Hematopoietic Stem Cells and Progenitors in Murine Autoimmune Arthritis

150 Pages·2017·4.43 MB·English

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All Theses and Dissertations (ETDs)
Spring 3-12-2013
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Kwadwo Asare Oduro Jr.
Washington University in St. Louis
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Oduro Jr., Kwadwo Asare, "Hematopoietic Stem Cells and Progenitors in Murine Autoimmune Arthritis"
(2013). All Theses and Dissertations (ETDs). 1052.
https://openscholarship.wustl.edu/etd/1052
This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has
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WASHINGTON UNIVERSITY IN ST. LOUIS
Division of Biology and Biomedical Sciences
Developmental Biology

Dissertation Examination Committee:
Kyunghee Choi, Chair
Marco Colonna
Daniel Link
Fanxin Long
Jason Mills
Deborah Novack

Hematopoietic Stem Cells and Progenitors in Murine Autoimmune Arthritis
by
Kwadwo Asare Oduro, Jr.

A dissertation presented to the
Graduate School of Arts and Sciences
of Washington University in
partial fulfillment of the
requirements for the degree
of Doctor of Philosophy

May 2013
St. Louis, Missouri
© 2013, Kwadwo Asare Oduro, Jr.
TABLE OF CONTENTS
List of Figures………………………………………………………………………………………………vi
List of Abbreviations………………………………………………………………………………………viii
Acknowledgements…………………………………………………………………………………….…..x
Abstract of the Dissertation………………………………………………………………………………xii

Chapters
Chapter 1: Introduction
• Physiological Maintenance of the Hematopoietic System………………………………………...2
• Lineage Priming and Phenotypic Identification of Hematopoietic Progenitors………………….3
• Primitive Progenitors in Non-Physiological States – Rationale for this study …………………..4
• KRNxG7 Arthritis Model………………………………………………………………………………6
• Importance of Myeloid Cells in Arthritis……………………………………………………………..7
• Relevance of K/BxN or KRNxG7 arthritis model to Human Disease…………………………….8
• References……………………………………………………………………………………………..9
• Figures………………………………………………………………………………………………..16

Chapter 2: General Characterization of Hematopoiesis in Arthritic Mice
• Overview………………………………………………………………………………………………19
• Primitive Progenitor Frequency Analysis………………………………………………………….19
o Similar Bone Marrow Primitive Progenitor Frequency and Turnover…………………19
o Increased Primitive Progenitors in Arthritic Spleen……………………………………..21
o Splenic Primitive Progenitor increase induced by T cell transfer arthritis…………….22
o Splenic Primitive Progenitor do not increase by mobilization from bone marrow……23
o Increased primitive progenitors in arthritic spleen is independent of TNFα………….25
o Summary of Primitive Progenitor Analysis……………………………………………….26
• Mature Cell and Committed Progenitor Frequency Analysis……………………………………26
ii
o Defective Bone Marrow B lymphopoiesis in arthritic marrow………………………….27
o Arthritic Marrow is myeloid skewed……………………………………………………….28
o Myeloid skewing in arthritic marrow is evident at the Committed Progenitor level….29
o Erythroid and B lymphoid lineage cells expanded in extramedullary sites…………...29
• References……………………………………………………………………………………………31
• Figures………………………………………………………………………………………………...37

Chapter 3: Cell Intrinsic Molecular Differences Between Arthritic and Control Primitive Progenitors
• Overview………………………………………………………………………………………………51
• Myeloid Priming in Arthritic KSL cells……………………………………………………………...52
o Arthritic KSL cells upregulate myeloid genes and downregulate erythroid genes…..52
o Induction of “Myeloid Inflammatory Signature” by Arthritogenic Serum Transfer……54
o “Myeloid Inflammatory Signature” genes are upregulated in Primitive HSCs………..55
• Features of Aging in Arthritic KSL cells……………………………………………………………56
o Elevation of IgG and P selectin proteins in Arthritic KSL cells…………………………57
• References……………………………………………………………………………………………60
• Figures………………………………………………………………………………………………..62

Chapter 4: Cell Intrinsic Functional differences between Arthritic and Control Primitive Progenitors
• Overview……………………………………………………………………………………………....77
• In vitro assessment of myeloid priming…………………………………………………………….77
o Arthritic KSL cells have Increased Myeloid Potential in vitro…………………………..77
o Arthritic KSL cells have diminished Lymphopoietic Propensity in vitro……………….79
o Increased myeloid and decreased lymphoid potentials induced by serum transfer...81
• In vivo assessment of myeloid priming…………………………………………………………….81
o Arthritic KSL cells have increased myeloid potential in vivo…………………………..81
o Myeloid priming occurs in the most immature HSC fraction of arthritic KSL cells….82
iii
• Molecular Mechanism of Myeloid Priming…………………………………………………………85
o Possible Roles for TLR4 and S100 proteins…………………………………………….85
• References……………………………………………………………………………………………88
• Figures………………………………………………………………………………………………...92

Chapter 5: Discussion and Future Directions
• Summary of Major Findings……………………………………………………………………….104
• Disease Relevance…………………………………………………………………………………104
o Role of primitive progenitor myeloid priming in disease………………………………104
o In situ involvement of primitive progenitors in arthritis………………………………...106
• Myeloid primed primitive progenitors in arthritis and normal aging……………………………109
• Primitive progenitor identification in other diseased states…………………………………….110

Chapter 6: Experimental Procedures
• Animals and Animal Treatment……………………………………………………………………118
o Animals
o Arthritis Transfer Models
o Mouse Antibody treatment
o Tissue Preparation
• Flow Cytometry……………………………………………………………………………………..121
o General Flow Cytometry
o Cell Cycle and Apoptosis Analyses
• Gene Expression Analysis…………………………………………………………………………123
o Microarray and Bioinformatics Analysis
o Quantitative Real Time PCR
• In vitro assays……………………………………………………………………………………….126
o Methylcellulose Colony Formation
iv
o In vitro Stromal Cell Free culture
o Osteoclast In vitro assay
o Differentiation on OP9 stromal cells
• In vivo assays……………………………………………………………………………………….128
o General Competitive Transplantation
o Young Recipient Competitive Transplantation – Supplemental Information
o Old Recipient Competitive Transplantation – Supplemental Information
o In situ Osteoclast Differentiaton of KSL cells
• Statistics……………………………………………………………………………………………..130
• Tables Associated with Experimental Proceedures…………………………………………….130
o Antibodies used in this study
o References for gene chips used for dendrogram analysis
o Quantitative RT-PCR primers used in this study
• References…………………………………………………………………………………………..133

v
LIST OF FIGURES
Chapter 1
Figure 1……………………………………………………………………………………………………16
Figure 2……………………………………………………………………………………………………17

Chapter 2
Figure 1…………………………………………………………………………………………………….37
Figure 2……………………………………………………………………………………………………39
Figure 3……………………………………………………………………………………………………41
Figure 4……………………………………………………………………………………………………43
Figure 5……………………………………………………………………………………………………44
Figure 6……………………………………………………………………………………………………46
Figure 7……………………………………………………………………………………………………47
Figure 8……………………………………………………………………………………………………48

Chapter 3
Figure 1……………………………………………………………………………………………………62
Figure 2……………………………………………………………………………………………………64
Figure 3……………………………………………………………………………………………………67
Figure 4……………………………………………………………………………………………………68
Figure 5……………………………………………………………………………………………………70
Figure 6……………………………………………………………………………………………………71
Figure 7……………………………………………………………………………………………………72

Chapter 4
Figure 1……………………………………………………………………………………………………92
Figure 2……………………………………………………………………………………………………93
vi
Figure 3……………………………………………………………………………………………………95
Figure 4……………………………………………………………………………………………………96
Figure 5……………………………………………………………………………………………………97
Figure 6……………………………………………………………………………………………………99
Figure 7…………………………………………………………………………………………………..100
Figure 8…………………………………………………………………………………………………..102

vii
LIST OF ABBREVIATIONS
B6 C57BL/6 mice
B6xG7 Progeny of crossing B6 and G7 mice
B6xKRN Progeny of crossing B6 and KRN mice
BM Bone Marrow
BrdU Bromodeoxyuridine
CLP Common Lymphoid Progenitor
c-Mpl Myeloproliferative Leukemia Virus Oncogene
EMH Extramedullary Hematopoiesis
FACS Fluorescence Activated Cell Sorting
G7 B6 congenic mice with MHC II I-Ag7 instead of I-Ab
G-CSF Granulocyte Colony Stimulating Factor
GFP Green Fluorescent Protein
GMP Granulocyte Monocyte Progenitor
HSC Hematpoietic Stem Cell
IFN Interferon
Ig Immunoglobulin
IgH Immunoglobulin Heavy Chain
IL Interleukin
K/BxN Progeny of crossing KRN and NOD mice
KRN A T cell receptor transgene or mice with this transgene
KRNxG7 Progeny of crossing KRN and G7 mice
KSL Kit+Sca1+Lin-
Lin Lineage (Mature Cell) Markers
LPS Lipopolysaccharide
LT-HSC Long Term Reconstituting Hematopoietic Stem Cell
viii