Roland Jurecic, Ph.D.

Associate Professor of Microbiology and Immunology
Room 403, Papanicolaou Building
1550 NW 10th Avenue
Telephone: 305- 243-6002
Fax: 305- 243-4623
Email: rjurecic@med.miami.edu

Research Interests:

Stem cells are defined as pluripotent cells with the capacity to divide asymmetrically (self-renewal), producing copies of itself and more committed daughter cells, which will generate a large number of mature cells. They are found in various tissues (bone marrow, intestines, skin, liver, brain, testis) where they participate in tissue homeostasis by replacing differentiated cells lost to physiological turnover, disease or injury. Stem cells hold a tremendous promise for development of cell therapies, stem cell transplantation and gene therapy protocols for treatment of various diseases. Research in the laboratory focuses on (a) elucidation of genetic mechanisms that regulate self-renewal, lineage commitment and differentiation of hematopoietic stem cells (HSC), and (b) developmental biology of hematopoietic and neural stem cells.

Molecular Genetics of Hematopoietic Stem Cell Differentiation

The life-long maintenance and regenerative capacity of the blood cell-forming (hematopoietic) system depend on self-renewal and differentiation of hematopoietic stem cells (HSC). Developmental cascade from HSC to mature blood cells (Fig. 1), defined by a series of commitment steps that gradually restrict developmental potential of intermediate progenitor cells, is regulated by an intricate network of genes (Fig. 2).

We have undertaken a comprehensive gene expression analysis and functional genomics approach to elucidate regulatory pathways that direct stem cell lineage commitment and differentiation (Fig. 3). Elucidation of the temporal and spatial network of gene expression during ontogeny and differentiation of HSC and progenitors could have far reaching implications for clinical bone marrow and HSC transplantation and gene therapy. Using differential screening and subtraction of full length cDNA libraries prepared from purified mouse Lin -Sca-1 + (containing HSC) and Lin -Sca-1 - (lacking HSC) bone marrow cells genes (Fig. 4), we have identified a number of novel candidate genes which are being studied in the lab.

One of the newly identified genes is Hepp (hematopoietic progenitor protein) (Fig. 5). Inactivation of Hepp leads to bone marrow cytopenia (Fig. 6), splenomegaly with follicular hyperplasia, decreased number of lineage-committed blood cell types (B200+, Gr-1+, Mac-1+ and TER-119+ cells), and significantly increased content of BM cell populations containing HSC (Lin-Sca-1+c-kit+) and progenitors (Lin-Sca-1-c-kit-) (Fig. 7). In addition, Hepp heterozygous mutants display skeletal defects, retarded growth (Fig. 8), and progressive neurodegenerative disease (Fig. 9). This data indicate that Hepp participates in regulation of progenitor cell number (expansion) and their subsequent differentiation.

Another example is FLRF (Rnf41) gene, a novel evolutionarily conserved C 3HC 4 RING finger gene (Fig. 10). Over-expression of FLRF in a multipotent HSC-like cell line EML leads to (a) significant decrease in production of lymphoid, myeloid and erythroid progenitors by EML cells and suppression of their multilineage differentiation, (b) decrease in proliferative capacity of EML cells, (c) significant ligand binding-independent down-regulation of endogenous receptors for cytokines Epo, IL-3, and GM-CSF, and (d) significant increase in the steady-state level of poly-ubiquitinated receptors in EML cells. These data indicate that during blood cell development FLRF acts as an E3 ubiquitin ligase and affects proliferation and differentiation of multipotential hematopoietic progenitors by regulating cytokine receptor levels through ligand-independent degradation (Fig. 11). By regulating steady-state levels of cytokine receptors FLRF could be maintaining optimal signaling for a proper cellular response (proliferation, lineage commitment, differentiation) of HSC and progenitors, while preventing oversignaling that could lead to leukemogenesis (Fig. 12).

Molecular Genetics of Stem Cell Self-renewal and Maintenance

Self-renewal of stem cells in diverse species and tissues suggests that evolutionarily conserved mechanisms regulate this common feature. Pumilio proteins are an evolutionarily highly conserved family of RNA-binding proteins that function as translational repressors during embryo development and cell fate specification and differentiation (Fig. 13). Based on their known functions in invertebrates and lower vertebrates it was proposed that a primordial function of Pumilio (Pum) proteins is to sustain proliferation and maintenance of stem cells. We have cloned mouse and human Pum genes (Fig. 14), and have shown that mouse Pum1 and Pum2 genes are transcribed preferentially in hematopoietic stem cells (HSC). To start analyzing their role during blood cell development we have over-expressed the RNA-binding domain of Pum2 in a stem cell factor (SCF)- dependent HSC-like cell line EML, which can differentiate into erythroid, myeloid and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and suppresses their mutilineage differentiation in the absence of SCF. This uncoupling of the survival and differentiation signals in EML cells is accompanied by (a) an increased expression of the full-length c-kit and a novel truncated c-kit receptor called tr-kit, and (b) cell intrinsic, SCF-independent activation of the c-kit, and its downstream signaling pathways (Fig. 15). These results indicate that Pum2 could be a part of an evolutionarily conserved intrinsic mechanism that plays an important role in supporting maintenance and self renewal of primitive multipotential hematopoietic cells through regulation of SCF/c-kit signaling pathway.

The observations that tr-kit expression is restricted to HSC and multipotent progenitors, and that an increased expression of tr-kit protein is associated with SCF-independent maintenance of EML cells, suggest a potentially important role for tr-kit in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. Since the Pum2 and tr-kit proteins are co-expressed in bone marrow cells enriched for HSC and early multipotent progenitors (e.g. Lin-Sca-1+c-kit+ cells), but not in later progenitors (e.g. Lin-Sca-1-c-kit- cells), we hypothesize that HSC and early multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways that could regulate their maintenance and differentiation. In contrast, more differentiated progenitors that lack self-renewal ability and do not express tr-kit, utilize only the canonical SCF-induced c-kit signaling. In this hypothetical model, the survival and maintenance of HSC and multipotent hematopoietic progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. These studies could provide important new insights into how stem cell self-renewal is controlled, and how the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation, can be uncoupled.

Developmental Biology and Plasticity of Hematopoietic Stem Cells

To study developmental potential of hematopoietic (HSC) in vivo we have developed a new in utero stem cell transplantation assay, named blastocyst engraftment assay (BEA). BEA is based on microinjection of transgenic stem cells (purified from the bone marrow of ROSAb-geo26 or GFP transgenic mice) into mouse 3.5 days old embryos (blastocysts). Microinjection of <100 Lin-Sca-1+ or Lin-Sca-1- cells (purified by FACS from the bone marrow of adult ROSA b-geo26 transgenic mice) into 3.5 dpc C57BL/6J blastocysts has demonstrated that only the population of more primitive Lin-Sca-1+ cells, but not Lin-Sca-1- cells, contains blastocyst colonizing cells (BCC), which can survive during embryo development and engraft yolk sac and fetal liver (<5% of transgenic cells) (Fig. 16). Furthermore, microinjection of transgenic HSC into blastocysts of immunodeficient RAG-2 mice leads to partial repopulation of lymphoid compartments in adult chimeric RAG-2 mice. Microinjection of highly enriched populations of HSC into blastocysts of knockout mice (GATA-2, SCL, AML-1, PU-1) with perturbed hematopoiesis could serve as a novel in vivo assay to analyze primitive hematopoietic cell homing and differentiation during early embryonic development.

Recent reports point to the possibility that tissue-specific stem cells may have the capacity to develop into cells of unrelated tissue(s) (Fig. 17). This discovery could have important implications for designing new stem cell transplantation and cell therapy protocols for treatment of various diseases. Using BEA assay we are studying developmental potential of hematopoietic (HSC) and mesenchymal stem cells (MSC), which reside in the bone marrow, and have important role in the production and regeneration of all types of mesenchymal cells (osteocytes, chondrocytes, adipocytes etc.). The aim of this project is to comparatively analyze whether mouse HSC and MSC possess the potential for differentiation into cell types other than that of blood and mesenchymal lineages.

Research Group:

• Ronald G. Nachtman, Ph.D., Research Associate
• Xin Jing, M.D., Ph.D., Research Associate
• Jie Yang, M.D., Ph.D. student
• Danislav Spassov, M.S., Ph.D. student
• Laila Mnayer, Ph.D. Postdoctoral Associate
• Luis Dabul (HHMI scholar)
• Candice Saltiel (summer student)

Former Lab Members :

• Jesus Esposito, HHMI fellow
• James M. Abdullah, B.S., Research Clinical Specialist
• Jorge Infante, M.D., ACS fellow

Selected Publications:

Danislav S. Spassov, Jie Yang, Ron G. Nachtman, Roland Jurecic (2005). Pum2 Protein Supports Maintenance and Suppresses Differentiation of Multipotent Hematopoietic Progenitors by Regulating the Function and Activation of c-kit Receptor. (in preparation).

Jie Yang, Danislav S. Spassov, Xin Jing, Ronald G. Nachtman, Roland Jurecic (2005). Expression of truncated c-kit receptor (tr-kit) during hematopoiesis is restricted to hematopoietic stem cell-enriched cell populations. (submitted).

Jing X., Dabul, L., Infante J., Nachtman R, Jurecic R. (2005). Novel ring finger FLRF (RNF41/Nrdp1) modulates differentiation of hematopoietic progenitors by regulating cytokine receptor levels through ubiquitination. (in preparation).

Huiling Liang, Qin Chen, Andrew H. Coles, Stephen J. Anderson, German Pihan, Allan Bradley, Rachel Gerstein, Roland Jurecic, and Stephen N. Jones (2003). Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue. Cancer Cell 4(5): 349-360.

Spassov DS, Jurecic R. (2003). Pumilio family of RNA-binding proteins: Does evolutionarily conserved structure equal conserved function? IUBMB Life 55: 359-366.

Spassov DS, Jurecic R. (2003). Mouse Pum1 and Pum2 Genes, Members of the Pumilio Family of RNA-binding Proteins, Show Differential Expression in Fetal and Adult Hematopoietic Stem Cells and Progenitors. Blood Cells Mol Dis. 30: 55-69.

Masanobu Komatsu, Michele Mammolenti, Monica Jones, Roland Jurecic, Thomas J. Sayers, Robert B. Levy (2003). Antigen-primed CD8+ T cells can mediate resistance preventing allogeneic marrow engraftment in the simultaneous absence of perforin, CD95L, TNFR1 and TRAIL dependent killing. Blood 101: 3991-3999.

Spassov DS, Jurecic R. (2002). Cloning and comparative sequence analysis of PUM1 and PUM2 genes, human members of the Pumilio family of RNA-binding proteins. Gene 299: 195-204.

Chen AJ, Zhou G, Juan T, Colicos SM, Cannon JP, Cabriera-Hansen M, Meyer CF, Jurecic R, Copeland NG, Gilbert DJ, Jenkins NA, Fletcher F, Tan TH, Belmont JW (2002). Dual-specificity phosphatase JKAP specifically activates the c-Jun N-terminal kinase pathway. J Biol. Chem. 277, 36592-36601.

Abdullah, JM., Jing, X., Spassov, D.S., Nachtman, RG, Jurecic R. (2001). Cloning and Characterization of HEPP, a Novel Gene Expressed Preferentially in Mouse Fetal and Adult Hematopoietic Progenitor Cells. Blood Cells, Molecules and Diseases 27(3): 667-676.

Abdullah, JM., Li X., Nachtman, RG, Jurecic R. (2001) FLRF, a Novel Evolutionarily Conserved RING Finger Gene, is Differentially Expressed in Mouse Fetal and Adult Hematopoietic Stem Cells and Progenitors. Blood Cells, Molecules and Diseases 27(1): 320-333.

Jurecic R, Belmont JW (2000). Long-distance DD-PCR and cDNA microarrays. Curr Opin Microbiol. 3(3): 316-21.

Jurecic R, Nachtman RG, Colicos SM, Belmont JW (1998). Identification and cloning of differentially expressed genes by long-distance differential display. Anal. Biochem. 259: 235-244.

Jurecic R, Nguyen T, Belmont JW (1996). Differential mRNA display using anchored oligo-dT and long sequence-specific primers as arbitrary primers. Trends Genet. 12(12): 502-504.

Simoneaux DK, Fletcher FA, Jurecic R, Shilling HG, Van NT, Pragna P, Belmont JW (1995). The receptor tyrosine kinase-related gene (ryk) demonstrates lineage and stage-specific expression in hematopoietic cells. J. Immunol. 154: 1157-1166.

Wakamiya M, Blackburn MR, Jurecic R, McArthur MJ, Geske RS, Cartwright J, Mitani K, Vaishnav S, Belmont JW, Kellems RE, Finegold MJ, Montgomery CA, Bradley A, Caskey CT (1995). Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice. Proc. Natl. Acad. Sci. USA 92: 3673-3677.

Jurecic R, Van NT, Belmont JW (1993). Enrichment and functional characterization of Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+ and Lin-Sca-1+WGA+ bone marrow cells from mice with a Ly-6a haplotype. Blood 82: 2673-2683.