Cell Types In Culture Research Articles

Dorothy Trump

<h3>Abstract</h3> Genetic changes acquired during <i>in vitro</i> culture pose a potential risk for the successful application of stem cells in regenerative medicine. To assess mutation accumulation risks induced by culturing,...

Biological Parameters Determining the Clinical Outcome of Autologous Cultures of Limbal Stem Cells

Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

Correction: Identification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture

The authors wish to retract this article for the following reasons: On re-examination of the data, some errors were identified which contradicted some of the main conclusions of the work. In particular, an error in the calculation of ∆(-) values for miR-193a-3p and miRPlus E1245 was identified, which affects the results reported in Figure 6, and hence the data does not support the conclusions originally drawn regarding the E1245. In addition, the interpretation of the presented PCA results is inaccurate since there is no convincing evidence of large or consistent variation related to treatment effect. Since the experiments were performed only twice, there is no statistical support for the reported miRNA expression level changes during XMRV infection of human cell, and therefore the conclusion that XMRV infection modulates miRNAs in host cells cannot be reported with confidence unless supported by additional experimental validation.

Detection and quantification of endoplasmic reticulum stress in living cells using the fluorescent compound, Thioflavin T

The endoplasmic reticulum (ER) plays a central role in the co- and post-translational modification of many proteins. Disruption of these processes can lead to the accumulation of misfolded proteins in the endoplasmic reticulum — a condition known as endoplasmic reticulum stress. In recent years, the association of endoplasmic reticulum stress with a number of disease pathologies has increased interest in the study of this condition. Current methods to detect endoplasmic reticulum stress are indirect and retrospective. Here we describe a new method to detect and quantify endoplasmic reticulum stress in live cells using Thioflavin T (ThT), a small molecule that exhibits enhanced fluorescence when it binds to protein aggregates. We show that enhanced ThT-fluorescence correlates directly with established indicators of unfolded protein response activation. Furthermore, enhanced ThT-fluorescence can be detected in living cells within 20min of application of an endoplasmic reticulum stress-inducing agent. ThT is capable of detecting endoplasmic reticulum stress induced by distinctly different conditions and compounds, in different cultured cell types as well as in mouse tissue samples. Pre-treatment with a potent endoplasmic reticulum stress-reducing agent, 4-phenylbutyric acid, mitigates the enhanced ThT signal. This new tool will be useful in future research investigating the role of protein misfolding in the development and/or progression of human diseases.

The Influence of Electric Field and Confinement on Cell Motility

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.

‘Poking’ microtubules bring about nuclear wriggling to position nuclei

Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg primordia. Although similar phenomena have been reported for a number of cultured cell types and some neurons in the zebra fish embryo, the mechanism and importance of the process remained unexplained. Wriggling involves the succession of sudden and random minor turns of the nuclei, about three twists in a minute with roughly 12° per twist, one of which lasts typically for 14 seconds. Wriggling is brought about by the growing microtubules seeded throughout the cell cortex, which, while poking the nuclei, buckle and exert 5–40 piconewtons over about 16 seconds - as it appears - through the nuclear pore complexes. While wriggling, the nuclei drift about 5 µm in a day in the immensely growing follicle cells along the apical-basal axis from the apical to the basal cell region. An over twofold excess of the microtubules nucleated in the apical cell region, as compared to those seeded in the basal cell cortex, makes the nuclei drift along the apical-basal axis. Nuclear wriggling and positioning appear to be tightly related processes: they cease simultaneously - when the nuclei become anchored by the actin cytoskeleton -, moreover, colchicine or taxol treatments eliminate both nuclear wriggling and positioning. We propose that the wriggling nuclei reveal a thus far not described nuclear positioning mechanism.

A Synthetic Dolastatin 10 Analogue Suppresses Microtubule Dynamics, Inhibits Cell Proliferation, and Induces Apoptotic Cell Death

We have synthesized eight analogues (D1-D8) of dolastatin 10 containing several unique amino acid subunits. Of these agents, D5 was found to be most effective in inhibiting both HeLa cell proliferation and microtubule assembly in vitro. At low nanomolar concentrations, D5 inhibited the proliferation of several types of cancer cells in culture. D5 bound to tubulin with a dissociation constant of 29.4 ± 6 μM. D5 depolymerized microtubules in cultured cells and produced mulitpolar spindles. At its half-maximal inhibitory concentration (15 nM), D5 strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. D5 increased the accumulation of checkpoint proteins BubR1 and Mad2 at the kinetochoric region and caused G2/M block in these cells. The blocked cells underwent apoptosis with the activation of Jun N-terminal kinase. The results suggested that D5 exerts its antiproliferative action by dampening microtubule dynamics.

Regulation of Postsynaptic Retrograde Signaling by Presynaptic Exosome Release

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Isolation and characterization of epithelial and myogenic cells by “fishing” for the morphologically distinct cell types in rat primary periodontal ligament cultures

The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin–EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the “cell fishing” method. The “fished” cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription–polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases.

Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal

To investigate the effects of Lizhong Tang, an herbal product used in traditional Chinese medicine, on mouse small intestine interstitial cells of Cajal (ICCs). Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. All of the experiments were performed at 30-32 °C. ICCs generated pacemaker potentials, and Lizhong Tang produced membrane depolarization in current-clamp mode. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang. Pretreatment with thapsigargin (a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang. However, pacemaker potentials were completely abolished in the presence of an external Ca²⁺-free solution, and under this condition, Lizhong Tang induced membrane depolarizations. Furthermore, When GDP-β-S (1 mmol/L) was in the pipette solution, Lizhong Tang still induced membrane depolarizations. In addition, membrane depolarizations were not inhibited by chelerythrine or calphostin C, which are protein kinase C inhibitors, but were inhibited by U-73122, an active phospholipase C inhibitors. These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.

The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform.

Regression Modeling to Inform Cell Incorporation Into Therapies for Craniosynostosis

Designing an appropriate tissue engineering solution for craniosynostosis (CS) necessitates determination of whether CS-derived cells differ from normal (wild-type, WT) cells and what assays are appropriate to test for differences. Traditional methodologies to statistically compare cellular behavior may not accurately reflect biologically relevant differences because they poorly address variation. Here, logistic regression was used to determine which assays could identify a biological difference between WT and CS progenitor cells. Quantitative alkaline phosphatase and MTS proliferation assays were performed on adipose, muscle, and bone marrow-derived cells from WT and CS rabbits. Data were stratified by assay, cell type, and days in culture. Coefficients of variation were calculated and assay results coded as predictive variables. Phenotype (WT or CS) was coded as the dependent variable. Sensitivity-specificity curves, classification tables, and receiver operating characteristic curves were plotted for discriminating models. Two data sets were utilized for subsequent analyses; one was used to develop the logistic regression models for prediction, and the other independent data set was used to determine the ability to predict group membership based on the predictive equation. The resulting coefficients of variation were high for all differentiation measures. Upon model implementation, bone marrow assays were observed to result in 72%-100% predictability for phenotype. We found predictive differences in our muscle-derived and bone marrow-derived cells suggesting biologically relevant differences. This data analysis methodology could help identify homogenous cells that do not differ between pathologic and normal individuals or cells that differ in their osteogenic potential, depending on the type of cell-based therapy being developed.

Adipose Tissue Engineering in Three-Dimensional Levitation Tissue Culture System Based on Magnetic Nanoparticles

White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet accumulation than 2D cultures. This indicates that 3D intercellular signaling better recapitulates WAT organogenesis. Combined, our studies show that adipospheres are appropriate for WAT modeling ex vivo and provide a new platform for functional screens to identify molecules bioactive toward individual adipose cell populations. This 3D methodology could be adopted for WAT transplantation applications and aid approaches to WAT-based cell therapy.

Highly Efficient Derivation of Skeletal Myotubes from Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are a promising model for the research of embryonic development and regenerative medicine. Since the first hESC line was established, many researchers have shown that pluripotent hESCs can be directed into many types of functional adult cells in culture. However, most of the reported methods have induced differentiation through the alteration of growth factors in the culture medium. These methods are time consuming; moreover, it is difficult to obtain a pure population of the desired cells because of the low efficiency of induction. In this study, we used a lentiviral-based inducible gene-expression system in hESCs to control the ectopic expression of MyoD, which is an essential transcription factor in skeletal muscle development. The induction of MyoD can efficiently direct the pluripotent hESCs into mesoderm in 24 h. The cells then become proliferated myoblasts and finally form multinucleated myotubes in vitro. The whole procedure took about 10 days, with an induction efficiency of over 90%. To our knowledge, this is the first time that hESCs have been induced into terminally differentiated cells with only one factor. In the future, these results could be a potential resource for cell therapy for diseases of muscle dysfunction.

Large T Antigens of Polyomaviruses: Amazing Molecular Machines

The large tumor antigen (T antigen) encoded by simian virus 40 is an amazing molecular machine because it orchestrates viral infection by modulating multiple fundamental viral and cellular processes. T antigen is required for viral DNA replication, transcription, and virion assembly. In addition, T antigen targets multiple cellular pathways, including those that regulate cell proliferation, cell death, and the inflammatory response. Ectopic T antigen expression results in the immortalization and transformation of many cell types in culture and T antigen induces neoplasia when expressed in rodents. The analysis of the mechanisms by which T antigen carries out its many functions has proved to be a powerful way of gaining insights into cell biology. The accelerating pace at which new polyomaviruses are being discovered provides a collection of novel T antigens that, like simian virus 40, can be used to discover and study key cellular regulatory systems.

Discrimination of NPC cell lines associated with malignant types using Raman spectroscopy

Nasopharyngeal carcinoma (NPC) is subdivided into four categories according to both the characterization of the NPC cell and their radiotherapy reaction. To discriminate these kinds of NPC, the representative cultured cell types (CNE1, CNE2, 5-8F, and 6-10B) are explored by single-cell micro-Raman spectroscopy (RS). The collected spectra are analyzed and compared by principal component analysis (PCA) and linear discriminate analysis (LDA). The algorithm is shown to have the lowest specificity and sensitivity of 89 and 92% for NPC division. These results demonstrate that it is a potentially clinical application for Raman spectroscopy to diagnosis NPC tissue types.

Cellular reprogramming: a new approach to modelling Parkinson's disease

iPSCs (induced pluripotent stem cells) offer an unparalleled opportunity to generate and study physiologically relevant cell types in culture. iPSCs can be generated by reprogramming almost any somatic cell type using pluripotency factors such as Oct4, SOX2, Nanog and Klf4. By reprogramming cells from patients carrying disease-associated mutations, and subsequent differentiation into the cell type of interest, researchers now have the opportunity to study disease-specific cell types which were previously inaccessible. In the case of PD (Parkinson's disease), reprogramming is advancing rapidly, and cell lines have been generated from patients carrying mutations in several disease-associated genes, including SNCA (α-synuclein), PARK2 (parkin), PINK1 (phosphatase and tensin homologue deleted on chromosome 10-induced putative kinase 1), PARK7 (DJ-1) and LRRK2 (leucine-rich repeat kinase 2), as well as idiopathic cases. Functional dopaminergic neurons have been differentiated from these cells and their physiology has been compared with control neurons. Human dopaminergic neurons had been previously inaccessible until post-mortem, when the disease is generally highly progressed into pathology. In comparison, iPSCs provide a living cell model with the potential to study early molecular changes which accumulate in cells and ultimately result in neurodegeneration. Although clear phenotypes have not yet been unambiguously identified in patient-derived dopaminergic neurons, there are suggested aberrations in cellular pathways involved in neurodegeneration. Overall, these cells offer a unique opportunity to study dopaminergic neurons carrying a 'Parkinsonian genome'. The present review discusses the advances in cellular reprogramming technologies and studies that have been carried out on PD-derived iPSCs and differentiated dopaminergic neurons.

Enzymatic Engineering of Polysialic Acid on Cells in Vitro and in Vivo Using a Purified Bacterial Polysialyltransferase

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST(Nm)) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST(Nm) synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST(Nm) and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST(Nm) requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST(Nm) was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST(Nm)-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST(Nm) occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST(Nm)-based approach may provide a valuable alternative to PST gene therapy.

Microfluidic Spinning of Flat Alginate Fibers with Grooves for Cell‐Aligning Scaffolds

Alginate microribbons with longitudinally grooved microstructures are continuously fabricated by means of a microfluidic system. The number and dimensions of the microgroovesare successfully controlled by regulation of the slit-shaped channel (yellow in figure). This method opens up the possibility of mass production of scaffolds for tissue engineering purposes, as it is proved that the grooved flat fibers can be used to align other types of cells in culture.

Primary Cultures of Glomerular Parietal Epithelial Cells or Podocytes with Proven Origin

Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis - demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology.