Assaying Cell Cycle Status Using Flow Cytometry

Kim, Kang Ho; Sederstrom, Joel M.

doi:10.1002/0471142727.mb2806s111

Cited by 225 publications

(173 citation statements)

References 25 publications

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“…Before investigating the relationship between cell cycle status and editing efficiency, we first explored the cell cycle progression of CD34+ cells in ex vivo culture using immunophenotyping combined with Hoechst 33342 (stains for DNA) and Ki67 (highly expressed in proliferating cells) staining. (Gerdes et al, 1984;Kim and Sederstrom, 2015). We found that more than 50% of cryopreserved mPB CD34+ HSPCs are quiescent (in G0) when thawed, but they gradually enter the cell cycle and are fully cycling by 3 days in SC culture (Figures 2A,2B, and S2).…”

Section: Establishing the Timing Of Cell Cycle Status In Cd34+ Subsetmentioning

confidence: 51%

Controlled cycling and quiescence enables homology directed repair in engraftment-enriched adult hematopoietic stem and progenitor cells

Wyman

Bray

et al. 2018

Preprint

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Hematopoietic stem cells (HSCs) are the source of all blood components, and genetic defects in these cells are causative of disorders ranging from severe combined immunodeficiency to sickle cell disease. However, genome editing of long-term repopulating HSCs to correct mutated alleles has been challenging. HSCs have the ability to either be quiescent or cycle, with the former linked to stemness and the latter involved in differentiation.Here we investigate the link between cell cycle status and genome editing outcomes at the causative codon for sickle cell disease in adult human CD34+ hematopoietic stem and progenitor cells (HSPCs). We show that quiescent HSPCs that are immunophenotypically enriched for engrafting stem cells predominantly repair Cas9-induced double strand breaks (DSBs) through an error-prone non-homologous end-joining (NHEJ) pathway and exhibit almost no homology directed repair (HDR). By contrast, non-quiescent cycling stem-enriched cells repair Cas9 DSBs through both error-prone NHEJ and fidelitous HDR. Pre-treating bulk CD34+HSPCs with a combination of mTOR and GSK-3 inhibitors to induce quiescence results in complete loss of HDR in all cell subtypes. We used these compounds, which were initially developed to maintain HSCs in culture, to create a new strategy for editing adult human HSCs. CD34+ HSPCs are edited, allowed to briefly cycle to accumulate HDR alleles, and then placed back in quiescence to maintain stemness, resulting in 6-fold increase in HDR/NHEJ ratio in quiescent, stem-enriched cells. Our results reveal the fundamental tension between quiescence and editing in human HSPCs and suggests strategies to manipulate HSCs during therapeutic genome editing.

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Section: Establishing the Timing Of Cell Cycle Status In Cd34+ Subsetmentioning

confidence: 51%

Controlled cycling and quiescence enables homology directed repair in engraftment-enriched adult hematopoietic stem and progenitor cells

Wyman

Bray

et al. 2018

Preprint

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“…Isolated CD4+ T cells were co-cultured at a 1:5 ratio with bone marrow-derived dendritic cells (BMDCs) for 3 days in the presence or absence of C. muridarum AR Nigg (5μg/mL). Expression of CD69 and Ki67 was determined by FACS surface and nuclear staining respectively, as described previously (36). Supernatants from dendritic-cell stimulated CD4+ T cells were collected and IL-2 concentrations determined by ELISA.…”

Section: Methodsmentioning

confidence: 99%

A Chlamydia-Specific TCR-Transgenic Mouse Demonstrates Th1 Polyfunctionality with Enhanced Effector Function

Poston

Girardi

et al. 2017

The Journal of Immunology

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Chlamydia is responsible for millions of new infections annually, and current efforts focus on understanding cellular immunity for targeted vaccine development. The Chlamydia-specific CD4 T cell response is characterized by the production of IFNγ, and polyfunctional Th1 responses are associated with enhanced protection. A major limitation in studying these responses is the paucity of tools available for detection, quantification, and characterization of polyfunctional, antigen-specific T cells. We addressed this problem by developing a TCR transgenic mouse with CD4 T cells that respond to a common antigen in Chlamydia muridarum and Chlamydia trachomatis. Using an adoptive transfer approach, we show that naïve transgenic CD4 T cells become activated, proliferate, migrate to the infected tissue, and acquire a polyfunctional Th1 phenotype in infected mice. Polyfunctional Tg Th1 effectors demonstrated enhanced IFNγ production compared to polyclonal cells, protected immune deficient mice against lethality, mediated bacterial clearance, and orchestrated an anamnestic response. Adoptive transfer of Chlamydia-specific CD4 TCR Tg T cells with polyfunctional capacity offers a powerful approach for analysis of protective effector and memory responses against chlamydial infection, and demonstrates that an effective monoclonal CD4 T cell response may successfully guide subunit vaccination strategies.

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“…As shown in Figure 3A-C, leukemia cells co-cultured with primary meningeal cells are less proliferative as shown by decreased Ki-67 staining, significantly decreased S phase, and increased G0/G1 phase. We next used Hoechst-Pyronin Y staining to better distinguish between G0 and G1 phases 29 . As shown in Figure 3D-F, leukemia cells in co-culture with primary meningeal cells exhibited increased G0 phase, indicative of quiescence, relative to leukemia cells in suspension.…”

Section: Resultsmentioning

confidence: 99%

“…Cell cycle and proliferation were assessed using a Click-iT Plus EdU flow cytometry kit (ThermoFisher). To differentiate G0 from G1 phases of the cell cycle, leukemia cells were stained with both Hoechst 33342 (Sigma-Aldrich) and pyronin Y (Sigma-Aldrich) and analyzed by flow cytometry 29 . To assess long-term quiescence in vivo , immediately prior to transplantation leukemia cells were labeled with the membrane dye DiR (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide; ThermoFisher) for microscopy or DiD (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; ThermoFisher) for flow cytometry 28,30–33 .…”

Section: Methodsmentioning

confidence: 99%

Disrupting the leukemia niche in the central nervous system attenuates leukemia chemoresistance

Jonart

Ebadi

Basile

et al. 2019

Preprint

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word count: 200 Text word count: 3955 Abstract 1Protection from acute lymphoblastic leukemia (ALL) relapse in the central nervous system (CNS) is 2 crucial to survival and quality of life for ALL patients. Current CNS-directed therapies cause significant 3 toxicities and are only partially effective. Moreover, the impact of the CNS microenvironment on 4 leukemia biology is poorly understood. Herein, we showed that leukemia cells associated with the 5 meninges of xenotransplanted mice, or co-cultured with meningeal cells, exhibit enhanced 6 chemoresistance due to effects on both apoptosis balance and quiescence. From a mechanistic 7 standpoint, we identified that leukemia chemoresistance is primarily mediated by direct leukemia-8 meningeal cell interactions and overcome by detaching the leukemia cells from the meninges. Next, 9 we used a co-culture adhesion assay to identify drugs that disrupted leukemia-meningeal adhesion. In 1 0 addition to identifying several drugs that inhibit canonical cell adhesion targets we found that 1 1Me6TREN, a novel hematopoietic stem cell (HSC) mobilizing compound, also disrupts leukemia-1 2 meningeal adhesion in vitro and in vivo. Finally, Me6TREN enhanced the efficacy of cytarabine in 1 3 treating CNS leukemia in xenotransplanted mice. This work demonstrates that the meninges exert a 1 4 critical influence on leukemia chemoresistance, elucidates mechanisms of CNS relapse beyond the 1 5well-described role of the blood-brain barrier, and identifies novel therapeutic approaches for 1 6 overcoming chemoresistance. 1 7 Introduction 1 8 Central nervous system (CNS) relapse is a common cause of treatment failure among patients with 1 9 acute lymphoblastic leukemia (ALL) 1-3 . Relapses occur despite CNS-directed therapies which include 2 0 high-dose systemic chemotherapy, intrathecal chemotherapy, and cranial irradiation in some high-risk 2 1 patients. These current CNS-directed therapies are also associated with significant acute and long-2 2 3 1 mice demonstrated that all, or most, B-cell ALL clones are capable of disseminating to the CNS 14,15 . 3 2 Third, CNS leukemia relapses occur despite high dose systemic and intrathecal chemotherapy. These 3 3therapies either overcome or bypass the blood-brain barrier. Fourth, it was shown that high Mer 3 4 kinase expressing, t(1;19) leukemia cells co-cultured with CNS-derived cells exhibit G0/G1 cell cycle 3 5 arrest, suggestive of dormancy or quiescence, as well as methotrexate resistance 16 . Similarly, Akers 3 6 et al. showed that co-culture of leukemia cells with astrocytes, choroid plexus epithelial cells, or 3 7meningeal cells enhanced leukemia chemoresistance 17 . Together these observations suggest that the 3 8 pathophysiology of CNS leukemia extends beyond the role of the blood-brain barrier. We hypothesize 3 9 that the ability of leukemia cells to persist in the unique CNS niche and escape the effects of 4 0 chemotherapy and immune surveillance likely also play critical roles in CNS leukemia and relapse. 4 1 4 2 However, while extensive...

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Assaying Cell Cycle Status Using Flow Cytometry

Cited by 225 publications

References 25 publications

Controlled cycling and quiescence enables homology directed repair in engraftment-enriched adult hematopoietic stem and progenitor cells

Controlled cycling and quiescence enables homology directed repair in engraftment-enriched adult hematopoietic stem and progenitor cells

A Chlamydia-Specific TCR-Transgenic Mouse Demonstrates Th1 Polyfunctionality with Enhanced Effector Function

Disrupting the leukemia niche in the central nervous system attenuates leukemia chemoresistance

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