cell cycle status
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2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danying Yi ◽  
Lijiao Zhu ◽  
Yuanling Liu ◽  
Jiahui Zeng ◽  
Jing Chang ◽  
...  

AbstractDeficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43− cells and derivative populations, but not that of CD34lowCD43− cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-β or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).


2021 ◽  
Author(s):  
Jonah J Klowss ◽  
Alexander P Browning ◽  
Ryan J Murphy ◽  
Elliot J Carr ◽  
Michael J Plank ◽  
...  

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.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3977-3977
Author(s):  
Kohei Shiroshita ◽  
Hiroshi Kobayashi ◽  
Shinichiro Okamoto ◽  
Keisuke Kataoka ◽  
Keiyo Takubo

Abstract Hematopoietic stem cells (HSCs) in steady-state are quiescent in cell cycle. CRISPR-Cas9 genome editing has revolutionized the HSC research and therapeutic application of HSCs for hematological diseases. Although these methods and clinical results are promising, keeping HSC function after highly efficient genome editing is still challenging because HSCs gradually lose their repopulation capacity following cell cycle activation. Preserving the function of HSCs after genome editing is an urgent issue. In this study, we interrogated the culture method following genome editing to reverse the cell cycle status of HSCs into a quiescent state to test how cell cycle status affects the genome editing efficiency and the HSC potential. To assess the relationship between genome editing efficiency and cell cycle status, we compared genome editing efficiency among the freshly-isolated or cultured HSCs and progenitors (HSPCs) at various time points. Ribonucleoprotein (RNP) complex was delivered into cells by electroporation. Genome-editing efficiency was evaluated by CD45 knockout rate comparing sgRNA for Rosa and CD45. All the HSPC fractions cultured displayed higher genome-editing efficiency following over-night preculture. Genome-editing efficiency of fresh HSC was lower than 20% while fresh granulocyte/monocyte progenitor (GMP) showed higher than 40%. Cell cycle analysis using EdU or Ki67 and Hoechst 33342 staining confirmed that genome-editing efficiency positively correlated with cell cycle activation. We further tested the effect of pre- and post-culture conditions for genome editing. While preculture with high cytokine concentration for a long period of time (> 16 hours) is required for the efficient genome editing, post-culture condition did not compromise the genome-editing efficiency. Given the quiescent nature of HSCs, we hypothesized that reverting activated HSCs back to quiescent state may improve the function of HSCs following genome editing. To test this, genome-edited HSCs were cultured in the quiescence-maintaining condition (SCF 1.5 ng/mL and TPO 1.0 ng/mL) under 1% O 2 atmosphere. After 7-day culture, more than 30% of cells kept the surface marker phenotype of CD150 + CD48 - LSK, and over 60% of cells were successfully underwent genome editing. Less than 10% of HSCs were EdU +, suggesting that HSCs reverted to cell cycle quiescence after genome editing. By contrast, HSCs cultured in a conventional high cytokine condition (proliferative condition) lost the surface marker phenotypes and highly incorporated EdU. To assess the long-term reconstitution potential of edited HSCs, the Evi1 expression level was evaluated using Evi1-IRES-GFP mice. The expression level of Evi1 was significantly higher in quiescent HSCs than proliferating HSCs after editing. These results suggest that, as post-electroporation culture, quiescence-maintaining condition reverts precultured HSCs back to a quiescent state in cell cycle. This protocol maintains phenotypic HSCs without compromising the genome-editing efficiency. To further determine the function of genome-edited HSCs, single cell colony assay was performed. Clonally sorted CD45 knockout HSCs cultured in the quiescence-maintaining condition after gene editing fully maintained colony-forming capacity, but HSCs cultured in the proliferating condition lost their capacity. We then performed transplantation assays using Ubc-GFP mice. GFP + HSCs were genome-edited for Rosa and transplanted into lethally-irradiated recipient mice with competitor cells. The donor-derived chimerism of edited HSCs with quiescence-maintaining condition in peripheral blood and bone marrow was generally superior to that of edited HSCs with proliferative condition. These data demonstrates that edited HSCs cultured in quiescence-maintaining condition maintain stem cell potential in vitro and in vivo. Altogether, we established an HSC-optimized, highly efficient genome-editing protocol. This study demonstrated that effectiveness of keeping HSC in a quiescent state even in the setting of genome editing. Our protocol is suitable for unveiling the function of genes distinguishing cycling and quiescent HSCs. Disclosures Kataoka: Celgene: Honoraria; Eisai: Honoraria; Astellas Pharma: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; MSD: Honoraria; Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Asahi Genomics: Current equity holder in publicly-traded company; Otsuka Pharmaceutical: Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding; Eisai: Other: Scholarship; Otsuka Pharmaceutical: Other: Scholarship; Ono Pharmaceutical: Other: Scholarship; Kyowa Kirin: Other: Scholarship; Shionogi: Other: Scholarship; Takeda Pharmaceutical: Other: Scholarship; Summitomo Dainippon Pharma: Other: Scholarship; Chugai Pharmaceutical: Other: Scholarship; Teijn Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Genetic Alterations: Patents & Royalties: PD-L1 abnormalties .


Author(s):  
Shanshan Jiang ◽  
Rong Wang ◽  
Lu Han ◽  
Kudelaidi Kuerban ◽  
Li Ye ◽  
...  

AbstractThis research aims to investigate the effect of gemcitabine (GEM) on various activities and functions of macrophages. Phagocytosis, cell autophagy and reactive oxygen species (ROS) were analysed by laser scanning confocal microscope. The cell cycle status and major histocompatibility complex II (MHC-II) expression were examined by flow cytometry. Inflammatory cytokine secretion such as tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) was detected by Elisa assay. The expression of proteins was analysed by western blot method. The results revealed that GEM-induced immune inhibition of M1-type RAW264.7 macrophages activated by interferon-γ (IFN-γ) and lipopolysaccharide (LPS). We also found that GEM inhibited autophagy, as evidenced by the reduced formation of autophagosome-like vacuoles and autophagosomes. Further study showed that incubation of activated macrophages with the autophagy inhibitor 3-MA induced immune suppression. In contrast, treatment with the autophagy inducer trehalose (Tre) restored phagocytosis, TNF-α and IL-6 secretion, and MHC-II expression in GEM-induced immune-inhibited macrophages. GEM reduced immune effect of M1-type RAW264.7 macrophages via inhibiting TNF-α, IL-6 and MHC-II expression. Furthermore, activation of autophagy by Tre reversed GEM-induced immune inhibition of RAW264.7 macrophages.


2021 ◽  
Author(s):  
Marina Capece ◽  
Anna Tessari ◽  
Joseph Mills ◽  
Gian Luca Rampioni Vinciguerra ◽  
Chenyu Liu ◽  
...  

AbstractThe OsTIR1/auxin-inducible degron (AID) system allows “on demand” selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. This new tool paves the way to studying the differential roles that target proteins may have in specific phases of the cell cycle.


Author(s):  
Xiao-Fang Zhou ◽  
Yang Liu ◽  
Jia-Ming Xu ◽  
Jin-Huo Wang ◽  
Zhen-Zhou Li ◽  
...  

Background: Pre-operative autologous blood donation (PABD) is one of the most widely distributed autologous blood donation means, which has positive effect on erythropoiesis. However, whether PABD can stimulate the bone marrow hematopoiesis after hepatectomy have not been reported. Methods: Totally 80 New Zealand rabbits were randomly divided into 4 groups that included control group, surgery group, hemodilutional autotransfusion (HA) group and PABD group. Automatic reticulocyte examination was performed to detect the content of reticulocyte and immature reticulocyte fractions (IRF). Flow cytometric analysis was employed to monitor the level of CD34+ cells and the cell cycle status. Southern blotting was conducted to determine the telomere length of CD34+ cells. Results: The content of high fluorescence reticulocytes (HFR) and IRF was decreased at 6 h and 24 h after autotransfusion. However, the level of CD34+ cells was upregulated after PABD. Cell cycle status analysis revealed that majority of the CD34+ cells in HA and PABD group were maintained in G0/G1 phase. The telomere length in HA and PABD group was shorten than that of control group and surgery group. Conclusion: PABD could promote the bone marrow hematopoietic functions in rabbits after hepatectomy via stimulating proliferation of CD34+ cells and shortening the telomere length of CD34+ cells, but the content of HFR was not increased immediately because of the stuck of CD34+ cells in G0/G1 phase.


Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1467
Author(s):  
Céline Ducloux ◽  
Bruno You ◽  
Amandine Langelé ◽  
Olivier Goupille ◽  
Emmanuel Payen ◽  
...  

Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage. Two of the clones tested, B12 and E2, reached sensitivity levels higher than those of UT7/Epo-S1 and CD36+ erythroid progenitor cells. These findings highlight the importance of cell cycle status for sensitivity to B19V, and we propose a promising new straightforward cell-based method for quantifying B19V infectious units.


2019 ◽  
Author(s):  
Rebekka Wegmann ◽  
Marilisa Neri ◽  
Sven Schuierer ◽  
Bilada Bilican ◽  
Huyen Hartkopf ◽  
...  

AbstractComprehensive benchmarking of computational methods for single-cell RNA sequencing (scRNA-seq) analysis is scarce. Using a modular workflow and a large dataset with known cell composition, we benchmarked feature selection and clustering methodologies for scRNA-seq data. Results highlighted a methodology gap for rare cell population identification for which we developed CellSIUS (Cell Subtype Identification from Upregulated gene Sets). CellSIUS outperformed existing approaches, enabled the identification of rare cell populations and, in contrast to other methods, simultaneously revealed transcriptomic signatures indicative of the rare cells’ function. We exemplified the use of our workflow and CellSIUS for the characterization of a human pluripotent cell 3D spheroid differentiation protocol recapitulating deep-layer corticogenesis in vitro. Results revealed lineage bifurcation between Cajal-Retzius cells and layer V/VI neurons as well as rare cell populations that differ by migratory, metabolic, or cell cycle status, including a choroid plexus neuroepithelial subgroup, revealing previously unrecognized complexity in human stem cell-derived cellular populations.


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