Quantifying the Dynamics of Hematopoiesis by In Vivo IdU Pulse-Chase, Mass Cytometry, and Mathematical Modeling.
ABSTRACT: We present a new method to directly quantify the dynamics of differentiation of multiple cellular subsets in unperturbed mice. We combine a pulse-chase protocol of 5-iodo-2'-deoxyuridine (IdU) injections with subsequent analysis by mass cytometry (CyTOF) and mathematical modeling of the IdU dynamics. Measurements by CyTOF allow for a wide range of cells to be analyzed at once, due to the availability of a large staining panel without the complication of fluorescence spillover. These are also compatible with direct detection of integrated iodine signal, with minimal impact on immunophenotyping based on the surface markers. Mathematical modeling beyond a binary classification of surface marker abundance allows for a continuum of cellular states as the cells transition from one state to another. Thus, we present a complete and robust method for directly quantifying differentiation at the systemic level, allowing for system-wide comparisons between different mouse strains and/or experimental conditions. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.
Project description:Primary glioblastomas are subdivided into several molecular subtypes. There is an ongoing debate over the cell of origin for these tumor types where some suggest a progenitor while others argue for a stem cell origin. Even within the same molecular subgroup, and using lineage tracing in mouse models, different groups have reached different conclusions. We addressed this problem from a combined mathematical modeling and experimental standpoint. We designed a novel mathematical framework to identify the most likely cells of origin of two glioma subtypes. Our mathematical model of the unperturbed in vivo system predicts that if a genetic event contributing to tumor initiation imparts symmetric self-renewing cell division (such as PDGF overexpression), then the cell of origin is a transit amplifier. Otherwise, the initiating mutations arise in stem cells. The mathematical framework was validated with the RCAS/tv-a system of somatic gene transfer in mice. We demonstrated that PDGF-induced gliomas can be derived from GFAP-expressing cells of the subventricular zone or the cortex (reactive astrocytes), thus validating the predictions of our mathematical model. This interdisciplinary approach allowed us to determine the likelihood that individual cell types serve as the cells of origin of gliomas in an unperturbed system.
Project description:Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID-19 patients and healthy controls, highlighting dynamic disease-associated changes in circulating immune cell frequency and phenotype.
Project description:Clonal hematopoiesis of indeterminate potential (CHIP) is a recently identified process where older patients accumulate distinct subclones defined by recurring somatic mutations in hematopoietic stem cells. CHIP's implications for stem cell transplantation have been harder to identify due to the high degree of mutational heterogeneity that is present within the genetically distinct subclones. In order to gain a better understanding of CHIP and the impact of clonal dynamics on transplantation outcomes, we created a mathematical model of clonal competition dynamics. Our analyses highlight the importance of understanding competition intensity between healthy and mutant clones. Importantly, we highlight the risk that CHIP poses in leading to dominance of precancerous mutant clones and the risk of donor derived leukemia. Furthermore, we estimate the degree of competition intensity and bone marrow niche decline in mice during aging by using our modeling framework. Together, our work highlights the importance of better characterizing the ecological and clonal composition in hematopoietic donor populations at the time of stem cell transplantation.
Project description:Chemoselective transformations that work under physiological conditions have emerged as powerful tools to label nucleic acids in cell-free and cellular environments. However, detailed studies investigating the influence of nucleic acid conformation on the performance of such chemoselective nucleic labeling methods are less explored. Given that nucleic acids adopt complex structures, it is highly important to study the scope of the chemical modification method in the context of nucleic acid conformations. Here we report a systematic study on the effect of local conformation on the postsynthetic Suzuki-Miyaura functionalization of human telomeric (H-Telo) DNA repeat oligonucleotide (ON) sequences, which form multiple G-quadruplex (GQ) structures. 5-Iodo-2'-deoxyuridine (IdU)-modified H-Telo ONs were synthesized by the solid-phase method, and when subjected to Suzuki-Miyaura cross-coupling reaction, its efficiency was found to depend on the type of conformation and the position of IdU label in different loops of the GQ structure. IdU-labeled GQs gave better yields as compared to single-stranded random coil structures. However, the IdU-labeled duplex under different ionic conditions did not undergo the coupling reaction. Further, using this method, we directly installed an environment-sensitive fluorescent probe, which photophysically reported the formation as well as distinguished different GQ topologies of telomeric repeat. Collectively, this systematic study underscores the influence of nucleic acid conformation, which has to be taken into account when establishing postsynthetic chemoselective functionalization strategies.
Project description:The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-?+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer.
Project description:Scientists have traditionally studied complex biologic systems by reducing them to simple building blocks. Genome sequencing, high-throughput screening, and proteomics have, however, generated large datasets, revealing a high level of complexity in components and interactions. Systems biology embraces this complexity with a combination of mathematical, engineering, and computational tools for constructing and validating models of biologic phenomena. The validity of mathematical modeling in hematopoiesis was established early by the pioneering work of Till and McCulloch. In reviewing more recent papers, we highlight deterministic, stochastic, statistical, and network-based models that have been used to better understand a range of topics in hematopoiesis, including blood cell production, the periodicity of cyclical neutropenia, stem cell production in response to cytokine administration, and the emergence of imatinib resistance in chronic myeloid leukemia. Future advances require technologic improvements in computing power, imaging, and proteomics as well as greater collaboration between experimentalists and modelers. Altogether, systems biology will improve our understanding of normal and abnormal hematopoiesis, better define stem cells and their daughter cells, and potentially lead to more effective therapies.
Project description:BACKGROUND:Injection drug use (IDU) is associated with multiple health harms. The vast majority of IDU initiation events (in which injection-naïve persons first adopt IDU) are assisted by a person who injects drugs (PWID), and as such, IDU could be considered as a dynamic behavioral transmission process. Data suggest that opioid agonist treatment (OAT) enrollment is associated with a reduced likelihood of assisting with IDU initiation. We assessed the association between recent OAT enrollment and assisting IDU initiation across several North American settings and used dynamic modeling to project the potential population-level impact of OAT scale-up within the PWID population on IDU initiation. METHODS AND FINDINGS:We employed data from a prospective multicohort study of PWID in 3 settings (Vancouver, Canada [n = 1,737]; San Diego, United States [n = 346]; and Tijuana, Mexico [n = 532]) from 2014 to 2017. Site-specific modified Poisson regression models were constructed to assess the association between recent (past 6 month) OAT enrollment and history of ever having assisted an IDU initiation with recently assisting IDU initiation. Findings were then pooled using linear mixed-effects techniques. A dynamic transmission model of IDU among the general population was developed, stratified by known factors associated with assisting IDU initiation and relevant drug use behaviors. The model was parameterized to a generic North American setting (approximately 1% PWID) and used to estimate the impact of increasing OAT coverage among PWID from baseline (approximately 21%) to 40%, 50%, and 60% on annual IDU initiation incidence and corresponding PWID population size across a decade. From Vancouver, San Diego, and Tijuana, respectively, 4.5%, 5.2%, and 4.3% of participants reported recently assisting an IDU initiation, and 49.4%, 19.7%, and 2.1% reported recent enrollment in OAT. Recent OAT enrollment was significantly associated with a 45% lower likelihood of providing recent IDU initiation assistance among PWID (relative risk [RR] 0.55 [95% CI 0.36-0.84], p = 0.006) compared to those not recently on OAT. Our dynamic model predicts a baseline mean of 1,067 (2.5%-97.5% interval [95% I 490-2,082]) annual IDU initiations per 1,000,000 individuals, of which 886 (95% I 406-1,750) are assisted by PWID. Based on our observed statistical associations, our dynamic model predicts that increasing OAT coverage from approximately 21% to 40%, 50%, or 60% among PWID could reduce annual IDU initiations by 11.5% (95% I 2.4-21.7), 17.3% (95% I 5.6-29.4), and 22.8% (95% I 8.1-36.8) and reduce the PWID population size by 5.4% (95% I 0.1-12.0), 8.2% (95% I 2.2-16.9), and 10.9% (95% I 3.2-21.8) relative to baseline, respectively, in a decade. Less impact occurs when the protective effect of OAT is diminished, when a greater proportion of IDU initiations are unassisted by PWID, and when average IDU career length is longer. The study's main limitations are uncertainty in the causal pathway between OAT enrollment and assisting with IDU initiation and the use of a simplified model of IDU initiation. CONCLUSIONS:In addition to its known benefits on preventing HIV, hepatitis C virus (HCV), and overdose among PWID, our modeling suggests that OAT scale-up may also reduce the number of IDU initiations and PWID population size.
Project description:The timing and duration of sleep results from the interaction between a homeostatic sleep-wake-driven process and a periodic circadian process, and involves changes in gene regulation and expression. Unraveling the contributions of both processes and their interaction to transcriptional and epigenomic regulatory dynamics requires sampling over time under conditions of unperturbed and perturbed sleep. We profiled mRNA expression and chromatin accessibility in the cerebral cortex of mice over a 3-d period, including a 6-h sleep deprivation (SD) on day 2. We used mathematical modeling to integrate time series of mRNA expression data with sleep-wake history, which established that a large proportion of rhythmic genes are governed by the homeostatic process with varying degrees of interaction with the circadian process, sometimes working in opposition. Remarkably, SD caused long-term effects on gene-expression dynamics, outlasting phenotypic recovery, most strikingly illustrated by a damped oscillation of most core clock genes, including Arntl/Bmal1, suggesting that enforced wakefulness directly impacts the molecular clock machinery. Chromatin accessibility proved highly plastic and dynamically affected by SD. Dynamics in distal regions, rather than promoters, correlated with mRNA expression, implying that changes in expression result from constitutively accessible promoters under the influence of enhancers or repressors. Serum response factor (SRF) was predicted as a transcriptional regulator driving immediate response, suggesting that SRF activity mirrors the build-up and release of sleep pressure. Our results demonstrate that a single, short SD has long-term aftereffects at the genomic regulatory level and highlights the importance of the sleep-wake distribution to diurnal rhythmicity and circadian processes.
Project description:Declarative modeling uses symbolic expressions to represent models. With such expressions, one can formalize high-level mathematical computations on models that would be difficult or impossible to perform directly on a lower-level simulation program, in a general-purpose programming language. Examples of such computations on models include model analysis, relatively general-purpose model reduction maps, and the initial phases of model implementation, all of which should preserve or approximate the mathematical semantics of a complex biological model. The potential advantages are particularly relevant in the case of developmental modeling, wherein complex spatial structures exhibit dynamics at molecular, cellular, and organogenic levels to relate genotype to multicellular phenotype. Multiscale modeling can benefit from both the expressive power of declarative modeling languages and the application of model reduction methods to link models across scale. Based on previous work, here we define declarative modeling of complex biological systems by defining the operator algebra semantics of an increasingly powerful series of declarative modeling languages including reaction-like dynamics of parameterized and extended objects; we define semantics-preserving implementation and semantics-approximating model reduction transformations; and we outline a "meta-hierarchy" for organizing declarative models and the mathematical methods that can fruitfully manipulate them.