Sanja Sekulovic
Wednesday, June 29, 2011, 12:30 pmRoom 200, Graduate Student Centre
“Characterization of Mouse Hematopoietic Stem Cells Primed to Actively Self-renew by NUP98-HOXA10hd Fusion Gene”
EXAMINING COMMITTEE:
Chair: Dr. John Hill (Pathology and Laboratory Medicine)
Supervisory committee: Dr. R. Keith Humphries, Research Supervisor (Medical Genetics), Dr. Fabio M. Rossi (Medical Genetics) & Dr. James M. Piret (Chemical & Biological Engineering)
University examiners: Dr. Kirk R. Schultz (Pediatrics) & Dr. Megan Levings (Surgery)
External examiner: Dr. Gerald Spangrude. Division of Hematology. University of Utah, Salt Lake City, Utah. USA
SUPERVISORY COMMITTEE: Dr. R. Keith Humphries (Medical Genetics), Dr. Connie J. Eaves (Medical Genetics) & Dr. Fabio M. Rossi (Medical Genetics)
High-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Recently, it has been demonstrated that engineered NUP98-HOXA10hd expression stimulates >1,000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin-Sca-1+c-kit+ cells. In this thesis I coupled such ability of engineered NUP98-HOXA10hd expression, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. I discovered that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at near unit efficiency in cultures initiated with single cells. The clonally expanded HSCs showed preservation of normal proliferation kinetics in vitro and consistent balanced contributions long-term to the lymphoid and myeloid lineages in vivo without evidence of leukemogenic transformation. Preservation of a normal proliferating HSC phenotype allowed their re-isolation in large numbers at 25% purity. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process. Although there is growing excitement about the prospect of in vitro expansion of HSCs and their use to enhance the safety and application of transplant-based therapies, deleterious consequences of such manipulations remain unknown. Thus, I further examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. I observed ~10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert-/- HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type or Tert-/- HSCs, suggesting that HSC replication post-transplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity upon prolonged self-renewal stimulation.
PUBLICATIONS:
Sekulovic S, Gasparetto M, Lecault V, Hoesli C, Kent DG, Rosten P, Wan A, Brookes C, Hansen CL, Piret JM, Smith C, Eaves CJ*, Humphries RK*. Ontogeny stage independent and high-level clonal expansion in-vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor. Blood submission #BLOOD/2011/350066; under revision. *Authors equally contributed to the work.
Sekulovic S*, Gylfadottir V*, Vulto I, Gasparetto M, Even Y, Brookes C, Smith C, Eaves CJ, Lansdorp PM, Rossi FM☼, Humphries RK☼. Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse hematopoietic stem cells. Blood submission #BLOOD/2010/319632; under revision.
*☼Authors equally contributed to the work.
Lecault V, Vaninsberghe M, Sekulovic S, Knapp DJ, Wohrer S, Bowden W, Viel F, McLaughlin T, Jarandehei A, Miller MM, Falconnet D, White AK, Kent DG, Copley MR, Taghipour F, Eaves CJ, Humphries RK, Piret JM, Hansen CL. High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nature Methods. Epub 2011 May 22.
Yung E, Sekulovic S, Argiropoulos B, Lai CK, Leung M, Berg T, Vollett S, Chang VC, Wan A, Wong S, Humphries RK. Delineating the key regions and functions of NUP98 contributing to the leukemogenic activity of NUP98-HOX fusions. Leuk Res. 2011 Apr;35(4):545-50. Epub 2010 Dec 3.
Even Y, Bennett JL, Sekulovic S, So L, Yi L, McNagny KM, Humphries RK, Rossi FM. NUP98-HOXA10hd-expanded hematopoietic stem cells efficiently reconstitute bone marrow of mismatched recipients and induce tolerance. Cell Transplant. 2010 Nov 19.
Petriv OI, Kuchenbauer F, Delaney AD, Lecault V, White A, Kent D, Marmolejo L, Heuser M, Berg T, Copley M, Ruschmann J, Sekulovic S, Benz C, Kuroda E, Ho V, Antignano F, Halim T, Giambra V, Krystal G, Takei CJ, Weng AP, Piret J, Eaves C, Marra MA, Humphries RK, Hansen CL. Comprehensive microRNA expression profiling of the hematopoietic hierarchy. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15443-8. Epub 2010 Aug 11.
Argiropoulos B, Yung E, Xiang P, Lo CY, Kuchenbauer F, Palmqvist L, Reindl C, Heuser M, Sekulovic S, Rosten P, Muranyi A, Goh SL, Featherstone M, Humphries RK. Linkage of the potent leukemogenic activity of Meis1 to cell-cycle entry and transcriptional regulation of cyclin D3. Blood. 2010 May 20;115(20):4071-82. Epub 2010 Mar 17.
Heuser M, Kuchenbauer F, Argiropoulos B, Sekulovic S, Leung M, Stasiak M, Ganser A, Humphries RK. Priming reloaded? Blood. 2009 Jul 23;114(4):925-6; author reply 926-7.
Sekulovic S, Imren S, Humphries K. High level in vitro expansion of murine hematopoietic stem cells. Curr Protoc Stem Cell Biol. 2008 Jan;Chapter 2:Unit 2A.7.
Ohta H*, Sekulovic S*, Bakovic S, Eaves CJ, Pineault N, Gasparetto M, Smith C, Sauvageau G, Humphries RK. Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions. Exp Hematol. 2007 May;35(5):817-30. *Authors equally contributed to the work.
PRESENTATIONS:
2009 Canadian Stem Cell Network Conference, Montreal, Canada
2008 International Society for Experimental Hematology Conference, Boston, USA
2007 International Society for Experimental Hematology Conference, Hamburg, Germany
2007 Canadian Stem Cell Network Conference, Toronto, Canada
2006 Keystone Symposia, Whistler, Canada
BIOGRAPHICAL NOTES:
Born: May 4, 1974, Belgrade, Serbia
Academic Studies: B. Sc. University of Belgrade, 2001. M. Sc. University of British Columbia, 2005
GRADUATE STUDIES:
Field of Study: Hematopoietic Stem Cell Biology
Courses:
MEDG 510: Advanced Immunogenetics. Dr. K. McNagny
MEDG 520: Advanced Human Molecular Genetics. Dr. R. McMaster
MEDG 521: Molecular and Cellular Biology of Cancer. Dr. G. Krystal
MEDG 530: Human Genetics. Dr. J. Friedman
MEDG 545: Current Topics in Medical Genetics. Dr. P. Hoodless
MEDG 548: Directed Studies. Dr. R. K. Humphries
AWARDS:
University of British Columbia PhD Tuition Award, 2006 – 2010
2008 International Society of Experimental Hematology Travel Award
2007 International Society of Experimental Hematology Travel Awa