Simulated microgravity impairs human NK cell cytotoxic activity against space radiation-relevant leukemic cells.

Natural killer (NK) cells are an important first-line of defense against malignant cells. Because of the potential for increased cancer risk from astronaut exposure to space radiation, we determined whether microgravity present during spaceflight affects the body's defenses against leukemogenesis. Human NK cells were cultured for 48 h under normal gravity and simulated microgravity (sµG), and cytotoxicity against K-562 (CML) and MOLT-4 (T-ALL) cells was measured using standard methodology or under continuous sµG. This brief exposure to sµG markedly reduced NK cytotoxicity against both leukemias, and these deleterious effects were more pronounced in continuous sµG. RNA-seq performed on NK cells from two additional healthy donors provided insight into the mechanism(s) by which sµG reduced cytotoxicity. Given our prior report of space radiation-induced human T-ALL in vivo, the reduced cytotoxicity against MOLT-4 is striking and raises the possibility that µG may increase astronaut risk of leukemogenesis during prolonged missions beyond LEO.

A Scoping Review of Animal Models for Development of Abdominal Adhesion Prevention Strategies.

INTRODUCTION: Abdominal adhesions represent a chronic postsurgical disease without reliable prophylaxis. Animal modeling has been a cornerstone of novel therapeutic development but has not produced reliable clinical therapies for prevention of adhesive small bowel obstruction. The purpose of this scoping review is to analyze animal models for abdominal adhesion generation by key considerations of external validity (i.e., fidelity, homology, and discrimination). METHODS: A literature review was performed in accordance with the Preferred Reporting Items for Systematic Reviews Extension for Scoping Reviews guidelines. Peer-reviewed publications were included that described the development or quality assessment of experimental animal models for abdominal adhesions with inclusion of a scoring system. Studies that focused on treatment evaluation, implantation of surgical devices, models of nonsurgical etiologies for abdominal adhesions, non-in vivo modeling, and investigations involving human subjects were excluded. RESULTS: Four hundred and fifteen (n = 415) articles were identified by prespecified search criteria. Of these, 13 studies were included for review. CONCLUSIONS: Translation of investigational therapeutics for abdominal adhesion prevention is dependent upon high-quality experimental animal models that reproduce the clinical adhesions seen in the operating room as a disease of the entire abdomen.

MicroRNA Analysis of In Vitro Differentiation of Spermatogonial Stem Cells Using a 3D Human Testis Organoid System.

Spermatogenesis produces male gametes from spermatogonial stem cells (SSC), beginning at puberty. Modern-day laboratory techniques allow for the long-term culture of SSC and in vitro spermatogenesis. The specific biochemical processes that occur during spermatogenesis remain poorly understood. One particular element of spermatogenesis that has yet to be characterized is the role of microRNAs (miRNA), short, non-transcribed RNAs that act as post-translational regulators of gene activity. In this study, we seek to describe the presence of miRNA in a two-dimensional (2D) SSC culture and a 3D human testis organoid (HTO) system. Testicular cells were isolated from the frozen tissue of three brain-dead subjects, propagated in cultures for four to five weeks, and used to form 3D HTOs. Following organoid formation, differentiation of testicular cells was induced. RNA was isolated from the whole testis tissue (WT) showing in vivo conditions, HTO Day Zero (2D SSC culture), Day 2 HTOs, and Day 23 differentiated HTOs, then analyzed for changes in miRNA expression using the Nanostring nCounter miRNA panel. One hundred ninety-five miRNAs met the criteria for expression in WT, 186 in 2D culture, 190 in Day 2 HTOs, and 187 in differentiated HTOs. One hundred thirty-three miRNAs were common across all conditions, and 41, 17, 6, and 11 miRNAs were unique for WT, 2D culture, Day 2 HTOs, and differentiated HTOs, respectively. Twenty-two miRNAs were similar between WT and differentiated HTOS. We evaluated the miRNA expression profiles of progressively complex stages of testicular cell culture, culminating in a 3D organoid model capable of meiotic differentiation, and compared these to WT. We identified a great variance between the native tissue and the culture system; however, some miRNAs are preserved. These data may provide avenues for deeper understanding of spermatogenesis and the ability to improve this process in the laboratory. Research on miRNA continues to be an essential avenue for understanding human spermatogenesis.

A comparative analysis of RNA-Seq and NanoString technologies in deciphering viral infection response in upper airway lung organoids.

In this study, we delved into the comparative analysis of gene expression data across RNA-Seq and NanoString platforms. While RNA-Seq covered 19,671 genes and NanoString targeted 773 genes associated with immune responses to viruses, our primary focus was on the 754 genes found in both platforms. Our experiment involved 16 different infection conditions, with samples derived from 3D airway organ-tissue equivalents subjected to three virus types, influenza A virus (IAV), human metapneumovirus (MPV), and parainfluenza virus 3 (PIV3). Post-infection measurements, after UV (inactive virus) and Non-UV (active virus) treatments, were recorded at 24-h and 72-h intervals. Including untreated and Mock-infected OTEs as control groups enabled differentiating changes induced by the virus from those arising due to procedural elements. Through a series of methodological approaches (including Spearman correlation, Distance correlation, Bland-Altman analysis, Generalized Linear Models Huber regression, the Magnitude-Altitude Score (MAS) algorithm and Gene Ontology analysis) the study meticulously contrasted RNA-Seq and NanoString datasets. The Magnitude-Altitude Score algorithm, which integrates both the amplitude of gene expression changes (magnitude) and their statistical relevance (altitude), offers a comprehensive tool for prioritizing genes based on their differential expression profiles in specific viral infection conditions. We observed a strong congruence between the platforms, especially in identifying key antiviral defense genes. Both platforms consistently highlighted genes including ISG15, MX1, RSAD2, and members of the OAS family (OAS1, OAS2, OAS3). The IFIT proteins (IFIT1, IFIT2, IFIT3) were emphasized for their crucial role in counteracting viral replication by both platforms. Additionally, CXCL10 and CXCL11 were pinpointed, shedding light on the organ tissue equivalent's innate immune response to viral infections. While both platforms provided invaluable insights into the genetic landscape of organoids under viral infection, the NanoString platform often presented a more detailed picture in situations where RNA-Seq signals were more subtle. The combined data from both platforms emphasize their joint value in advancing our understanding of viral impacts on lung organoids.

In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

The Application of Ultrasmall Gold Nanoparticles (2 nm) Functionalized with Doxorubicin in Three-Dimensional Normal and Glioblastoma Organoid Models of the Blood-Brain Barrier.

Among brain tumors, glioblastoma (GBM) is very challenging to treat as chemotherapeutic drugs can only penetrate the brain to a limited extent due to the blood-brain barrier (BBB). Nanoparticles can be an attractive solution for the treatment of GBM as they can transport drugs across the BBB into the tumor. In this study, normal and GBM organoids comprising six brain cell types were developed and applied to study the uptake, BBB penetration, distribution, and efficacy of fluorescent, ultrasmall gold nanoparticles (AuTio-Dox-AF647s) conjugated with doxorubicin (Dox) and AlexaFluor-647-cadaverine (AF647) by confocal laser scanning microscopy (CLSM), using a mixture of dissolved doxorubicin and fluorescent AF647 molecules as a control. It was shown that the nanoparticles could easily penetrate the BBB and were found in normal and GBM organoids, while the dissolved Dox and AF647 molecules alone were unable to penetrate the BBB. Flow cytometry showed a reduction in glioblastoma cells after treatment with AuTio-Dox nanoparticles, as well as a higher uptake of these nanoparticles by GBM cells in the GBM model compared to astrocytes in the normal cell organoids. In summary, our results show that ultrasmall gold nanoparticles can serve as suitable carriers for the delivery of drugs into organoids to study BBB function.

Simulated Microgravity Impairs Human NK Cell Cytotoxic Activity Against Space Radiation-Relevant Leukemic Cells.

Natural killer (NK) cells are important effectors of the innate immune system. Unlike T cells, NK cells do not require antigen-priming, making them an important first-line of defense against malignant cells. Because of the potential for increased cancer risk as a result of astronaut exposure to space radiation, we performed studies to determine whether conditions of microgravity present during spaceflight affects the body's natural defenses against leukemogenesis. Human NK cells were cultured for 48 hours under normal gravity and simulated microgravity (sµG), and cytotoxicity against K-562 (CML) and MOLT-4 (T-ALL) cell lines was measured using standard methodology or under continuous conditions of sµG. Even this brief exposure to sµG markedly reduced NK cytotoxicity against both leukemic cells using standard assay procedures, and these deleterious effects were even more pronounced in continuous sµG. RNA-seq performed on NK cells from two healthy donors provided insight into the mechanism(s) by which sµG reduced cytotoxicity. Given our prior report that human HSC exposed to simulated space radiation gave rise to T-ALL in vivo , the reduced cytotoxicity against MOLT-4 is striking and raises the possibility that µG may add to astronaut risk of leukemogenesis during prolonged missions beyond LEO.

Correction: Understanding the Role of Growth Factors in Modulating Stem Cell Tenogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0083734.].

Prevention of post-operative adhesions: Model development and pilot outcomes of human placental stem cell-based interventions.

BACKGROUND: Abdominal adhesions are the most common surgical complication and without reliable prophylactics. This study presents a novel rat model for abdominal adhesions and reports pilot results of human placental stem cell (hPSC)-based therapies. METHODS: Forty-four (n = 44) male Sprague-Dawley rats (250-350 g) were used in the experiment. Of these, thirty-eight (n = 38) were included in a preliminary data set to determine a minimum treatment effect. Adhesions were created in a reproducible model to the abdominal wall and between organs. Experimental groups included the control group (Model No Treatment, MNT), Plasmalyte A (Media Alone, MA, 10 mL), hPSC (5 × 106 cells/10 mL Plasmalyte A), hPSC-CM (hPSC secretome, conditioned media) in 10 mL Plasmalyte A, Seprafilm™ (Baxter, Deerfield, IL), and sham animals (laparotomy only). Treatments were inserted intraperitoneally (IP) and the study period was 14 days post-operation. Results are reported as the difference between means of an index statistic (AIS, Animal Index Score) and compared by ANOVA with pairwise comparison. RESULTS: The overall mean AIS was 23 (SD 6.16) for the MNT group with an average of 75% of ischemic buttons involved in abdominal adhesions. Treatment groups MA (mean overall AIS 17.33 SD 6.4), hPSC (mean overall AIS 13.86 SD 5.01), hPSC-CM (mean overall AIS 13.13 SD 6.15), and Seprafilm (mean overall AIS 13.43 SD 9.11) generated effect sizes of 5.67, 9.14, 9.87, and 9.57 decrease in mean overall AIS, respectively, versus the MNT. DISCUSSION: The presented rat model and scoring system represent the clinical adhesion disease process. hPSC-based interventions significantly reduce abdominal adhesions in this pilot dataset.

Analysis of gene expression dynamics and differential expression in viral infections using generalized linear models and quasi-likelihood methods.

Introduction: Our study undertakes a detailed exploration of gene expression dynamics within human lung organ tissue equivalents (OTEs) in response to Influenza A virus (IAV), Human metapneumovirus (MPV), and Parainfluenza virus type 3 (PIV3) infections. Through the analysis of RNA-Seq data from 19,671 genes, we aim to identify differentially expressed genes under various infection conditions, elucidating the complexities of virus-host interactions. Methods: We employ Generalized Linear Models (GLMs) with Quasi-Likelihood (QL) F-tests (GLMQL) and introduce the novel Magnitude-Altitude Score (MAS) and Relaxed Magnitude-Altitude Score (RMAS) algorithms to navigate the intricate landscape of RNA-Seq data. This approach facilitates the precise identification of potential biomarkers, highlighting the host's reliance on innate immune mechanisms. Our comprehensive methodological framework includes RNA extraction, library preparation, sequencing, and Gene Ontology (GO) enrichment analysis to interpret the biological significance of our findings. Results: The differential expression analysis unveils significant changes in gene expression triggered by IAV, MPV, and PIV3 infections. The MAS and RMAS algorithms enable focused identification of biomarkers, revealing a consistent activation of interferon-stimulated genes (e.g., IFIT1, IFIT2, IFIT3, OAS1) across all viruses. Our GO analysis provides deep insights into the host's defense mechanisms and viral strategies exploiting host cellular functions. Notably, changes in cellular structures, such as cilium assembly and mitochondrial ribosome assembly, indicate a strategic shift in cellular priorities. The precision of our methodology is validated by a 92% mean accuracy in classifying respiratory virus infections using multinomial logistic regression, demonstrating the superior efficacy of our approach over traditional methods. Discussion: This study highlights the intricate interplay between viral infections and host gene expression, underscoring the need for targeted therapeutic interventions. The stability and reliability of the MAS/RMAS ranking method, even under stringent statistical corrections, and the critical importance of adequate sample size for biomarker reliability are significant findings. Our comprehensive analysis not only advances our understanding of the host's response to viral infections but also sets a new benchmark for the identification of biomarkers, paving the way for the development of effective diagnostic and therapeutic strategies.