Cell Types In Culture Research Articles

Kinetic model of Clostridium beijerinckii's Acetone-Butanol-Ethanol fermentation considering metabolically diverse cell types

Clostridium beijerinckii population branches into metabolically diverse cell types in batch cultures. Here, we present a new kinetic model of C. beijerinckii’s Acetone-Butanol-Ethanol fermentation that considers three cell types: producers of acids (acidogenic), consumer of acids and producers of solvents (solventogenic), and spores cells. The model accurately recapitulates batch culture data. Also, the model estimates cell type-specific kinetic parameters, which can be helpful to improve the operation of the ABE fermentation and give a framework to study acidogenic and solventogenic metabolic pathways. To exemplify the latter, we used a constraint-based model to study how the ABE pathways are used among acidogenic and solventogenic cell types. We found that among both cell types, glycolytic production of ATP and consumption of NAD+ varies widely during the fermentation, with their maximum production/consumption rates happening when acidogenic and solventogenic growth rates were at their highest. However, acidogenic cells use the ABE pathway to contribute with an extra 12.5% of the total production of ATP, whereas solventogenic cell types use the ABE pathway to supply more than 75% of the demand for NAD+, alternating between the production of lactate and butyrate, being both coupled to the production of NAD+.

Infection of Glia by Human Pegivirus Suppresses Peroxisomal and Antiviral Signaling Pathways.

Human pegivirus (HPgV) infects peripheral leukocytes but was recently shown to be a neurotropic virus associated with leukoencephalitis in humans. In the present study, we investigated the neural cell tropism of HPgV as well as its effects on host immune responses. HPgV wild type (WT) and a mutant virus with a deletion in the HPgV NS2 gene (ΔNS2) were able to productively infect human astrocytes and microglia but not neurons or an oligodendrocyte-derived cell line. Of note, the ΔNS2 virus replicated better than WT pegivirus in astrocytes, with both viruses being able to subsequently infect and spread in fresh human astrocyte cultures. Infection of human glia by HPgV WT and ΔNS2 viruses resulted in suppression of peroxisome-associated genes, including PEX11B, ABCD1, PEX7, ABCD3, PEX3, and PEX5L, during peak viral production, which was accompanied by reduced expression of IFNB, IRF3, IRF1, and MAVS, particularly in ΔNS2-infected cells. These data were consistent with analyses of brain tissue from patients infected with HPgV in which we observed suppression of peroxisome and type I interferon gene transcripts, including PEX11B, ABCD3, IRF1, and IRF3, with concurrent loss of PMP70 immunoreactivity in glia. Our data indicate that human astrocytes and microglia are permissive to HPgV infection, resulting in peroxisome injury and suppressed antiviral signaling that is influenced by viral diversity. IMPORTANCE Human pegiviruses are detected in 1 to 5% of the general population, principally infecting leukocytes, although their effects on human health remain uncertain. Here, we show that human pegivirus infects specific neural cell types in culture and human brain and, like other neurotropic flaviviruses, causes suppression of peroxisome and antiviral signaling pathways, which could favor ongoing viral infection and perhaps confer susceptibility to the development of neurological disease.

Phenotypical modifications of immune cells are enhanced by extracellular matrix

Immune cells not only constitute tumour microenvironment but they may even affect disease prognosis as a result of dual functional roles that they may play in tumour tissues. Two frequently used established immune cell lines (lymphocytic Jurkat and monocytic THP-1) were used to test whether microenvironmental factors, especially molecular components of extracellular matrix, can shape the phenotype of immune cells. Proliferation, morphological and phenotypical analyses were applied to compare behaviour of the immune cells, typically cultured as suspensions in culture medium, with their behaviour in collagen type I-based and Matrigel-based 3D cultures. Density of both immune cell types in routine suspension cultures affected their subsequent proliferation in extracellular matrices. THP-1 cells appeared to be more sensitive to their surrounding microenvironment as judged from extracellular matrix type-dependent changes in their cell doubling times and from slight increase in their diameters in both extracellular matrix-containing cell cultures. Moreover, even chemically uninduced monocytic THP-1 cells were present in a minor fraction as CD68 positive cell population in collagen type I matrix indicating their partial differentiation to macrophages. Observed modifications of immune cells by microenvironmental factors may have profound implications for their roles in healthy and pathological tissues.

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium.

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry. To this end, we confirmed SARS-CoV-2 entry factor and antigen expression in post-mortem COVID-19 patient ocular surface tissue and observed productive viral replication in cadaver samples and eye organoid cultures, most notably in limbal regions. Transcriptional analysis of ex vivo infected ocular surface cells and hESC-derived eye cultures revealed robust induction of NF-κB in infected cells as well as diminished type I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicate limbus as a portal for viral entry.

Lef-1 controls cell cycle progression in airway basal cells to regulate proliferation and differentiation.

The mammalian airways are lined by a continuous epithelial layer that is maintained by diverse populations of resident multipotent stem cells. These stem cells are responsible for replenishing the epithelium both at homeostasis and following injury, making them promising targets for stem cell and genetic-based therapies for a variety of respiratory diseases. However, the mechanisms that regulate when and how these stem cells proliferate, migrate, and differentiate remains incompletely understood. Here, we find that the high mobility group (HMG) domain transcription factor Lef-1 regulates proliferation and differentiation of mouse tracheal basal cells. We demonstrate that conditional deletion of Lef-1 stalls basal cell proliferation at the G1/S transition of the cell cycle, and that Lef-1 knockout cells are unable to maintain luminal tracheal cell types in long-term air-liquid interface culture. RNA sequencing analysis revealed that Lef-1 knockout (Lef-1KO) results in downregulation of key DNA damage response and cell cycle progression genes, including the kinase Chek1. Furthermore, chemical inhibition of Chek1 is sufficient to stall basal cell self-renewal in a similar fashion as Lef-1 deletion. Notably, the cell cycle block imposed by Lef-1KO in vitro is transient and basal cells eventually compensate to proliferate normally in a Chek1-independent manner. Finally, Lef-1KO cells were unable to fully regenerate tracheal epithelium following injury in vivo. These findings reveal that Lef-1 is essential for proper basal cell function. Thus, modulating Lef-1 function in airway basal cells may have applications in regenerative medicine.

Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105.

BackgroundScrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKβ-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined.Principal findingsWestern blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen’s ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm.ConclusionsO. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine.

An impressive percentage of biomedical advances were achieved through animal research and cell culture investigations. For drug testing and disease researches, both animal models and preclinical trials with cell cultures are extremely important, but present some limitations, such as ethical concern and inability of representing complex tissues and organs. 3D cell cultures arise providing a more realistic in vitro representation of tissues and organs. Environment and cell type in 3D cultures can represent in vivo conditions and thus provide accurate data on cell-to-cell interactions, and cultivation techniques are based on a scaffold, usually hydrogel or another polymeric material, or without scaffold, such as suspended microplates, magnetic levitation, and microplates for spheroids with ultra-low fixation coating. This review aims at presenting an updated summary of the most common 3D cell culture models available, as well as a historical background of their establishment and possible applications. Even though 3D culturing is incapable of replacing other current research types, they will continue to substitute some unnecessary animal experimentation, as well as complement monolayer cultures. In this aspect, 3D culture emerges as a valuable alternative to the investigation of functional, biochemical, and molecular aspects of human pathologies.

Isolation and Electron Microscopic Analysis of Liver Cancer Cell Mitochondria.

Isolation of mitochondria is a crucial method for examining molecular details of this organelle's manifold functions. Historically, mitochondrial isolations required large amounts of sample material which impeded their isolation from cultured cells. We have therefore developed a method allowing for controlled and reproducible isolation of intact and functional mitochondria from diverse cell types in culture. Here we provide a methodological update of this approach together with a protocol for the subsequent analysis of such isolated mitochondria by electron microscopy. Combining the isolation procedure with this powerful imaging method can reveal ultrastructural mitochondrial peculiarities in disease settings that might not be evident in intact cells and allows for assessment of mitochondrial membrane integrity and sample purity.

Non-destructive quality monitoring of 3D printed tissue scaffolds via dielectric impedance spectroscopy and supervised machine learning

Majority of methods currently used for quality assessment of tissue engineered medical products (TEMPs) are offline and destructive in nature, which is one of the factors impeding the scale up and translation of these technologies. In this study, we investigate quality assessment of TEMP via dielectric impedance spectroscopy (DIS) and supervised machine learning (ML) as a non-destructive alternative that requires minimal human intervention. 3D printed, NaOH-treated polycaprolactone (PCL) scaffolds seeded with human adipose-derived stem cells (hASC), NIH 3T3, MG63, and human chondrocyte cells were assessed via DIS over 4 days of in vitro culture. The results showed that the cell type and duration in culture had a significant effect on the delta permittivity (Δε, an important DIS metric. Five supervised ML algorithms – K Nearest Neighbors (KNN), Logistic Regression, Random Forest Classifiers, Support Vector Machines, and artificial neural network – were then used to analyze the comprehensive structured permittivity datasets to determine their ability to discern between different cell types and culture durations. The KNN algorithm demonstrated the best accuracy (99%). The outcomes of this study demonstrate the approach of using DIS and supervised ML in conjunction for assessment of TEMPs in an automated manufacturing system.

Circulating Exosomal miRNAs Signal Circadian Misalignment to Peripheral Metabolic Tissues

Night shift work increases risk of metabolic disorders, particularly obesity and insulin resistance. While the underlying mechanisms are unknown, evidence points to misalignment of peripheral oscillators causing metabolic disturbances. A pathway conveying such misalignment may involve exosome-based intercellular communication. Fourteen volunteers were assigned to a simulated day shift (DS) or night shift (NS) condition. After 3 days on the simulated shift schedule, blood samples were collected during a 24-h constant routine protocol. Exosomes were isolated from the plasma samples from each of the blood draws. Exosomes were added to naïve differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed. ChIP-Seq analyses for BMAL1 protein, mRNA microarrays and exosomal miRNA arrays combined with bioinformatics and functional effects of agomirs and antagomirs targeting miRNAs in NS and DS exosomal cargo were examined. Human adipocytes treated with exosomes from the NS condition showed altered Akt phosphorylation responses to insulin in comparison to those treated with exosomes from the DS condition. BMAL1 ChIP-Seq of exosome-treated adipocytes showed 42,037 binding sites in the DS condition and 5538 sites in the NS condition, with a large proportion of BMAL1 targets including genes encoding for metabolic regulators. A significant and restricted miRNA exosomal signature emerged after exposure to the NS condition. Among the exosomal miRNAs regulated differentially after 3 days of simulated NS versus DS, proof-of-concept validation of circadian misalignment signaling was demonstrated with hsa-mir-3614-5p. Exosomes from the NS condition markedly altered expression of key genes related to circadian rhythm in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Our results indicate that a simulated NS schedule leads to changes in exosomal cargo in the circulation. These changes promote reduction of insulin sensitivity of adipocytes in vitro and alter the expression of core clock genes in peripheral tissues. Circulating exosomal miRNAs may play an important role in metabolic dysfunction in NS workers by serving as messengers of circadian misalignment to peripheral tissues.

Abstract 3990: Tri-compartment (epithelial, immune, fibroblast) patient-derived models of the tumor microenvironment from an immuno-genomic profiled cohort of early stage colorectal cancers

Abstract Background: Immunotherapy has so far had limited success in colorectal cancer (CRC), with its efficacy restricted to a subset of microsatellite instability high (MSI-H) tumors. A comprehensive interrogation of the CRC tumor immune microenvironment (TME) is urgently needed. We present here an ongoing multi-platform study on early stage colon and rectal cancers, where immuno-genomic profiling of tumors and patient-derived cell models of tumor epithelia, cancer-associated fibroblasts and tumor-infiltrating lymphocytes (TIL) complement each other, with opportunities for mutual cross-validation between experimental and bioprofiling data. Methods: At the time of writing, 21 of a planned 50 early stage CRC patients have been recruited. Surgically resected tumors are processed for 4 broad classes of analyses: 1) Bulk tissue profiling by RNA and whole exome sequencing; 2) High resolution protein and transcriptome analysis comprising scRNA-seq and flow cytometry/CyToF; 3) H&E analysis and multiplex immunohistochemistry for TME-specific proteins; 4) Culture of epithelial, fibroblast, and TIL lines, and generation of patient-derived xenografts for functional studies. Results: Four tumors were MSI-H and 17 were microsatellite stable (MSS), with 1 POLE-mutant MSS patient harboring over 6000 mutations. Bulk genomics analysis revealed the most common mutations to be in TP53, APC, MUC17, and TGFBR2. The most frequently altered pathways were WNT, followed in order by p53, TGFβ, PI3K, and RAS-MAPK. scRNA-seq and flow cytometry/CyToF analyses are being performed to examine immune phenotypes, mediators of cell migration, and immune suppressive populations, which complement data on transcriptomic profile, histopathology, and spatial localization of TME cellular components. Of the 3 cultured cell types, 16 patients have lines of at least one cell compartment established in vitro. Characteristics of individual models will be reported at the meeting. Establishing the 3 cultured cell types from the same patient will enable us to develop an autologous patient-derived co-culture system to evaluate all 3 pairwise interactions, including TIL cytotoxicity toward epithelial cells, mutual modulation by fibroblast and epithelial cells in co-culture and their phenotypic alterations, and fibroblast regulation of TIL cytotoxic function. Autologous co-cultures with all 3 cell types are also planned. Conclusions: The generation of well-annotated multi-platform profiling data from CRC tumors, complemented by matched tri-compartment patient-derived cell cultures, enables mutual cross-validation between experimental models of the TME and bioprofiling data. Citation Format: Lindsay H. Kua, Fiona Y. Lee, Christine L. Eng, Harini Srinivasan, Rahul Nahar, Janice H. Oh, Nicole Ann L. Gunn, Kai Xian Thng, Ashley S. Yong, Adrian C. Sim, Rebecca Lim, Nicole Boo, Simeen Malik, Michael T. Wong, Tze Guan Tan, Shu Wen S. Ho, Shirleen Soh, Wan Jun Lim, Macalinao Dominique Camat, Joe P. Yeong, Clarinda W. Chua, Wei Qiang Leow, Ramanuj DasGupta, Si-Lin Koo, Lewis Hong, Brian Henry, Tony K. Lim, Iain B. Tan. Tri-compartment (epithelial, immune, fibroblast) patient-derived models of the tumor microenvironment from an immuno-genomic profiled cohort of early stage colorectal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3990.

Molecular mechanisms of coronary disease revealed using quantitative trait loci for TCF21 binding, chromatin accessibility, and chromosomal looping

BackgroundTo investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we create a catalog of genetic variants associated with three stages of transcriptional cis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMCs).ResultsWe use a pooling approach with HCASMC lines to map regulatory variants that mediate binding of the CAD-associated transcription factor TCF21 with ChIPseq studies (bQTLs), variants that regulate chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with Hi-C methods (clQTLs). We examine the overlap of these QTLs and their relationship to smooth muscle-specific genes and transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlate to lead SNPs where they show allelic effects. By utilizing genome editing, we verify that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease-associated TGFB1-SMAD3 pathway to the CAD-associated FN1 gene through a response QTL that modulates both chromatin accessibility and chromosomal looping.ConclusionsTogether, these studies represent the most thorough mapping of multiple QTL types in a highly disease-relevant primary cultured cell type and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.

Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells.

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

Studying Adipocyte and Immune Cell Cross Talk Using a Co-culture System.

The co-culture of adipocytes and immune cells, such as macrophages or T cells (CD4+ or CD8+ subsets), is a novel experimental approach used to study paracrine interactions (or the cross talk) between cultured cell types in isolation, in order to understand their role in obese adipose tissue (AT) inflammation and dysfunction. Here we describe the general methodologies required for the co-culture of mature adipocytes (differentiated 3T3-L1 pre-adipocyte cell line) with primary immune cell subsets purified from mouse splenic mononuclear cells using a magnetic MicroBead positive selection, wherein multiple immune cell populations can be purified sequentially from a single mouse spleen, thereby providing diversity in the types of immune cells that can be co-cultured with adipocytes. Additionally, we describe experimental procedures for co-culturing adipocytes and immune cells in two different co-culture systems, including a cell contact-dependent co-culture system, wherein the cells are in direct physical contact, and a cell contact-independent, soluble mediator-driven co-culture system wherein the cells are physically separated by a trans-well semipermeable membrane. Finally, we discuss how these co-culture models can be utilized to recapitulate the AT microenvironment in obesity by utilizing physiologically relevant ratios of adipocytes:immune cells (specifically CDllb+ macrophages, CD4+ T cells, or CD8+ T cells) and lipopolysaccharide stimulation that mimics endotoxin concentrations observed in obesity.

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions.

A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials.

Measuring Absolute Cell Volume Using Quantitative-Phase Digital Holographic Microscopy and a Low-Cost, Open-Source, and 3D-Printed Flow Chamber

Although cell volume is a critical cell parameter and acute changes of its regulation mechanisms can reveal pathophysiological states, its accurate measurement remains difficult. On the other hand, custom-made devices are now possible through the application of 3D printing that meet the needs of certain specific applications. Therefore, in this article we describe the development of a low-cost, open-source, and 3D-printed flow chamber optimized to perform non-invasive accurate measurements of important cell biophysical parameters. This measurement development includes the absolute cell volume, mean cell thickness, and whole-cell refractive index using quantitative-phase digital holographic microscopy. Specifically, this advancement makes it possible to develop a flow chamber that preserves cell viability while exhibiting a fast washout process for the two-liquid decoupling procedure - an experimental procedure allowing to separately measure the intracellular refractive index and cell thickness from the quantitative-phase signal - characterized by a homogeneous return of the extracellular refractive index over the entire imaging slit toward the known value of the last perfusion solution. Such a fast washout process has been extensively characterized by monitoring the time required to obtain a stabilization of the quantitative-phase signal following the perfusion of the two liquids. A more rapid quantitative-phase signal stabilization time results in fewer cell changes, including important cell position and shape modifications. These changes likely take place over the washout process time and ultimately result in important artefacts to all cell biophysical measurements. The proposed flow chamber has been used to perform measurements of the whole-cell refractive index, mean cell thickness, and absolute cell volume of three cell types in culture during resting state, and hypo-osmotic and hyper-osmotic challenges. For a typical cell body, these biophysical parameters are measured with a precision of 0.0006, 100 nm, and 50 µm3 respectively. Finally, the design files of the flow chamber will be shared with the scientific community through an open-source model.

Higginsianins A and B, two fungal diterpenoid α-pyrones with cytotoxic activity against human cancer cells

Two new diterpenoid α-pyrones, named higginsianins A and B, were isolated from the mycelium of the microbial fungus Colletotrichum higginsianum grown in liquid culture. In previous studies, we have shown that both compounds reduce viability of different types of cancer cells in culture. Here, we extend our previous observations and explore, at a deeper level, the cellular effects of higginsianins treatment. Higginisianins A and B reduce viability of A431, HeLa and H1299 cancer cells. Both compounds increase the level of the cell cycle inhibitor p21WAF and reduce the rate of cell proliferation. Cell cycle analyses reveal that higginsianins arrest cancer cells in S-phase. Furthermore, cells incubated with higginsianins reveal discrete γ-H2AX positive nuclear foci indicating the occurrence of DNA lesions. At longer incubation times, higginsianins induce massive cell detachment and non-apoptotic cell death. Human primary keratinocytes and spontaneously immortalized Hacat cells, a preneoplastic cell line model, are less sensitive to higginsianins effects. These findings suggest that higginsianins exhibit considerable cytotoxicity against a wide spectrum of malignant cells and may be considered as promising anticancer agents.

Cultured cell-derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells.

Cell-derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro-environment. Therefore, cultured cell-derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro-environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell-derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)-derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.

Abstract 2645: Quantitative measurement of multiple signal transduction pathway activities in cell and tissue culture, including cancer, fibroblast, and immune cell types

Abstract To improve pathophysiology research, biomarker discovery and drug development, cell culture models should adequately mimic human (patho)physiology and provide reproducible results. This requires comparison between cultured cells/tissue and actual histopathology in the patient, as well as standardization of culture experiments to ensure experimental reproducibility, preferably in a quantitative manner1. 10-15 signal transduction pathways govern major cellular processes, e.g. cell division, differentiation and migration. The past decade we developed tests to quantitatively measure functional activity of signal transduction pathways in individual cell/tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the respective signalling pathway. Tests provide quantitative pathway activity scores and are intended to be used for diagnostics and life sciences research2-4. Method: Tests have been developed for androgen (AR) and estrogen receptor (ER), Hedgehog (HH), Wnt, TGFβ, Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK pathways. After calibration and freezing of the models, extensive biological test validation was performed on healthy/diseased cell and tissue types, including multiple cancer, fibroblast and immune cell types. Using Affymetrix expression microarray data (GEO database) >900 cell lines of most cell and cancer types were analyzed, as well as primary cultures of most immune cell types. LnCaP (prostate), MCF7, BT474 (breast), HCC827 (lung) and A2780 (ovarian) cancer cell lines were compared across laboratories. Results: Typical expected single or combined pathway activities were confirmed, e.g. ER activity in breast, AR activity in prostate, and Wnt activity in colon cancer; HH activity in soft tissue tumor, NFκB activity in lymphoma, frequently combined with PI3K and/or MAPK and/or JAK-STAT growth factor pathways. Quantitative pathway activities were reproducible within studies, but highly variable between labs, and dependent on culture conditions. Conclusion: Our pathway tests measure signaling pathway activity in many cell and tissue types, and can be used as quantitative readout for cell/tissue culture. Applications are: standardization of cell/tissue culture to ensure reproducibility; comparison between culture-based disease model and patient histopathology; quantitative assessment of drug efficacy on disease models; assessment of toxicity on healthy cell/tissue models. A number of tests have been adapted to qPCR, enabling use on FFPE tissue and small samples. 1 Ben-David U, et al. Nature, 2018;560(7718):325; 2 Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 3 Verhaegh W, Stolpe A van de. Oncotarget, 2014:5(14):5196-7; 4 Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972 Citation Format: Anja Van De Stolpe, Marcia Alves de Inda, Eveline den Biezen-Timmermans, Laurent Holtzer, Henk van Ooijen, Wim Verhaegh. Quantitative measurement of multiple signal transduction pathway activities in cell and tissue culture, including cancer, fibroblast, and immune cell types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2645.

Conversion of human and mouse fibroblasts into lung-like epithelial cells

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.