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
Tsvee Lapidot 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.
Orit Kollet 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
Alexander Kalinkovich multi-lineage (lymphoid and myeloid) hematopoiesis in transplanted immune deficient
NOD/SCID and NOD/SCID/B2mnull mice.
Polina Goichberg 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
Eike Buss 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
Ayelet Dar 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
Asaf Spiegel studied. 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.
Shoham Shivtiel
Published Results:
Melania Tesio
SDF-1/CXCR4 interactions are involved in stem cell homing and
mobilization
Yaron Vagima
Homing of human stem cells from the circulation to the bone marrow (BM) of
transplanted immune deficient mice, and their subsequent proliferation and differentiation
Amir Shachanovitz
depend on interactions between SDF-1, which is expressed in the host bone marrow,
and its receptor CXCR4, which is expressed on the donor homing cells. Interfering with
Kfir Lapid SDF-1/CXCR4 interactions by pretreatment of immature human CD34+ progenitors with
neutralizing CXCR4 Ab blocked their in vivo homing and repopulation, while untreated
cells could home within hours into the BM of recipient mice.

Department of
Immunology

972 8 934 2481

972 8 934 4141

Tsvee.Lapidot@weizmann.ac.il

www.weizmann.ac.il/immunology/
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Fig. 1 SDF-1/CXCR4 interactions and other regulators of stem cell homing

Life Science Open Day | 2006 | Weizmann Institute of Science


Fig. 2 A model for stress-induced stem cell mobilization
In contrast, increasing CXCR4 expression by in vitro cytokine
stimulation enhanced responses to SDF-1, demonstrating
improved homing and engraftment.
Release and mobilization of stem cells from the marrow
to the circulation are induced via stress and DNA damaging
agents in order to harvest stem cells for clinical transplantation.
Multiple daily stimulations with cytokines such as G-CSF recruit
human stem cells to the circulation, however the mechanism
is unknown. Proteolytic enzymes such as Neutrophil Elastase
released during mobilization, degrade SDF-1 in the bone marrow
after G-CSF administration. Concurrently, the levels of CXCR4
expressed on hematopoietic cells within the bone marrow
increase prior to their mobilization. Neutralizing Ab for CXCR4
or SDF-1 reduced human and mouse stem cell mobilization,
demonstrating SDF-1/CXCR4 signaling in cell egress. Our results
reveal a partial overlap in regulation of stem cell homing and
release/mobilization and that in both processes SDF-1/CXCR4
interactions play a major role.
SDF-1/CXCR4 interactions also regulate the function
of leukemic stem cells
SDF-1 also plays an important role in the migration of leukemic
human stem cells. While normal and leukemic cells share
similar mechanisms of migration, different homing patterns as
well as SDF-1 signaling pathways were found when comparing
malignant human Pre-B ALL cells to normal immature CD34+
cells.
In another malignant disease, AML, we found high levels of
intracellular CXCR4 and SDF-1 in all leukemic cells, including cells
that do not express surface CXCR4. SDF-1 regulates the survival
and proliferation of these cells, and CXCR4 is essential for their
homing into the BM of immune deficient mice, demonstrating
dynamic regulation of CXCR4 in these cells. AML cells secrete
high levels of the proteolytic enzyme elastase. Specific inhibition
of elastase reduced in vitro migration, proliferation and in vivo
homing of cells from AML patients, in contrast to normal CD34+
progenitor cells.
These results suggest that abnormal secretion and regulation
of proteolytic enzymes such as elastase by primitive myeloid
leukemia cells are important for their in vivo behavior and
invasive phenotype.
Stem cells / blood vessel wall interactions
The expression of SDF-1 by endothelial cells of the blood
vessels is crucial for inducing cell arrest under shear flow,
an essential step for a successful transendothelial migration
from the circulation into the bone marrow. In addition, SDF-1
activated the major adhesion molecules such as CD44, LFA-1,
VLA-4 and VLA-5 on migrating human stem and progenitor cells
as part of the multi step process of homing and transendothelial
migration. CXCR4 expressed by endothelial cells also serves to
bind circulating SDF-1, internalize and translocate it to the BM,
supporting the cell homing process and revealing how peripheral
organs can communicate with the bone marrow reservoir. This
chemokine-transporter function of CXCR4 was a characteristic
of endothelial and stromal cells but not of hematopoietic cells.
Thus, chemokine translocation across the blood-bone marrow
barrier allows effective transfer of functional SDF-1 from the
periphery to the stem cell niche in the bone marrow during
both homeostasis and ‘alarm’ situations.
CXCR4/SDF-1 signaling
Signal transduction pathways are triggered by binding of SDF-
1 to CXCR4. Activation of PI3K, but not MAPK, is required for
motility of enriched immature human CD34+ cells. The atypical
PKCzeta isoform was found to be essential for the process
of migration and stem cell retention in the bone marrow.
Moreover, activation of PKCzeta by SDF-1 was PI3K dependent.
Functional expression of CXCR4 on human CD34+ progenitors
was increased by sustained elevation in cellular cAMP, an effect
specifically mediated by PKCzeta, involving Rap1 and Rac1
signaling. Functional cAMP elevation led to increased ability of
human CD34+ cells to transmigrate the BM endothelial layer,
and adhere to BM stroma in vitro, and augmented homing
potential to the BM and spleen of immune deficient mice in a
Rac1 and PKCz-dependent manner.
Lentiviral vectors for gene transfer
Overexpression of CXCR4 on normal CD34+ cells induced
increased SDF-1 mediated migration, signaling and more
important, in vivo repopulation and retention in the BM of
immune deficient mice. Unexpectedly, over expression of CXCR4
on human progenitors also increased their in vitro proliferation
and provides powerful tools to study directional migration and
regulation of CXCR4 expression.
CD44 is essential for stem cell homing and migration
CD44 is the principal receptor of hyaluronic acid (HA), which is
the major component of the extracellular matrix. CD44 blocking
prevents homing and engraftment of human CD34+ progenitors
in immune deficient mice. We find a cross talk between CD44 and
SDF-1/CXCR4 pathway, since SDF-1 increases cell adhesion and
spreading on HA, and formation of cell protrusions expressing
CXCR4, while blocking of CD44 vetoes this process. Our data
suggest that SDF-1 mediates adhesion and anchorage of stem
cells to the extracellular matrix of the BM niches by altering the
cytoskeleton and relocating surface CD44 expression.
Stress-induced SDF-1 and MMP-9 recruit human
CD34+ cells to the injured liver
SDF-1 is expressed by liver bile-duct epithelium. Human
CD34+ progenitors home to the irradiated murine liver in a
CXCR4 dependent manner. Engrafted human cells localized in
clusters surrounding the bile ducts, in close proximity to SDF-
1 expressing epithelial cells, and infrequently differentiated to
albumin-producing cells. Radiation or inflammation induced
by infections by the hepatitis virus increased SDF-1 levels and
hepatic injury induced MMP-9 activity, leading to increased
CXCR4 expression and SDF-1-mediated recruitment of
hematopoietic progenitors and leukocytes to the liver as part
of host defense and organ repair. Thus, stress-induced signals
such as increased expression of SDF-1 and MMP-9 recruit
human CD34+ progenitors to the liver of NOD/SCID mice
shifting the stem cell niche to the ischemic site. Our results
suggest the potential of hematopoietic CD34+ cells to respond
to stress signals from non-hematopoietic injured tissues, as an
important mechanism for tissue targeting and repair.

Ongoing Projects:
1. The interplay between chemokines, cytokines, proteolytic
enzymes, adhesion molecules and stromal cells in regulation
of the stem cell niche.
2. Mechanism of stress induced mobilization and recruitment
of stem cells as well as maturing leukocytes from the bone
marrow reservoir to the circulation during inflammation and
injury as part of host defense and repair.
3. The role of bone-resorbing Osteoclasts and their proteolytic
enzymes, Cathepsin K and MMP-9, in regulation of stem cell
mobilization and proliferation.
4. CXCR4/SDF-1 signaling in migration of normal versus leukemic
human progenitor cells. Similarities and differences.
5. Homing of normal and leukemic progenitor cells: in vivo
imaging of the homing process.
6. The membrane bound proteolytic enzyme MT1-MMP: its role
in stem cell homing and mobilization.
7. In vivo migration and dissemination of leukemic human
AML and Pre B ALL stem cells, the role of the proteolytic
enzyme elastase, the Focal Adhesion Kinase (FAK), and the
chemokine SDF-1.
8. CD45 signaling and function in stem cell development,
homing and mobilization.

Selected Publications
Peled & Lapidot et al (1999) Dependence of Human Stem Cell
Engraftment and Repopulation of NOD/SCID Mice on CXCR4.
Science, 283, 845-848.
Petit & Lapidot et al (2002) G-CSF induces stem cell mobilization
by decreasing bone marrow SDF-1 and upregulating CXCR4. Nat
Immunol, 3, 687–694.
Dar & Lapidot et al (2005) Chemokine receptor CXCR4-dependent
internalization and resecretion of functional chemokine SDF-1
by bone marrow endothelial and stromal cells, Nat Immunol, 6,
1038-46.