Prof. Tsvee Lapidot

Tsvee Lapidot Prof. Tsvee Lapidot
The Edith Arnoff Stein Professorial Chair in Stem Cell Research

Research Interests Members Selected Publications

Research Interests:
Mechanism of Stem Cell Migration and Immune Development: regulation of the bone marrow reservoir of immature and maturing leukocytes as part of host defense and repair during homeostasis and alarm situations.
The Role of Stromal Cells, Cytokines, Chemokines, Adhesion Molecules and Proteolytic Enzymes in Normal and Leukemic Human Hematopoiesis
Hematopoietic stem cells are a rare population within the bone marrow which actively maintain continuous production of all mature blood cell-lineages throughout life, including major components of the immune system such as T and B lymphocytes, myeloid neutrophils and monocytes, while maintaining s small pool of undifferentiated stem and progenitor cells.
During development, or in experimental and clinical transplantations, stem cells migrate through the blood circulation and home to the bone marrow, repopulating it with immature and maturing blood cells, which in turn are released into the circulation. We have previously developed a preclinical in vivo model that identifies normal and leukemic human CD34+CD38-/low stem cells based on their functional ability to initiate multi-lineage (both lymphoid and myeloid) hematopoiesis in transplanted immune deficient NOD/SCID and NOD/SCID/B2mnull mice.
The process of hematopoietic stem cell homing, bone marrow repopulation and release back to the circulation which are crucial for stem cell function and for development of the immune system, is not well understood. Our major goal is to determine the molecular mechanisms which govern stem cell migration, proliferation and differentiation and to identify the interplay between bone remodeling via osteoclast/osteoblast interactions and regulation of the stem cell niche and hematopoiesis. The unique roles of stromal cells, hematopoietic cells, chemokines, cytokines, adhesion molecules and proteolytic enzymes which regulate these processes and their relationship are currently investigated.
Our research is focused on interactions between the bone marrow reservoir of leukocytes and peripheral organs during steady sate homeostasis as well as during alarm situations in which immature and maturing leukocytes are recruited from the bone marrow to the circulation as part of host defense and repair mechanisms. In particular, the chemokine stromal derived factor one (SDF-1), which is produced by many cell types and also by bone marrow stromal and endothelial cells, is the only powerful chemoattractant for immature human and murine stem cells which express its receptor CXCR4, is extensively studied. This chemokine is highly preserved throughout evolution since human and mouse SDF-1 are cross-reactive and differ in one amino acid only. SDF-1 also serves as a survival factor for stem and progenitor cells, and both the ligand and its receptor are regulated by HIF-1 which is activated by hypoxia. Our results reveal a partial overlap between regulation of stem cell homing, and release with stem cell proliferation and differentiation. In addition both similarities and differences exist between regulation of normal and leukemic human stem cell migration and development.

Figure 1. SDF-1 expression on human bone marrow

(J Clin Invest 2000;106:133). SDF-1 expressing immature BM osteoblasts lining the endosteum region (OB) and stromal cells (ST) (a, arrow, x200), venule (b, arrow), and arteriole (c, arrow) in human BM sections (x1000).

Figure 2. A model for stress-induced stem cell mobilization

(Hematology 2003;419). DNA-damaging chemotherapy and inflammatory cytokines such as G-CSF induce a transient increase in SDF-1 levels within the BM as part of the alarm situation. G-CSF triggers neutrophils to proliferate and release proteases (elastase, cathepsin G, and proteinase 3). Concurrently, proliferation and activation of osteoclasts, which release the mobilizing chemokine IL-8 and secrete MMP-9 in response to SDF-1 stimulation, take place. The massive inflammatory proteolytic enzyme activity leads to degradation of stem cell anchorage and retention signals (VCAM-1 and SDF-1), inactivation of G-CSF, and remodeling of the BM extracellular matrix. MMP-9 mediates shedding of membrane-bound SCF, which together with proteinase 3 induces progenitor cell proliferation and CXCR4 upregulation, followed by partial inactivation of CXCR4 and c-kit by the proteolytic machinery. These sequential events, which are repeated and intensified after each cycle of G-CSF stimulation, orchestrate the egress of progenitors from the BM into the circulation.

Figure 3. Polarization and pseudopodia formation in human CD34+ progenitors migrating toward SDF-1 gradient

(Blood 2004; 103(8):2981). Time-lapse videomicroscopy of enriched human CD34+ cells migrating on HA substrate toward a gradient of SDF-1. Non-stimulated progenitors have only limited numbers of pseudopodia and move randomly (Video 1).
CD34+ progenitors migrating toward SDF-1 gradient, become progressively more polarized and display multiple CD44-positive protrusions at their leading edge (Video 2).

Figure 4. SDF-1/CXCR4 interactions and other regulators of stem cell homing

(Blood 2000; 95:3289). (a) Stem cell rolling interactions on constitutively expressed endothelial E and P selectins. Following rolling, CXCR4+ stem cells (blue cells) are activated by SDF-1, which is secreted from bone marrow endothelial cells and triggers LFA-1/ICAM-1 and VLA-4/VCAM-1 interactions, supporting firm adhesion to endothelial cells. (b) Cells that do not express sufficient levels of CXCR4 (purple) will detach from the endothelial layer and return to the blood stream. (c) The arrested human CXCR4+ stem cells, in response to SDF-1, will extravasate and migrate through the underlying basal lamina ECM using VLA-4 and VLA-5 integrin receptors to FN. (d) Migrating stem cells will eventually reach the "stem cell niches," which consist of stromal cells that present the proper set of adhesion molecules (e.g. VCAM-1 and FN), SDF-1, and growth stimulatory factors.

Figure 5. Bone-resorbing osteoclasts induce stem cell mobilization in response to inflammatory and other stress signals.

Inflammatory signals, RANKL expression, and SDF-1 production by (a) MM cells, (b) synovial T cells of RA patients, (c) mast cells, or (d) breast and prostate cancer cells metastasizing the bone increase recruitment of osteoclast progenitors to the bone and their activation (e). Proteolytic enzymes secreted by bone-resorbing osteoclasts temporarily alter the endosteal stem cell niche and induce proliferation and mobilization of hematopoietic stem and progenitor cells (f) and their recruitment to high SDF-1-expressing injured tissues or organs (g) as part of host defense and repair mechanisms.

Figure 6. Hematopoietic stem cells line the inner surface of the bone, attached by components of the extracellular matrix.

(Nat Med, 2006. Jun;12(6):657-664). Osteoclasts, when induced to differentiate by osteoblasts, secrete various factors that cleave the extracellular matrix, resulting in the mobilization of stem cells from the bone marrow. Thus, bone homeostasis and hematopoiesis have been shown to be linked. The cover depicts a primary osteoclast stained for CXCR4 (blue), polymerized actin (red) and nuclear DNA (green).

Figure 7: Dopamine receptor agonists increase the polarization and motility of CD34+ cells.

(Nat Immunol. 2007 10:1123-31) Microscopy of cells immunolabeled for Dopamine receptor-5 (green) and stained for polymerized actin (red). Arrows indicate clustering of DR5 in polarized cells. (Scale bar, 10 µm)

Group Members:

Staff Scientists: Orit Kollet
Alexander Kalinkovich
Polina Goichberg

Post-doctoral Fellows: Eike Buss

Ayelet Dar
Shoham Shivtiel

Asaf Spiegel

Melania Tesio

Ph.D. Students: Yaron Vagima

Amir Schajnovitz

Kfir Lapid

Aya Ludin

M.Sc. Students: Tomer Itkin

Cristina Botez (Karin)

Selected Publications

Figure 1. SDF-1 expression on human bone marrow (J Clin Invest 2000;106:133). SDF-1 expressing immature BM osteoblasts lining the endosteum region (OB) and stromal cells (ST) (a, arrow, x200), venule (b, arrow), and arteriole (c, arrow) in human BM sections (x1000).		Figure 2. A model for stress-induced stem cell mobilization (Hematology 2003;419). DNA-damaging chemotherapy and inflammatory cytokines such as G-CSF induce a transient increase in SDF-1 levels within the BM as part of the alarm situation. G-CSF triggers neutrophils to proliferate and release proteases (elastase, cathepsin G, and proteinase 3). Concurrently, proliferation and activation of osteoclasts, which release the mobilizing chemokine IL-8 and secrete MMP-9 in response to SDF-1 stimulation, take place. The massive inflammatory proteolytic enzyme activity leads to degradation of stem cell anchorage and retention signals (VCAM-1 and SDF-1), inactivation of G-CSF, and remodeling of the BM extracellular matrix. MMP-9 mediates shedding of membrane-bound SCF, which together with proteinase 3 induces progenitor cell proliferation and CXCR4 upregulation, followed by partial inactivation of CXCR4 and c-kit by the proteolytic machinery. These sequential events, which are repeated and intensified after each cycle of G-CSF stimulation, orchestrate the egress of progenitors from the BM into the circulation.
Figure 3. Polarization and pseudopodia formation in human CD34+ progenitors migrating toward SDF-1 gradient (Blood 2004; 103(8):2981). Time-lapse videomicroscopy of enriched human CD34+ cells migrating on HA substrate toward a gradient of SDF-1. Non-stimulated progenitors have only limited numbers of pseudopodia and move randomly (Video 1). CD34+ progenitors migrating toward SDF-1 gradient, become progressively more polarized and display multiple CD44-positive protrusions at their leading edge (Video 2).
Figure 4. SDF-1/CXCR4 interactions and other regulators of stem cell homing (Blood 2000; 95:3289). (a) Stem cell rolling interactions on constitutively expressed endothelial E and P selectins. Following rolling, CXCR4+ stem cells (blue cells) are activated by SDF-1, which is secreted from bone marrow endothelial cells and triggers LFA-1/ICAM-1 and VLA-4/VCAM-1 interactions, supporting firm adhesion to endothelial cells. (b) Cells that do not express sufficient levels of CXCR4 (purple) will detach from the endothelial layer and return to the blood stream. (c) The arrested human CXCR4+ stem cells, in response to SDF-1, will extravasate and migrate through the underlying basal lamina ECM using VLA-4 and VLA-5 integrin receptors to FN. (d) Migrating stem cells will eventually reach the "stem cell niches," which consist of stromal cells that present the proper set of adhesion molecules (e.g. VCAM-1 and FN), SDF-1, and growth stimulatory factors.		Figure 5. Bone-resorbing osteoclasts induce stem cell mobilization in response to inflammatory and other stress signals. Inflammatory signals, RANKL expression, and SDF-1 production by (a) MM cells, (b) synovial T cells of RA patients, (c) mast cells, or (d) breast and prostate cancer cells metastasizing the bone increase recruitment of osteoclast progenitors to the bone and their activation (e). Proteolytic enzymes secreted by bone-resorbing osteoclasts temporarily alter the endosteal stem cell niche and induce proliferation and mobilization of hematopoietic stem and progenitor cells (f) and their recruitment to high SDF-1-expressing injured tissues or organs (g) as part of host defense and repair mechanisms.
Figure 6. Hematopoietic stem cells line the inner surface of the bone, attached by components of the extracellular matrix. (Nat Med, 2006. Jun;12(6):657-664). Osteoclasts, when induced to differentiate by osteoblasts, secrete various factors that cleave the extracellular matrix, resulting in the mobilization of stem cells from the bone marrow. Thus, bone homeostasis and hematopoiesis have been shown to be linked. The cover depicts a primary osteoclast stained for CXCR4 (blue), polymerized actin (red) and nuclear DNA (green).		Figure 7: Dopamine receptor agonists increase the polarization and motility of CD34+ cells. (Nat Immunol. 2007 10:1123-31) Microscopy of cells immunolabeled for Dopamine receptor-5 (green) and stained for polymerized actin (red). Arrows indicate clustering of DR5 in polarized cells. (Scale bar, 10 µm)

Staff Scientists:	Orit Kollet
	Alexander Kalinkovich
	Polina Goichberg
Post-doctoral Fellows:	Eike Buss
	Ayelet Dar
	Shoham Shivtiel
	Asaf Spiegel
	Melania Tesio
Ph.D. Students:	Yaron Vagima
	Amir Schajnovitz
	Kfir Lapid
	Aya Ludin
M.Sc. Students:	Tomer Itkin
	Cristina Botez (Karin)