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Generation of eosinophils from cryopreserved murine bone marrow cells.

ABSTRACT: Eosinophils are produced in the bone marrow from CD34+ eosinophil lineage-committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage-committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage-committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage-committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage-committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5.

SUBMITTER: Schollaert KL

PROVIDER: S-EPMC4281061 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Generation of eosinophils from cryopreserved murine bone marrow cells.

Schollaert Kaila L KL Stephens Michael R MR Gray Jerilyn K JK Fulkerson Patricia C PC

PloS one 20141231 12

Eosinophils are produced in the bone marrow from CD34+ eosinophil lineage-committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage-committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage-committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinoph ...[more]

PMID: 25551463

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Project description:Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.

Dataset Information

Generation of eosinophils from cryopreserved murine bone marrow cells.

Publications

Generation of eosinophils from cryopreserved murine bone marrow cells.

Similar Datasets

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