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:Chondrogenic differentiation of hMSC has been investigated by this study using different growth conditions including control (INC), TGFβ1 (T), TGFβ1 + BMP2 (TB), TGFβ1 + GDF5 (TG). For each condition triplicate time course expression measurements were performed for 10 different time points. Osteogenic differentiation of hMSC has been investigated by this study using different growth conditions including control (MD), dexamethasone (DX), dexamethasone + BMP2 (DB), dexamethasone + VitaminD3 (DV). For each condition triplicate time course expression measurements were performed for 10 different time points. Adipogenic differentiation of hMSC has been investigated by this study using different growth conditions including proliferation medium (PR), dexamethasone + IBMX + rosiglitazone (AD), dexamethasone (DX), dexamethasone + BMP2 (OS). For each condition triplicate time course expression measurements were performed for 10 different time points.
Project description:Ouyang2014 - photomorphogenic UV-B signalling
This model is described in the article:
UV-B signaling network captured by mathematical modeling.
Ouyang X, Huang X, Jin X, Chen Z,
Yang P, Ge H, Li S, Deng XW.
Proc. Natl. Acad. Sci. U.S.A. 2014 Aug;
Long-wavelength and low-fluence UV-B light is an
informational signal known to induce photomorphogenic
development in plants. Using the model plant Arabidopsis
thaliana, a variety of factors involved in UV-B-specific
signaling have been experimentally characterized over the past
decade, including the UV-B light receptor UV resistance locus
8; the positive regulators constitutive photomorphogenesis 1
and elongated hypocotyl 5; and the negative regulators cullin4,
repressor of UV-B photomorphogenesis 1 (RUP1), and RUP2.
Individual genetic and molecular studies have revealed that
these proteins function in either positive or negative
regulatory capacities for the sufficient and balanced
transduction of photomorphogenic UV-B signal. Less is known,
however, regarding how these signaling events are
systematically linked. In our study, we use a systems biology
approach to investigate the dynamic behaviors and correlations
of multiple signaling components involved in Arabidopsis
UV-B-induced photomorphogenesis. We define a mathematical
representation of photomorphogenic UV-B signaling at a temporal
scale. Supplemented with experimental validation, our
computational modeling demonstrates the functional interaction
that occurs among different protein complexes in early and
prolonged response to photomorphogenic UV-B.
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Project description:Human mesenchymal stem cells (hMSCs) are defined as multi-potent colony-forming cells expressing a specific subset of plasma membrane markers when grown on flat tissue culture polystyrene. However, as soon as hMSCs are used for transplantation, they are exposed to a 3D environment, which can strongly impact cell physiology and influence proliferation, differentiation and metabolism. Strategies to control in vivo hMSC behavior, for instance in stem cell transplantation or cancer treatment, are skewed by the un-physiological flatness of the standard well plates. We used micrometer-scale defined surface topographies as a model to describe the phenotype of hMSCs during adaptation to their new environment. Compared to hMSCs cultured on flat polystyrene, we observed dramatically changed cell morphologies accompanied by shrinkage of cytoplasm and nucleus, a decreased overall cellular metabolism, and slower cell cycle progression resulting in a lower proliferation rate in cells exposed to surface topographies. We hypothesized that this reduction in proliferation rate effects their sensitivity to certain cancer drugs, which was confirmed by higher survival rate of hMSCs cultured on topographies exposed to paclitaxel. Thus, micro-topographies can be used as a model system to mimic the natural cell micro-environment, and be a powerful tool to optimize cell treatment in vitro. For more information check:https://cbit.maastrichtuniversity.nl/
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.