The Pennsylvania State University. The Graduate School. Huck Institute of Life Sciences STUDY OF GROWTH OF STRESS ERYTHROID PROGENITORS FROM

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1 The Pennsylvania State University The Graduate School Huck Institute of Life Sciences STUDY OF GROWTH OF STRESS ERYTHROID PROGENITORS FROM PERIPHERAL BLOOD MONONUCLEAR CELLS IN MURINE AND HUMAN ANEMIC MODEL SYSTEMS. A Thesis in Molecular, Cellular and Integrative Biosciences by Sneha Hariharan 2016 Sneha Hariharan Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2016

2 ii The thesis of Sneha Hariharan was reviewed and approved* by the following: Robert F. Paulson Professor of Veterinary and Biomedical Sciences Thesis Advisor Melissa Rolls Associate Professor of Biochemistry and Molecular Biology Chair of the Molecular, Cellular and Integrative Biosciences Graduate Program K. Sandeep Prabhu Professor of Immunology and Molecular Toxicology Pamela A. Hankey Giblin Professor of Immunology Immunology and Infectious Disease Undergraduate Advisor *Signatures are on file in the Graduate School

3 ABSTRACT Chronic anemia is one of the predominant blood disorders that causes morbidity and mortality in patients and severely affects the quality of life in patients. Current treatments for anemia include blood transfusions, recombinant erythropoietin treatment and in severe cases bone marrow transplantation, which aim at restoring normal hemoglobin and erythrocyte levels. However, research shows that prolonged use of any of these treatments can lead to infectious and noninfectious complications in case of blood transfusions, venous thromboembolism and hyporesponsiveness in case of Epo treatment. These drawbacks call for an alternative treatment that can be used in the long term with minimal complications, ideally using a natural defense mechanism of the body to anemia. Steady State erythropoiesis occurs in the bone marrow and is primarily homeostatic. In response to anemic stress the need for new erythrocytes quickly outpaces the erythropoietic capacity of steady state erythropoiesis. At these times, stress erythropoiesis predominates. Unlike steady state erythropoiesis, which generates erythrocytes at a constant rate, BMP4 dependent stress erythropoiesis relies on mobilization, expansion and differentiation of distinct stress erythroid progenitors, leading to generation of a larger number of erythrocytes. Stress erythropoiesis is best understood in mice where this process is primarily extra-medullary occurring in the adult spleen and liver and in the fetal liver during development. Although stress erythroid progenitors develop in the adult spleen, they originate in the bone marrow and once they migrate to the spleen they acquire the characteristics of stress progenitors. This thesis presents an in depth study of the migration of stress erythroid progenitors from the bone marrow to peripheral blood and ultimately the spleen. Here, we demonstrate the growth and identification of stress erythroid progenitors from peripheral blood mononuclear cells in mice with acute anemia and study the mechanism regulating the migration of these stress progenitor populations. In addition, we extended this study to the analysis of stress progenitors in human sickle cell anemia patients. iii

4 iv TABLE OF CONTENTS List of Figures..... vi Acknowledgements..... vii Chapter 1 Introduction.1 Research Background.2 Impact..6 Chapter 2 Murine anemic model: Functional assay analysis Background...7 Experimental Method Results 10 Chapter 3 Cxcl12 expression in concert with stress erythroid progenitors migration Background...14 Experimental Method Results 15 Chapter 4 Effect of Sympathetic Nervous System (SNS) on mobilization of stress erythroid progenitors...17 Background...17 Experimental Method Results OHDA Isoproterenol. 20 Chapter 5 Stress erythroid progenitors in Sickle Cell Disease patient PBMCs in human Model Background...22

5 v Experimental Method Results 24 Chapter 6 Conclusion.. 28 Future work 30 Appendix : Supplementary data..32 Appendix A: Flow cytometry analysis of PBMCs obtained from SCD patient samples and control samples before and after culture.32 Appendix B: Flow Cytometry analysis of murine anemic PBMC stress progenitors...38 Bibliography..39

6 vi LIST OF FIGURES Figure 1-1: Model for stress erythropoiesis Figure 1-2: Stress erythropoiesis progenitors... 4 Figure 2-1: Model for stress erythropoiesis Figure 2-2: Schematic of progenitor population generated during stress erythropoietic recovery in mice...8 Figure 2-3. Experimental set up for study of migration of stress erythroid progenitors to peripheral blood in acute anemic model in mice. 9 Figure 2-4: Induction of acute anemia in C57BL/6 mice and isolation of Peripheral Blood Mononuclear Cells at different time points...11 Figure 2-5: Stress BFU-E colonies developed from expanded progenitors derived from peripheral blood cells.12 Figure 3-1: Changes in relative mrna expression of Cxcl12 (SDF-1) and its cognate receptors Cxcl4 and Cxcl7 during the course of PHZ treatment..16 Figure 4-1: Analysis of migration of stress erythroid progenitors under conditions of ablation of SNS with administration of norepinephrine analog 6-OHDA..19 Figure 4-2: Analysis of migration of stress erythroid progenitors under conditions of stimulation of SNS with administration of non selective β-adrenergic agonist Figure 5-1: Schematic representation of experimental protocol for study of stress erythroid progenitors in PBMCs of human Sickle Cell Disease patient samples...23 Figure 5-2: Functional colony assay to characterize stress BFU-Es identified from expanded PBMCs of multiple human anemic patients Figure 5-3: Flow cytometry analysis of PBMCs obtained from normal human Peripheral blood sample (control).26 Figure 5-4: Flow cytometry analysis of PBMCs obtained from SCD patient sample expressing high numbers of stress BFU-E colonies after culture Figure 6-1. Schematic representation of migration of stress progenitors during acute anemia in mice 29

7 vii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Robert F Paulson, for his unfailing encouragement and guidance throughout my graduate studies. I am especially thankful for his unwavering faith and patience during periods of slow progress and when tough decisions had to be made. Thank you to Dr. Jane Little for her enthusiasm about the project and the opportunity to work with human anemic patient samples. My sincere thanks to Dr. Pamela Hankey for being an amazing committee chair, whose unflagging support of my work and progress has been inspirational. I am particularly grateful to Dr. K Sandeep Prabhu, friend and collaborator to all at Paulson Lab, whose support has carried me through many experiments over the years. I am very grateful to Dr. Ross Hardisson, whose enthusiasm has resulted in many fruitful collaborations with his lab, in addition to making him a great committee member. My special thanks to Ruth Nissy and the entire flow cytometry lab for the many, many of patience while helping me with the running of my experiments. Finally, none of this would have been possible without the unwavering love and support of my family and friends, to whom I will always be grateful.

8 1 Chapter 1 Introduction Anemia is one of the most prominent blood disorders. Studies show that anemia is independently associated with patient morbidity and mortality. It can be manifested as the primary disorder or as a secondary pathology to other diseases including chronic disorders 1,4,5,25. Chronic anemia can be the primary disease as in the case of congenital anemic disorders like Sickle Cell Anemia (SCA) and Thalassemia 1, 11 or a secondary pathology that is associated with chronic conditions like cancer or Chronic Kidney Disease (CKD). Current treatment strategies for anemia focus on restoring normal red blood cell and hemoglobin levels in order to regulate oxygen supply to tissues. These treatments include blood transfusions and stem cell transplantations in severe cases. Arguably, blood transfusion is the fastest and most effective way of treating anemia. Yet, various studies have shown that prolonged transfusions can lead to infectious and non-infectious complications. 2,3,4. An alternate therapy used to treat non-congenital anemia involves injection of human recombinant erythropoietin (Epo) in order to stimulate erythropoiesis 6. Even though it is effective and reduces the requirement of blood transfusions in patients, Epo treatment has been shown to increase incidents of venous thromboembolism in patients and is less effective in cancer patients due to the possible interference with chemotherapy. Furthermore there is a possibility of hypo-responsiveness to Epo treatment over prolonged periods of time These drawbacks in the prolonged use of current treatments call for an alternate mechanism that was more efficient in the long-term treatment of chronic anemia. An alternative approach to treating chronic anemia could be to understand the response of the body to anemia and develop therapies that take advantage of this mechanism. Recent studies have shown that, in mice, anemia induces a specific bone

9 2 morphogenic protein 4 (BMP4) dependent physiological stress response pathway, which generates a large number of erythrocytes in order to increase oxygen delivery to tissues. Unlike steady state erythropoiesis, which generates erythrocytes at a constant rate, BMP4 dependent stress erythropoiesis relies on mobilization, expansion and differentiation of specific stress erythroid progenitors, leading to generation of a much larger number of erythrocytes. Work in our lab showed that stress erythroid progenitors initially expand without differentiation. Once the pool of progenitors is large enough, the cells transition to differentiating progenitors and synchronously generate new erythrocytes. Recently, these general themes associated with stress erythropoiesis have been identified in human anemic responses, suggesting the possibility of a similar system in humans 12,13. A thorough understanding of this stress erythropoietic pathway could lead to novel long-term treatment of chronic anemia. This thesis aims at understanding the migration of stress erythroid progenitors to peripheral blood. Here, we demonstrate the growth and identification of stress erythroid progenitors from peripheral blood mononuclear cells in mice with acute anemia. It also aims at translating the understanding of this system to human stress erythropoiesis and potentially identifying stress erythroid progenitors in the peripheral blood of anemic human patients. Research Background: Stress erythropoiesis is a mechanism by which the body produces a large number of erythrocytes in response to acute anemia. Unlike steady state bone marrow erythropoiesis, which is a homeostatic process that produces erythrocytes at a constant rate, stress erythropoiesis attends to the need for a higher number of erythrocytes to counteract anemic stress. In mice, this is an extramedullary response that occurs in fetal liver during development and in adult spleen and liver. In adult mice, this process occurs predominantly in the spleen. The microenvironment of the spleen induces rapid proliferation and differentiation of specific stress erythroid progenitor cells. Current

10 3 work in the lab has led to the development of a model for stress erythropoiesis 13. Tissue hypoxia caused by acute anemia induces BMP4 expression, which promotes the expansion and differentiation of specific stress erythroid progenitors 22. Study of erythroid recovery from bone marrow transplantation experiments has expanded the current model of BMP4 dependent stress erythropoiesis 19. Initially, after transplantation, the short-term reconstituting hematopoietic stem cells (STR-HSCs) migrate to the spleen, where, in response to HedgeHog (HH) 17 and BMP4 they become specified as stress erythroid progenitors. Early stress progenitors undergo a step of rapid amplification that allows the progenitors to generate sufficient progeny so that, when they differentiate, they produce enough erythrocytes to ensure the survival of the mouse. During this amplification stage the stress progenitors are unable to differentiate. The current model for stress erythropoiesis is illustrated in Figure 1-1. Using bone marrow transplant as an assay we showed that donor derived stress erythroid progenitors are capable of establishing a durable stress erythroid response compartment that responded to subsequent anemic challenges, thereby suggesting that stress erythropoiesis has a potential to treat chronic anemia in the long term. Figure 1-1: Model for stress erythropoiesis: A schematic representation of the steps in the development and differentiation of stress erythroid progenitors.

11 4 Using CD45 alleles to distinguish donor and recipient cells, different populations of stress erythroid progenitors have been identified. The STR-HSCs are characterized by CD34+Kit+Sca1+Lin- (34KSL) cell surface markers 15,16,18. This population of cells, though identified as precursors for stress erythropoiesis progenitors, is not capable of forming BFU-Es (Burst Forming Units-Erythroid) indicating the role of signals from other cells in the unfractionated bone marrow in specifying the lineage. After migration to the spleen, these cells respond to BMP4, HH and GDF15 18,20,21 signaling from the spleen microenvironment, producing the amplifying stress progenitor population characterized by Kit+Sca1+CD34+CD133+ markers. These expanding stress progenitors are unable to form stress BFU-Es. Anemia and tissue hypoxia leads to an up-regulation of Epo expression by kidney. The increase in serum Epo coupled with BM' Spleen' Epo'Receptor'nega1ve' CD34ACD133+' Kit+Sca1+CD71+' CD34ACD133A' Kit+Sca1ACD71+' Ter119 lo' CD34+KSL' CD34+CD133+' Kit+Sca1+CD71+' CD34ACD133A' Kit+Sca1+' CD71+Ter119 lo' hypoxia Figure'2:'Stress'erythropoie1c'progenitors'' act in concert with BMP4 and stem cell factor to induce the transition of amplifying progenitors to differentiating progenitors. These differentiating progenitors are characterized by kit+sca1+cd34- CD133- cell surface markers and are capable of producing stress BFU-Es. (Figure 1-2). Stress'erythropoie1c'Stem'cells ' Erythroid'restricted,'self'renewing' Provide'erythroid'ShortAterm'radioprotec1on' when'transplanted' Figure 1-2: Stress erythropoiesis progenitors BMP4'responsive'' Stress'BFUAE ' Another aspect of stress erythropoiesis to be studied is the mobilization of progenitors. Previous work has established that cells destined to become BMP4 dependent stress progenitors are mobilized to migrate out of the bone marrow. Initially, bone marrow cells home to spleen and in

12 5 response to HedgeHog signal in the splenic microenvironment, they develop into BMP4 responsive stress progenitors. In response to acute anemia these cells are induced to differentiate. In fact, there is an almost complete differentiation of these cells during the recovery from anemia. New stress progenitors are replenished in the spleen leading to the development of a new wave of BMP4 responsive stress progenitors, which can differentiate in response to the next anemic challenge 17. BMP4 dependent stress erythropoietic progenitors have not been characterized in peripheral blood. However, analysis of Sickle cell mice identified a compensatory hematopoietic mechanism in these mice. Analysis of bone marrow, spleen and peripheral blood samples in sickle cell mice showed that bone marrow erythropoiesis showed a marked depletion in BFU-E progenitors despite an increase in proliferation. This observation is highly suggestive of the fact that the compensatory mechanism they were observing was extra medullary erythropoiesis. Interestingly, the study also showed mobilization of BFU-E early progenitors into the peripheral blood and identified these progenitors by the expression of Sca + Lin - in the peripheral blood 27. This situation is similar to progenitors identified in peripheral blood samples of sickle cell anemia and thalassemia patients, which identified a population of progenitors marked by the expression of CD34, Glycophorin A (GpA), CD71 and KIT. This observation suggests that this progenitor population may be the human equivalent of early expanding stress progenitor cells population (CD34+CD133+Kit+Sca1+). In addition, analysis of these progenitors from patients showed that they express high levels of fetal hemoglobin (HbF). Study of bone marrow transplant patients and some patients of chronic anemic diseases such as sickle cell anemia and thalassemia have shown that erythrocytes generated in response to anemic stress exhibit characteristics of fetal erythrocyte antigens and express HbF. Increase in HbF expression has also been observed in studies done on non-human primates, such as baboons after phenylhydrazine induced acute anemia. Expression of HbF in sickle cell anemia and thalassemia patients is therapeutic. This proposal aims to further understand the migration of murine BMP4

13 6 dependent stress erythropoietic progenitors and their characterization in peripheral blood. It also aims to extend the study to human anemic model and correlate stress erythropoiesis to the clinical parameters in human anemia. Impact: This thesis characterizes the mobilization of stress erythroid progenitors from the bone marrow into peripheral blood during BMP4 dependent stress erythropoiesis in mice and extends the understanding of this process to identify stress progenitor in peripheral blood of anemic human patients in an attempt to manipulate the stress erythropoiesis pathway as a viable therapy to treat chronic anemia.

14 7 Chapter 2 Murine anemic model: Functional assay analysis Background: Work in our laboratory established a new model for stress erythropoiesis, where a specialized population of stress erythroid progenitors is rapidly mobilized in response to anemia to produce large numbers of new erythrocytes (Figure 2-1). In the mouse, this response is extra-medullary in that it occurs in the fetal liver during development and in the adult spleen and liver 15. Following recovery, new progenitors are replenished in the spleen by cells migrating from the bone marrow. Figure 2-1: Model for stress erythropoiesis. The Figure depicts the stages of stress erythropoiesis with the signals involved depicted below each step. Severe anemia leads to the complete mobilization of BMP4 dependent stress erythroid progenitors. Following recovery, a new population of progenitors is replenished through the

15 8 migration of bone marrow short term reconstituting hematopoietic stem cells (STR-HSCs) into the spleen. Once in the spleen, they interact with Hedgehog (HH) ligands, which specify the stress erythroid fate 15. The stress progenitors next rapidly expand but are unable to differentiate. The increase in serum epo concentration leads to a transition from expanding progenitors to differentiating progenitors. As cells go through these developmental stages they change their cell surface markers (Figure 2-2). Figure 2-2: Schematic of progenitor population generated during stress erythropoietic recovery in mice. Populations of cells that can provide erythroid rescue of anemic mice are shown in the pink box. Previous work studying stress erythropoiesis has been effective in characterizing the distinct populations of stress erythroid progenitors originating in the bone marrow and their development from early expanding progenitor population to later erythroid progenitor populations capable of differentiation in spleen. However, we are yet to understand the process of migration of these early stress erythroid precursors from the bone marrow to spleen where they maintain the progenitor population and the potential mechanisms involved in the regulation of the same. In this chapter, we study the migration of these stress progenitors and attempt to characterize the mobilized stress progenitors.

16 9 Experimental Method: Figure 2-3: Experimental set up for study of migration of stress erythroid progenitors to peripheral blood in acute anemic model in mice. 6 to 8 weeks old wild type C57BL/6 mice were injected with a dose of Phenyl Hydrazine at 100mg/kg. After induction of acute anemia, Peripheral blood was isolated by performing cardiac puncture at different time points (12 hrs, 24 hrs, 36 hrs, 48 hrs, 3 days, 5 days, 7 days and 10 days) with 6 replicates for each time point. The isolation of PBMCs from the peripheral blood obtained was performed using density gradient protocol of Histopaque 1083 (murine PBMC isolation). Once isolated, the PBMCs were cultured in Differentiation media containing IMDM media containing 20% fetal bovine serum + Shh(25ng/mL) + BMP4(15ng/mL) + GDF15(30ng/mL) + SCF(15ng/mL) + Epo(3ng/mL) and 2%O 2 for 5 days. The functional assay for stress BFU-Es was then performed by plating 0.5 x 10 5 expanded cells/ ml of methylcellulose media (StemCell Technologies, Vancouver, BC, Canada) containing 3 U/mL Epo (Figure 2-3).

17 10 Flow cytometry analyses of murine PBMCs in acute anemic murine model: The flow cytometry assay was performed using fluorochromes APC (C-Kit), PE (Sca1), FITC (CD34) and PE-Cy5 (CD133) in the flow cytometer Fortessa. The results were analyzed using the Flow Jo software. Results: The hematocrit was determined for every time point including 0 (control) to confirm the induction of anemia with PHZ injection. Figure 2-4a shows that at 0 hrs. the mice have normal hematocrit. With progression of time the hematocrit steadily decreases representing anemia with the lowest value observed at 48 hrs. after injection. We also observe that by days 6-7 the mice have recovered from acute anemia, which is in line with previous studies from the lab and represents the activation of the BMP4 dependent stress erythropoiesis. In figure 2-4b the data shows the isolation of peripheral blood mononuclear cell at different time points. The results indicate that there is a steady increase in the number of PBMCs isolated from the same volume of blood in the initial stages of acute anemia which increases up to almost 2 fold at 3 days post PHZ treatment (Figure 2-4c). As the mice recover from the anemia the number of PBMCs decreases and by day 10 it reaches levels comparable with the control (day 0). Figure 2-4c represents the ratio of expanded cells obtained after culture in differentiation media with respect to PBMCs obtained at every time point. The result shows that the number of expanded cells steadily decreases as anemia is induced until 60. As the mice begin to recover from anemia using the stress erythropoietic pathway the expanded cells begin to increase again.

18 11 A Hematocrit days 5 days 6 days 7 days 10 days B PBMC/ml Blood days 5 days 6 days 7 days 10 days C 1 Expanded cells/pbmc days 5 days 6 days 7 days 10 days Figure 2-4: Induction of acute anemia in C57BL/6 mice and isolation of Peripheral Blood Mononuclear Cells at different time points. A). Hematocrit at various time points after injection representing induction of acute anemia B)number of PBMNCs per ml of blood at the indicated time points post PHZ treatment C)Expansion of stress erythroid progenitors from isolated PBMCs in vitro

19 12 Figure 2-5 represents the functional assay demonstrating the identification of stress erythroid progenitor cells derived from culture of PBMCs. Figure 2-5a shows the number of stress BFU-Es obtained from expanded cells with respect to PBMCs obtained. The data shows that stress BFU- Es can be derived from PBMCs, but precursor cells are only observed after the recovery from anemia is well underway and after the peak expansion of stress BFU-Es in the spleen at 36. We observe an approximately 900 fold increase in stress BFU-E potential at 3 days post PHZ treatment. Figure 2-5b shows the number of BFU-Es as a function of the expanded cells. Once again we observe the greatest increase at 3 days post PHZ treatment. Taken together, these data support the idea that migration of stress progenitor precursors is regulated and is initiated once the recovery from anemia is under way. Flow cytometric analysis of the stress progenitors was performed at different time points. However, due to the in vivo treatment with phenylhydrazine, the analysis of the results was not significant. PHZ is known to interfere with the fluorescence in FACS analysis. Despite in vitro culture of the PBMCs in a culture devoid of PHZ, severe autofluorescence at all different time points compromised the significance of the flow data (Appendix B) A BFUEs/PBMC frequency days 5 days 6 days 7 days 10 days

20 13 B BFUEs/expanded cells days 5 days 6 days 7 days 10 days Figure 2-5: Stress BFU-E colonies developed from expanded progenitors derived from peripheral blood cells A) Frequency of Stress BFU-Es as a function of peripheral blood mononuclear cells B). Stress BFU-E frequency as a function of stress progenitors expanded from PBMCs. Both data are calculated as (average no. of BFU-E colonies * No. of PBMCs or Expanded cells)/ (No..f cells plated)

21 14 Chapter 3 Cxcl12 expression in concert with stress erythroid progenitors migration Background: Having established the identification of stress progenitor cells in expanded PBMCs, we ventured to understand the mechanism controlling the migration of stress erythroid progenitors. Previous work shows that under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the activation of β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation is elevated, leading to an increased output of neutrophils and inflammatory monocytes. 36 Interaction of nervous system in controlling the migration of hematopoietic stem and progenitor cells (HSPC) via Cxcl12 has also been studied previously. HSPC, attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). They demonstrated that enforced HSPC egress from BM niches depends critically on the nervous system. 37 Studies show that CXCL12/SDF-1 dynamically regulates hematopoietic stem cell (HSC) attraction in the bone marrow. Circadian regulation of bone formation and HSC traffic is relayed in bone and BM by β -adrenergic receptors (β -AR) expressed on HSCs, osteoblasts and mesenchymal stem /progenitor cells. 40 The lowest Cxcl12 expression levels in murine bone marrow coincide with the peak of blood HSCs in the morning, and the highest Cxcl12 levels in the evening match the lowest circulating HSC counts. 38, 39. Studies also show the effect of expression of Cxcl12 cognate receptors CXCR4 and CXCR7 on the egression of HSCs. The disruption of CXCL12 interaction with CXCR4, its cognate receptor, using specific small CXCR4 inhibitor molecule is sufficient to

22 15 induce HSC mobilization from the BM to the peripheral circulation. While circadian release of norepinephrine (NE) by nerve terminals in the BM leads to rhythmic Cxcl12 downregulation, possibly via reduced Sp1 nuclear content in stromal cells, The expression of CXCR4 in HSCs also follows circadian oscillations, suggesting that a coordinated expression of secreted molecules and ligands regulates the steady-state traffic of HSCs. 40 Based on the literature review demonstrating the importance of Cxcl12 expression in migration of steady state hematopoietic stem cells can be affected by the circadian rhythm and the regulation of Cxcl12 under chronic variable stress in mice, we hypothesize that acute anemia affects the expression of Cxcl12 and its cognate receptors CXCR4 and CXCR7, which in turn inversely effects the migration of early hematopoietic stress progenitors from the bone marrow. Experimental Method: Mice were treated with PHZ (100mg/kg) and on the indicated days (chapter 3), RNA from bone marrow cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer s instructions. Relative expression of Cxcl12, Cxcr4 and Cxcr7 mrna was analyzed by reversetranscribed PCR (RT-PCR) using Taqman Probes. Results: The relative mrna expression levels of Cxcl12 and its cognate receptors Cxcr4 and Cxcr7 indicate a correlation with mobilization of stress erythroid progenitors when observed in relation with the stress BFU-Es formation as observed in Figure 2-5b. In figure 3-1a, the low Cxcl12 mrna expression until day 3 correlates with the identification of BFU-E potential migrated progenitors identified in chapter 2 with the exception of the peak at 60. However, this aberration in the result can be explained in combination with the mrna expression of the cognate receptors. The relative mrna expression of cognate

23 16 receptors CXCR4 and CXCR7 fits well with our hypothesis. The downregulation of the receptor mrna until day 3 interferes with the activity of Cxcl12 in the bone marrow, thereby aiding the mobilization of stress erythroid progenitors from the bone marrow. This also explains the observation of stress BFU-Es in expanded anemic PBMCs (Figure 2-5). Additionally, the small spike in stress BFU-Es observed at day 10 corresponds with the downregulation of Cxcl12 and its cognate receptors at that time point which may be indicative of a second wave of stress erythroid progenitor cells migration in the anemic murine model (Figure 3-1b). A Cxcl12 qpcr - PHZ treatment D0 36H 48H 60H d3 d5 d6 d7 d10 B cxcr7 cxcr D0 36H 48H 60H d3 d5 d6 d D0 36H 48H 60H d3 d5 d6 d7 Figure 3-1: Changes in relative mrna expression of Cxcl12(SDF-1) and its cognate receptors Cxcl4 and Cxcl7 during the course of PHZ treatment. A). qrt-pcr analysis of Cxcl12 expression in bone marrow following induction of acute anemia in mice. B) Relative changes in Cxcl12 receptors.

24 17 Chapter 4 Effect of Sympathetic Nervous System (SNS) on mobilization of stress erythroid progenitors Background: We have established in the previous chapter that Cxcl12 and its cognate receptors CXCL4 and CXCL7 are involved in the regulation of mobilization of stress erythroid progenitors under acute anemic conditions in murine model. This chapter further explores the mechanism involved in the mobilization of stress progenitors. It has been shown that under homeostasis, small numbers of hematopoietic stem cells (HSCs) are detectable in the bloodstream of mammals. A recent study exploring the mechanism of their trafficking showed that the cyclical release of HSCs and expression of Cxcl12 are regulated by core genes of the molecular clock through circadian noradrenaline secretion by the sympathetic nervous system. These adrenergic signals are locally delivered by nerves in the bone marrow, transmitted to stromal cells by the β3-adrenergic receptor, leading to a decreased nuclear content of Sp1 transcription factor and the rapid downregulation of Cxcl12. They used isoprenaline (isoproterenol), a non-selective β-adrenergic agonist, to show the decrease CXCL12 production in a dose-dependent manner 41 Another study explored the possibility that signals emanating from the nervous system participate in HSPC mobilization. To test this hypothesis, they disrupted catecholaminergic neurons by serial perinatal injections of 6-hydroxydopamine (6OHDA) and found that the number of HSPCs mobilized by G-CSF was dramatically reduced in 6OHDA-lesioned mice compared to littermates injected with vehicle control. 6OHDA is a norepinephrine (NE) analog 6- hydroxydopamine (6-OHDA) that mediates chemical sympathectomy 37

25 18 These studies show that the Sympathetic Nervous System is involved in the regulation of HSC mobilization in steady state. The results from Chapter 5 show that Cxcl12 and its cognate receptors CXCR4/7 may affect mobilization of stress erythroid progenitors under conditions of acute stress. Since we know that under steady state condition Cxcl12 expression is regulated by noradrenaline secretion by the sympathetic nervous system, we explore the effect of SNS on mobilization of stress erythroid progenitors under conditions of acute anemia using β3 adrenergic analog, 6OHDA, which would lead to sympathectomy (ablation of SNS) and a non selective β adrenergic agonist, isoproterenol, which stimulates the SNS. Experimental Method: 1) 6-8 weeks old 6 to 8 weeks old wild type C57BL/6 mice (3/group) were injected with 200 mg/kg 6-hydroydopamice (6-OHDA) (Sigma-Aldrich) in 0.9% NaCl plus 10-7 M ascorbic acid (Sigma-Aldrich) on day-3 and 100 mg/kg on day-5 and day-7. The control group was injected with the vehicle. On day 7, the mice were injected with a weight dependent dose of Phenyl Hydrazine at 100mg/kg. After induction of acute anemia, Peripheral blood was isolated by performing cardiac puncture at different time points (12 hrs, 24 hrs, 36 hrs, 48 hrs, 3 days, 5 days, 7 days and 10 days). The isolation of PBMCs from the peripheral blood obtained was performed using density gradient protocol of Histopaque 1083 (murine PBMC isolation). Once isolated, the PBMCs were cultured in Differentiation media containing IMDM media containing 20% fetal bovine serum + Shh(25ng/mL) + BMP4(15ng/mL) + GDF15(30ng/mL) + SCF(15ng/mL) + Epo(3ng/mL) and 2%O 2 for 5 days. The functional assay for stress BFU-Es was then performed by plating 0.5 x 10 5 expanded cells/ ml of methylcellulose media (StemCell Technologies, Vancouver, BC, Canada) containing 3 U/mL Epo. 2) 6-8 weeks old 6 to 8 weeks old wild type C57BL/6 mice (3/group) were injected with 100 mg/kg of isoproterenol in 0.9% NaCl. The control group was injected with the vehicle. 3 days

26 later, the mice were injected with Phenyl Hydrazine at 100mg/kg. PBMC isolation and analysis at different time points was performed as described with 6-OHDA protocol. 19 Results: 6-OHDA The migration of stress erythroid progenitors in the peripheral blood was functional assay for stress BFU-Es in expanded cells from acute anemic mice after ablation of the sympathetic nervous system by injection of 6-hydroxydopamine (6OHDA). Compared to Stress BFU-Es from observed from expanded PBMCs in acute anemic mice (Figure 2-5), there is ~900 fold decrease in stress progenitor colonies in mice with ablated SNS (Figure 4-1a) when graphed as a function of PBMCs and ~300 fold decrease when graphed as a function of expanded cells (Figure 4-1b). Some colonies are observed around the 3-5 days mark but they are minimal and do not contribute to migration of stress erythroid progenitors. This experiment also observed a high rate of mortality amongst the experimental mice (~70%), which can be explained by the lack of migration of stress erythroid progenitors, which would be crucial for the recovery of the mice from acute anemia using the BMP4 dependent stress erythropoietic pathway or by severe dehydration caused by 6-OHDA treatment (Figure 4-1c). A BFU-Es/PBMC frequency with and without 6- OHDA treatment OHDA +PHZ treatment PHZ treatment days 5 days 7 days

27 20 B BFU-Es/expanded cells frequency with and without 6-OHDA treatment days 5 days 7 days 6-OHDA +PHZ treatment PHZ treatment C Mice survival ajer treatment with 6- OHDA and PHZ h 12h 24h 36h 48h 3days 5days 7days Figure 4-1: Analysis of migration of stress erythroid progenitors under conditions of ablation of SNS with administration of nor epinephrine analog 6-OHDA. A) Frequency of stress BFU-Es cultured from expanded PBMCs of anemic mice with respect to peripheral blood mononuclear cells with and without 6-OHDA treatment B). Stress BFU-E frequency as compared to stress progenitors expanded from PBMCs with and without 6-OHDA treatment (Calculated as shown in figure 2-5). C) Survival curve showing the number of mice surviving after treatment with 6-OHDA leading to SNS ablation followed with treatment of PHZ inducing acute anemia. Isoproterenol The stimulation of SNS using a nonselective β-adrenergic analog isoproterenol induces the migration of stress erythroid progenitors from the bone marrow to the peripheral blood a little

28 21 earlier at 36 as compared to treatment with PHZ alone as seen (Figure 4-2). Consistent with previous data, the migration of stress erythroid progenitors in expanded PBMCs peaks at 3 days with the first wave of stress erythropoiesis under anemic conditions. The injection of saline alone did not affect the migration of stress erythroid progenitors (data not shown). The number of stress BFU-Es with respect to expanded progenitors is ~100 fold higher than those observed with ablated SNS (Figure 4-1). A BFUEs/PBMC frequency - Isoproterenol days 5 days 7 days B BFUEs/Expanded cells - Isoproterenol days 5 days 7 days Figure 4-2: Analysis of migration of stress erythroid progenitors under conditions of stimulation of SNS with administration of non-selective β-adrenergic agonist. A) Frequency of stress BFU-Es cultured from expanded PBMCs of anemic mice with respect to peripheral blood mononuclear cells B). Stress BFU-E frequency as compared to stress progenitors expanded from PBMCs (Calculated as shown in figure 2-5).

29 22 Chapter 5 Stress erythroid progenitors in Sickle Cell Disease patient PBMCs in human Model Background: Our lab has extended the analysis of stress erythropoiesis to cultures of human bone marrow, where we observed the expansion of a similar population of stress erythroid progenitors (CD34+CD133+Kit+) that form stress BFU-E colonies in vitro. Human stress erythropoiesis is thought to be similar to fetal erythropoiesis and we observe that human stress BFU-E express fetal Hb. 42, 43 Study of bone marrow transplant patients and some patients of chronic anemic diseases such as sickle cell anemia and thalassemia have shown that erythrocytes generated in response to anemic stress exhibit characteristics of fetal erythrocyte antigens and express HbF. Increase in HbF expression has also been observed in studies done on non-human primates, such as baboons after Phenylhydrazine induced acute anemia. Expression of HbF in sickle cell anemia and thalassemia patients is therapeutic. Our analysis of murine stress erythroid progenitors showed that they could provide erythroid short-term radioprotection when transplanted into irradiated recipients. It is not possible to test the ability of human stress erythroid progenitors in a clinical transplant setting, but human cells can be transplanted into irradiated immunocompromised mice (NOD.Cg-PrkdcscidIl2rgtm1Sug/J; termed NOG). NOG mice show greatest development of CD235a+cells when compared to other immunocompromised strains. 44 Despite all the inferences that can be obtained from these observations that strongly suggest a parallel stress erythropoietic pathway in human anemic models, it is difficult to characterize the progenitors in human anemic patient samples. Based on the study described in this thesis, we propose to extend the characterization of stress erythroid progenitors from expanded PBMCs to human anemic model. Human peripheral blood samples of Sickle cell disease patients, obtained

30 23 from Dr. Jane Little in Case Western Reserve University Hospital. It is hypothesized that stress erythroid progenitor populations can be identified in human peripheral blood mononuclear cell populations, which can be cultured in vitro, and these cells are present in the peripheral blood of anemic patients. Experimental Set up: Human peripheral blood samples from patients suffering from sickle cell disease was obtained from Case Western Reserve University Hospital as single-sided blind study. The control Peripheral Blood samples were obtained from ReachBio Labs. The PBMCs were then isolated from peripheral blood using Histopaque 1077 using density gradient. The PBMCs obtained were then counted and 1 x 10 6 cells/ml were plated in human differentiation media for 5 days with Epo and hypoxia. The remaining cells were tagged with fluorochrome and flow cytometry assay was performed on them. 5 days after culture in DM, the expanded cells were further counted and 1x10 5 cells/ml were plated in human methylcellulose media for functional assay of stress BFU-Es and the remaining cells were tagged with fluorochromes to perform flow cytometry assay. (Figure 5-1) Figure 5-1: Schematic representation of experimental protocol for study of stress erythroid progenitors in PBMCs of human Sickle Cell Disease patient samples.

31 24 Results: From the PBMCs isolated from the peripheral blood and cultured in differentiation media, the number of expanded cells for each patient sample was calculated relative to the number of Peripheral Blood Mononuclear Cells as well as for control human samples (Figure 5-2a). Even though the degree of anemia in each patient is different and unknown to us in a blind study, it can be observed that the control samples show very little expansion as compared to patient samples. Similarly no BFU-Es are observed in control human sample as it fits with the hypothesis that stress anemia is required to induce the migration and expansion of stress progenitors (Figure 5-2b). In samples UPN1 and UPN3 1x10 6 cells were plated for stress BFU-E that led to uncountable number of BFU-Es which could not be characterized graphically. From Figure 5-2 we can see that for human samples expressing high level of stress progenitors from expanded human anemic PBMCs (UPN4, UPN5, UPN10, UPN15), the expansion of progenitor cells from anemic human PBMCs are correspondingly high whereas for patient samples and control samples with low/ nil BFU-E counts, the population of expanded progenitors are also low, as expected. A 0.25 Expanded cells /PBMC Expanded cells /PBMC 0 UPN1 UPN3 UPN4 UPN5 UPN7 UPN8 UPN9 UPN10 UPN11 UPN12 UPN15 UPN16 HC1 HC2

32 25 B 900 BFUEs/Expanded cells BFUEs/Expanded cells * * UPN1 UPN3 UPN4 UPN5 UPN7 UPN8 UPN9 UPN10 UPN11 UPN12 UPN15 UPN16 HC1 HC2 Figure 5-2:Functional colony assay to characterize stress BFU-Es identified from expanded PBMCs of multiple human anemic patients. A) Frequency of stress BFU-Es cultured from expanded PBMCs of anemic mice with respect to peripheral blood mononuclear cells B). Stress BFU-E frequency as compared to stress progenitors expanded from PBMCs. *- Too many to count At Day 0, the PBMCs obtained were analyzed to characterize the stress progenitors. The cells were first gated on Kit cell marker. Gated on the Kit+ cells, CD34/CD133 cells are characterized. In the control sample, cells marked with the stem cell marker Kit were minimal at day 0 and at day 5. Looking at stress progenitors, even on day 5 the population of CD34+/CD133+; CD34- /CD133- stress progenitors are minimal (Figure 5-3), which corresponds with the functional assay analysis.

33 26 HC1 control sample Gated on Kit posi=ve cells Human control without culture CD 34 With culture c-kit CD 133 Figure 5-3: Flow cytometry analysis of PBMCs obtained from normal human Peripheral blood sample (control). The top panel represents the flow analysis of fresh PBMCs derived from peripheral blood without culture. The lower panel represents the analysis of PBMCs after 5 days culture in differentiation media. On the other hand, UPN15, which exhibited high stress BFU-Es and a high expansion of progenitors, has a very low value of Kit + cells/ of the Kit positive cells. After being cultured in DM, a 300-fold increase in Kit positive cells are observed with an exponential increase in CD34+/CD133+ stress progenitor population as well (Figure 5-4). The same analysis was performed for each of the patient samples (Appendix).

34 27 UPN15 Gated on Kit + cells CD 34 C-Kit CD 133 Figure 5-4: Flow cytometry analysis of PBMCs obtained from SCD patient sample expressing high numbers of stress BFU-E colonies after culture. The top panel represents the flow analysis of fresh PBMCs derived from peripheral blood without culture. The lower panel represents the analysis of PBMCs after 5 days culture in differentiation media

35 28 Chapter 6 Conclusions Using an acute anemic model for mice (phenylhydrazine injection) we have showed that peripheral blood cells can be cultured, expanded, identified and functionally assayed for stress BFU-E formation. Use of recent findings in the field of stress erythropoiesis has made it possible for us to culture and characterize stress erythroid progenitors in peripheral blood for the first time. This is a crucial discovery in the field as it opens a wide arena of application for using this robust alternate erythropoietic pathway for therapeutic treatment of anemia. This analysis has also helped us to further understand the process of migration of stress erythroid progenitors in mice model (Figure 6-1). From these results we can infer that onset of recovery from acute anemia induces mobilization of potential stress progenitors which home to spleen. Therefore, the expansion and differentiation of stress progenitors in the spleen appears to stimulate the migration of these progenitors to the peripheral blood. By day 7, the mice recover from the acute anemia, which correlates with the upregulation of Cxcl12 in the bone marrow that inhibits the egression of stress progenitors. By day 10 a new wave of stress erythroid progenitors are further replenished, again characterized by downregulation of Cxcl12. These results help understand the process of stress erythroid migration along with a preliminary understanding of the pathway involved in the regulation of the same.

36 29 PHZ treatment Day 0 24 Hrs 36 Hrs Day 3 Day 7 Day 10 Day 21 BMP4 induc5on stress progenitors in spleen Migra5on of stress progenitors to peripheral blood Recovery. No stress progenitors in Peripheral blood Replenishment of new wave of stress progenitors Full recovery Figure 6-1. Schematic representation of migration of stress progenitors during acute anemia in mice. We examined the potential mechanism involved in regulating the migration of these stress erythroid progenitors. We observe that cells get mobilized out of the BM in what appears to be a regulated fashion. These cells contain a population of stress erythropoietic precursors that when cultured become stress BFU-Es. We show that stress erythroid progenitors mobilization maybe regulated by changes in expression of Cxcl12 and its receptors CXCR4 and CXCR7. The ablation of the SNS using 6-OHDA severely interfered with the migration of stress erythroid progenitors from the bone marrow as confirmed by the exponential decrease in colony forming ability by the expanded stress erythroid progenitors migrated to the peripheral blood. The decrease in overall volume of blood collected (~50% decrease) from the mice via cardiac puncture and high level of mortality in these mice as compared to those only treated with PHZ could possibly be explained by severe dehydration caused by 6-OHDA treatment. In contrast, injection of mice with β- adrenergic agonist, isoproterenol, which stimulated the SNS leading to an earlier migration of stress progenitors, which is also consistent with the survival of treated mice. The identification of stress erythroid progenitors in expanded peripheral blood mononuclear cells in acute anemic model allowed us to extend this study to human congenital anemic model. We were able to successfully identify and functionally assay stress erythroid progenitor colonies from the expansion of PBMCs obtained from Sickle cell disease patients (Dr. Jane Little, Case

37 30 Western Reserve University Hospital). The identification of stress erythroid progenitors from flow cytometry was also consistent with the functional assay data. This study of human sickle cell anemia patients also shows that expansion of PBMCs in stress erythropoiesis differentiation media correlates with potential to form stress BFU-Es. This is a highly exciting result as it opens abundant opportunities to study stress erythropoiesis in humans using peripheral blood a compared to bone marrow samples. Future work: In order to circumvent the difficulty faced with characterizing the stress erythroid progenitors with flow cytometry due to PHZ interference in murine model, a different mode of induction of anemia can be used such as Murine model system of thalassemia. Bone marrow transplantation assays to rescue lethally irradiated mice can be of use here. Alternatively, we could look at inflammatory anemia model to study and characterize the migration of distinct stress erythroid progenitor population. We also need to further understand the effect of sympathetic nervous system on migration of stress progenitors. We could use ceramide galactosyltransferase (Cgt) knockout mice to understand this process better. Cgt -/- mice have severe neurological abnormalities and fail to mobilize bone marrow HSPCs in steady state in vivo or use antagonists of β-adrenergic receptors such as propranolol (β blocker) to study the mechanism in vitro. The preliminary work in human anemic patients opens the door for further in depth characterization of stress erythropoiesis in humans. Future work involves correlating the mobilization of stress progenitors with other clinical parameters of the disease. Human stress erythropoiesis often exhibits properties of fetal erythropoiesis. Fetal erythrocyte characteristics and antigens as well as the expression of fetal hemoglobin (HbF) are observed during the

38 31 recovery from erythropoietic stress. 42, 43 Hence, these stress erythroid progenitors need to be further characterized for parameters such as expression of HbF, Glycophorin A, CD34, KIT and CD71 to name a few. We must also extend these studies to test patients with other anemic diseases such as thalassemia or myeloid dysplastic syndrome to name a few.

39 32 Appendix Supplementary data Appendix A Flow cytometry analysis of PBMCs obtained from SCD patient samples and control samples before and after culture. The top panel represents the flow analysis of fresh PBMCs derived from peripheral blood without culture. The lower panel represents the analysis of PBMCs after 5 days culture in differentiation media. The Left hand side panes represent expression of c-kit cell surface markers on the cells. The Right hand side panel represents CD34 (y-axis) /CD133 (x-axis) expression gated in Kit+ cells. Human control without culture With culture UPN 1, Day 0

40 33 Upn3 day 0 Upn3 day 5 Upn 4

41 34 UPN 5 Upn5 day 0 Upn5 day 5 Upn 7

42 35 UPN 8 UPN 9

43 36 UPN 10 UPN 11

44 37 UPN 12 UPN 16

45 38 Appendix B Flow Cytometry analysis of murine anemic PBMC stress progenitors: Flow cytometry analysis of PBMCs derived from PHZ treated mice at two representative time points of Day 3 an Day 7. The top panel represents flow cytometry at Day 0 of culture in Differentiation media and the bottom panel represents the same at Day 5. Day 3 Day 7 C-Kit CD34 C-Kit CD34 Sca CD133 Sca CD133