Variation In Cell Populations Research Articles

Abstract 15156: Statistical Modeling of Cardiac-Derived Progenitor Cell Extracellular Vesicles to Predict Clinical Trial Outcomes

Introduction: Cardiac-derived c-kit+ progenitor cells (CPCs) are under investigation in the CHILD phase I clinical trial (NCT03406884) for the treatment of hypoplastic left heart syndrome (HLHS). Therapeutic efficacy of CPCs can be attributed to the release of extracellular vesicles (EVs), carrying beneficial RNA cargo. Despite some successes with pediatric cardiac cell therapy, large variation in cell populations and patient outcomes remains a problem. To investigate these discrepancies and understand sources of cell therapy variability we took a machine learning approach: combining bulk CPC-derived EV (CPC-EV) RNA sequencing and cardiac-relevant in vitro experiments to build a predictive model. Methods: We isolated CPCs from cardiac biopsies of congenital heart disease patients (n=30) and the lead-in HLHS patients in the CHILD trial (n=7). We cultured CPCs, collected EVs from the media, and sequenced EV miRNA and total RNA. We treated cardiac endothelial cells and fibroblasts with CPC-EVs and measured inflammatory or fibrotic gene expression with qRT-PCR. We treated mesenchymal stromal cells with CPC-EVs and assessed migration in a Boyden chamber system. We treated cardiac endothelial cells seeded on Matrigel with CPC-EVs and measured tube formation. Finally, we put together the CPC-EV sequencing and experimental data in partial least squares and regularized regression models. Results: With our models we determined that the most important CPC-EV RNA signals involved in pro-reparative outcomes had significant fit to cardiac development and signaling pathways. Furthermore, using a model trained on previously collected CPC-EVs, we were able to predict in vitro outcomes for the CHILD clinical samples. Finally, we formed rank-based predictions for the CHILD lead-in patients, based on the in vitro assays. Conclusions: The CHILD phase I trial is set to conclude next year. We will validate our predictions with the release of these clinical data. With this work, we (1) investigated the EV RNA signals involved in pediatric cardiac repair and (2) will use our in vitro results to predict CHILD clinical trial outcomes. Our results highlight the importance of quantitative studies in determining the underlying variance in pediatric cardiac cell therapy.

Comparative computational RNA analysis of cardiac-derived progenitor cells and their extracellular vesicles

Stem/progenitor cells, including cardiac-derived c-kit+ progenitor cells (CPCs), are under clinical evaluation for treatment of cardiac disease. Therapeutic efficacy of cardiac cell therapy can be attributed to paracrine signaling and the release of extracellular vesicles (EVs) carrying diverse cargo molecules. Despite some successes and demonstrated safety, large variation in cell populations and preclinical/clinical outcomes remains a problem. Here, we investigated this variability by sequencing coding and non-coding RNAs of CPCs and CPC-EVs from 30 congenital heart disease patients and used machine learning methods to determine potential mechanistic insights. CPCs retained RNAs related to extracellular matrix organization and exported RNAs related to various signaling pathways to CPC-EVs. CPC-EVs are enriched in miRNA clusters related to cell proliferation and angiogenesis. With network analyses, we identified differences in non-coding RNAs which give insight into age-dependent functionality of CPCs. By taking a quantitative computational approach, we aimed to uncover sources of CPC cell therapy variability.

Partitioning stable and unstable expression level variation in cell populations: A theoretical framework and its application to the T cell receptor.

Phenotypic variation in the copy number of gene products expressed by cells or tissues has been the focus of intense investigation. To what extent the observed differences in cellular expression levels are persistent or transient is an intriguing question. Here, we develop a quantitative framework that resolves the expression variation into stable and unstable components. The difference between the expression means in two cohorts isolated from any cell population is shown to converge to an asymptotic value, with a characteristic time, τT, that measures the timescale of the unstable dynamics. The asymptotic difference in the means, relative to the initial value, measures the stable proportion of the original population variance . Empowered by this insight, we analysed the T-cell receptor (TCR) expression variation in CD4 T cells. About 70% of TCR expression variance is stable in a diverse polyclonal population, while over 80% of the variance in an isogenic TCR transgenic population is volatile. In both populations the TCR levels fluctuate with a characteristic time of 32 hours. This systematic characterisation of the expression variation dynamics, relying on time series of cohorts’ means, can be combined with technologies that measure gene or protein expression in single cells or in bulk.

Abstract 1915: In vivo prostate tumor tissue stiffness differs by tumor region and can be recapitulated in bioengineered prostate tumor tissues

Abstract Throughout the tumorigenic process, locational heterogeneities in tumor tissue microarchitecture develop as a result of aberrant angiogenesis and a subsequently induced oxygen and nutrient gradient within the three-dimensional (3D) mass. This phenomenon often results in differential tissue stiffness between the necrotic, quiescent, and proliferative tumor regions. In vitro, strong correlations have been found to exist between cell culture platform stiffness and acquired chemoresistance and varied drug response. Therefore, to accurately recapitulate the tumor microenvironment, biomimetic models must provide a mechanically similar scaffold. This study reports novel quantification of the in vivo prostate tumor stiffness and the ensuing development of tunable 3D bioengineered tumor tissue (BioTT) to successfully recapitulate in vivo mechanical cues in vitro. In vivo samples were generated by subcutaneously injecting Matrigel-suspended metastatic prostate cancer cells (PC-3) into the flank of athymic NCr nude mice. Resultant tumors (300 – 1,500 mm3) were excised from the murine host and geometrically dissected to provide samples from the tumor core, midpoint, and periphery. The Young’s modulus was quantified via parallel plate compression under physiological conditions. The 3D BioTT model is comprised of poly(ethylene glycol)-fibrinogen (PF) with varying amounts of excess poly(ethylene glycol) diacrylate (PEGDA) to modulate the mechanical properties of the scaffold. PC-3 cancer cells and BJ-5ta human fibroblasts were encapsulated within the covalently crosslinkable biomaterial and co-cultured for 29 days in vitro. Cell viability was assessed by LIVE/DEAD staining and cellular morphology was visualized with Hoechst 33342, Phalloidin, and the anti-fibroblast immunomarker, TE-7. Temporal variations in cell populations were quantified by flow cytometry and mechanical stiffness characterization was again performed by parallel plate compression. In vivo prostate cancer tumors presented a wide range of tissue stiffness heterogeneity (200 – 5,750 Pa), characterized by an increasing modulus with respect to locational progression from the core to the periphery (n = 48 per tumor region). The BioTT model successfully recapitulated the full tumor stiffness range through biomaterial composition modulation; the addition of excess PEGDA significantly stiffened the PF scaffold (p ≤ 0.05, n = 3). PC-3 and BJ-5ta cells survived the encapsulation process and remained viable throughout long-term co-culture. Visualization of the 3D cellular microenvironment revealed both cancer and stromal cells maintained characteristic morphology. In future studies, the BioTT will be extended to a microfluidic chip platform, thus augmenting the physiological relevancy of the model by incorporating dynamic shear conditions and the ability to monitor cancer cell metastasis. Citation Format: Nicole L. Habbit, Benjamin Anbiah, Luke S. Anderson, Joshita Suresh, Iman Hassani, Matthew Eggert, Shanese L. Jasper, Balabhaskar Prabhakarpandian, Robert D. Arnold, Elizabeth A. Lipke. In vivo prostate tumor tissue stiffness differs by tumor region and can be recapitulated in bioengineered prostate tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1915.

Abstract 4649: Duplex Sequencing detects rare subclonal variants that mark early carcinogenesis and preneoplastic clonal evolution

Abstract Cancer is a disease of somatic evolution, characterized by the natural selection of genomic mutations that facilitate enhanced cell survival and proliferation. Although many thousands of tumor genomes have now been sequenced, our ability to identify early genetic patterns of clonal selection in both humans and model organisms have been hampered by inadequately sensitive methodologies for identifying mutations during the long period between their occurrence and the final outgrowth of a clinically apparent tumor. Sensitive molecular tests are capable of measuring minute levels of cancer cells in tissue samples, however no existing method is satisfactory at scaling to high-throughput detection at the rate of one cancer-associated variant per more than a million normal cells. NGS is used to study the genetic variation in cell populations, although the accuracy of standard NGS methods limit our ability to detect sub-clones below ~1%. Duplex Sequencing (DS) is a sensitive NGS error-correction method which increases the accuracy of base calls by more than 100,000-fold. DS enables precise reconstruction of the original double-stranded source molecule while overcoming both chemical and technical artifacts that arise during library preparation and sequencing. Here we present the use of Duplex Sequencing, in both human and mouse tissues, for measuring sub-clones at allelic fractions less than 1×10-4 for an early glimpse into pre-neoplastic evolution. We illustrate how, merely weeks after mutagen exposure, we observe emerging clones of cells bearing cancer-driving mutations in histologically normal mouse tissue. Furthermore, we illustrate how similar patterns of clonal selection can be seen in multiple otherwise healthy tissues of humans as part of normal aging. We discuss how variations in the pattern and rate of accumulation of rare cancer-associated mutations offers a new preclinical tool for evaluating carcinogenicity of chemicals, as well as a potential clinical tool for assessing life-integrated carcinogenic processes and cancer risk in humans. Citation Format: Charles C. Valentine, Mark Fielden, Robert Young, Jake Higgins, Lindsey Williams, Tan Li, Rohan Kulkarni, Sheroy Minocherhomji, Rosana Risques, Patrick Danaher, Jesse Salk. Duplex Sequencing detects rare subclonal variants that mark early carcinogenesis and preneoplastic clonal evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4649.

Analysis of cellular autofluorescence in touch samples by flow cytometry: implications for front end separation of trace mixture evidence

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650–670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor’s contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650–670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures.Graphical Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

Radiation therapy and neutropenia

Acute radiation injury typically manifests as cytopenias: anemia, neutropenia, and thrombocytopenia; however, the measurement of peripheral blood counts belies the complex mechanism and timing of RT effects on hematopoietic stem cell (HSC) populations. There are several potential mechanisms for RT effects on the bone marrow, which are not mutually exclusive and may be complimentary. These include direct damage to HSC and reduction in their number and function, changes to the surrounding bone marrow stroma and microenvironment, and injury to ancillary cell populations that serve to regulate the hematopoietic process (eg, colony-stimulating factor [CSF] or erythropoietin-secreting cells). These effects are most likely with large volume irradiation of the pelvis or spine, as the primary site of functional bone marrow is the pelvis and vertebrae, which account for approximately 60% of total volume. Most of the data for detailed hematopoietic effects of RT are derived from large single fraction total body radiation exposures or from extended field administrations that are no longer used in routine practice. Even very low RT doses (as low as 0.3 Gy) can lead to measurable changes in peripheral blood counts (primarily lymphocytes at these doses) owing to the exquisite radiosensitive nature of these cells. With larger doses, lymphopenia typically occurs almost immediately due to radiation-induced apoptosis in interphase, followed by granulocytopenia, then thrombocytopenia, and ultimately anemia. Granulocyte counts may actually have an initial transient rise owing to mobilization from the periphery into the circulation, followed by a subsequent rapid decline. After an intermediate dose of total body radiation, initial decline in cell counts occurs approximately in 1, 4, 8, and 12 days for lymphocytes, granulocytes, platelets, and red blood cells, respectively. Clinical cytopenia occurs in approximately 1 day, 1 week, 2-3 weeks, and 2-3 months, respectively. These differences between cell lineages are owing to inherent radiosensitivity differences, variations in cell population reserves and mobilization, time required for hematopoietic cell replacement, and the life span of the differentiated peripheral cell (with red blood cells have the longest life cycle).

Erythrocyte deformation in high-throughput optical stretchers

Optical stretchers can be used to quantify elastic and homeostatic properties of cells. Because they can apply forces to cells without requiring direct contact, they may noninvasively measure mechanical properties related to cell and membrane health. Present-day optical stretchers are, however, limited to measurements on individual stationary cells, limiting throughput. To overcome this limitation and allow study of variations in cell populations, we recently developed and tested a microfluidic chamber that measures optical stretching parameters for erythrocytes under dynamic flowing conditions. The method uses a single linear diode laser bar and permitted measurements at low flow rates and higher throughput. Here, we numerically investigate the feasibility of further increasing the measurement rates of the optical stretcher in parameter domains where hydrodynamic and optical forces are of comparable magnitude. To do this we couple a recently implemented dynamic optical ray-tracing technique with a fluid-structure interaction solver to simulate the deformation of osmotically swollen erythrocytes in fluid flow of variable rate. Our results demonstrate that a detectable steady-state stretch is induced at nominal optical powers and flow rates. In addition, we find that flow rates can be increased significantly with no major effect on net cell stretch showing the feasibility of application of this technique at greatly increased throughputs.

In Situ Metabolic Profiling of Single Cells by Laser Ablation Electrospray Ionization Mass Spectrometry

Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO(2)-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.

Darwin's selection within the human body

It has been widely accepted that Darwin's natural selection plays a basic role in carcinogenesis and some other disease processes, but is not relevant to the normal multicellular life within the human body. The hypothesis proposed here is that selection and other evolutionary mechanisms not only normally or physiologically exist within the body of human beings and other multicellular organisms, but also persist and ‘function’ throughout the entire life span of the organism, from the embryo development to the aging process. This is based on the concept of cell individuality and the fact that there are a great deal of variations in cell populations, in particular possible existence of somatic genetic variations within the body. To realize the existence of natural selection in the multicellular life and study how it operates within organisms in both physiological and pathological conditions are fundamentally important to our understanding of human biology and medicine.

Chapter 14 High-Resolution Analysis of Nuclear DNA Employing the Fluorochrome DAPI

Protocols are available for high-resolution analysis of nuclear DNA that enable to discriminate somatic cells from male and female donors and to characterize precisely the ploidy state of tumors This chapter discusses protocols developed for use in cells obtained from cell suspensions and solid tissues. The procedures include fixation with 70% ethanol. Thus, they are applicable in specimens that have to be stored before measurement. The chapter focuses on the use of 6-diamidino-2-phenylindole (DAPI) for flow cytometry (FCM). DAPI proved to be a specific, highly fluorescent stain, very well suited for FCM of DNA in whole cells, nuclei, and chromosomes. The protocols are recommended for accurate and reproducible measurements of the nuclear DNA, especially for the assessment of DNA content variations in cell populations affected by chemical or physical mutagens and for the reliable detection and characterization of aneuploid cell clones in tumors.

Mouse 3T3 cell filtrability correlating with concanavalin A agglutinability

In a previous study of the dextran gel sphere model system, a possible correlation between cell deformability and agglutinability by concanavalin A was indicated. Cell deformability was evaluated as filtrability, using polycarbonate membrane filtration. With 25-mm diameter filters and 5-ml cell suspensions at (0.8–16) · 10 5 cells/ml, the filtrability at a given filter pore size was highly reproducible and was not affected by variations in cell population, viability, washings of cells retained on filter, or temperature. The filtrability of EDTA-dissociated 3T3 cells through 12-μm pore size filter was 8%, and a suspension of 10 6 cells/ml was not agglutinated by 600 μg concanavalin A. The filtrability of trypsin-dissociated 3T3 cells was 95%, and these cells were agglutinated by 200 μg of the lectin. EDTA-dissociated SV-3T3 cells had a filtrability of 73% and were also highly agglutinable. Formalin fixation reduced the high filtrability to 6%, and also abolished the agglutinability. As a further test of the correlation, trypsin-dissociated 3T3 cells were admixed with the fixed cells. The agglutinability varied with the proportions of the two cell components, and the admixtures could be separated according to filtrability into the original components with distinctly different agglutinability. Furthermore, 25% of a random population of EDTA-dissociated SV-3T3 cells retained by the filter were found to be non-agglutinable. The separated SV-3T3 cell fractions could also form admixtures of different agglutinability. It is concluded that the agglutinability of mouse 3T3 and SV-3T3 cells by concanavalin A can be correlated with the predicted by cell filtrability.

Mechanisms Regulating the Concentration and the Conformation of Progesterone Receptor(s) in the Uterus

Abstract After administration of estradiol to castrated guinea pigs, a rise in the uterine concentration of specific progesterone binding sites (receptor) was observed as early as at 6 hours, and the peak of concentration (8-fold over basal values) was obtained within approximately 24 hours. The effect of the estrogen was prevented by RNA and protein synthesis inhibitors administered 15 min before estrogen. On the other hand, these compounds had no effect when administered 20 hours after estradiol. These results were obtained by using animals with a stable low concentration of receptor (as observed after castration of the cycling guinea pig at diestrus, a time when the concentration is the lowest). From the rate of the decay of binding site concentration during a period of time when no more synthesis of the receptor occurs, the approximate half-life of the receptor was found to be 5 days. When progesterone was administered 20 hours after the estrogen, it provoked a rapid fall in receptor concentration, so that less than 20% remained 1 day after the injection. This decrease was neither due to masking by unlabeled progesterone nor due to the accumulation of receptor-steroid complexes in the nucleus. The progesterone effect was not prevented by protein synthesis inhibitors. After hormone injection, there were also variations in the sedimentation coefficient of the progesterone receptor. After estradiol, initially only the 4.5 S form increased, but secondarily the 6.7 S receptor became predominant. During the decay in receptor concentration, the 6.7 S binder disappeared faster, so that 7 days after estrogen injection equal amounts of both molecular forms were observed. These data suggest that the progesterone receptor in the guinea pig uterus is under a double control: a positive control by estrogen, requiring RNA and protein synthesis, and a negative control by progesterone, possibly due to an increased inactivation rate. In vitro experiments have indicated that the rate of inactivation of receptor molecules located in the nuclei is faster than that of the cytosol receptor. The relevance of this observation to the physiological situation, however, is still undecided. The effects of estradiol and progesterone on progesterone receptor content in the uterus are compatible with the cyclic changes observed physiologically in intact animals. It is not known whether these changes in the concentration of uterine receptor are due to molecular variations in individual cells or to variations in cell population.

Variations in cell population of intestinal lamina propria in relation to age.

Variations in cell population of intestinal lamina propria in relation to age.