scholarly journals A stochastic mathematical model of 4D tumour spheroids with real-time fluorescent cell cycle labelling

2021 ◽  
Author(s):  
Jonah J Klowss ◽  
Alexander P Browning ◽  
Ryan J Murphy ◽  
Elliot J Carr ◽  
Michael J Plank ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Nutrient Availability ◽  
Quantitative Information ◽  
Fluorescent Cell ◽  
Tumour Spheroid ◽  
Tumour Spheroids ◽  
Cell Cycle Status ◽  
Growing Population

In vitro tumour spheroid experiments have been used to study avascular tumour growth and drug design for the last 50 years. Unlike simpler two-dimensional cell cultures, tumour spheroids exhibit heterogeneity within the growing population of cells that is thought to be related to spatial and temporal differences in nutrient availability. The recent development of real-time fluorescent cell cycle imaging allows us to identify the position and cell cycle status of individual cells within the growing population, giving rise to the notion of a four-dimensional (4D) tumour spheroid. In this work we develop the first stochastic individual-based model (IBM) of a 4D tumour spheroid and show that IBM simulation data qualitatively and quantitatively compare very well with experimental data from a suite of 4D tumour spheroid experiments performed with a primary human melanoma cell line. The IBM provides quantitative information about nutrient availability within the spheroid, which is important because it is very difficult to measure these data in standard tumour spheroid experiments. Software required to implement the IBM is available on GitHub.

scholarly journals Immune regulation of in vitro murine megakaryocyte development. Role of T lymphocytes and Ia antigen expression.

1985 ◽  
Vol 162 (6) ◽  
pp. 2053-2067 ◽  
Author(s):  
M W Long ◽  
D N Shapiro
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
Immune Modulation ◽  
Immune Recognition ◽  
Activated T Cells ◽  
Ia Antigen ◽  
Cell Cycle Status

Mitogen-activated murine T lymphocytes or T cell hybridomas produce an activity (megakaryocyte [Mk] potentiator activity) that enhances the in vitro growth and development of Mk colonies. This activity was found in optimal concentrations (2.5%) in T cell hybridoma-conditioned medium, and was also produced by feeder layers of concanavalin A-activated T cells. A subpopulation of murine Mk progenitor cells (colony-forming units; CFU-Mk) bears the Ia antigen. Separate experiments indicated that T cell products stimulate CFU-Mk by increasing their basal levels of Ia expression as well as the frequency of cells actively synthesizing DNA. The hypothesis that the expression of this antigen was related to the cell cycle status of these progenitor cells was confirmed in studies that indicated that ablation of actively cycling cells in vivo abrogated the cytotoxic effects of anti-Ia monoclonal antibodies. The interdependence of T cell lymphokine regulation of both Ia expression and cell cycle status was also seen in in vitro experiments in which Ia+ progenitor cells were eliminated by complement-dependent cytotoxicity. The removal of Ia+ cells prevented 5-hydroxyurea-mediated inhibition of cells in S phase. We hypothesize that immune modulation of megakaryocytopoiesis occurs via soluble T cell products that augment Mk differentiation. Further, the mechanism of immune recognition/modulation may occur via Ia antigens present on the surface of these progenitor cells.

scholarly journals Mathematical Model of Tumour Spheroid Experiments with Real-Time Cell Cycle Imaging

2021 ◽  
Vol 83 (5) ◽  
Author(s):  
Wang Jin ◽  
Loredana Spoerri ◽  
Nikolas K. Haass ◽  
Matthew J. Simpson
Keyword(s):  
Mathematical Model ◽  
Cell Cycle ◽  
Real Time ◽  
Tumour Spheroid

scholarly journals Quantitative analysis of tumour spheroid structure

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexander P Browning ◽  
Jesse A Sharp ◽  
Ryan J Murphy ◽  
Gency Gunasingh ◽  
Brodie Lawson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tumour Microenvironment ◽  
Culture Conditions ◽  
Experimental Models ◽  
Seeding Density ◽  
Modelling Framework ◽  
Tumour Spheroid ◽  
Tumour Spheroids ◽  
Wide Range

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
K. M. Whitworth ◽  
W. G. Spollen ◽  
S. M. Blake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Background ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Illumina Genome Analyzer ◽  
Similar Genetic Background

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1505-1505
Author(s):  
Wendy W. Pang ◽  
Elizabeth A. Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Expression Profiles ◽  
Frequency Function ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

scholarly journals Effect of interleukin-11 on cycling status and clonogenic maturation of fetal and adult hematopoietic progenitors

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 900-903
Author(s):  
KR Schibler ◽  
YC Yang ◽  
RD Christensen
Keyword(s):  
Cell Cycle ◽  
Tritiated Thymidine ◽  
Single Agent ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Colony Forming Units ◽  
Cell Cycle Status ◽  
Interleukin 11 ◽  
Human Cytokine

A recently cloned human cytokine, interleukin-11 (IL-11), has functional similarities to IL-6. We tested the hypothesis that the hematopoietic actions of IL-11 in vitro also resemble those of IL-6. The effect of IL-11 on the cell cycle status of fetal and adult hematopoietic progenitors was assessed using serum-free incubations followed by tritiated thymidine suicide studies. Its effect on clonogenic maturation was assessed by including IL-11, either as a single agent or with subplateau or plateau concentrations of other recombinant cytokines, in cultures that contained neutralizing monoclonal antibodies directed against relevant growth factors. Similar to IL-6, IL-11 resulted in accelerated cycling of fetal colony-forming units-mixed (CFU-MIX), CFU-granulocyte macrophage (CFU-GM), and erythroid burst-forming units (BFU-E). This effect was additive to that of submaximal, but not to plateau, concentrations of IL-6. However, no effect of IL-11 was observed on cycling status of adult progenitors. As a single agent, IL-11 failed to support clonal maturation of either fetal or adult progenitors. IL-11 was additive to GM-CSF in supporting clonal maturation of CFU-GM from adult marrow but not from fetal blood. We conclude that the in vitro hematopoietic actions of IL-11 on cell cycle status of hematopoietic progenitors resemble those of IL-6. However, unlike IL-6, IL-11 as a single agent failed to support clonal maturation of fetal CFU-GM, BFU-E, and CFU-MIX.

scholarly journals Mitochondria-targeted triphenylphosphonium-based compounds do not affect estrogen receptor α

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8803
Author(s):  
Ludmila A. Zinovkina ◽  
Alina K. Galivondzhyan ◽  
Anastasia S. Prikhodko ◽  
Ivan I. Galkin ◽  
Roman A. Zinovkin
Keyword(s):  
Cell Cycle ◽  
Estrogen Receptor ◽  
Real Time ◽  
Cell Cycle Progression ◽  
Expression Vector ◽  
Estrogen Receptor Α ◽  
Luciferase Expression ◽  
Cycle Progression ◽  
Mcf 7

Background Targeting negatively charged mitochondria is often achieved using triphenylphosphonium (TPP) cations. These cationic vehicles may possess biological activity, and a docking study indicates that TPP-moieties may act as modulators of signaling through the estrogen receptor α (ERα). Moreover, in vivo and in vitro experiments revealed the estrogen-like effects of TPP-based compounds. Here, we tested the hypothesis that TPP-based compounds regulate the activity of ERα. Methods We used ERa-positive and ERα-negative human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231, respectively). Cell proliferation was measured using a resazurin cell growth assay and a real-time cell analyzer assay. Cell cycle progression was analyzed using flow cytometry. Real-time PCR was used to assess mRNA expression of endogenous estrogen-responsive genes. Luciferase activity was measured to evaluate transcription driven by estrogen-responsive promoters in cells transfected with an estrogen response element (ERE)3-luciferase expression vector. Results The TPP-based molecules SkQ1 and C12TPP, as well as the rhodamine-based SkQR1, did not increase the proliferation or alter the cell cycle progression of MCF-7 cells. In contrast, 17β estradiol increased the proliferation of MCF-7 cells and the proportion of cells in the S/G2/M-phases of the cell cycle. TPP-based compounds did not affect the induction of transcription of an ERE-luciferase expression vector in vitro, and SkQ1 did not alter the levels of expression of estrogen-dependent genes encoding GREB1, TFF1, COX6, and IGFBP4. Conclusion TPP-based compounds do not possess properties typical of ERα agonists.

scholarly journals Immature megakaryocytes in the mouse: physical characteristics, cell cycle status, and in vitro responsiveness to thrombopoietic stimulatory factor

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 569-575 ◽  
Author(s):  
MW Long ◽  
N Williams ◽  
S Ebbe
Keyword(s):  
Cell Cycle ◽  
Cell Types ◽  
Equilibrium Density ◽  
Early Maturation ◽  
Hydroxyurea Treatment ◽  
Cell Groups ◽  
Stimulatory Factor ◽  
Cell Cycle Status

Abstract The heterogeneity among immature megakaryocytes has been examined by physical properties, cell cycle status, and responsiveness to thrombopoietic stimulatory factor. Three types of immature megakaryocytes exist that can be recognized by acetylcholinesterase staining, nuclear shape, high nucleus/cytoplasm ratio, and small size (8--18 mu) with respect to mature megakaryocytes (greater than 18 mu). These three acetylcholinesterase-containing cell types are distinguished by their nuclear configuration: a round, indented, and lobed nucleus. The lobed cell type was found to overlap with and enhance detection of megakaryoblasts (stage I megakaryocytes). These cells had a sedimentation velocity range of 3.5--19.0 mm hr-1 and a density range of 1.072--1.095 g cm-3. Separation of these three classes of immature megakaryocytes was achieved by equilibrium density centrifugation with modal buoyant densities of 1.079 g cm-3 (round), 1.084 g cm-3 (indented), and 1.089 g cm-3 (lobed). In the presence of thrombopoietic stimulatory factor, the round nucleated cells, but not the indented or lobed nuclei morphology, were observed to develop into large mature megakaryocytes in 60-hr semisolid cell cultures. Development of two cell groups, or colonies of megakaryocytes, was not observed during this in vitro incubation period. In vivo treatment with hydroxyurea indicated that 57.5% +/- 19% of the round nucleus form were actively synthesizing DNA. No reduction in the numbers of indented or lobed nucleus forms were observed following hydroxyurea treatment. The data in this report strongly support the concept that these three types of immature megakaryocytes reflect the early maturation stages occurring in megakaryocyte differentiation.

scholarly journals Immature megakaryocytes in the mouse: physical characteristics, cell cycle status, and in vitro responsiveness to thrombopoietic stimulatory factor

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 569-575 ◽  
Author(s):  
MW Long ◽  
N Williams ◽  
S Ebbe
Keyword(s):  
Cell Cycle ◽  
Cell Types ◽  
Equilibrium Density ◽  
Early Maturation ◽  
Hydroxyurea Treatment ◽  
Cell Groups ◽  
Stimulatory Factor ◽  
Cell Cycle Status

The heterogeneity among immature megakaryocytes has been examined by physical properties, cell cycle status, and responsiveness to thrombopoietic stimulatory factor. Three types of immature megakaryocytes exist that can be recognized by acetylcholinesterase staining, nuclear shape, high nucleus/cytoplasm ratio, and small size (8--18 mu) with respect to mature megakaryocytes (greater than 18 mu). These three acetylcholinesterase-containing cell types are distinguished by their nuclear configuration: a round, indented, and lobed nucleus. The lobed cell type was found to overlap with and enhance detection of megakaryoblasts (stage I megakaryocytes). These cells had a sedimentation velocity range of 3.5--19.0 mm hr-1 and a density range of 1.072--1.095 g cm-3. Separation of these three classes of immature megakaryocytes was achieved by equilibrium density centrifugation with modal buoyant densities of 1.079 g cm-3 (round), 1.084 g cm-3 (indented), and 1.089 g cm-3 (lobed). In the presence of thrombopoietic stimulatory factor, the round nucleated cells, but not the indented or lobed nuclei morphology, were observed to develop into large mature megakaryocytes in 60-hr semisolid cell cultures. Development of two cell groups, or colonies of megakaryocytes, was not observed during this in vitro incubation period. In vivo treatment with hydroxyurea indicated that 57.5% +/- 19% of the round nucleus form were actively synthesizing DNA. No reduction in the numbers of indented or lobed nucleus forms were observed following hydroxyurea treatment. The data in this report strongly support the concept that these three types of immature megakaryocytes reflect the early maturation stages occurring in megakaryocyte differentiation.

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