556442, and {type:entrez-nucleotide,attrs:{text:FN813492,term_id:307133353}}FN813492. Another patient was also infected with A. variabilis fungi. The patient was a 45-year-old woman with diabetes from Derabassi (Punjab), India, who was hospitalized because of swelling in her right breast and blackening of overlying skin. A diagnosis of right breast gangrene was made. Therefore, local debridement of the swelling was conducted, and tissue samples were tested by microbiologic culture and histopathologic analysis. A KOH wet mount showed broad aseptate hyphae with right-angled branching. The fungal isolate was tentatively identified as A. elegans. Histopathologic analysis confirmed a diagnosis of zygomycosis. The patient was treated under local anesthesia by debridement of infected tissue and some of the healthy surrounding tissue (Figure, panel B). However, an antifungal regimen could not be given because she had disturbed renal function. Her condition deteriorated, septicemia was observed, and she died from sudden cardiac arrest on the sixth day after admission. The fungal isolate was also identified as A. variabilis (98.9% identity, GenBank accession no. {type:entrez-nucleotide,attrs:{text:FN556443,term_id:306480969}}FN556443). Although most cases of infection with A. variabilis fungi have been reported in India (5), infections with this fungus may have a wider distribution. A recent study demonstrated that this species represented 0.5% of fungi of the order Mucorales isolated from clinical samples in the United States (6). Furthermore, a high mortality rate and the fact that most of these infections involve otherwise healthy patients make this a serious infection. The number of infections with Apophysomyces spp. is underestimated because these fungi do not usually sporulate on standard fungal culture media used in clinical laboratories. These fungi require special nutrient-deficient growth medium (Czapek agar), a high temperature in comparison to other human pathogens (37°C–42°C), and prolonged incubation (7–10 days) (7). A difference in mortality rate was observed when we compared patients in our study (80%) with those reported by Chakrabarti et al. (5) (28.5%) in India, even though treatment was generally similar, i.e., local debridement and amphotericin B. Other differences in our study were that the infection in 4 patients was preceded by intramuscular injection, and these 2 patients had diabetes mellitus. In conclusion, A. variabilis is an emerging pathogenic fungus that can cause rapid and fatal infections in humans. As more isolates of Apophysomyces fungi become available, molecular typing studies must be conducted to better understand the epidemiology and distribution of different Apophysomyces spp. Figure A) Sporangiophore (center) and sporangiospores of Apophysomyces variabilis fungi. Scale bar = 10 μm. B) Clinical manifestations in a woman infected with A. variabilis fungi in the upper part of the chest and the breast. A color version of this ...

Selective 15N labeling of barstar in a T7 polymerase system

The T7 RNA polymerase system for selective protein labeling, previously optimized for the time of introducing the 15N amino acid relative to the inducer (IPTG) and host polymerase inhibitor (rifampicin), was further improved by the use of a special growth medium enriched in particular amino acids and by administration of an aminotransferase inhibitor (α-aminooxyacetic acid); the latter prevented isotope redistribution (as required by NMR studies) even with readily metabolized leucine. The approach is shown to be successful for selective labeling of barstar with [15N]Trp and [15N]Leu; it can be used with any proteins expressed in the T7 system.

FACS technology used in a new rapid bacterial detection method

Culture methods for bacterial detection (BacT/ALERT or Pall eBDS) are currently implemented in blood donor screening procedures in many countries. Experience in the first years after implementation of these detection assays showed that although the analytical sensitivity was extremely high (about 1 CFU mL(-1)), the majority of these platelets were still transfused before a positive screening result was attained. Rapid technologies were developed to more effectively prevent transfusion-transmitted bacterial infection. In this study, a new rapid bacterial detection method based on fluorescence-activated cell sorting (FACS) technology was developed. Bacteria were stained with thiazole orange dye for 5 min and measurement was taken immediately after staining. The entire process took only 30 min. Six transfusion-relevant bacteria strains were tested in a spiking study. Without pre-incubation in a special bacteria growth medium, analytical sensitivity ranged between 10(5) CFU mL(-1) (Klebsiella oxytoca and Serratia marcesens) and 10(3) CFU mL(-1) (Escherichia coli). Sensitivity could be improved to 10(1) CFU mL(-1) for all tested bacteria by adding a pre-incubation step (6 h at 37 degrees C). Although preliminary in nature, results of our study suggest that bacterial detection by FACS technology in conjunction with a pre-incubation step offers a sensitive alternative technology to culture methods. Additionally, it provides the benefit of a rapid test time and the opportunity of preventing bacterial transmitted infections more effectively.

Cultivation of fetal erythroid precursors from maternal blood: Isolation and characterization by PCR and FISH

Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce sufficient fetal cells for prenatal molecular genetic and cytogenetic analysis. For in vitro cultivation, 10 ml of blood was collected from 22 women carrying a male fetus. After triple-density gradient centrifugation, the mononucleated cells were cultivated for 10 to 14 days in special hematopoietic growth medium. Red and white colored cell colonies were individually collected by micromanipulation. A representative sample from each colony was characterized by chromosome Y-specific polymerase chain reaction (PCR) systems. The remaining cells of the Y-positive colonies were used to perform chromosome preparations and fluorescence in situ hybridization (FISH) to detect XY-positive interphase nuclei and metaphases. Y-positive signals could be detected in 15 (68%) of the 22 analyzed blood samples. With SRY PCR 10.5% (40/379) of the collected red colonies were determined to be of fetal origin and 6.1% (32/522) of the colonies analyzed by amelogenin PCR were Y-positive. All collected white cell colonies were Y-negative. FISH analyses of PCR-positive colonies revealed that less than 30% of the cells within a colony are of fetal origin and reflect more precisely the actual situation within a single colony. Moreover the successful preparation of fetal metaphases in non-invasive prenatal diagnosis is presented.

Lymphocyte Activation- 31P Magnetic Resonance Studies of Energy Metabolism and Phospholipid Pathways

31P NMR spectra of perfused lymphocytes embedded in alginate capsules and activated by interleukin-2 (IL-2) are remarkably different from those of control lymphocytes. The main differences are the appearance and gradual increase of phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occur following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and are not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibits the effects of PHA, but not of IL-2. There are no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine (PC) and phosphatidylethanolamine is an inherent feature of the activation process. 31P NMR spectra of lymphocytes are characterized by a low phosphocholine signal. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of phosphocholine cytidyltransferase, indicate that choline kinase plays a key role in regulating PC synthesis in human lymphocytes.

Lymphocyte activation and phospholipid pathways. 31P magnetic resonance studies

31P NMR spectra of perfused lymphocytes, embedded in alginate capsules and activated by interleukin-2, were remarkably different from those of control lymphocytes. The main differences were the appearance and gradual increase in phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occurred following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and were not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibited the effects of phytohemagglutinin, but not of interleukin-2. There were no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine and phosphatidylethanolamine is an inherent feature of the activation process. 31P NMR spectra of lymphocytes are characterized by a low signal of phosphocholine. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of cytidylyltransferase, indicated that choline kinase plays a key role in regulating phosphaditylcholine synthesis in human lymphocytes.