Hematopoietic and Cardiovascular Regeneration

Hal E. Broxmeyer, PhD
Loren J. Field, PhD
Keith March, PhD
Christie M. Orschell, PhD
David G. Skalnik, PhD
Edward F. Srour, PhD
Mervin C. Yoder, Jr, MD

Hal E. Broxmeyer, PhD

Positions

• Distinguished Professor
• Chairman & Mary Margaret Walther
• Professor of Microbiology/Immunology
• Professor of Medicine
• Scientific Director of the Walther Oncology Center

Address
Walther Oncology Center
Research Institute #2
950 West Walnut Street, Room-302
Indiana University-Purdue University Indianapolis
Indianapolis, IN 46202-5181

Phone: (317) 274-7510
Fax: (317) 274-7592
Email: hbroxmey@iupui.edu

Research Interests

Dr. Broxmeyer's laboratory is interested in the study of blood cell production (hematopoiesis) in the context of normal and abnormal cell regulation as assessed in vitro and in vivo at the level of proliferation/self-renewal/differentiation/apoptosis and homing/migration of hematopoietic stem and progenitor cells and also of the immunology of cord blood and adult immune cells. As a clinical correlate, his laboratory is interested in the use of hematopoietic stem and progenitor cells, especially those from umbilical cord blood, for transplantation purposes, and the use of cytokines for treatment of hematological disorders. His group has focused recently on the family of molecules called chemokines for enhancement of proliferation, survival, or movement (homing and mobilization) of early subsets of blood cells, alone and in the presence of colony stimulating factors and the potent co-stimulating ligands, stem cell factor and Flt3-ligand for tyrosine kinase receptors. His laboratory recently demonstrated that inactivation/deletion of the peptidase activity of CD26 on murine stem cells greatly enhances their homing and engraftment capability, and studies on human cells are ongoing as a prelude to clinical translation efforts. He has also done studies with the SDF-1/CXCL12-CXCR4 antagonist AMD3100 that helped demonstrate the potent stem and progenitor cell mobilizing capacity of AMD3100 that led the clinical trials with AMD3100 in this context. The laboratory also studies the growth and hematopoietic differentiation of murine embryonic stem cells. Intracellular signal transduction studies are utilized to evaluate the mechanisms of cell proliferation and survival, mobilization of cells from the marrow to the blood and chemotaxis. This includes gene transfer approaches. Mice in which specific genes are either deleted or inserted are utilized to determine dominant regulatory effector proteins. Recent studies have focused on mice in which the following genes involved in intracellular signaling have been "knocked-out": p21cip1, p18INK4C, p27kip1, p18/p27, p18/p21, SHIP, SHP-1, Stat4, Stat6, Stat 5a, Stat 5b, CIITA, BCL-6 and BAZF. Studies with CD1d deleted mice are also ongoing as are those with Stat-3 deletions and IL-31 receptor deletions. Studies are ongoing in the area of the immune reactivity of cord blood vs. adult T lymphocytes, monocytes and dendritic cells, and the differentiation of these cells.

Recent Publications

• Christopherson, K.W. II, Hangoc, G., Mantel, C., and Broxmeyer, H.E. 2004. Modulation of hematopoietic stem cell homing and engraftment by CD26. Science. 305:1000-1003.
• Li, G., Kim, Y.J., and Broxmeyer, H.E. 2005. Macrophage colony-stimulating factor drives cord blood monocyte differentiation into IL-10highIL-12absent dendritic cells with tolerogenic potential. J. Immunol. 174:4706-4717.
• Broxmeyer, H.E., Orschell, C.M., Clapp, D.W., Hangoc, G., Cooper, S., Plett, A., Liles, W.C., Li, X., Graham-Evans, B., Campbell, T.B., Calandra, G., Bridger, G., Dale, D.C., and Srour, E.F. 2005. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J. Exp. Med. 201:1307-1318.
• Basu, S., and Broxmeyer, H.E. 2005. Transforming growth factor-b1 modulates responses of CD34+ cord blood cells to stromal cell derived factor-1/CXCL12. Blood. 106:485-493.
• Guo, Y., Graham-Evans, B., and Broxmeyer, H.E. 2006. Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells. Stem Cells. 24:850 – 856.

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Loren J. Field, PhD

Positions

• Professor of Medicine
• Professor of Cellular and Integrative Physiology

Address
Herman B. Wells Center
R4, W376
702 Barnhill Drive
Indiana University School of medicine
Indiana University-Purdue University Indianapolis
Indianapolis, IN 46202

Phone: (317) 274-5085
Fax: (317) 274-8679
E-mail: ljfield@iupui.edu

Research Interests

Use of transgenic models, molecular biology and cell culture approaches to understand the molecular regulation of the cardiomyocyte cell cycle and terminal differentiation.

During embryonic and early neonatal development the myocardium undergoes a period of hyperplastic growth which results in an exponential increase in the number of myocytes that constitute the heart. Soon after birth cardiomyocyte proliferation ceases and subsequent increases in myocardial mass is accomplished by cellular hypertrophy. The molecular basis for the transition from hyperplastic to hypertrophic myocardial growth remains largely unknown. Anomalies in the regulation of cardiomyocyte proliferation can give rise to congenital heart defects (ie hypoplastic ventricle syndromes). Moreover, because cardiomyocyte cell cycle withdrawal is irreversible, cell death with an ensuing loss of myocardial function is observed in many forms of adult cardiovascular disease. The ability to therapeutically program cardiomyocyte proliferation might provide a means to treat some forms of pediatric and adult cardiovascular disease. Work in our laboratory is focused on developing strategies with which to augment cardiomyocyte proliferation in both developing and adult hearts. Several current approaches are:

1. Characterize genes which impact upon cardiomyocyte proliferation and terminal differentiation during development. There are many descriptive studies examining the expression of known cell cycle regulatory genes during cardiomyocyte terminal differentiation. However, gene transfer experiments are need to establish if a given candidate gene plays a causative role in the process. Accordingly, we have used transgenic mice to test the role of a number of cell cycle regulatory genes, and have identified several candidates which might be useful for engendering therapeutic myocardial growth. We have also performed structure:function analyses on the Tuberous Sclerosis (TS) genes: TS is a childhood cancer syndrome wherein ca. 50% of the effect children develop benign myocardial tumors. We have generate several dominate interfering TS mutants which alter cardiomyocyte terminal differentiation in transgenic animals.

2. Identification of genes which program cardiomyocyte proliferation in a genetic model of cardiac tumorigenesis. We have generated transgenic mice which express the SV40 Large T Antigen oncoprotein in the heart. These mice develop myocardial tumors comprised of differentiated, proliferating cardiomyocytes. We have used cell lines derived from the transgenic mouse tumors to identify the myocardial proteins which bind to T-Antigen: binding proteins identified by analogous approaches with other cell types have proven to be important cell cycle regulators. Current efforts are centered on cloning these T-Antigen binding proteins, and establishing their function using both in vitro (cell transfection and molecular analyses) and in vivo (transgenic and knock-out mice) approaches.

3. Determining if intracardiac engraftment of donor myocytes can be used to augment cardiac function. We have recently shown that fetal cardiomyocytes can be used to form stable grafts in adult hearts using murine and canine models. Clinical application of this approach is dependent upon the identification of a suitable source of donor cells. Potential candidates include genetically modified skeletal myoblasts and cardiomyocytes derived from ES cells. Current efforts are focused on determining the spectrum of cardiomyocytes which can differentiate from ES cells in vitro (ie atrial, ventricle, purkinje and pacemaker cells), as well as establishing the molecular determinants of donor cell survival and proliferation following engraftment.

Recent Publications

• Murry, C.E., Soonpaa, M.H., Reinecke, H., Nakijima, H. Nakijima, H.O., Rubart, M., Pasumarthi, K.B.S., Virag, J.I., Bartelmez, J.H., Poppa, V., Bradford, G., Dowell, J.D., Williams, D.A. and Field, L.J. (2004) Absence of Cardiac Transdifferentiation after Direct Injection of Adult Hematopoietic Stem Cells into Myocardial Infarcts. Nature 428:664-668.
• Rubart, M., Soonpaa, M.H., Nakajima, H. and Field, L.J. (2004) Spontaneous and Evoked Intracellular Calcium Transients in Donor-Derived Myocytes Following Intra-cardiac Myoblast Transplantation. Journal of Clinical Investigation 114:775-783.
• Pasunarthi, K.B.S., Nakajima, H., Nakajima, H.O., Soonpaa, M.H. and Field, L.J. (2005) Targeted expression of cyclin D2 results in cardiomyocyte cell cycle activation and concomitant regression of myocardial infarct size in transgenic mice. Circulation Research 96:110-118.
• Murry, C.E., Field, L.J. and Menasché, P. (2005) Cell-Based Cardiac Repair Reflections at the 10-Year Point. Circulation 112:3174-3183.
• Nakajima, H., Nakajima, H.O., Dembowsky, K. Pasumarthi, K.B.S. and Field, L.J. (2006) Cardiomyocyte cell cycle activation ameliorates fibrosis in the atrium. Circulation Research 98:141-148.

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Keith March, PhD

Positions

• Professor of Medicine, Cellular & Integrative Physiology and Biomedical Engineering
• Director, Indiana Center for Vascular Biology and Medicine

Address
Indiana University School of Medicine
Indiana University-Purdue University Indianapolis
975 West Walnut Street, IB 441
Indianapolis, IN 46202

Phone: (317) 278-0130
Fax: (317) 278-0089
Email: kmarch@iupui.edu

Research Interests

Research areas include 1.) circulating and tissue-resident progenitor cells involved in vascular growth and repair, with a particular interest in cells found in high abundance in adipose stroma (adipose stromal cells), which may represent a particular type of endothelial progenitor cells; 2.)the cell biology and molecular mechanisms of vascular remodeling and its relationship to smooth muscle cell cycle control; and 3.) local therapeutic interventions to treat the heart and blood vessels, involving device and drug or gene combinations.

Recent Publications

• Sindermann JR, March KL. Balancing luminal size and smooth muscle proliferation—a key control point in atherosclerosis and arteriogenesis. EXS. 2005;(94):193-205.
• Hou D. Narciso H. Kamdar K. Zhang P. Barclay B. March KL. Stent-based nitric oxide delivery reducing neointimal proliferation in a porcine carotid overstretch injury model. Cardiovascular & Interventional Radiology. 28(1):60-5, 2005
• Sindermann, JR Köbbert , Skaletz-Rorowski A, Eschert H, Breithardt G, Plenz G, March KL. Vascular injury response in mice is dependent on the genetic background. American Journal of Physiology - Heart & Circulatory Physiology (2006), in press.
• Hou Y, Plett PA, Ingram DA, Rajashekhar G, Orschell CM, Yoder MC, March KL, Clauss M (2006). Endothelial-monocyte-activating polypeptide II induces migration of endothelial progenitor cells va the chemokine receptor CXCR3. Exp Hematol. 34(8): 1125-32
• Bell LN, Ward JL, Degawa-Yamauchi M, Bovenkerk JE, Jones RM, Cacucci BM, Gupta CE, Sheridan C, Sheridan K, Shankar SS, Steinberg HO, March KL, Considine RV. Adipose Tissue Production of Hepatocyte Growth Factor Contributes to Elevated Serum HGF in Obesity. American Journal of Physiology: Endocrinology & Metabolism (2006), in press

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Christie M. Orschell, PhD

Positions

• Associate Research Professor of Medicine
• Director, Indiana University School of Medicine GLP Stem Cell Testing Facility

Address
Department of Medicine
Indiana University School of Medicine
Indiana University-Purdue University Indianapolis
1044 W. Walnut Street, R4-202
Indianapolis, IN 46202

Phone: (317) 274-3589
Fax: (317) 274-0396
Email: corschel@iupui.edu

Research Interests

Research in my laboratory is focused on two areas of hematopoietic stem cell biology: 1) transplantation and engraftment, and 2) endogenous reconstitution after lethal radiation. The first focuses on understanding the homing and activation of hematopoietic stem cells after transplantation and how these early events impact long-term multi-lineage hematopoietic reconstitution. The second examines parameters affecting endogenous reconstitution and aims to identify new radiomitigators and radioprotectants to be used as medical countermeasures against potential radiological threats, as well as for recovery from myelosuppressive therapy for malignancy. It is anticipated that both these areas of research will result in new information regarding hematopoietic and immune reconstitution pathways, and will contribute to our knowledge base of hematopoiesis, thymopoiesis, and hematopoietic stem cell biology.

Recent Publications

• PA Plett, SM Frankovitz, and CM Orschell-Traycoff. In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients. BLOOD 100(10):3545-3552, Nov. 2002.
• J Rehman, J Li, CM Orschell, and KL March. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation107:35-40, Feb. 2003.
• PA Plett, SM Frankovitz, and CM Orschell. Distribution of marrow repopulating cells between bone marrow and spleen early after transplantation. BLOOD 102(6):2285-2291, Sept. 2003.
• PA Plett, R Abonour, SM Frankovitz, CM Orschell. Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells. Hematology, 32:773-781, Aug 2004
• EF Srour, X Tong, KW Sung, PA Plett, S Rice, J Daggy, CT Yiannoutsos, R Abonour, CM Orschell. Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0. Blood, 105(8):3109-16, April 15, 2005.

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David G. Skalnik, PhD

Positions

• Professor of Pediatrics
• Professor of Biochemistry and Molecular Biology

Address
Wells Center for Pediatric Research
Cancer Research Institute, Room 327
Indiana University School of Medicine
Indiana University-Purdue University Indianapolis
1044 W. Walnut St.
Indianapolis, IN 46202

Phone: (317) 274-8977
Fax: (317) 274-8928
Email: dskalnik@iupui.edu

Research Interests

Research in my laboratory is focused on the basic biology of chromatin structure, epigenetic modifications, and regulation of gene expression during development. Much of our current focus in on CpG binding protein, a factor that is required for appropriate regulation of cytosine methylation and histone modifications. This factor is also required for stem cell differentiation. Current interests include a gaining a detailed molecular understanding of how this factor intersects with and regulates the known epigenetic enzymatic machinery, how this factor functions during post-gastrulation development, and how epigenetic modifications are altered in the absence of this protein.

Recent Publications

• Lee, J.-H., Voo, K.S., and Skalnik, D.G. (2001) Identification and characterization of the DNA-binding domain of CpG binding protein. Journal of Biological Chemistry 276, 44669-44676.
• Lee, J.-H., and Skalnik, D.G. (2002) CpG binding protein is a nuclear matrix- and euchromatin-associated protein localized to nuclear speckles containing human trithorax: Identification of nuclear matrix targeting signals. Journal of Biological Chemistry 277, 42259-42267.
• Carlone, D.L. Lee, J-H., Young, S.R.L., Dobrota, E., Butler, J.S., Ruiz, J., and Skalnik, D.G. (2005) Reduced cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein. Molecular and Cellular Biology 25, 4881-4891.
• Lee, J.-H. and Skalnik, D.G. (2005) CpG binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex. Journal of Biological Chemistry 280, 41725-41731.
• Young, S.R.L., Mumaw, C., Marrs, J.A., and Skalnik, D.G. (2006) Antisense targeting of CXXC finger protein 1 results in reduced cytosine methylation and disruption of hematopoiesis in Zebrafish. Journal of Biological Chemistry, 281, 37034-37044.

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Edward F. Srour, PhD

Positions

• Professor of Medicine
• Professor of Pediatrics and Microbiology/Immunology
• Director, Flow Cytometry Resource Facility, Indiana University Cancer Center

Address
Department of Medicine, Section of Hematology/Oncology
R4 202
Indiana University School of Medicine
Indiana University-Purdue University Indianapolis
Indianapolis, IN 46202

Phone: (317) 274-3589
Fax: (317) 274-0396
Email: esrour@iupui.edu

Research Interests

The research program in the laboratory of Dr. Srour focuses on stem cell biology in general. In particular, three areas of interest are pursued, hematopoietic stem cells, adult stem cells, and cancer stem cells. Investigations concerning hematopoietic stem cells examine the relationship between cell cycle status and homing and engraftment of these cells as well as the impact of the hematopoietic microenvironment through ontogeny on these properties. Using a murine model, the laboratory investigates the differentiation and lineage specification potential of a group of common pluripotent adult stem cells detected in multiple tissues and the possible utilization of these cells in tissue repair and regenerative medicine. In collaboration with other investigators at Indiana University, Dr. Srour is working on identifying cancer stem cells in breast cancer and determining the relationship between putative cancer stem cells and metastasis.

Recent Publications

• Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li X, Graham-Evans B, Calandra G, Bridger G, Dale DC, Srour EF. Rapid Mobilization of Murine and Human Hematopoietic Stem and Progenitor Cells with AMD3100, a CXCR4 Antagonist. Journal of Experimental Medicine. J. Exp Med 201(8):1307-1318, 2005.
• Srour, EF, Tong X, Sung KW, Plett AP, Rice S, Daggy J, Yiannoutsos CT, Abonour R, Orschell CM. Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual CD34+CD38-/lo cells in G0. Blood 105(8):3109-3116, 2005.
• Kondo T, Case J, Srour EF, Hashino E. Skeletal muscle-derived progenitor cells have the potential to differentiate into neurons. Neuroreport. 17:1-4, 2006.
• Hall K, Horvath TL, Abonour R, Cornetta K, Srour EF. Decreased homing of retrovirally transduced human bone marrow CD34+ cells in the NOD/SCID mouse model. Experimental Hematology 34(4):433-442, 2006.
• Zuba-Suma EK, Abdel-Latif A, Case J, Tiwari S, Hunt G, Kucia M, Vincent RJ, Ranjan S, Ratajczak MZ, Srour EF, Bolli R, Dawn B. Sca-1 expression is associated with decreased cardiomyogenic differentiation potential of skeletal muscle-derived adult primitive cells. Journal of Molecular and Cellular Cardiology, 41(4):650-660, 2006.

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Mervin C. Yoder, Jr, MD

Positions

• Richard and Pauline Klingler Professor of Pediatrics
• Professor of Biochemistry

Address
Herman B. Wells Center
702 Barnhill Drive
Indiana University School of Medicine
Indiana University-Purdue University Indianapolis
Indianapolis, IN 46202

Phone: (317) 274-4738
Fax: (317) 274-8679
Email: myoder@iupui.edu

Research Interests

Cellular and molecular approaches are combined to elucidate the role of hematopoietic stromal cells in regulating the proliferation and differentiation of hematopoietic stem cells throughout murine ontogeny. The precise temporal and spatial appearance of hematopoietic stem cells in the murine embryo remains controversial. It is well known that blood cells first appear in the yolk sak and become evident sequentially in the fetal liver, spleen, and bone marrow compartments. Recent investigations implicate hematopoietic c stem cell origin within the murine embryos in the region of the aorta-gonad-mesonephros region. We have identified hematopoietic stem cells in the murine yolk sac prior to the formation of the fetal liver. Our research efforts are focused on the following questions: 1) Where and when do hematopoietic stem and progenitor cells originate in the murine embryos? 2) Do yolk sac endothelial cells play a role in the formation, proliferation, and engraftment of yolk sac hematopoietic stem cells in vivo? 3 ) Will embryonic endothelial cells facilitate hematopoietic stem cell proliferation ex vivo with or without the addition of recombinant cytokines? These questions are being addressed using cell sorting and transplant assays with donor cells isolated from a variety of transgenic mice.

In related work, we are investigating the role played by several integrin molecules in supporting the adhesion, proliferation, and differentiation of primitive hematopoietic cells from the murine yolk sac. We have identified several integrin alpha and beta chains on populations of cells enriched for hematopoietic stem cell activity. Using monoclonal antibodies, flow cytometry, and our novel transplantation assay we will determine whether the expression of specific integrin molecules can be used to further enrich for hematopoietic stem cells. We are investigating which molecules play a role in the adhesion of hematopoietic progenitor cells to endothelial cells or to purified extracellular matrix molecules in vitro. Using activating or blocking antibodies we are determining whether signaling through the integrin molecules affects progenitor cell growth and differentiation.

We are collaborating with Dr. Eddy Srour to characterize a murine skeletal muscle-derived stem cell that possesses hematopoietic repopulating ability in vivo. This stem cell has been partially characterized using monoclonal antibodies and FACS and can be enriched via cell sorting. This stem cell gives rise to skeletal but not cardiac muscle upon in vitro differentiation though no evidence of skeletal muscle commitment is present in the freshly isolated stem cell population. Our current focus is to examine the repopulating ability of the transplanted stem cells at a clonal level and to optimize gene transfer in the cells ex vivo.

Finally, we are collaborating with Dr. Arun Srivastava in the department of Microbiology and Immunology to determine the feasibility of using adeno-associated virus 2 (AAV) based recombinant vectors as gene transfer vehicles for delivery of human globin molecules into thalassemic mice. Stem cell populations are isolated, transduced with the AAV particles, and transplanted into congenic mice. Sequential blood analysis is used to document the level of gene expression in the recipient mice.

Recent Publications

• Broxmeyer HE, Srour E, Orschell C, Ingram DA, Cooper S, Plett PA, Mead LE,Yoder MC. Cord blood stem and progenitor cells. Methods Enzymol. 2006;419:439-73.
• Yoder MC, Mead LE, Prater D, Krier TR, Mroueh KN, Li F, Krasich R, Temm CJ, Prchal JT, Ingram DA. Re-defining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 2006 Oct 19; [Epub ahead of print]
• Zou GM, Luo MH, Reed A, Kelley MR, Yoder MC. Ape1 regulates hematopoietic differentiation of embryonic stem cells through its redox functional domain. Blood. 2006 Oct 19; [Epub ahead of print]
• Ingram DA, Krier TR, Mead LE, McGuire C, Prater DN, Bhavsar J, Saadatzadeh MR, Bijangi-Vishehsaraei K, Li F, Yoder MC, Haneline LS. Clonogenic Endothelial Progenitor Cells are Sensitive to Oxidative Stress. Stem Cells. 2006 Oct 5; [Epub ahead of print]
• Wu X, Estwick SA, Chen S, Yu M, Ming W, Nebesio TD, Li Y, Yuan J, Kapur R, Ingram D, Yoder MC, Yang FC. Neurofibromin plays a critical role in modulating osteoblast differentiation of mesenchymal stem/progenitor cells. Hum Mol Genet. 2006 Oct 1;15(19):2837-45. Epub 2006 Aug 7.

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