Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance

Zhuang, Lihui; Pound, John D.; Willems, Jorine J.L.P.; Taylor, Anne Marion; Forrester, Lesley M.; Gregory, Christopher D.

doi:10.1016/j.jim.2012.06.008

Cited by 23 publications

(24 citation statements)

References 36 publications

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“…Furthermore, in addition to the apoptotic neutrophils, bone marrow derived macrophages begin to appear, likely as a result of secretion of GM-CSF by eosinophils in the culture (Levi-Schaffer et al, 1995). Consistent with a recent description of embryonic stem cell-derived macrophages (Zhuang et al, 2012), we observed bone marrow derived macrophages that were proficient at phagocytosis of apoptotic cells (Figure 3A). On day 6 of the culture, the proportion of eosinophils in the culture outweighs the proportion of neutrophils.…”

Section: Resultssupporting

confidence: 92%

Purification of functional eosinophils from human bone marrow

Wong

Jelinek

2013

Journal of Immunological Methods

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Eosinophils are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states. More recently, they have also been shown to play a role in regulation of murine plasma cell homeostasis in the bone marrow, which prompted our investigation of human bone marrow eosinophils. However, effective methods to isolate eosinophils from human bone marrow thereby allowing comparisons with circulating eosinophils have not yet been described. Herein we describe the development of a novel, cost effective protocol for the purification of eosinophils from human bone marrow that allows us to obtain bone marrow eosinophils of near 100% purity after an 8-day culture system. Furthermore, we demonstrate that bone marrow eosinophils have characteristics similar to blood eosinophils, including the expression of IL-5Rα, the presence of eosinophil-specific granules, and similar activation kinetics upon phorbol myristate acetate and high-dose IL-5 stimulation. While migratory responses toward the chemokine CXCL12 differed between purified bone marrow and freshly isolated blood eosinophils, migratory responses were similar upon comparison of bone marrow eosinophils with blood eosinophils cultured ex vivo for 8 days prior to assay. Interestingly, a concurrent upregulation of CXCR4 expression was not observed in these cultured blood eosinophils. Taken together, we have overcome the existing challenges to the study of bone marrow eosinophils through our novel strategy for cell purification and have thus enabled future investigations of these cells and their role(s) in human health and disease.

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Section: Resultssupporting

confidence: 92%

Purification of functional eosinophils from human bone marrow

Wong

Jelinek

2013

Journal of Immunological Methods

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“…To differentiate mouse ES cells to macrophages, we adapted and modified the approach of Zhuang et al 8 ( Fig. 1a ).…”

Section: Resultsmentioning

confidence: 99%

“…Unlike many other cell lines, ES cells are normal diploid cells with virtually unlimited proliferative abilities and have the capacity to differentiate into many different cell types in vitro . Tremendous progress has been made in defining culture conditions to promote differentiation of mouse ES cells along various specific lineages 8 25 26 . Coupled with the success in achieving complete gene inactivation in ES cells, valuable information can now be gained on gene function.…”

Section: Discussionmentioning

confidence: 99%

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

Yeung

Hale

Xia

et al. 2015

Sci Rep

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The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

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“…Hematopoietic progenitors generated by these protocols on day 9 give rise to a variety of myeloid lineages when plated in MethoCult semisolid media containing SCF, IL3, IL6 and EPO (Stem Cell Technologies) (Fig 2c). Conventional feeder free protocols for macrophage differentiation from ES or iPS cells employ embryoid body formation using pre-selected fetal calf sera that support hematopoietic differentiation [14], [21]. Here we searched for combinations of cytokines and growth factors that direct macrophage differentiation in microculture under serum- and feeder- free conditions.…”

Section: Resultsmentioning

confidence: 99%

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

et al. 2014

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We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

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Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance

Cited by 23 publications

References 36 publications

Purification of functional eosinophils from human bone marrow

Purification of functional eosinophils from human bone marrow

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

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