Biology subjects

Steill, J.

Publications and source records attributed to Steill, J..

5 recordsLinked to original sources

bioRxiv2024

Diminished Immune Cell Adhesion in Hypoimmune ICAM-1 Knockout Pluripotent Stem Cells

Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g., T cell) immune responses, but have largely not addressed the innate immune cells (e.g., monocytes, neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies, we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding, thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. HighlightsO_LIAntibody blocking of ICAM-1 on human pluripotent stem cell-derived cells inhibits immune cell adhesion C_LIO_LICRISPR/Cas9 knock-out of ICAM-1 ablates surface and secreted ICAM-1 protein and inhibits immune adhesion C_LIO_LIICAM-1 knock-out results in decreased T cell proliferative responses to human pluripotent stem cell-derived grafts in vitro, and resistance to immune-mediated graft loss in vivo C_LIO_LIAddition of ICAM-1 knock-out to first generation MHC knock-out human pluripotent stem cells confers protection against immune adhesion C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/597791v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@890b56org.highwire.dtl.DTLVardef@147449borg.highwire.dtl.DTLVardef@1e94f08org.highwire.dtl.DTLVardef@43c55c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC - pluripotent stem cell-derived endothelial cells; KO - knock-out; dSMAC - distal supramolecular activation complex; pSMAC - peripheral supramolecular activation complex; cSMAC - central supramolecular activation complex. C_FIG

immunology↗

bioRxiv2023

Macrophages undergo functionally significant reprograming of nucleotide metabolism upon classical activation

During an immune response, macrophages specifically rewire their metabolism to support functional changes. Using a multi-omics approach, we identified nucleotide metabolism as one of the most significantly rewired pathways across the metabolic network in classically activated macrophages. Further isotopic tracing studies revealed the substantial changes in nucleotide de novo synthesis, degradation, and salvage fluxes in stimulated macrophages, as well as the key reactions where metabolic regulation occurs: 1) de novo synthesis of purine nucleotides is shut down and particularly blocked at the last step of IMP synthesis catalyzed by ATIC; 2) de novo synthesis of pyrimidines is maintained up to UMP, but further synthesis of CTP (catalyzed by CTPS) and dTMP (catalyzed by TYMS) is greatly reduced; 3) Nucleotide degradation to nitrogenous bases is increased, but further oxidation of purine bases (catalyzed by XOR) is inhibited, causing a great accumulation of nucleosides and bases; and 4) cells switch to salvaging the nucleosides and bases as the primary means to maintain purine nucleotides. Mechanistically, we found these changes are driven by a combination of transcriptional regulation and enzyme inhibition. Nitric oxide (NO) was identified as a major regulator, driving the strong inhibition of ATIC and XOR, and the transcriptional downregulation of Tyms. To understand the functional impact of the activation-induced switch from purine de novo synthesis to salvage, we knocked out the purine salvage enzyme Hprt. Hprt knockout significantly alters functional gene expression in activated macrophages, suppresses macrophage migration, and increases pyroptosis. Furthermore, knocking out Hprt or Xor increases the proliferation of the intracellular parasite Toxoplasma gondii in macrophages. Together, these results comprehensively uncovered the dynamic rewiring of nucleotide metabolism in classically activated macrophages, elucidated the key regulatory mechanisms, and identified the functional significance of such rewiring.

cell biology↗

bioRxiv2023

Serial KinderMiner (SKiM) Discovers and Annotates Biomedical Knowledge Using Co-Occurrence and Transformer Models

BackgroundThe PubMed database contains more than 34 million articles; consequently, it is becoming increasingly difficult for a biomedical researcher to keep up-to-date with different knowledge domains. Computationally efficient and interpretable tools are needed to help researchers find and understand associations between biomedical concepts. The goal of literature-based discovery (LBD) is to connect concepts in isolated literature domains that would normally go undiscovered. This usually takes the form of an A-B-C relationship, where A and C terms are linked through a B term intermediate. Here we describe Serial KinderMiner (SKiM), an LBD algorithm for finding statistically significant links between an A term and one or more C terms through some B term intermediate(s). The development of SKiM is motivated by the the observation that there are only a few LBD tools that provide a functional web interface, and that the available tools are limited in one or more of the following ways: 1) they identify a relationship but not the type of relationship, 2) they do not allow the user to provide their own lists of B or C terms, hindering flexibility, 3) they do not allow for querying thousands of C terms (which is crucial if, for instance, the user wants to query connections between a disease and the thousands of available drugs), or 4) they are specific for a particular biomedical domain (such as cancer). We provide an open-source tool and web interface that improves on all of these issues. ResultsWe demonstrate SKiMs ability to discover useful A-B-C linkages in three control experiments: classic LBD discoveries, drug repurposing, and finding associations related to cancer. Furthermore, we supplement SKiM with a knowledge graph built with transformer machine-learning models to aid in interpreting the relationships between terms found by SKiM. Finally, we provide a simple and intuitive open-source web interface (https://skim.morgridge.org) with comprehensive lists of drugs, diseases, phenotypes, and symptoms so that anyone can easily perform SKiM searches. ConclusionsSKiM is a simple algorithm that can perform LBD searches to discover relationships between arbitrary user-defined concepts. SKiM is generalized for any domain, can perform searches with many thousands of C term concepts, and moves beyond the simple identification of an existence of a relationship; many relationships are given relationship type labels from our knowledge graph.

bioinformatics↗

bioRxiv2020

SKiM - A generalized literature-based discovery system for uncovering novel biomedical knowledge from PubMed

Literature-based discovery (LBD) uncovers undiscovered public knowledge by linking terms A to C via a B intermediate. Existing LBD systems are limited to process certain A, B, and C terms, and many are not maintained. We present SKiM (Serial KinderMiner), a generalized LBD system for processing any combination of A, Bs, and Cs. We evaluate SKiM via the rediscovery of discoveries by Don Swanson, who pioneered LBD. Using only literature from the 19th century up to a year before Swansons discoveries, SKiM uncovers all five discoveries. We apply SKiM to repurposing drugs for 26 conditions of high prevalence. Manual analysis confirmed 65 discoveries useful for four diseases from Swansons discoveries from one to 31 years prior to their first validation by clinical trials. SKiM predicts many new potential drug candidates representing prime targets for wet lab validation. SKiM can be applied to any biomedical inquiry sufficiently mentioned in the literature.

bioinformatics↗

bioRxiv2020

Interspecies Chimeric Conditions Affect the Developmental Rate of Human Pluripotent Stem Cells

Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. Differences in the timing and expression levels of genes corresponding to neuron cell types and brain region identity under chimeric conditions were also observed. The altered developmental rates and lineage outcomes described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation. Author SummaryHuman pluripotent stem cells often require long in vitro protocols to form mature cell types of clinical relevance for potential regenerative therapies, a ramification of a nine-month developmental clock in utero that also runs ex utero. What controls species-specific developmental time and whether the timer is amenable to acceleration is unknown. Further, interspecies chimeric embryos are increasingly being created to study early human development or explore the potential growth of human organs for transplantation. How the conflicting developmental speeds of cells from different species co-differentiating together affect each other is not understood. Here, using genome-wide transcriptional analysis of RNA-sequencing time courses, we show that 1) co-differentiating human embryonic stem cells intermixed with mouse stem cells accelerated elements of human developmental programs, 2) the acceleration was dose-dependent on the proportion of mouse cells, and 3) human cells in chimeric samples correlated to in utero samples earlier than human only samples. Our results provide evidence that some components of species-specific developmental clocks may be susceptible to acceleration.

developmental biology↗