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Effects of Simulated Microgravity on Human Umbilical Cord Blood Hematopoietic Stem Cells

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Title:
Effects of Simulated Microgravity on Human Umbilical Cord Blood Hematopoietic Stem Cells
Creator:
PONIATOWSKI, ADAM F. ( Author, Primary )
Copyright Date:
2008

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Antibodies ( jstor )
Blood ( jstor )
Cell cycle ( jstor )
Cell growth ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Microgravity ( jstor )
Progenitor cells ( jstor )
Stem cells ( jstor )
Viability ( jstor )

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University of Florida
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University of Florida
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Copyright Adam F. Poniatowski. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
6/30/2005
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436098636 ( OCLC )

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EFFECTS OF SIMULATED MICROGR AVITY ON HUMAN UMBILICAL CORD BLOOD HEMATOPOIETIC STEM CELLS By ADAM F. PONIATOWSKI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by ADAM F. PONIATOWSKI

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This thesis is for my mother, Laura, for her constant optimism and passionate interest, and of course for Katharine, who supports my chosen path unconditionally.

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ACKNOWLEDGMENTS I would first like to acknowledge Dr. Roger Tran-Son-Tay, Dr. Vijay Reddy, and Dr. Chris Batich for their constant support and dedication to my research project. Additionally, I would like to acknowledge Dr. Reddy and Dr. Tran-Son-Tay for the novel idea for this project in studying the effects of microgravity on human cord blood stem cells, thus bridging the biomedical field with engineering. I also thank Dr. Reddy for obtaining the Institutional Review Board (IRB) approval that provided cord blood samples for this research. I would like to deeply thank my parents, Laura Poniatowski and William Poniatowski, for providing me with the guidance necessary to reach this pinnacle in my academic career. Special thanks go to my mother, who has always managed to foster a harmony of focus and dreams in my life. Warmest thanks go to Katharine, my fiance, for remaining steady and loving through all of the joys and pitfalls of this difficult journey. She has and always will be my foundation and motivation to succeed. Additional appreciation goes to Neal Benson of the Flow Cytometry Core Laboratory, and the technologists of the Shands Hospital Stem Cell Laboratory, including Michele Sugrue, Diann Fisk, and Cheryl Roberts. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii NOMENCLATURE ............................................................................................................x ABSTRACT ......................................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 2 OBJECTIVES...............................................................................................................3 Rationale.......................................................................................................................3 Objective.......................................................................................................................5 Specific Aims................................................................................................................6 3 BACKGROUND..........................................................................................................7 Hematopoietic Stem Cells............................................................................................7 Differentiation Pathways.......................................................................................8 Cellular Markers....................................................................................................8 Growth Conditions................................................................................................9 Simulated Microgravity Technology............................................................................9 Hematopoietic Cell Culture in Microgravity..............................................................12 4 MATERIALS AND METHODS...............................................................................14 Cell Preparation..........................................................................................................14 Umbilical Cord Blood Collection........................................................................14 Nucleated Cell Separation...................................................................................15 Nucleated Cell Viability Assays..........................................................................16 CD34 + Cell Separation........................................................................................17 Flow Cytometric Assays.............................................................................................18 CD34 + Cell Staining............................................................................................18 v

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Viability Staining.................................................................................................20 Cell Cycle Staining..............................................................................................20 Flow Cytometry...................................................................................................21 Determination of Optimal Static Culture Conditions.................................................23 Unpurified Nucleated Cell Fraction Cultures......................................................23 CD34 + -Purified Cell Fraction Cultures...............................................................24 Short-Term Simulated Microgravity Culture Assessment.........................................24 Simulated Microgravity Cultures........................................................................25 Control Cultures..................................................................................................26 Secondary Long-Term Static Cultures.......................................................................27 Hematopoietic Progenitor Cell Assays.......................................................................27 Statistics......................................................................................................................28 5 RESULTS AND DISCUSSION.................................................................................29 Determination of Optimal Static Culture Conditions.................................................29 Short-Term Simulated Microgravity Culture Assessment.........................................30 Secondary Long-Term Static Culture Assessment.....................................................38 6 CONCLUSIONS AND FUTURE WORK.................................................................42 APPENDIX A STATIC CULTURE DATA AND FIGURES...........................................................45 B HARV CULTURE DATA AND FIGURES..............................................................50 C SECONDARY LONG-TERM STATIC CULTURE DATA AND FIGURES.........57 LIST OF REFERENCES...................................................................................................61 BIOGRAPHICAL SKETCH.............................................................................................66 vi

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LIST OF TABLES Table page A-1 Initial Culture Data (Day 0), Unpurified Fraction Cultures, Media A......................47 A-2 Culture Data, Unpurified Fractions, Media A...........................................................47 A-3 Initial Culture Data (Day 0), Unpurified Fraction Cultures, Media B.......................48 A-4 Culture Data, Unpurified Fractions, Media B...........................................................48 A-5 Initial Culture Data (Day 0), CD34 + -Purified Fraction Cultures, Media B...............49 A-6 Culture Data, CD34 + -Purified Fractions, Media B....................................................49 B-1 Initial Culture Data (Day 0), HARV and Control Cultures.......................................53 B-2 Culture Data, HARV Cultures...................................................................................53 B-3 Cell Cycle Data, HARV Cultures..............................................................................54 B-4 Culture Data, Control Cultures..................................................................................55 B-5 Cell Cycle Data, Control Cultures.............................................................................56 C-1 Initial Culture Data (Day 0 Post-Initial Culture), Post-HARV Cultures...................59 C-2 Culture Data, Post-HARV Cultures...........................................................................59 C-3 Progenitor Cell Culture Data, Post-HARV Cultures.................................................59 C-4 Initial Culture Data (Day 0 Post-Initial Culture), Post-Static Cultures.....................60 C-5 Culture Data, Post-Static Cultures.............................................................................60 C-6 Progenitor Cell Culture Data, Post-Static Cultures....................................................60 vii

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LIST OF FIGURES Figure page 3-1 Renewal and differentiation pathways of PHSC..........................................................7 3-2 Free-body diagram of a particle suspended in microgravity......................................10 3-3 The Rotary Cell Culture System (RCCS)...................................................................11 4-1 Density separation of umbilical cord blood................................................................16 4-2 The structure and cellular interaction of antibodies used for CD34 + cell separation.18 4-3 Morphological gate dot-plots of fresh HUCB cells....................................................21 4-4 Viability gate dot-plot of a CD34 + -purified fraction of fresh HUCB cells................22 4-5 CD34 + gate dot-plots of fresh HUCB cells................................................................22 4-6 Injecting cells into the HARV using a sterile syringe................................................25 5-1 Nucleated cell and CD34 + cell growth, purified vs. unpurified fractions..................31 5-2 Nucleated cell progression, CD34 + -purified fraction.................................................32 5-3 NC progression, two-day HARV culture...................................................................33 5-4 CD34 + cell progression, CD34 + -purified fraction......................................................34 5-5 Cell cycle activity, CD34 + -purified fraction...............................................................35 5-6 Viable cell cycle activity, CD34 + -purified fraction....................................................36 5-7 Cell growth, secondary long-term static cultures.......................................................39 5-8 CD34 + purity, secondary long-term static cultures....................................................40 5-9 Progenitor growth, 14-day cultures............................................................................41 A-1 Absolute nucleated cell and CD34 + cell growth, purified vs. unpurified fractions...46 B-1 Absolute nucleated cell progression, CD34 + -purified fraction..................................51 viii

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B-2 Absolute CD34 + cell progression, CD34 + -purified fraction......................................52 C-1 Absolute cell growth, secondary long-term static cultures........................................58 ix

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NOMENCLATURE 3-D.Three-Dimensional 7-AAD....7-Amino-actinomycin D BFU-EBurst-Forming Unit Erythroid CFUColony-Forming Unit CFU-GMColony-Forming Unit Granulocyte-Macrophage CFU-Mk Colony-Forming Unit Megakaryocyte EDTA..Ethylenedinitrilotetraacetic Acid EPO.Erythropoietin Fab..Fragment, Antigen-Binding FBS.Fetal Bovine Serum FcFragment, Crystallizable FcR..Fc Receptor FLFlt3-Ligand G-CSF.Granulocyte-Colony Stimulating Factor GVHDGraft-Versus-Host Disease HARV.High Aspect Ratio Vessel HSC.Hematopoietic Stem Cell HUCB.Human Umbilical Cord Blood IgG..Immunoglobulin G x

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IL-3.Interleukin-3 IL-6.Interleukin-6 IMDM.Iscove’s Modified Dulbecco’s Medium LTSC..Long Term Static Culture NC...Nucleated Cell PBS.Phosphate Buffered Saline PHSC..Pluripotent Hematopoietic Stem Cell R-PE...R-Phycoerythrin RCCS..Rotary Cell Culture System RWV...Rotating-Wall Vessel SCF.Stem Cell Factor STLV..Slow Turning Lateral Vessel WBC...White Blood Cell xi

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF SIMULATED MICROGRAVITY ON HUMAN UMBILICAL CORD BLOOD HEMATOPOIETIC STEM CELLS By Adam F. Poniatowski December 2004 Chair: Roger Tran-Son-Tay Major Department: Biomedical Engineering One major issue surrounding the use of human umbilical cord blood (HUCB) stem cells for transplantation is the lack of nucleated cells needed to engraft an adult of average weight. Though many researchers have been able to efficiently expand HUCB stem cell populations in vitro, others have found that stem cell frequency declines rapidly during this growth, suggesting that rapid differentiation may cause a decrease in self-renewal rate and slow long-term growth. Based on evidence that simulated microgravity culture induces a state of quiescence in hematopoietic cells, causing reduced differentiation and allowing increased long-term expansion, the current study addressed whether or not culture of HUCB stem cells in simulated microgravity would result in similar findings. Presently, no work has been done which observes the effects of simulated microgravity on cord blood stem cells. A protocol for culturing and characterizing HUCB cells in conventional culture and in simulated microgravity (using a high aspect ratio vessel, or HARV) was developed. xii

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To assess proliferation and differentiation, nucleated cell counts were performed using a hemacytometer and CD34 + cell counts were performed using antigen labeling and detection via antibody staining and flow cytometry. Colony-forming unit (CFU) cultures were grown post-culture in a specialized media and enumerated to assess differentiation potential. In order to monitor cell cycle activity and quiescence, DNA quantization was performed via nucleic staining and flow cytometry. After establishing a method for growing HUCB stem cells in a conventional, static environment, the effects of a short-term simulated microgravity culture on nucleated cell growth, CD34 + cell growth and purity, and cell cycle activity were observed. HUCB stem cells cultured in simulated microgravity exhibited significant nucleated cell and CD34 + cell death, reduced CFU production, and prolonged quiescence over a three-day culture period. Establishment of a post-microgravity long-term static culture (LTSC) led to complete hematopoietic exhaustion within fourteen days. Thus culture in simulated microgravity under the conditions tested proved to be too damaging to HUCB stem cells to allow improved long-term hematopoietic expansion. It is possible that technical problems, including incorrect vessel rotation speed, may have led to cell death in the HARV, while cell-related issues, including low initial cell concentrations and high initial cell primitivity, may have hindered cell growth. xiii

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CHAPTER 1 INTRODUCTION In 1939, the first bone marrow transplant was attempted to treat aplastic anemia, a pathological condition in which the bone marrow fails to produce adequate numbers of blood cells. Though initially unsuccessful, the procedure has evolved to become an established treatment for various degenerative diseases and hematological deficiencies (Leger and Nevill 2004). The primary purpose for transplant is to restore or accelerate the regenerative capabilities of the body’s hematopoietic system through the introduction of healthy stem cells to deficient bone marrow. Early in vivo mice studies revealed that populations of bone marrow derived stem cells were capable of self-regeneration and repletion of all blood cell lineages in secondary hosts (Siminovitch, et al. 1963), laying the foundation for future investigations of various human stem cell products, including bone marrow, peripheral blood, and umbilical cord blood. Human umbilical cord blood (HUCB) stem cell products present an attractive alternative to bone marrow or peripheral blood products due to their ease of procurement, increased availability, and relatively high progenitor frequency. Unfortunately, the extremely low number of nucleated cells (NCs) contained in a healthy cord blood sample hinders engraftment. Though many researchers have been able to efficiently expand cord blood stem cell populations in vitro, others have found that stem cell frequency declines rapidly during this growth, suggesting that rapid differentiation, or committal to a particular blood cell, may cause a decrease in self-renewal rate and slow long-term 1

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2 growth. It may be possible to counteract this effect by inducing an initial proliferation rate reduction via cell culture in simulated microgravity. The simulated microgravity cell culture system, or rotating-wall vessel (RWV), was developed in 1991 by NASA scientists to examine the effects of space on human tissue in vitro and explain the bone and muscle loss suffered by astronauts during spaceflight (Schwarz, et al. 1992). Since its inception, hundreds of studies have been documented which test the effects of the RWV on various types of cells. While many studies have shown that simulated microgravity promotes growth of various human cells, including colon (Jessup, et al. 1997) and prostatic cancer (Zhau, et al. 1997) cell lines, other investigations have shown that simulated microgravity reduces the proliferation rates and differentiation rates of various cells, including human mesenchymal stem cells (Zayzafoon, et al. 2004) and hematopoietic stem cells (HSC) (Plett, et al. 2004, Plett, et al. 2001). Plett et al. (Plett, et al. 2001) have further shown that the reduction of proliferation and differentiation rates caused by simulated microgravity lead to a more efficient preservation of the primitive potential of human bone marrow-derived stem cells in secondary long-term static cultures (LTSCs), promoting long-term hematological growth. These studies, along with multiple transplant-related advantages, make HUCB an interesting candidate for culture in simulated microgravity. Presently, no work has been done which observes the effects of simulated microgravity on cord blood stem cells.

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CHAPTER 2 OBJECTIVES A severe lack of white blood cells (WBCs) can exhaust the body’s ability to fight infection. The causes of WBC deficiency can include non-malignant conditions, such as aplastic anemia or other bone marrow deficiencies, malignant conditions, such as leukemia, lymphoma, or myeloma, and blood cell depleting treatments for these pathologies, such as chemotherapy and radiation therapy. Patients affected with non-malignant conditions are unable to produce normal numbers of WBCs to combat infection, while those affected with malignant conditions exhibit uncontrolled myelogenous or lymphocytic WBC growth, conditions that impede the normal production and function of blood cells, result in the accumulation of tumor masses in lymph nodes and other sites, and weaken bone via cytokine release (The Leukemia and Lymphoma Society 2004). Two common forms of treatment for hematological cancer (malignant conditions) include chemotherapy and radiotherapy, both of which involve the destruction of cancerous cells through the administration of large doses of drugs (chemotherapy) and/or radiation (radiotherapy). One major disadvantage of these treatments involves the destruction of healthy blood cells at targeted sites, a problem requiring the regeneration of normal hematological populations via stem cell transplantation. Rationale Various methods of stem cell transplantation are used to directly treat non-malignant conditions and to promote healthy blood cell regeneration after destructive 3

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4 treatment of malignant conditions. Stem cells can be collected autologously or allogeneicly from three different sources: bone marrow, peripheral blood, or umbilical cord blood, each uniquely associated with both advantages and drawbacks. Bone marrow stem cells can be aspirated directly from the bone marrow, assuring fraction purity; however, the procedure is extremely invasive and requires the patient to be under general or spinal anesthetic. Additional disadvantages include the lack of suitable HLA-matched donors and complications due to immune rejection, or graft-versus-host disease (GVHD) (Gluckman 2000). Alternatively, peripheral blood can be harvested without the use of anesthesia, requiring only the administration of hematopoietic growth factors to the patient’s blood stream in order to mobilize stem cells and facilitate proliferation, allowing collection via venipuncture and immediate leukapheresis (Bensinger, et al. 1993). Furthermore, allogeneic transplantation of peripheral blood from matched siblings instead of bone marrow has been shown to lead to faster hematologic recovery, similar risk of rejection, and improved survival in patients exhibiting various conditions (Couban, et al. 2002). However, the limited availability of donor samples and generally low progenitor cell, or colony-forming unit (CFU) content coupled with the existing risk for immune rejection due to HLA-matching disparities presents a major clinical setback. Umbilical cord blood stem cell transplants have become increasingly popular due to ease of procurement, increased availability, relatively high progenitor frequency, and decreased lymphocyte alloreactive potential. The immaturity of the immune system at birth may be the principle reason for this increased clonogenic potential and the reduced incidence and severity of GVHD upon transplantation (Gluckman 2000). One major issue however surrounding the use of cord blood stem cells for transplantation is the lack

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5 of nucleated cells needed to engraft an adult of average weight. Current research focused on the in vitro expansion of cord blood stem cells has shown favorable results that may help to alleviate this major setback. Many investigators have shown that under appropriate conditions, NC (Lazzari, et al. 2001), stem cell (Piacibello, et al. 1997), and progenitor cell growth (Piacibello,et al.1998) can be sustained and greatly expanded in vitro for several months. Shpall et al. (2002) went on to show in a clinical study that transplantation of expanded cord blood stem cells did not significantly delay engraftment compared to freshly isolated cells. However, there is some evidence that stem cell frequency and self-renewal rate significantly decline over time due to rapid differentiation (Fietz, et al. 1999, Lam, et al. 2001, Tanavde, et al. 2002). A reversal of this phenomenon may be possible through culture in an environment previously shown to reduce proliferation and differentiation rates of various cells, such as that created by the mechanics of simulated microgravity. Objective The simulated microgravity culture environment created by the RWV has been shown to significantly reduce growth rates of many cells, including human bone marrow stem cells (Plett, et al. 2001). Several factors may contribute to these observed growth rate reductions in microgravity, including affected signal transduction (Cooper and Pellis 1998), locomotion and cell adhesion (Pellis, et al. 1997), and slow cell cycle kinetics (Cogoli-Greuter, et al. 1996). Plett et al. (2001) observed a decrease of NC and stem cell growth accompanied by a slight increase in stem cell frequency after a fourto six-day culture in the RWV, followed by significant increases in NC and progenitor cell growth in secondary long-term static cultures over twelve weeks. These favorable long-term effects may have been a result of the initial prolonged quiescence exhibited by cells

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6 cultured in microgravity which remained in the G o /G 1 phase of the cell cycle for extended periods of time, allowing them to avoid rapid differentiation and thus retain a greater potential for self-renewal and long-term growth than conventionally cultured cells (Plett, et al. 2001). The goal of the current study is to induce a similar state of quiescence in HUCB stem cells via culture in simulated microgravity in order to improve long-term hematopoietic expansion through prolonged maintenance of the primitive phenotype. Specific Aims The specific aims of the project were to: 1. Establish, implement, and assess a method for growing NCs and stem cells from HUCB in a static environment for up to fourteen days. NC growth was quantified using a hemacytometer. Stem cell purity and growth were assessed via flow cytometry. 2. Observe the effects of a short-term simulated microgravity culture of HUCB stem cells on NC growth, stem cell growth and purity, and cell cycle activity, and determine an optimal HARV culture time based on these parameters. Simulated microgravity cultures were seeded using a high aspect ratio vessel (HARV). Cell cycle activity was assessed via flow cytometry. 3. Implement the pre-determined optimal HARV culture time and assess the effects of a fourteen-day post-microgravity LTSC on NC growth, stem cell growth and purity, and development of hematopoietic CFUs derived from HUCB. CFU growth was quantified following fourteen-day progenitor cell cultures.

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CHAPTER 3 BACKGROUND Hematopoietic Stem Cells A stem cell is a cell that has the ability to divide (self-renew) and, under the right conditions, give rise (differentiate) to the many different cell types that make up an organism, including heart cells, skin cells, blood cells, and nerve cells. Pluripotent hematopoietic stem cells (PHSCs) are mononuclear blood-forming stem cells found in the red bone marrow of certain bones in the human body and in blood retrieved from the umbilical cord of a fetus. Over the course of an entire lifetime, these stem cells are capable of constantly renewing or differentiating into any one of the diverse types of blood cells, including erythrocytes (red blood cells), thrombocytes (platelets), and various leukocytes (WBCs) (Goldsby, et al. 1992). Figure 3-1 shows a general schematic of the renewal and differentiation pathways of PHSC. Lymphoid Progenitor Cell Myeloid Progenitor Cell Pluripotent HSC Erythrocytes Monocytes (WBC) Thrombocytes Granulocytes (WBC) Lymphocytes (WBC) Figure 3-1. Renewal and differentiation pathways of PHSC. 7

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8 Differentiation Pathways Differentiating stem cells first become mononuclear progenitor cells, or CFUs, through lymphopoiesis (formation of lymphoid progenitor cells), or myelopoiesis (formation of myeloid progenitor cells). Lymphoid progenitors differentiate directly into nucleated WBCs, while myeloid progenitors form various blood cells. The three main types of myeloid CFUs include CFU-Mks (colony-forming unit megakaryocytes), BFU-Es (burst-forming unit erythroids), and CFU-GMs (colony-forming unit granulocyte-macrophages). BFU-Es differentiate further into erythrocytes, the primary cells responsible for transporting oxygen through the circulatory system. CFU-Mks differentiate further into megakaryocytes, which can then each give rise to over 6,000 thrombocytes, adhesive cell fragments that clump together following injury and release chemicals to the lining of blood vessels, stimulating contraction of the injured vessels to minimize blood loss. CFU-GMs are a very important component in the body’s immune response, as they differentiate further into myeloblasts and monoblasts, both of which are nucleated WBC precursors (Rhoades and Pflanzer 2003). Cellular Markers All PHSCs and CFUs derived from the human blood system express a single-chained trans-membrane glycoprotein known as the CD34 antigen (Lanza, et al. 2001, Tavian, et al. 1996). CD34 antigen density is highest on the most primitive PHSCs and decreases as cells mature, fading completely when cells differentiate fully. CD34 + cells can be identified using antigen-binding antibodies, allowing for purification and enumeration of the stem cell and progenitor cell populations.

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9 Growth Conditions Researchers have shown that PHSCs, NCs, and CFUs derived from HUCB can be grown using various cytokine cocktails. Human recombinant flt3-ligand (FL) has been shown to be an early acting cytokine supporting the growth of very primitive HSCs only when synergized with other growth factors (Piacibello, et al. 1997), including stem cell factor (SCF) (De Felice, et al. 1999), interleukin-3 (IL-3) (Su, et al. 2002), interleukin-6 (IL-6) (Lazzari, et al. 2001, Rappold, et al. 1999), granulocyte-colony stimulating factor (G-CSF) (Fietz, et al. 1999), erythropoietin (EPO) (Fietz, et al. 1999), and thrombopoietin (TPO) (Kawada, et al. 1999, Piacibello, et al. 1997) , a ligand expressed on both early and committed hematopoietic progenitors (Kaushansky 1995). These cytokines have been used in several combinations with different media to provide a hospitable environment for long-term expansion of HUCB cells. Piacibello et al. (1997, 1999) showed that a simple cocktail of FL and TPO in Iscoves’s Modified Dulbecco’s Medium (IMDM) supplemented with serum was able to support large hematological expansions of CD34 + -purified cultures over twenty-five weeks. Fietz et al. (1999) showed that expansion of unpurified NC cultures was possible using IMDM supplemented with SCF, IL-3, IL-6, G-CSF, and EPO, arguing that CD34 + purification entails costly and excessive cell manipulation and loss of stem cells. Cultures initiated with both unpurified NC fractions and CD34 + -purified fractions will be tested for growth potential in the current study. Simulated Microgravity Technology The basis of simulated microgravity cell culture technology was developed in 1991 by NASA scientists at the Johnson Space Center in Houston, Texas to examine the effects of space on human tissue in vitro (Schwarz, 1992). The environment of

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10 microgravity is simulated by the horizontal rotation of a cylindricalor disc-shaped vessel, or bioreactor, completely filled with media, creating a low shear, low turbulence fluid flow in which cells eventually migrate toward an equilibrium state, maintaining their position over an average of time and space in the inertial frame of reference (Wolf and Schwarz 1991). This particle suspension is maintained by balancing gravity-induced sedimentation with rotation-induced centrifugation and fluid drag, promoting the formation of 3-dimensional (3-D) tissue-like assemblages of cells (Gao, et al. 1997). The free-body diagram of a particle suspended in microgravity is shown in Figure 3-2. P F B F S r Particle Path G F D P = particle F S = force of gravity-induced sedimentation F B = liquid buoyant force F D = liquid drag force G = gravity vector = angular velocity r = distance to center of vessel Figure 3-2. Free-body diagram of a particle suspended in microgravity. Synthecon Incorporated (Houston, TX) produces a commercially available RWV bioreactor and motor-driven base: the Rotary Cell Culture System (RCCS) (Figure 3-3). Two bioreactor designs have been developed. The high aspect ratio vessel (HARV) (Figure 3-3) is a rotating disc-shaped vessel that allows oxygenation through a posteriorly

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11 attached flat silicone rubber gas transfer membrane, while the slow turning lateral vessel (STLV), a cylindrical-shaped vessel, allows oxygenation through a similar center core membrane (Synthecon Incorporated 2000). C D A B Figure 3-3. The Rotary Cell Culture System (RCCS). A) High aspect ratio vessel (HARV), base, and motor. B) Open HARV, with white silicone rubber gas transfer membrane exposed. C) Sealed HARV. D) Injecting media into sealed HARV via syringe. The rotation of the RWV allows for gentle mixing of the cellular media and in concert with the oxygenation membrane provides a continuous delivery of large volumes of undissolved gases to the cells (Hammond and Hammond 2001). The bioreactor must be completely filled in order to eliminate the occurrence of boundary layers between the media and vessel wall (Schwarz, et al. 1992), while the initial rotational speed must be set so that cells and media rotate synchronously, providing an optimal, low shear

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12 environment for nutrient and waste transfer (Unsworth and Lelkes 1998). Because these properties must be maintained in order to simulate microgravity, waste must be replaced with media daily (Unsworth and Lelkes 1998). Hematopoietic Cell Culture in Microgravity While cultivation in microgravity has been shown to enhance the growth, differentiation, and function of various cell types, including several human cancer cell lines (Ingram, et al. 1997, Jessup, et al. 1997, Zhau, et al. 1997), several studies observing the behavior of healthy hematopoietic cells in both true and simulated microgravity have shown inhibitory effects. True microgravity has been shown to severely suppress the activation of human lymphocytes (Cogoli-Greuter, et al. 1996, Cooper, et al. 2001), hinder differentiation of bone marrow-derived macrophages (Armstrong, et al. 1995), and reduce the proliferation of lymph node-derived lymphocytes (Nash and Mastro 1992) in on-board spaceflight studies. Davis et al. (1996) showed that bone marrow-derived CD34 + cell proliferation rates were dramatically reduced in microgravity cultures. Research on bone marrow-derived stem cells has since been expanded upon to include cultures in simulated microgravity, revealing similar results. Plett et al. (2001) observed a decrease of bone marrow-derived NC and CD34 + cell growth after a fourto six-day culture in the RWV. Upon initiation of secondary LTSCs however, converse observations were made, as significant increases in NC and CFU-GM growth were observed over twelve weeks. In a later study, Plett et al. (2004) confirmed the initial microgravity-induced proliferation rate reduction after a twoto threeday HARV culture, followed by a similar increase of CFU-GM production as compared to static cultures. The investigators concluded that the sustained self-renewal potential

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13 observed may have been a result of the initial prolonged quiescence exhibited by cells cultured in microgravity which remained in the G o /G 1 phase of the cell cycle for extended periods of time as compared to conventionally cultured cells over a two day period (Plett, et al. 2001). No studies have been performed which test the effects of true or simulated microgravity on the primitive potential of HUCB stem cells.

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CHAPTER 4 MATERIALS AND METHODS The present study investigated the effects of simulated microgravity on the proliferation and differentiation rates of HUCB stem cells. It was proposed that a microgravity-induced reduction of proliferation and differentiation would cause a prolonged maintenance of primitive potential, promoting long-term hematopoietic expansion. A protocol for culturing and characterizing HUCB cells in conventional culture and in simulated microgravity was developed. Microgravity was simulated using the HARV. To assess proliferation and differentiation, NC counts were performed using a viability stain assay and a hemacytometer, and CD34 + cell counts were performed using antigen labeling and detection via antibody staining and flow cytometry. CFU cultures were grown post-culture in a specialized media and enumerated to assess differentiation potential. In order to monitor cell cycle activity and quiescence, DNA quantization was performed via nucleic staining and flow cytometry. Cell Preparation Umbilical Cord Blood Collection Umbilical cord blood samples were collected for donation or research with written consent from the mother. Samples were collected at either North Florida Regional Medical Center or Shands at Alachua General Hospital in Gainesville, Florida, by healthcare personnel using the required protocol provided by LifeSouth Community Blood Centers. After collection, blood samples were stored in 300-ml blood bags pre-filled with 35 ml CPD (citric-phosphate-dextrose solution) to prevent coagulation. The 14

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15 samples were transported on wet ice via courier to the Bone Marrow Transplant Unit’s Stem Cell Laboratory at Shands Hospital in Gainesville, Florida, at which time they were analyzed for NC content by laboratory technologists. Only samples containing less than 810 8 NCs were used, as higher NC counts provide an adequate source for transplantation, rendering the samples bankable for donation. Upon confirmation of research status, the samples used in the present study were stored overnight in the laboratory refrigerator (3 – 5 C) and picked up the following morning for further processing. On average, samples were processed for culture 33 hours after collection. It has been shown that there is no significant difference in CD34 + cell recovery from umbilical cord blood samples collected in a CPD solution after 24 or 72 hours of storage at 4 C as compared to fresh samples, while CFU-GM recovery decreased only after 72 hours of storage (Hubel, et al. 2003, Koenigbauer, et al. 2002). These studies differ in NC recovery findings, as Koenigbauer et al. (2002) report no significant difference in recovery after 24 and 72 hours of storage, while Hubel et al. (2003) report a decline in recovery to 95% after 24 hours and 81% after 72 hours. These studies suggest that the short-term storage of samples used in the present study may not have affected cell recovery and growth potential. Nucleated Cell Separation Blood samples were extracted from blood bags and diluted in a 1:1 volume ratio with 1X Phosphate Buffered Saline (PBS), prepared at a pH of 7.4 with 10X PBS (GIBCO, Long Island, NY). In 50-ml centrifuge tubes (Becton Dickinson Biosciences Discovery Labware, Franklin Lakes, NJ), 15 ml aliquots of the blood solution were layered either over or under 10 ml of Lymphoprep (Axis-Shield, Oslo, Norway), a solution containing compounds which aggregate erythrocytes and increase their

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16 sedimentation rate for efficient separation. The tubes were centrifuged at 300 g for 30 minutes at room temperature. As shown if Figure 4-1, centrifugation of the solution along with the aid of Lymphoprep (Axis-Shield, Oslo, Norway) causes the components of the mixture to separate by density, allowing easy removal of the NC layer using a pipette. Plasma Nucleated Cells Lymphoprep Erythrocytes Figure 4-1. Density separation of umbilical cord blood. Centrifugation causes the components of the blood solution to separate by density. The NC fractions were combined (2 fractions into one new tube), diluted with a 1:1 volume ratio of 1X PBS, and washed via centrifugation (250 g, 10 minutes, 4 C). The supernatant was poured off, and the cell pellets were resuspended in 1X PBS and combined for counting. Nucleated Cell Viability Assays Aliquots of cell solutions were diluted with Trypan Blue Stain (GIBCO, Long Island, NY), a blue dye capable of penetrating lysed cell membranes to assist in viability counts. Approximately 10 l of this solution was loaded onto a Bright-Line Hemacytometer (Hausser Scientific, Horsham, PA) and analyzed for live NC content with the aid of an inverted light microscope (Leica Microsystems Inc., Bannockburn, IL).

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17 CD34 + Cell Separation CD34 + cell separation was performed as described by Miltenyi Biotec protocols. NC fractions were centrifuged (250 g, 10 minutes, 4 C) and resuspended in a 1275 mm polystyrene tube (Becton Dickinson Biosciences Discovery Labware, Franklin Lakes, NJ) at a concentration of 110 8 cells/0.3 ml MACS buffer, a degassed solution consisting of 1X PBS, 0.5% Bovine Serum Albumin (Fisher Scientific, USA), and 2 mM ethylenedinitrilotetraacetic acid, or EDTA (Fisher Scientific, USA). After resuspension, 100 l aliquots of FcR Blocking Reagent (Miltenyi Biotec, Auburn, CA) and CD34 MicroBeads (Miltenyi Biotec, Auburn, CA) per 110 8 total cells were added to the cell suspension. The Fc fragments (crystallizable, non-specific fragments) of antibodies in the blocking reagent bind to Fc receptors on all nucleated cells, blocking any non-specific binding of CD34 MicroBeads (Miltenyi Biotec, Auburn, CA). CD34 MicroBeads (Miltenyi Biotec, Auburn, CA) consist of magnetic beads (50 nm in diameter) directly coated with CD34-specific antibodies (clone QBEND/10) for magnetic labeling of cells which express the CD34 antigen. Figure 4-2 illustrates the structures and cellular action of the antibodies. The cells were incubated for 30 minutes at 4 C, washed with 1 – 2 ml of MACS buffer, centrifuged (200 g, 5 minutes, 4 C), and resuspended in MACS buffer at a concentration of 110 8 cells/ml. Cells were then passed through a Type LS High Gradient Magnetic Separation Column (Miltenyi Biotec, Auburn, CA) positioned in a MidiMACS Separation Unit (Miltenyi Biotec, Auburn, CA). The magnetic field created by the interaction between the separation unit and the column allows unlabeled cells to elute through the column while positively labeled cells (CD34 + cells) remain in the

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18 column’s magnetic shaft. After negative cell elution, the column was washed with 9 ml MACS buffer, removed from the separation unit, and plunged with 5 ml MACS buffer. The column separation step was repeated to ensure fraction purity. Fc Receptors CD34 Antigen CD34 + Cell FcR Blocking CD34 Specific withMicroBead Fc Fab Antibodies Interaction Figure 4-2. The structure and cellular interaction of antibodies used for CD34 + cell separation. Both antibodies used consist of a non-specific Fc fragment, and a specific antigen-binding, or Fab fragment. The FcR blocking antibody will bind with non-specific FcRs on all nucleated cells, while the CD34 antibody will bind only to CD34 antigens on CD34 + cells. The CD34 antibody is attached to a paramagnetic bead which is required for magnetic separation. Flow Cytometric Assays Staining a diverse population of cells with fluorochrome-conjugated antibodies or dyes aids in the identification of specific cells. Cells can be tagged with specific antibodies or stained with nucleic dyes and enumerated using a flow cytometer. CD34 + Cell Staining In order to enumerate CD34 + cells via flow cytometry, two sets of cells from a population must first be stained with one of two fluorochrome-conjugated antibodies: an isotype-matched control antibody or a CD34-specific antibody. The isotype control

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19 antibody has no specificity for CD34 + cells, but exhibits all of the same non-specific binding properties as the CD34 antibody; additionally, the isotype antibody is conjugated with an identical color fluorochrome as the CD34 antibody. Thus the isotype antibody serves as a check on specificity and sets the background level of fluorescence emission above which cells are counted as CD34 + . Fresh cells. Aliquots of fresh cells designated isotype controls were removed from the original NC solution (done prior to magnetic labeling and CD34 + separation in order to spare purified cells) and washed in 1275 mm polystyrene tubes (Becton Dickinson Biosciences Discovery Labware, NJ) with 1 – 2 ml of a staining buffer consisting of 1X PBS, 0.5% Bovine Serum Albumin (Fisher Scientific, USA), and 0.01% Sodium Azide (Fisher Scientific, USA). The cells were centrifuged (200 g, 5 minutes, 4 C), resuspended in staining buffer with 10% Human AB Serum (Sigma, St. Louis, MO), and incubated for 20 minutes at 4 C. The Fc fragment of the human immunoglobulin G (IgG) antibody in the serum binds to FcRs on all nucleated cells in the same fashion as the FcR Blocking Reagent (Miltenyi Biotec, Auburn, CA) used during magnetic labeling, blocking non-specific, FcR-mediated binding of other antibodies. The cells were then stained using 20 l R-Phycoerythrin (R-PE)-conjugated mouse IgG 1 , isotype control antibody (clone MOPC-21) (Becton Dickinson Biosciences PharMingen, San Diego, CA) per 110 6 cells, and incubated for 20 minutes at 4 C. All remaining NCs were stained after magnetic labeling with 100 l R-PE-conjugated mouse anti-human CD34 antibody (clone 581) (Becton Dickinson Biosciences PharMingen, San Diego, CA) per 110 8 cells and incubated for 10 minutes at 4 C, as recommended by the manufacturer.

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20 Cultured cells. Aliquots of cultured cells were washed with staining buffer, centrifuged (200 g, 5 minutes, 4 C), resuspended in staining buffer with 10% Human AB Serum (Sigma, St. Louis, MO) to block non-specific binding, and incubated for 20 minutes at 4 C. The cells were then stained using 20 l of either R-Phycoerythrin (R-PE)-conjugated mouse IgG 1 , isotype control antibody (clone MOPC-21) (Becton Dickinson Biosciences PharMingen, San Diego, CA) or R-PE-conjugated mouse anti-human CD34 antibody (clone 581) (Becton Dickinson Biosciences PharMingen, San Diego, CA) per 110 6 cells or less, and incubated for 20 minutes at 4 C. Viability Staining After CD34 + cell staining, cells were washed, resuspended in staining buffer with 20% Via-Probe (Becton Dickinson Biosciences PharMingen, San Diego, CA), and incubated for 10 minutes at 4 C. The Via-Probe stain contains 7-Amino-actinomycin D (7-AAD), a nucleic acid dye which penetrates the cell membrane of dead cells and binds to their DNA, allowing for the exclusion of nonviable cells in flow cytometric assays. Cell Cycle Staining Aliquots of fresh (post-magnetic labeling and CD34 + separation) or cultured cells were washed, resuspended in equal volumes of ribonuclease A (2 mg/ml) (Sigma, St. Louis, MO) and cell cycle staining buffer, consisting of 1X PBS, 0.6% Igepal CA-630 detergent (Sigma, St. Louis, MO), and 0.1 mg/ml propidium iodide (Sigma, St. Louis, MO), and incubated for 30 minutes at 4 C. The detergent lyses cell membranes so that the propidium iodide stain can bind to DNA, allowing quantization via flow cytometry. The ribonuclease molecule destroys cellular RNA to eliminate RNA staining. All cells were stored in the dark at 4 C until flow cytometric analysis was performed.

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21 Flow Cytometry Cells were characterized via flow cytometry on a FACScan (BD Biosciences Immunocytometry Systems, San Jose, CA) and analyzed using CellQuest (BD Biosciences Immunocytometry Systems, San Jose, CA) and ModFit (Verity Software House, Topsham, ME) software. CD34 + cell enumeration and viability analysis. Cells were characterized for CD34 + purity and viability through analysis of laser light-scattering properties and fluorescence emission. For each sample, 5,000 events were recorded by the FACScan (BD Biosciences Immunocytometry Systems, San Jose, CA), and cells were gated based on their morphology (forward and side light scatter), viability (7-AAD stain intensity), and CD34 presentation (PE stain intensity) using CellQuest (BD Biosciences Immunocytometry Systems, San Jose, CA) software (Figures 4-3 through 4-5). B A Figure 4-3. Morphological gate dot-plots of fresh HUCB cells. A) Nucleated cell fraction. B) CD34 + -purified cell fraction. Cells are plotted as a function of their light scattering properties and gated (R1 region) to include the population of cells which exhibit morphological similarities to non-granular, nucleated blood cells, including lymphocytes and CD34 + cells. As shown in Figure 4-3, cells are gated (R1) to include the largest population of nucleated blood cells in the sample, or those which exhibit morphological similarities to

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22 non-granular cells, including lymphocytes and CD34 + cells. This gate thus serves only as an aid for determining CD34 + purity in the sample; it is not absolute. Figure 4-4. Viability gate dot-plot of a CD34 + -purified fraction of fresh HUCB cells. Cells previously gated in the R1 region are plotted as a function of their fluorescence emission (7-AAD or ViaProbe stain intensity vs. PE stain intensity) and gated (R2 region) to include the population of cells which do not display the viability stain, detectable only in dead cells. A B Figure 4-5. CD34 + gate dot-plots of fresh HUCB cells. A) Isotype control sample, nucleated cell fraction. B) CD34-tagged sample, CD34 + -purified fraction. Cells previously gated in the R1 and R2 regions are plotted as a function of their fluorescence emission (7-AAD or ViaProbe stain intensity vs. PE stain intensity) and gated (R3 region) to include the population of cells which display the CD34 stain. Cell cycle analysis. Cells were characterized for cell cycle activity through analysis of fluorescence emission (propidium iodide stain). For each sample, up to

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23 30,000 events were recorded by the FACScan (BD Biosciences Immunocytometry Systems, San Jose, CA). DNA content was plotted on histograms using CellQuest (BD Biosciences Immunocytometry Systems, San Jose, CA) software and samples were analyzed for cell cycle activity using ModFit (Verity Software House, Topsham, ME) software. Determination of Optimal Static Culture Conditions Cultures initiated with both unpurified NC fractions and CD34 + -purified fractions were tested for growth potential. Unpurified Nucleated Cell Fraction Cultures Nucleated cells were initiated in culture in 24-well plates (Becton Dickinson Biosciences Discovery Labware, NJ) at concentrations of 1 – 1.5 10 6 NCs/ml in 1.2 ml of two separate medias. Media A consisted of IMDM (GIBCO, Long Island, NY) supplemented with SCF (130 ng/ml) (Sigma, St. Louis, MO), IL-3 (8 ng/ml) (R&D Systems, Minneapolis, MN), IL-6 (20 ng/ml) (Sigma, St. Louis, MO), G-CSF (12 ng/ml) (Berlex Laboratories, Inc., Richmond, CA), EPO (0.4 U) (Amgen Inc., Thousand Oaks, CA) and FL (50 ng/ml) (Sigma, St. Louis, MO) as previously described by Fietz et al. (1999). Media B consisted of IMDM (GIBCO, Long Island, NY) and 10% fetal bovine serum (FBS) (GIBCO, Long Island, NY) supplemented with FL (PeproTech, Inc., Rocky Hill, NJ) (50 ng/ml), and TPO (PeproTech Inc., Rocky Hill, NJ) (5 ng/ml) as previously described by Piacibello et al. (1997). All cells were cultured in a 37 C and 5% CO 2 – 95% air environment. Cytokines were added every three to four days, and cells were demidepopulated weekly for characterization by removal of half the cells, followed by the addition of fresh

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24 media and cytokines to match the original well volume of 1.2 ml. Cells were assessed for live NC content (hemacytometer), CD34 + purity (flow cytometer), and live CD34 + cell content (hemacytometer and flow cytometer) over a culture period of fourteen days. Live NC content was calculated using the following equation, Live NC Content = Live Cells Counted 2 n (4-1) where n = number of demidepopulations performed. After CD34 + purity was assessed using a flow cytometer, live CD34 + cell content was calculated using the following equation, Live CD34 + Content = Live Cells Counted CD34 + Purity 2 n (4-2) where n = number of demidepopulations performed. CD34 + -Purified Cell Fraction Cultures CD34 + cells were initiated in culture in 24-well plates (Becton Dickinson Biosciences Discovery Labware, NJ) at concentrations of 6 – 12 10 3 CD34 + cells/ml in 1.2 ml of IMDM (GIBCO, Long Island, NY) and 10% fetal bovine serum (FBS) (GIBCO, Long Island, NY) supplemented with FL (PeproTech, Inc., Rocky Hill, NJ) (50 ng/ml), and TPO (PeproTech Inc., Rocky Hill, NJ) (5 ng/ml) in a 37 C and 5% CO 2 – 95% air environment as previously described by Piacibello et al. (1997). Cytokines were added every three to four days, and cells were demidepopulated weekly and assessed for live NC content, CD34 + purity, and live CD34 + cell content over a culture period of fourteen days as previously described for unpurified NC fraction cultures. Short-Term Simulated Microgravity Culture Assessment CD34 + -purified fractions were cultured in a simulated microgravity or static (control) environment for up to three days at concentrations of 1 – 8 10 4 CD34 +

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25 cells/ml in complete media consisting of IMDM (GIBCO, Long Island, NY) and 10% FBS (GIBCO, Long Island, NY) supplemented with FL (PeproTech, Inc., Rocky Hill, NJ) (50 ng/ml), and TPO (PeproTech Inc., Rocky Hill, NJ) (5 ng/ml) in a 37 C and 5% CO 2 – 95% air environment. The unique cell availabilities of each sample coupled with the dynamics of each experiment contributed to the range of initial concentrations used. In each experiment, cultures were initiated with the largest possible CD34 + cell concentration. Simulated Microgravity Cultures Cells were subjected to simulated microgravity in the HARV. The anterior and posterior portions of the HARV were screwed together and 10 ml of cells in complete media with cytokines were injected into one open port using a sterile 12 cc syringe (Sherwood, Davis, and Geck, St. Louis, MO) (Figure 4-6). Figure 4-6. Injecting cells into the HARV using a sterile syringe. An empty syringe was screwed on to the adjacent port and used to remove air bubbles by gently mixing. After filling the HARV, the open ports were capped using

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26 sterile stopcocks. The HARV was then loaded onto the rotary base and rotated at a speed of 10 rpm as suggested by the manufacturer for suspension cells. At daily intervals during culture, the HARV was purged of waste using a sterile syringe filled with approximately 0.5 ml of complete media. Cells were harvested after one, two, or three days by complete removal from culture and assessed for live NC content (hemacytometer), CD34 + purity (flow cytometer), overall viability (flow cytometer), cell cycle activity (flow cytometer), and live CD34 + cell content (hemacytometer and flow cytometer). The HARV was emptied and washed twice with 3 ml complete media to remove all remaining cells. Control Cultures Cells designated as control samples were cultured in triplicate in 24-well plates (Becton Dickinson Biosciences Discovery Labware, NJ) in 1.2 ml of complete media with cytokines per well at identical CD34 + cell concentrations as in the HARV. The amount of cell volume per static well was calculated in order to ensure that cells in each culture were allowed equal gas transfer (identical number of cells per unit surface area in each culture) using the following equation, HHWWANAN (4-3) where N W = number of cells in well, A W = well surface area available for gas transfer, N H = number of cells in HARV, and A H = HARV surface area available for gas transfer. Equating the cell concentrations of each culture gives HHWWVNVN (4-4)

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27 where V W = cell volume in well, and V H = cell volume in HARV. Because the allowable cell volume in the HARV is fixed, the well volume (V W ) can be found by combining Equations 4-3 and 4-4 and rearranging, giving HWHWAAVV (4-5) Cells were harvested after one, two, or three days by complete removal from culture and assessed for live NC content, CD34 + purity, overall viability, cell cycle activity, and live CD34 + cell content as previously described for HARV-cultured cells. Wells were emptied and washed twice with 1 ml complete media to remove all remaining cells. Secondary Long-Term Static Cultures When secondary LTSCs were established, all cells from HARV and well plate cultures were re-plated separately in 24-well plates at identical NC concentrations of 1 – 2 10 4 NCs/ml in 1.2 ml complete media consisting of IMDM (GIBCO, Long Island, NY) and 10% FBS (GIBCO, Long Island, NY) supplemented with FL (PeproTech, Inc., Rocky Hill, NJ) (50 ng/ml), and TPO (PeproTech Inc., Rocky Hill, NJ) (5 ng/ml) in a 37 C and 5% CO 2 – 95% air environment. Cells were demidepopulated every three to four days and assessed for live NC content, CD34 + purity, overall viability, cell cycle activity, and live CD34 + cell content over a culture period of fourteen days post-initial culture as described previously. Hematopoietic Progenitor Cell Assays Fresh cells, cells from initial two-day HARV and static cultures, and cells from secondary LTSCs were seeded in duplicate in plastic 35-mm tissue culture dishes (Corning Incorporated, Corning, NY) in 1.1 ml of MethoCult methylcellulose media

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28 (StemCell Technologies, British Columbia, Canada) at concentrations of either 1,375 CD34 + cells/ml or 12,500 NC/ml as recommended by the manufacturer. After fourteen days of culture in a 37 C and 5% CO 2 – 95% air environment, BFU-Es and CFU-GMs were enumerated using an inverted light microscope (Leica Microsystems Inc., Bannockburn, IL). CFU content was calculated using the following equation, CFU Content = Colonies Counted 2 n (4-6) where n = number of demidepopulations performed in secondary LTSCs. Statistics Statistical analysis was performed to determine significance between two culture populations. Students’ t-tests were performed using Microsoft Excel (Microsoft Corporation, USA) software to determine significant differences in data. Significance was taken at p 0.05 and p 0.01.

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CHAPTER 5 RESULTS AND DISCUSSION The goal of the present study was to induce a state of quiescence in HUCB stem cells via culture in simulated microgravity in order to improve long-term hematopoietic expansion through prolonged maintenance of the primitive phenotype. After establishing a method for growing cells in a conventional, static environment, the effects of a short-term simulated microgravity culture on NC growth, stem cell growth and purity, and cell cycle activity were observed, and an optimal HARV culture time was determined based on these parameters. The optimal HARV culture time was then implemented, followed by the establishment of a fourteen-day post-microgravity LTSC, which was further assessed for NC growth, stem cell growth and purity, and development of hematopoietic CFUs. Static cultures initiated in triplicate wells, simulated microgravity cultures initiated in individual HARVs, and CFU cultures initiated in duplicate dishes were considered individual experiments, even if derived from the same cord blood sample; thus donor samples were not compared. While donor variability may cause data discrepancies, assessing variability was beyond the scope of this project. Determination of Optimal Static Culture Conditions A method for growing NCs and CD34 + cells in a static environment was developed. Cultures initiated with both unpurified NC fractions and CD34 + -purified fractions were tested for NC and CD34 + cell growth potential (Figure 5-1). Unpurified fractions grown in Media A exhibited an average Day 0 CD34 + purity of 0.45 % (n=5), 29

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30 while those grown in Media B exhibited an average Day 0 CD34 + purity of 0.70 % (n=3). CD34 + -purified fractions grown in Media B exhibited an average Day 0 CD34 + purity of 30 % (n=3). Because experiments were performed over ranging cell concentrations due to cell availability and protocol requirements (Fietz, et al. 1999, Piacibello, et al. 1997), cell number data on each day was normalized by initial (Day 0) cell numbers specific to each experiment. Figures describing absolute cell number change can be found in Appendix A along with tabulated data. The cellular growth trends of unpurified fractions observed by Fietz et al. (1999) were not reproducible in the current study, possibly due to donor variability. The cellular growth trends of purified fractions observed by Piacibello et al. (1997) however were similar to the results of the current study. By comparison, the purified fraction exhibited significantly greater cell growth than the unpurified fractions over the culture period. The difference in purity of stem cells and progenitor cells is the most likely explanation for superior purified fraction growth. The method used to grow cells from purified fractions in these experiments was implemented for samples in HARV experiments. Short-Term Simulated Microgravity Culture Assessment The effects of simulated microgravity on one-, two-, and three-day cultures of CD34 + -purified fractions (average CD34 + purity of 94 %) on NC growth, stem cell growth and purity, and cell cycle activity were observed. An optimal HARV culture time was then determined based on these parameters. As before, cell number data on each day was normalized by initial (Day 0) cell numbers specific to each experiment due to the range of concentrations used, limited cell availability, and volume constraints of the HARV. Figures describing absolute cell number change can be found in Appendix B along with tabulated data.

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31 Nucleated Cell and CD34+ Cell GrowthPurified vs. Unpurified Fractions05101520253005-712-14DayLive NC Ratio Compared to Day 0 012305-712-14DayLive CD34+ Cell Ratio Compared to Day 0 Unpurified Fraction, Media A Unpurified Fraction, Media B Purified Fraction, Media B A B Figure 5-1. Nucleated cell and CD34 + cell growth, purified vs. unpurified fractions. A) Nucleated cell progression. B) CD34 + cell progression. The live cell ratios (y-axis) are defined as (live cells on Day X/live cells on Day 0). NC counts were performed using a hemacytometer, while CD34 + cell counts were performed using a hemacytometer and flow cytometer. NC and CD34 + growth from the purified fraction is significant over 14 days (p 0.05). By comparison, live NC ratios are significantly greater in purified cultures on Days 5 – 7 and Days 12 – 14 (p 0.01). Live CD34 + cell ratios are significantly greater in purified cultures on Days 12 – 14 (p 0.01).

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32 Figure 5-2 shows the nucleated cell progression of cultures over a three day culture period (Day 0, n=6; Day 1, n=3; Day 2, n=6; Day 3, n=4). HARV-cultured cells exhibited a significant NC death on each day as compared to Day 0 and significantly smaller live NC ratios than controls on Days 1 – 3. This suggests that HARV-cultured CD34 + cells are ceasing to differentiate while NCs are ceasing to proliferate and are dying over a three-day culture period. Nucleated Cell ProgressionCD34+-Purified Fraction 00.20.40.60.811.20123DayLive NC Ratio Compared to Day 0 Static HARV Figure 5-2. Nucleated cell progression, CD34 + -purified fraction. NC counts were performed using a hemacytometer. NC death of HARV-cultured cells is significant on all days as compared to Day 0 (Day 1, p 0.05; Days 2 and 3, p 0.01), while NC death of static-cultured cells is significant only on Day 2 (p 0.01). By comparison, HARV-cultured cells exhibited significantly smaller live NC ratios than controls on Days 1 – 3 (Day 1, p 0.05; Days 2 and 3, p 0.01). The effects of initial CD34 + cell concentration on NC progression of a two-day HARV culture were assessed (n=1 for each concentration) (Figure 5-3). Although the low number of experiments performed for each concentration makes it difficult to draw solid conclusions based on these data, initial concentration appears to have no affect on NC progression within the ranges tested. There may however be a threshold

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33 concentration value below which cells will not proliferate, as shown in HARV-cultured bone marrow stem cells to be 1 10 5 CD34 + cells/ml (Plett, et al. 2001), a concentration difficult to achieve in HARV-culture using HUCB cells due to limited availability and volume requirements. NC Progression Two-Day HARV Culture00.10.20.30.40.50.6Increasing ConcentrationLive NC Ratio Compared to Day 0 1.0 E4 CD34+ cells/ml 1.5 E4 CD34+ cells/ml 2.0 E4 CD34+ cells/ml 4.0 E4 CD34+ cells/ml 6.0 E4 CD34+ cells/ml 8.0 E4 CD34+ cells/ml Figure 5-3. NC progression, two-day HARV culture. Figure 5-4 shows the CD34 + cell progression and CD34 + purity of cultures over a three-day culture period (Day 0, n=6; Day 1, n=3; Day 2, n=5; Day 3, n=2). HARV-cultured cells exhibited a significant CD34 + cell death on each day as compared to Day 0 and significantly smaller live CD34 + cell ratios than controls on Days 2 and 3. This suggests that HARV-cultured CD34 + cells are ceasing to proliferate and are dying over a three-day culture period. HARV-cultured cells also exhibited a significantly smaller CD34 + purity than controls on Day 2, refuting the hypothesis that HARV-cultured cells would retain a larger stem cell frequency than static cultures.

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34 CD34+ Cell ProgressionCD34+-Purified Fraction 00.20.40.60.811.20123DayLive CD34+ Ratio Compared to Day 0 Static HARV CD34+Purity 0.020.040.060.080.0100.0120.00123Day% of Non-Granular NCs Static HARV A B Figure 5-4. CD34 + cell progression, CD34 + -purified fraction. A) CD34 + cell progression. B) CD34 + purity. CD34 + purity was assessed using a flow cytometer, while CD34 + cell counts were performed using a hemacytometer and flow cytometer. CD34 + death of HARV-cultured cells is significant on all days as compared to Day 0 (Day 1, p 0.05; Days 2 and 3, p 0.01), while CD34 + death of static-cultured cells is significant only on Day 2 (p 0.01). By comparison, HARV-cultured cells exhibited significantly smaller live CD34 + cell ratios than controls on Days 2 and 3 (p 0.01) and significantly smaller CD34 + purity than controls on Day 2 (p 0.05). Figure 5-5 shows the cell cycle activity of cultures over a three day culture period (Day 0, n=6; Day 1, n=3; Day 2, n=5; Day 3, n=2). The data shows that HARV-cultured

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35 cells never became significantly active, while static-cultured cells became significantly active on Day 2 and remained active through Day 3, supporting the hypothesis that the HARV would induce a state of quiescence in cells. The data also shows that HARV-cultured cells exhibited significantly lower activity levels than controls on Days 2 and 3. This data may be misleading however, as all cells, including dead cells, were stained due to the nature of the DNA stain used to quantify active and inactive cells. In an attempt to solve this problem and assess the cell cycle activity of live cells, overall viability was assumed evenly distributed over all cell cycles and factored into the active cell data (Figure 5-6). Cell Cycle ActivityCD34+-Purified Fraction 0102030400123Day% of All Cells Exhibiting Activity Static HARV Figure 5-5. Cell cycle activity, CD34 + -purified fraction. The percentage of all cells which have exited the G o /G 1 phase of the cell cycle is plotted. Cell cycle activity was assessed using a flow cytometer. Cell cycle activity of HARV-cultured cells is not significantly different on any day as compared to Day 0, while activity of static-cultured cells is significantly greater on Days 2 and 3 as compared to Day 0 (p 0.05). By comparison, HARV-cultured cells exhibited significantly lower activity levels than controls on Days 2 (p 0.05) and 3 (p 0.01).

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36 The modified cell cycle data shows similar results to the unmodified data in that live HARV-cultured cells never became significantly active, while live static-cultured cells became significantly active on Day 2 and remained active through Day 3, further supporting the hypothesis that the HARV would induce a state of quiescence in cells. The HARV and control cultures however did not exhibit significantly different viable activity levels on any day, contradictory to the unmodified results. It may be difficult to draw conclusions from these data alone due to the questionable validity of the assumption of evenly distributed viability across all cell cycles. Viable Cell Cycle ActivityCD34+-Purified Fraction 010203040500123Day% of Live CellsExhibiting Activity Static HARV Figure 5-6. Viable cell cycle activity, CD34 + -purified fraction. The percentage of live cells which have exited the G o /G 1 phase of the cell cycle is plotted. Cell cycle activity was assessed using a flow cytometer. Cell cycle activity of HARV-cultured cells is not significantly different on any day as compared to Day 0, while activity of static-cultured cells is significantly greater on Days 2 and 3 as compared to Day 0 (p 0.05). By comparison, HARV-cultured cells do not exhibit significantly different activity levels as compared to controls on any day. Although both sets of cell cycle data support the conclusion that HARV-cultured cells remain quiescent while static-cultured cells become active over a three-day culture

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37 period, the unique shortcomings of each method of data assessment make it difficult to firmly draw this conclusion. The same holds true for the case of contradictory results in activity differences between cultures. Under the conditions tested, the HARV environment has proven to cause significant NC and CD34 + cell death in one-, two-, and three-day cultures. Control cultures show significant cell death on Day 2, but rebound by Day 3, most likely due to the acclamation time of cells to a new environment. The significant increase in cell cycle activity exhibited by control cultures after two and three days supports this theory. The case of “rebounding” may be argued for the HARV cells, although the prolonged quiescence exhibited by these cells suggests that they show no signs of renewed growth in the environment of simulated microgravity; thus a HARV culture of longer than three days under the conditions tested may not result in positive outcomes. As a whole, the data suggest that a HARV culture of two or three days under the conditions tested is equally damaging to the viability and activity of cells. Cells cultured in the HARV for one day however do not exhibit significant differences from static-cultured cells in CD34 + viability, purity, or cell cycle activity, suggesting that the effects of simulated microgravity may not be fully pronounced after only one day of culture. The effects of simulated microgravity on the parameters measured appear to become apparent only after two days of culture. Because the data do not show any evidence of HARV cells rebounding by Day 3, a HARV culture of two days was employed before initiating LTSCs in hopes of increasing long-term hematopoietic expansion. There are several possibilities for the observed cell death in HARV cultures. First, the employed HARV rotation speed of 10 rpm may have been too slow to allow the

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38 necessary suspension of cells, causing sedimentation and thereby preventing the formation of a 3-D culture environment and hindering proper gas transfer. Conversely, the rotation speed may have been too fast, causing cells to strike the vessel walls and become damaged. Second, the low initial cell concentrations used may have inhibited the normal cell-to-cell interaction that might be seen in vivo. Finally, the high initial primitivity of HUCB cells may have led to decreased adaptation to HARV cultures. Secondary Long-Term Static Culture Assessment A two-day HARV culture time was implemented, followed by the establishment and assessment of fourteen-day post-microgravity LTSCs on NC growth, stem cell growth and purity, and development of hematopoietic CFUs. As before, cell number data on each day was normalized by initial (Day 0) cell numbers specific to each experiment due to the range of concentrations used. Figures describing absolute cell number change can be found in Appendix C along with tabulated data. Figure 5-7 shows the nucleated cell and CD34 + cell progression of secondary LTSCs over a fourteen-day post-initial culture period (n=2). Cells cultured initially in the HARV did not recover, as complete cell death was recorded by Day 14 post-HARV culture in all experiments, while control cultures exhibited significant NC growth by Day 14 post-initial static culture. This may prove that a two-day HARV culture of HUCB stem cells under the conditions tested may be too damaging to allow rejuvenation and improved long-term hematopoietic expansion. Figure 5-8 shows the CD34 + purity of secondary LTSCs over a fourteen-day post-initial culture period (n=2). The significantly lower long-term CD34 + purity of cells cultured initially in the HARV refutes the hypothesis that HARV-cultured cells initiated

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39 in secondary LTSCs would retain a larger long-term stem cell frequency than static cultures. Cell GrowthSecondary Long-Term Static Cultures0510152025047 811 1214Days Post-Initial CultureLive NC RatioCompared to Day 0 0.00.51.01.52.02.53.0047 811 1214Days Post-Initial CultureLive CD34+ Cell Ratio Compared to Day 0 Post-Static Post-HARV B A Figure 5-7. Cell growth, secondary long-term static cultures. A) NC progression. B) CD34 + cell progression. Day 0 post-initial culture represents the end of the two-day initial culture. NC counts were performed using a hemacytometer, while CD34 + cell counts were performed using a hemacytometer and flow cytometer. Post-static cultures exhibit significant NC growth over 14 days (p 0.05), while post-HARV cultures exhibit significant NC and CD34 + cell death over 14 days (p 0.01). By comparison, live NC ratios are significantly

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40 greater in post-static cultures on Days 7 – 8 (p 0.01), Days 11 – 12 (p 0.05), and Day 14 (p 0.01), while live CD34 + cell ratios are significantly greater in post-static cultures on Days 7 – 8 (p 0.01), Days 11 – 12 (p 0.01), and Day 14 (p 0.05). CD34+ PuritySecondary Long-Term Static Cultures 0102030405060708090047 811 1214Days Post-Initial Culture% of Non-Granular NCs Post-Static Post-HARV Figure 5-8. CD34 + purity, secondary long-term static cultures. By comparison, HARV-cultured cells exhibited significantly smaller CD34 + purity than controls on Days 7 – 8 (p 0.05) and Day 14 (p 0.01) post-initial culture. A two-day HARV culture time was implemented and assessed for hematopoietic CFU production (n=2) (Figure 5-9). Cells cultured initially in the HARV for two days were not capable of generating CFU-GMs or BFU-Es, even after seven days of LTSC. These data suggest that HARV-cultured cells are too unhealthy after two days of culture in the HARV under the conditions tested to promote colony formation. It should be noted that although significant differences were apparent between cultures in several assays involving LTSCs, the low number of experiments performed may make it difficult to draw solid conclusions from the data.

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41 CFU-GM Growth14-Day Cultures050100150200250027Culture Initiation DayCFU-GM # Static HARVInitial CulturePost-Initial Culture 048121620027Culture Initiation DayBFU-E # Static HARVInitial CulturePost-Initial Culture A B Figure 5-9. Progenitor growth, 14-day cultures. A) CFU-GM progression. B) BFU-E progression. Cells from initial two-day cultures (Day 0 and 2, initial culture) and secondary long-term static cultures (Day 7, post-initial culture) were seeded in progenitor cultures and enumerated after fourteen days. By comparison, cells cultured initially in the HARV exhibit significantly lower CFU-GM and BFU-E numbers than cells cultured initially in static on Day 2 (initial culture) and Day 7 (post-initial culture) (p 0.01).

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CHAPTER 6 CONCLUSIONS AND FUTURE WORK HUCB stem cells cultured in simulated microgravity exhibited significant NC and CD34 + cell death and reduced CFU production over a three-day culture period. These effects became apparent only after two days of culture as compared to static controls. Because cells were not visible to the naked eye, it was difficult to determine whether or not the employed HARV rotation speed of 10 rpm caused cells to attain the desired 3-D suspension, preventing sedimentation or cell-to-wall collision, and allowing sufficient gas transfer. It is therefore difficult to conclude that the cell death observed in the HARV was not purely due to a technical issue. In order to find the rotation speed which provides optimal suspension, it may be possible to focus an intense light source on the cells, assess their position by opacity, and fine tune the rotation speed accordingly. While technical issues may have played a key role in cell death in the HARV, cell-related issues may have also hindered growth. Although concentration did not appear to have an affect on NC progression in this study, it is possible that the dynamics of the 3-D culture environment created by the HARV coupled with low cell concentrations impeded cell-to-cell interaction, which may have hindered growth; there may be a threshold concentration value below which cells will not proliferate, as seen with bone marrow stem cells (Plett, et al. 2001). Seeding cultures with larger cell concentrations than those used in this study may be difficult however due to the limited availability of CD34 + cells derived from a single sample of umbilical cord blood and HARV volume requirements. Future studies should focus on the development of an intra-HARV culture well and 42

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43 scaffold designed to minimize the cell volume requirements for culture while still modeling the environment of microgravity, thus allowing increased initial cell concentrations and possibly improving cell-to-cell interaction. The effects of cell death exhibited by HARV-cultured cells appeared to be accompanied by a prolonged state of quiescence, as hypothesized, in which cells did not significantly exit the G 0 /G 1 phase of the cell cycle. The discrepancies of data assessment however make it difficult to confirm that only live cells were assessed for cell cycle activity. In order to solidify the results found in the current study, a future study should involve staining only live cells using Hoescht dyes or DRAQ-5, DNA stains designed to penetrate the membranes of live cells. The extremely primitive state of fresh HUCB cells may have further contributed to the observed cell death in HARV cultures. The percentage of active cells in fresh bone marrow stem cell populations successfully grown in simulated microgravity (Plett, et al. 2001) was over 10% greater than that of fresh HUCB stem cell populations used in this study, suggesting that cell maturity may be a factor in HARV culture adaptation. Future studies should assess the effect of initial primitivity on hematopoietic expansion, comparing the growth potential of HUCB cells to that of bone marrow stem cells and peripheral blood stem cells grown in simulated microgravity. HARV-cultured HUCB stem cells were not able to rejuvenate in LTSC, exhibiting complete hematopoietic exhaustion within fourteen days post-initial culture. While the effects of simulated microgravity may have not been fully pronounced after one day of culture, a two-day HARV culture under the conditions tested has proven to be too damaging to cells to allow improved long-term hematopoietic expansion. Thus future

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44 studies should focus on solving the technical and cell-related problems involved with HARV culture of HUCB stem cells to allow seeding of a more healthy population of cells in LTSC.

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APPENDIX A STATIC CULTURE DATA AND FIGURES

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Absolute Nucleated Cell and CD34+ Cell GrowthPurified vs. Unpurified Fractions012345605-712-14DayNC # (X 106) 025507510012515005-712-14DayCD34+ Cell # (X 103) Unpurified Fraction, Media A Unpurified Fraction, Media B Purified Fraction, Media B B A Figure A-1. Absolute nucleated cell and CD34 + cell growth, purified vs. unpurified fractions. A) Nucleated cell progression. B) CD34 + cell progression. 46

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47 Table A-1. Initial Culture Data (Day 0), Unpurified Fraction Cultures, Media A n NC Concentration ( 10 6 NCs/ml) Live CD34 + Cell Purity (%) CD34 + Cell Concentration ( 10 3 CD34 + /ml) 1 1.0 0.36 3.6 2 1.0 0.54 5.4 3 1.0 0.54 5.4 4 1.5 0.54 8.3 5 1.5 0.54 8.1 Table A-2. Culture Data, Unpurified Fractions, Media A n Day Live NCs ( 10 6 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio 0 3.60 1.00 0.36 12.96 1.00 5 0.93 0.26 3.51 32.80 2.53 1 12 0.63 0.18 0.00 0.00 0.00 0 3.60 1.00 0.54 19.44 1.00 5 2.50 0.69 1.32 33.00 1.70 2 12 0.26 0.07 0.00 0.00 0.00 0 3.60 1.00 0.54 19.44 1.00 5 2.80 0.78 1.86 52.08 2.68 3 12 0.56 0.16 0.00 0.00 0.00 0 5.56 1.00 0.54 30.02 1.00 5 4.70 0.85 0.78 36.66 1.22 4 12 0.48 0.09 0.00 0.00 0.00 0 5.56 1.00 0.54 30.02 1.00 5 5.60 1.01 1.12 62.72 2.09 5 12 1.30 0.23 0.00 0.00 0.00

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48 Table A-3. Initial Culture Data (Day 0), Unpurified Fraction Cultures, Media B n NC Concentration ( 10 6 NCs/ml) Live CD34 + Cell Purity (%) CD34 + Cell Concentration ( 10 3 CD34 + /ml) 1 1.0 0.72 7.2 2 1.0 0.72 7.2 3 1.0 0.72 7.2 Table A-4. Culture Data, Unpurified Fractions, Media B n Day Live NCs ( 10 6 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio 0 3.60 1.00 0.72 25.92 1.00 7 2.22 0.62 0.12 2.66 0.10 1 12 0.45 0.13 0.42 1.89 0.07 0 3.60 1.00 0.72 25.92 1.00 7 2.60 0.72 0.10 2.60 0.10 2 12 0.39 0.11 0.05 0.19 0.00 0 3.60 1.00 0.72 25.92 1.00 7 2.13 0.59 0.10 2.13 0.08 3 12 0.44 0.12 1.61 7.15 0.28

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49 Table A-5. Initial Culture Data (Day 0), CD34 + -Purified Fraction Cultures, Media B n NC Concentration ( 10 3 NCs/ml) Live CD34 + Cell Purity (%) CD34 + Cell Concentration ( 10 3 CD34 + /ml) 1 20.0 29.08 5.8 2 40.0 29.08 11.6 3 27.4 31.80 8.7 Table A-6. Culture Data, CD34 + -Purified Fractions, Media B n Day Live NCs ( 10 3 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio 0 72.00 1.00 29.08 20.94 1.00 7 286.88 3.98 11.53 33.08 1.58 1 14 2126.25 29.53 2.34 49.75 2.38 0 144.00 1.00 29.08 41.88 1.00 7 534.75 3.71 7.52 40.21 0.96 2 14 2636.25 18.31 4.17 109.93 2.63 0 98.71 1.00 31.80 31.39 1.00 6 332.00 3.36 33.28 110.49 3.52 3 13 2100.00 21.27 2.60 54.60 1.74

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APPENDIX B HARV CULTURE DATA AND FIGURES

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Absolute Nucleated Cell ProgressionCD34+-Purified Fraction 0501001502002500123DayNC # (X 103) 01002003004005006000123DayNC # (X 103) 0100200300400500600012DayNC # (X 103) Static HARV A B C Figure B-1. Absolute nucleated cell progression, CD34 + -purified fraction. A) Cultures assessed on Days 1, 2, and 3 (n=3). B) Cultures assessed on Days 2 and 3 (n=4). C) Cultures assessed on Day 2 (n=6). Individual experiments with similar assessment days are combined into groups for accurate representation of data. 51

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52 CD34+ Cell ProgressionCD34+-Purified Fraction 040801201602000123DayCD34+ Cell # (X 103) 050100150200250012DayCD34+ Cell # (X 103) 0100200300400500600700012DayCD34+ Cell # (X 103) Static HARV A B C Figure B-2. Absolute CD34 + cell progression, CD34 + -purified fraction. A) Cultures assessed on Days 1, 2, and 3 (n=2). B) Cultures assessed on Days 1 and 2 (n=3). C) Cultures assessed on Day 2 (n=5). Individual experiments with similar assessment days are combined into groups for accurate representation of data.

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53 Table B-1. Initial Culture Data (Day 0), HARV and Control Cultures n NC Concentration ( 10 3 NCs/ml) Live CD34 + Cell Purity (%) CD34 + Cell Concentration ( 10 3 CD34 + /ml) 1 15.5 94.95 14.7 2 21.0 96.75 20.3 3 10.4 94.95 9.9 4 63.5 96 61.0 5 82.0 93.06 76.3 6 44.0 86.29 38.0 Table B-2. Culture Data, HARV Cultures* n Day Live NCs ( 10 3 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio Overall Viability (%) 0 154.5 1.00 94.95 146.70 1.00 95.96 1 122.5 0.79 81.61 99.97 0.68 95.12 2 27.3 0.18 42.45 11.59 0.08 22.42 1 3 24.0 0.16 50.35 12.08 0.08 28.77 0 209.7 1.00 96.75 202.88 1.00 94.84 1 149.5 0.71 80.38 120.17 0.59 96.28 2 85.0 0.41 91.56 77.83 0.38 95.12 2 3 47.5 0.23 0 103.8 1.00 94.95 98.56 1.00 93.19 1 55.0 0.53 72.28 39.75 0.40 97.61 2 21.3 0.20 66.31 14.09 0.14 44.96 3 3 10.0 0.10 61.90 6.19 0.06 17.29 0 635.0 1.00 96.00 609.60 1.00 93.59 1 2 350.0 0.55 4 3 150.0 0.24 0 820.0 1.00 93.06 763.09 1.00 98.31 1 2 150.0 0.18 56.72 85.08 0.11 12.62 5 3 0 440.0 1.00 86.29 379.68 1.00 94.14 1 2 59.4 0.14 33.44 19.86 0.05 12.63 6 3 *Entries left blank indicate that data was not recorded.

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54 Table B-3. Cell Cycle Data, HARV Cultures* n Day G 0 /G 1 (%) G 2 /M (%) S (%) Active (%) Overall Viability (%) Viable and Active (%) 0 99.04 0.14 0.82 0.96 95.96 1.00 1 99.41 0.00 0.59 0.59 95.12 0.62 2 98.83 0.93 0.24 1.17 22.42 5.22 1 3 98.97 0.34 0.69 1.03 28.77 3.58 0 98.51 0.17 1.31 1.49 94.84 1.57 1 97.74 0.32 1.94 2.26 96.28 2.35 2 93.95 0.07 5.98 6.05 95.12 6.36 2 3 0 98.61 0.00 1.39 1.39 93.19 1.49 1 97.64 0.75 1.62 2.36 97.61 2.42 2 90.86 1.13 8.01 9.14 44.96 20.33 3 3 96.93 0.00 3.07 3.07 17.29 17.76 0 98.33 0.23 1.44 1.67 93.59 1.78 1 2 4 3 0 98.59 0.77 0.64 1.41 98.31 1.43 1 2 99.51 0.00 0.49 0.49 12.62 3.88 5 3 0 97.87 0.90 1.23 2.13 94.14 2.26 1 2 99.52 0.00 0.48 0.48 12.63 3.80 6 3 *Entries left blank indicate that data was not recorded.

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55 Table B-4. Culture Data, Control Cultures* n Day Live NCs ( 10 3 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio Overall Viability (%) 0 55.6 1.00 94.95 52.81 1.00 95.96 1 56.3 1.01 80.27 45.15 0.85 96.72 2 41.0 0.74 88.79 36.36 0.69 97.69 1 3 48.0 0.86 89.20 42.82 0.81 97.28 0 75.5 1.00 96.75 73.04 1.00 94.84 1 71.3 0.94 78.78 56.13 0.77 98.30 2 51.0 0.68 89.48 45.63 0.62 96.98 2 3 78.0 1.03 0 37.4 1.00 94.95 35.48 1.00 93.19 1 32.5 0.87 67.68 22.00 0.62 97.20 2 30.9 0.83 73.69 22.75 0.64 97.06 3 3 35.6 0.95 69.06 24.60 0.69 94.29 0 228.6 1.00 96.00 219.46 1.00 93.59 1 2 198.0 0.87 4 3 257.0 1.12 0 295.2 1.00 93.06 274.71 1.00 98.31 1 2 209.4 0.71 76.04 159.23 0.58 97.21 5 3 0 158.4 1.00 86.29 136.68 1.00 94.14 1 2 75.0 0.47 83.29 62.47 0.46 96.16 6 3 *Entries left blank indicate that data was not recorded.

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56 Table B-5. Cell Cycle Data, Control Cultures* n Day G 0 /G 1 (%) G 2 /M (%) S (%) Active (%) Overall Viability (%) Viable and Active (%) 0 99.04 0.14 0.82 0.96 95.96 1.00 1 98.58 0.17 1.25 1.42 96.72 1.47 2 72.94 3.74 23.32 27.06 97.69 27.70 1 3 63.39 0.00 36.61 36.61 97.28 37.63 0 98.51 0.17 1.31 1.49 94.84 1.57 1 95.78 0.13 4.10 4.22 98.3 4.29 2 90.36 0.69 8.96 9.64 96.98 9.94 2 3 0 98.61 0.00 1.39 1.39 93.19 1.49 1 94.88 0.20 4.93 5.12 97.2 5.27 2 71.1 2.38 26.52 28.90 97.06 29.78 3 3 67.53 0.94 31.53 32.47 94.29 34.44 0 98.33 0.23 1.44 1.67 93.59 1.78 1 2 4 3 0 98.59 0.77 0.64 1.41 98.31 1.43 1 2 90.99 2.06 6.95 9.01 97.21 9.27 5 3 0 97.87 0.90 1.23 2.13 94.14 2.26 1 2 89.79 1.56 8.65 10.21 96.16 10.62 6 3 *Entries left blank indicate that data was not recorded.

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APPENDIX C SECONDARY LONG-TERM STATIC CULTURE DATA AND FIGURES

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Absolute Cell GrowthSecondary Long-Term Static Cultures050010001500200025003000047 811 1214Days Post-Initial Culture NC # (x 103) 020406080100120140160180047 811 1214Days Post-Initial CultureCD34+ Cell # (x 103) Post-Static Post-HARV B A Figure C-1. Absolute cell growth, secondary long-term static cultures. A) Nucleated cell progression. B) CD34 + cell progression. 58

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59 Tabulated data below from secondary LTSC experiments n=1 and n=2 correspond to two-day initial culture data from Appendix B, experiments n=5 and n=6, respectively. Table C-1. Initial Culture Data (Day 0 Post-Initial Culture), Post-HARV Cultures n NC Concentration ( 10 3 NCs/ml) Live CD34 + Cell Purity (%) CD34 + Cell Concentration ( 10 3 CD34 + /ml) 1 20 56.72 11.344 2 10 33.44 3.344 Table C-2. Culture Data, Post-HARV Cultures n Day PIC* Live NCs ( 10 3 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio Overall Viability (%) 0 96.0 1.00 56.72 54.45 1.00 12.62 4 52.9 0.55 3.61 1.91 0.04 12.23 7 59.9 0.62 0.00 0.00 0.00 18.79 11 39.9 0.42 0.00 0.00 0.00 7.45 1 14 0.0 0.00 0.00 0.00 0.00 0.00 0 36.0 1.00 33.44 12.04 1.00 12.63 4 28.2 0.78 6.04 1.70 0.14 10.72 8 27.6 0.77 0.00 0.00 0.00 12.31 12 0.0 0.00 0.00 0.00 0.00 0.00 2 14 0.0 0.00 0.00 0.00 0.00 0.00 *PIC: Post-initial culture. Table C-3. Progenitor Cell Culture Data, Post-HARV Cultures n Day PIC* CFU-GM # BFU-E # -2 45.0 15.5 0 0.0 0.0 1 7 0.0 0.0 -2 61.5 13.0 0 0.0 0.0 2 7 0.0 0.0 *PIC: Post-initial culture.

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60 Table C-4. Initial Culture Data (Day 0 Post-Initial Culture), Post-Static Cultures n NC Concentration ( 10 3 NCs/ml) Live CD34 + Cell Purity (%) CD34 + Cell Concentration ( 10 3 CD34 + /ml) 1 20 76.04 15.208 2 10 83.29 8.329 Table C-5. Culture Data, Post-Static Cultures n Day PIC* Live NCs ( 10 3 ) Live NC Ratio Live CD34 + Cell Purity (%) Live CD34 + Cells ( 10 3 ) Live CD34 + Cell Ratio Overall Viability (%) 0 96.0 1.00 76.04 73.00 1.00 97.21 4 98.8 1.03 30.35 31.88 0.44 98.38 7 717.4 7.47 5.91 42.40 0.58 98.26 11 972.4 10.13 1.53 14.88 0.20 97.54 1 14 2160.0 22.50 6.54 141.26 1.94 89.33 0 24.0 1.00 83.29 29.98 1.00 96.16 4 83.2 3.47 51.38 64.12 2.14 98.52 8 224.4 9.35 4.58 15.42 0.51 99.08 12 370.8 15.45 0.90 5.01 0.17 99.11 2 14 462.0 19.25 5.46 37.84 1.26 92.43 *PIC: Post-initial culture. Table C-6. Progenitor Cell Culture Data, Post-Static Cultures n Day PIC* CFU-GM # BFU-E # -2 45.0 15.5 0 100.5 9.5 1 7 220.0 10.0 -2 61.5 13.0 0 88.5 7.5 2 7 210.0 12.0 *PIC: Post-initial culture.

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BIOGRAPHICAL SKETCH Adam F. Poniatowski obtained his Bachelor of Science degree in Chemical Engineering with honors from the University of Florida in May of 2002. He conducted research as an Engineering Research Center Scholar under Dr. Dinesh O. Shah in the Chemical Engineering Department in surfactant studies and interfacial phenomenon, during which time he co-authored a paper with Dr. James Kanicky. Mr. Poniatowski continued his academic career at the University of Florida, working toward a Master of Science degree in Biomedical Engineering with advisors Dr. Tran-Son-Tay, Dr. Vijay Reddy, and Dr. Chris Batich, studying the effects of simulated microgravity on human umbilical cord blood stem cells. Mr. Poniatowski aided in the establishment of collaboration between his advisors in the Biomedical Engineering Department and the Department of Hematology and Oncology and in June of 2004 was awarded the Tarr Family Scholarship from the Tarr Family Foundation for his contributions to biomedical research. 66