Gene Expression Profiling of Human Mesenchymal Stem Cells for Tissue Engineering

Tissue engineering is a combination of advanced technologies with a goal to fabricate better materials for the repair or replacement of damaged tissue. This is accomplished with the help of cells, biomaterials, and other physiological and chemical cues. In tissue engineering, human mesenchymal stem cells (hMSCs) have huge potential as they can differentiate into many cell types, reduce immune response, be isolated from adult bone marrow, and are easily available. The present study analyzes the gene expression profile of hMSCs when cultured under different conditions: two-dimensional (2D) versus three-dimensional (3D) culture systems; presence or absence of radicals, and exposure to ultra violet (UV) radiation. In our experiments, cells were cultured on 2D substrates and in 3D hydrogel systems fabricated with and without free-radicals. Total RNA was extracted from cells cultured under each condition and the differential expression of genes was analyzed using the Human U133 plus 2.0 Affymetrix gene chip. We observed that the largest difference in gene expression occurs in cells cultured in 3D as compared to 2D systems. UV radiation did not have a significant effect on gene expression but, when combined with the free-radicals generated during fabrication, significant variations in the hMSC gene expression profile were observed. Therefore, can conclude that, while all three factors (2D vs. 3D, free-radicals, and UV light) can influence the gene expression profile of hMSCs, switching from 2D to 3D cell culture system results in the largest change in gene expression

Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells

<div><p>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 (315 nm–400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce 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 encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying.</p></div

Principle components analysis shows tightly segregated clustering based on culture condition, not by UV exposure.

<p>(alternate viewing angle and axis values available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139307#pone.0139307.s002" target="_blank">S2 Fig</a>). PCA is a statistical analysis tool which reduces the dimensionality of data by determining the key variables resulting in differences seen between samples[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139307#pone.0139307.ref025" target="_blank">25</a>]. Each axis of this PCA map represents a linear combination of expression levels from many thousands of gene transcripts such that, combined, the maximum variation among all data points is achieved on only three axes. The result gives a visual representation of which samples behave similarly to each other by their physical closeness in three dimensions, while including information from many thousands of variables (gene expression levels). This is a bird’s eye view of the entire set of gene array data, for which absolute units and values can be considered arbitrary. The following in-depth analysis of pathway enrichment and specific gene expression provide insight into how each group differs from the others in their gene expression.</p

UV effects in 2D groups are insignificant.

<p>(a) This Venn diagram shows that whether combining raw data across months or using repetition power between months, no significant pathways or biological functions are found in the differentially expressed genes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) or Gene Ontology (GO) except for a slight change in cell cycle. (b) The most extreme gene changes for each comparison have relatively weak fold changes, and a heat map (unsupervised hierarchical clustering) of the 148 gene transcripts (103 unique genes) differentially expressed between 2D₂UV vs. 2D₂ shows that none of the other UV comparisons in 3D have any differences in expression level for these genes. (red is upregulated, blue is downregulated, genes included in the heat map are those differentially expressed between denoted * groups) Also of note is the stark similarity between samples 2D₁ and 2D₂. The 3D groups look very similar to each other, while they are very different from the 2D groups. The most repeated changes of significance were in the genes DHRS3 and FOXQ1. Their individual scatter plots, (c) and (d), reinforce the similarities between the months as well as the direction of change. However, for so few genes and so small a change, the effects could easily be found by chance. (RMA = robust multi-array average).</p

Experimental groups illustrated.

<p>A single vial of P0 hMSCs were expanded at low density to greater than 25 million cells at P1. These cells were trypsinized and a portion of them replated at 500,000 cells / 75cm² flask for 2D samples. Another portion of those cells was encapsulated in poly(ethylene glycol) diacrylate (MW 4000 Da). Two encapsulation methods were used, radical polymerization with ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and conjugate addition with a pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) crosslinker. Several samples of each type were created and half from each type were irradiated with a Black Ray UV bench lamp, peak wavelength 365nm.</p

IPA summary for 3D_R±UV vs. 3D_C±UV, (>2fold, p<0.05).

<p>^aIPA analysis returned several categories of known networks, molecular and cellular functions, and upstream regulators that could be considered important differences between radical polymerization and conjugate addition. This table summarizes the specific changes between these two polymerization groups at each level of evaluation (whole network activity, group function, specific protein activation) and gives the relative strengths of these components in the relevant statistical measure.</p><p>^bIPA network score: For networks, the IPA network score is a relative measure of relevance, with the two highest scores reported.</p><p>^c<u>p-value range (# molecules):</u> For molecular and cellular functions, each function, such as “Cellular Development”, represents a combination of lower level functions, each with a p-value. Thus the significance of these higher level functions is given as a range that covers the p-values of the lower level functions. The # molecules is the number of user input dataset molecules associated with that higher level function.</p><p>^d<u>p-value (activation z-score, prediction):</u> For individual upstream regulators, the expected cascade of transcriptional changes in downstream molecules is evaluated to determine if a particular upstream regulator is acting, and directions and magnitudes of particular changes can be used to determine the activation z-score. Positive z-scores above 2 predict activation while negative z-scores below -2 predict inhibition.</p><p>IPA summary for 3D_R±UV vs. 3D_C±UV, (>2fold, p<0.05).</p

Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19

Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-Cov-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.<br /

Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry

Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug's therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions

Premature skewing of T cell receptor clonality and delayed memory expansion in HIV-exposed infants

Abstract While preventing vertical HIV transmission has been very successful, HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations in T cell memory differentiation between iHEU and iHUU being significant from week 15 of life. The altered memory T cell differentiation in iHEU was preceded by lower TCR Vβ clonotypic diversity and linked to TCR clonal depletion within the naïve T cell compartment. Compared to iHUU, iHEU had elevated CD56loCD16loPerforin+CD38+CD45RA+FcεRIγ+ NK cells at 1 month postpartum and whose abundance pre-vaccination were predictive of vaccine-induced pertussis and rotavirus antibody responses post 3 months of life. Collectively, HIV/ARV exposure disrupted the trajectory of innate and adaptive immunity from birth which may underlie relative vulnerability to infections in iHEU