Low-dose, long-wave UV light does not affect gene expression of human mesenchymal stem cells
ABSTRACT: This experiment was conducted to test multiple hypotheses: 1) long-wave 365 nm UV light exposure at low fluences does not alter gene expression of hMSC, 2) presence of radical species during polymerization causes DNA damage in hMSC, 3) 3D encapsulation of hMSC causes changes in gene expression of hMSC compared with traditional 2D culture, 4) Differencesin 3D hydrogel networks induce gene expression changes in hMSC The first publication derived from this data set concerns UV exposure and reactive radical species. Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light is an easily accessible and commonly used wavelength. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies only investigate cell viability, ignoring other possible non-toxic effects. Since light exposure could potentially induce phenotypic changes (i.e. if damage repair mechanisms are activated), we examined changes in gene expression of human mesenchymal stem cells exposed to light under various 2D and 3D culture conditions. While exposure to long-wave UV light did not induce any significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus 3D scaffolds. In total, 24 samples were analyzed. Three different culture conditions were created: 2D(plated), 3DR (encapsulated, radical polymerization), and 3DC (encapsulated, conjugate addition). Each culture condition was further subjected to UV radiation or no UV radiation, for 6 total experimental groups. Each experimental group was performed in triplicate. The 2D experimental groups, with and without UV, were additionally performed twice, once simultaneously with the 3DR samples, and once simultaneously with the 3DC samples. 3DR: encapsulated cells using radical polymerization (APS/TEMED) in a poly(ethylene glycol) (MW=4,000 g/mol) hydrogel in PBS. 3DC: encapsulated cells using conjugate addition with a four-arm PEG-Thiol (pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) ) as the cross-linker in PBS.
Project description:Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells’ 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells’ reciprocal interactions with their pericellular matrix when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their pericellular environment through degradation and/or protein secretion, imparting them with similar pericellular stiffnesses, regardless of initial hydrogel properties. These cell-secreted pericellular matrices play a role in regulating hMSC fate, with secretion of a stiff proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.
Project description:2 Breast Cancer Cell lines were seeded onto TCPS, on 2D PEG-PC gels, and encapsulated in 3D PEG-Maleimide gels as single cells or spheroids to determine transcript level changes based on culture platform Overall design: Examination of transriptome data of 2 cell lines in several biomaterial platforms
Project description:The influence of 2D and 3D cell culture platforms on vascular function was investigated by comparing gene expression for human pluripotent stem cell-derived endothelial cells (H1-ECs), primary human brain vascular pericytes (pericytes), and human umbilical vein endothelial cells (HUVECs) cultured on tissue culture polystyrene (TCP, “2D”), on or in poly(ethylene glycol) (PEG) hydrogels formed via “thiol-ene” photopolymerization, and on or in gelled Matrigel. ECs cocultured with pericytes in PEG formed vascular networks with global gene expression that was highly correlated to a standard 3D Matrigel assay (Spearman’s coefficients ≥ 0.98). H1-ECs, HUVECs, and pericytes were characterized gene expression signatures associated with the cell cycle and mitosis when cultured on TCP surfaces compared to cells cultured on top of or encapsulated in PEG hydrogels or Matrigel. The proliferative signature was not necessarily a function of the 2D format, since endothelial cells cultured on PEG hydrogels were not characterized by increased proliferation or a proliferative gene signature compared to cells encapsulated in PEG hydrogels. The proliferative phenotype for H1-ECs on TCP was regulated by FAK-ERK activity, and inhibition or knockdown of ERK pathway signaling decreased proliferation and cell cycle genes while increasing expression of “3D-like” vasculature development genes. Our results suggest that cells in 2D culture adopt a highly proliferative state that interferes with normal vascular function and provides unique insight into the importance of cellular and extracellular context for in vitro tissue modeling. Overall design: 30 samples for RNA-Seq Experiment #1 ("RNAseq1": H1 human embryonic stem cells, “H1-ESC-1”; H1 ES cells-derived endothelial cells, "H1-EC1"; HUVECs, "HUV1"; pericytes, "PC"; and H1-EC1 cocultured with PC, H1-EC1-PC cocultures). Poly(ethylene glycol) (PEG) hydrogels : 4 million endothelial cells/mL (H1-EC1 or HUVECs) and/or 2 million pericytes/mL were encapsulated in 30 µL PEG hydrogels in the bottom of 24-well transwell inserts . Matrigel (MG): 2 million endothelial cells/mL and 1 million pericytes/mL were encapsulated in 30 µL gelled Matrigel (4.5 mg/mL). Endothelial cells, pericytes, and cocultures encapsulated in PEG or Matrigel were maintained under hypoxic conditions (1.5% O2) in E7V medium supplemented with 1% bovine serum albumin. H1-ECs were also seeded on gelled Matrigel (4.5 mg/mL, “2D on Matrigel”) at a density of 60,000 cells/cm2 and incubated for one day in E7V medium with 1% BSA under normoxic conditions. Tissue culture polystyrene (TCP): TCP surfaces were coated with Matrigel at a dilute concentration that does not form a gel (H1-ES cells), recombinant vitronectin (H1-ECs or HUVECs), or poly-l-lysine (pericytes, as per manufacturer’s instructions) and cultured under normoxic conditions. 18 samples for RNA-Seq Experiment #2 ("RNAseq2": H1-ECs, “H1-EC2”; or HUVECs “HUV2”; cultured using PEG hydrogels, gelled Matrigel (6.4 mg/mL), or on Matrigel-coated TCP surfaces. For 3D culture, 4 million endothelial cells/mL were encapsulated in 30 µL PEG hydrogels (50% crosslinking, 2 mM CRGDS) or gelled Matrigel formed in single wells of 24-well TCP plates. For 2D culture, 200,000 endothelial cells per well of a 12-well TCP plates were seeded on Matrigel-coated surfaces (“MG-TCP”) or on 300 µL PEG hydrogels (50% crosslinking, 2 mM CRGDS) or gelled Matrigel formed in the bottom of the well. H1-ES cells were included as a control for both experiments. Duplicate samples are provided for each condition.
Project description:Seven-day-old white-light-grown wild-type, cop1-4 or hy5-1 mutant Arabidopsis seedlings were exposed for fifteen minutes to polychromatic radiation with decreasing short-wave cut-off in the UV range (WG305 = +UV-B, WG327 = -UV-B) and samples were taken 1 h after the onset of irradiation.
Project description:Analysis of 2D (transwell) and 3D (collagen type I) cultured MDCK cells and HGF (a MAPK activator). Traditional 2D cultures are fast and inexpensive but do no mimic natural niche/cell environment as well as the more laborious and costly 3D-cultures. 3D cultures, arguably, are better models for the study of developmental processes, such as tubulogenesis. Epithelial organs (such as kidney) develop via tubulogenesis, a process, at least in part, regulated by MAPK signaling. Therefore, 2D and 3D cells also treated with HGF plus MAPK inhibitors. Results provide insights into differential response to HGF-induced tubulogenesis depending on cell culture conditions (2D vs. 3D). 29 samples total: 2D and 3D control (untreated) in quadruplicate, respectively; 2D and 3D + HGF in quadruplicate, respectively; 2D + HGF + PD-98059 in quadruplicate; 3D + HGF + PD-98059 in triplicate; 2D + HGF + U0126 in triplicate; and 3D + HGF + U0126 in triplicate.
Project description:Seven-day-old white-light-grown Arabidopsis seedlings were exposed for 15 minutes to polychromatic radiation with decreasing short-wave cut-off in the UV range, transferred back to the standard growth chamber and samples were taken 1 and 6 hours after the start of irradiation.
Project description:TGFbeta/TNFalpha treated spheroid A549 cultures are a model of the epithelial-mesenchymal transition (EMT). These experiments capture the changes in global gene expression that result from cells being induced to undergo EMT (3D control vs 3D treated), but also the differences in gene expression when A549 is grown in spheroid cultures (2D control vs 3D untreated). EMT is efficiently induced only in the spheroid culture model. A total of 8 samples are analyzed, corresponding to 4 conditions (2D control, 2D treated, 3D control, 3D treated) and 2 biological replicates.
Project description:Transcriptional profiling of the Fischer Rat Thyroid (FRT) cells comparing polarizing cells grown as a confluent two-dimensional monolayer (2D culture system) with cells grown in matrigel where they acquire a three-dimensional follicular structure (3D culture system).The goal was to identify regulators of 3D epithelial thyroid polarization and follicle formation. Overall design: Two condition experiment, 3D vs. 2D FRT cell cultures. Biological replicates: 4 for both conditions. A pool of the 2D replicates was compared with each 3D replicate. One dyeswap replicate comparing 2D pool with 3D pool.
Project description:To identify the expression profiles of zebra mussel byssus unique genes with the regeneration of the byssal threads, a time-course study was performed. The RNA samples from the feet of fully attached zebra mussels (maintained attachment for more than 4 weeks) were extracted as common reference. The detached mussels were allowed to re-attach on underwater surface. Then the RNA samples from feet of the mussels at different time points post detachment were taken, namely, 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, and 21 days post detachment. The comparisons of the genes expression profiles were made between different time points and reference, as well as the any two connected time points (expect for 7 days and 21 days). The dual-channel microarray hybridizations were performed as following: 12h→R, 12h←R, 1D→R, 1D←R, 2D→R, 2D←R, 3D→R, 3D←R, 4D→R 4D←R, 7D→R, 7D←R, 21D→R, 21D←R, 12h→1D, 12h←1D, 1D→2D, 1D←2D, 2D→3D, 2D←3D, 3D→4D, 3D←4D, 4D→7D, 4D←7D, 7D→21D, 7D←21D. The direction of the arrows stand for the way of labeling: Alexa555→Alexa647. Six biological replicates of 1D, 2D, 3D, 4D, and 7D were used; four biological repllicates of 12h and 21D were taken; fourteen biological replicates of reference were applied. The whole experiment includes 26 microarray slide hybridizations.
Project description:The development of more complex but reliable systems for compound testing in a pharmaceutical context is a challenging task to date. Three-dimensional (3D), organ mimetic cell culture is aiming to become an alternative to common two-dimensional (2D) cell culture or animal testing in that field. We developed a biocompatible 3D cell culture environment for a hepatocellular carcinoma (HCC) model that enables cellular maintenance in a polycarbonate scaffold structure. Albumin, regarded as a differentiation marker, was elevated in statically 3D cultivated HepG2 cells. Expression of HCC tumor marker alpha-fetoprotein (AFP) was reduced compared to immunofluorescence stainings of 2D cultivated cells. Remarkably, expression of cytokeratin and pathophysiologically relevant beta-1 integrin (ITGB1) was found enhanced in nonperfused 3D cell culture. Changes in gene expression induced by the 3D cultivation environment were investigated using Ingenuity Pathway Analysis (IPA). Our findings revealed involvement of the insulin growth factor (IGF) signaling pathway in upregulation of matrix metalloproteinases (MMP) and ITGB1. The experimental data indicate a more differentiated state in 3D cultivated HepG2 cells than in the respective 2D experiments. Hence, scaffold-supported 3D cultivation of HepG2 cells may lead to a gain of information valuable for both drug testing and cancer research. HepG2 cells were cultivated for five days under 2D and 3D statical and perfused conditions. Cultivation was started with 0.25x10^6 cells in 2D and with 1x10^6 vital cells for the 3D experiments. The day of seeding was defined as d0. The groups were classified as follows: 2D, i.e., monolayer cultures, 3D, i.e., statical 3D cultures and BR, which denotes perfused 3D culture of HepG2 cells. The perfusable bioreactor system was operated using a peristaltic pump. It houses the MatriGrid, a polycarbonate-based microporous cellular support. For 3D static cultivation, cell-inoculated MatriGrids were placed in wells of a 24-well plate. Microarray experiments of three 2D (i.e., control), three 3D statically and three actively perfused 3D cultivations were performed at SIRS-Lab GmbH (SIRS-Lab GmbH, Jena, Germany) according to the manufacturer's instructions (Illumina, San Diego, CA). Altogether, 9 RNA samples of hepatocyte cultures and an internal control RNA were hybridized on two HumanHT-12 v4 Expression BeadChips.