Education:
MB 1982 Medicine- University of Western Australia, Australia
BS 1982 Medicine- University of Western Australia, Australia
Started at USC: 1993
Research Topics: Gene Therapy, Stem Cell Biology
Research Description
Hematopoietic Stem Cell Biology and Gene Transfer
The central research interest in our laboratory is the study of the basic biology of human hematopoietic stem cells (HSC) and how this biology relates to the transfer of genes (transduction) into HSC for the purposes of gene therapy. HSC can be isolated from bone marrow, umbilical cord blood and peripheral blood using flow cytometry (FACS). HSC are largely in a resting (GO) state and this fact, combined with their rarity among other hematopoietic cells, has made the assay of HSC very difficult. Much of our research has therefore concentrated on developing new assays to identify and study HSC in vitro. The quiescence of HSC is also a major obstacle to transduction using the strategies currently available for gene therapy ie the use of retroviral vectors based on the Moloney Murine Leukemia Virus (MoMuLV) which integrates only into target cells which are actively cycling. We have therefore studied the means by which HSC can be induced to cycle, to be transduced and to remain functionally primitive. More recently, we have published data supporting the use of Lentiviral (HIV-1) based vectors to transduce non-dividing HSC.
We have developed two main assays to measure key characteristics of HSC. Both are based on the initial isolation of cells which express the antigen CD34 but do not express the differentiation associated antigen CD38 ie CD34+CD38– cells. The Extended Long Term Culture-Initiating Cell (ELTC-IC) assay measures cells which are quiescent for long periods in vitro but when stimulated to cycle have enormous generative or proliferative capacity, greater than any previously identifiable hematopoietic populations. This assay however measures only cells of the erythroid and myeloid lineages. One of the defining characteristics of HSC is their pluripotentiality ie ability to generate cells of all lineages. Until recently, no system has existed to generate lymphoid cells from human HSC in vitro. We have therefore developed an assay using a murine stromal line S17 which allows differentiation of CD19+ B lymphoid progenitors from immunophenotypically uncommitted CD34+CD38– cells. Modification of the S17 assay using a “switch culture” approach has allowed us to identify single CD34+CD38– cells with the ability to differentiate into both B lymphoid, NK, dendritic and myeloid lineages. With the combination of the ELTC-IC and pluripotent cell asssays we can now determine the means by which true HSC can be efficiently transduced. The effects of growth factor stimulation, co-cultivation with adherent cell layers or fibronectin, serum concentration, envelope protein on viral particles and vector backbone (lenti-viral versus MoMuLV) on transduction and function of HSC are under investigation. The results of these studies will directly impact clinical gene therapy trials in development at CHLA.
With the ability to now assay the complete lineage potential of single cells we are now able to identify not only pluripotent HSC but also more lineage restricted cellls such as Common Lymphoid Progenitors. The identification and study of CLP in human cord blood and bone marrow has been a major focus of the laboratory recently. Using the immunophenotype and culture conditions developed in these studies, we have been able to critically analyze the characteristics of lymphoid and myleoid dendritic cells.
A related focus is to study the roles of certain genes in lineage commitment decisions at the stem cell and progenitor levels. Using the assays and techniques developed in our gene transfer/stem cell biology research, we have studyied the effects on hematopoiesis of the homeobox genes HOXA5. Because of our interest in the regulation of differentiation from stem cells, we have now embarked on studies of so-called “Stem Cell Plasticity”. The combined ability of the laboratory to isolate stem cells, to constitutively express genes of interest using retroviral vectors, and to apply lineage-specific assays will allow us to study the potential of cells of the cord blood and bone marrow to differentiate into non-hematopoietic tissues.
Selected Publications
Chu M, Siegmund KD, Hao QL, Crooks GM, Tavaré S, Shibata D. - Inferring relative numbers of human leucocyte genome replications. - Br J Haematol [ 2008 ] Apr 10; . PubMed
Abdel-Azim H, Zhu Y, Hollis R, Wang X, Ge S, Hao QL, Smbatyan G, Kohn DB, Rosol M, Crooks GM. - Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl. - Blood [ 2008 ] Apr 15;111(8):4064-74 . PubMed
Rountree CB, Senadheera S, Mato JM, Crooks GM, Lu SC. - Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice. - Hepatology [ 2008 ] Apr;47(4):1288-97 . PubMed
Hao QL, George AA, Zhu J, Barsky L, Zielinska E, Wang X, Price M, Ge S, Crooks GM. - Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7- lympho-myeloid thymic progenitors. - Blood [ 2008 ] Feb 1;111(3):1318-26 . PubMed
Berg T, Rountree CB, Lee L, Estrada J, Sala FG, Choe A, Veltmaat JM, De Langhe S, Lee R, Tsukamoto H, Crooks GM, Bellusci S, Wang KS. - Fibroblast growth factor 10 is critical for liver growth during embryogenesis and controls hepatoblast survival via beta-catenin activation. - Hepatology [ 2007 ] Oct;46(4):1187-97 . PubMed
Rountree CB, Barsky L, Ge S, Zhu J, Senadheera S, Crooks GM. - A CD133-expressing murine liver oval cell population with bilineage potential. - Stem Cells [ 2007 ] Oct;25(10):2419-29 . PubMed
Payne KJ, Crooks GM. - Immune-cell lineage commitment: translation from mice to humans. - Immunity [ 2007 ] Jun;26(6):674-7 . PubMed
Rountree CB, Wang X, Ge S, Barsky L, Zhu J, Gonzales I, Crooks GM. - Bone marrow fails to differentiate into liver epithelium during murine development and regeneration. - Hepatology [ 2007 ] May;45(5):1250-60 . PubMed
Shah AJ, Kapoor N, Crooks GM, Weinberg KI, Azim HA, Killen R, Kuo L, Rushing T, Kohn DB, Parkman R. - The effects of Campath 1H upon graft-versus-host disease, infection, relapse, and immune reconstitution in recipients of pediatric unrelated transplants. - Biol Blood Marrow Transplant [ 2007 ] May;13(5):584-93 . PubMed
Garon EB, Marcu L, Luong Q, Tcherniantchouk O, Crooks GM, Koeffler HP. - Quantum dot labeling and tracking of human leukemic, bone marrow and cord blood cells. - Leuk Res [ 2007 ] May;31(5):643-51 . PubMed
