Dietary regulation of myelopoiesis and stromal cell isolation

Ousley, Carey

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https://hdl.handle.net/2142/112974 Copy

Description

Title

Dietary regulation of myelopoiesis and stromal cell isolation

Author(s)

Ousley, Carey

Issue Date

2021-06-29

Director of Research (if dissertation) or Advisor (if thesis)

McKim, Daniel B

Committee Member(s)

• Inoue, Makoto
• Woods, Jeffrey

Department of Study

Animal Sciences

Discipline

Animal Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• Myelopoiesis
• Atherosclerosis
• Mesenchymal Stromal Cell
• Endothelial Cell
• Hematopoiesis
• Flow Cytometry

Abstract

Chapter 1 Lifestyle factors such as stress and diet contribute to the pathogenesis of atherosclerosis in part by promoting increased myelopoiesis. Previous work showed that high dietary salt intake may enhance myelopoiesis. This suggests that salt-induced myelopoiesis may be a novel pro atherosclerotic mechanism. Here, the effect of two NaCl-supplemented diets on myelopoiesis was assessed in mice. Chow supplemented with either 5% or 10% sodium chloride did not alter monocyte, granulocyte, T cell, B cell, or NK cell distributions in blood. In spleen, 10% sodium chloride did reduce the presence of NK cells but had minimal effect on other cell populations. In bone marrow, 10% sodium chloride increased the presence of T cells and both diets reduced the presence of B cells but had little effect on other populations. Cell cycle analysis showed that both diets reduced the presence of proliferating hematopoietic progenitors in the spleen, while neither diet influence hematopoietic progenitor proliferation in the bone marrow. In contrast to some reports, high salt did not influence myelopoiesis in normocholesteremic mice. Chapter 2 Stromal cells are critical regulators of bone marrow hematopoietic niches, but assessment of their regulatory roles has been impeded by difficult and ineffective dissociation methods. Here, we methodically address bone marrow stromal cell dissociation. Yield of bone marrow CD45-/Ter119-/CD31+/CD202b+ endothelial cells (ECs) and CD45-/Ter119-/CD44-/PDGFR+mesenchymal stromal cells (MSCs) were determined by flow cytometry. Liberase DL, Collagenase D, and Dispase II (all supplemented with DNase) enhanced EC and MSC yields, with Dispase II + DNase proving most effective. Combinations of these enzymes did not exhibit additive benefits, nor did the addition of Elastase, TrypLE, Hyaluronidase, or Accutase. Similarly, common mechanical dissociation approaches also proved ineffective. However, the combination of gentle Dispase II + DNase dissociation with magnetic sorting dramatically enriched both ECs and MSCs. This work methodically addressed common approaches for bone marrow stromal dissociation and established an effective approach for enrichment.

Graduation Semester

2021-08

Type of Resource

Thesis

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http://hdl.handle.net/2142/112974

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Copyright 2021 Carey Ousley

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