8/14/2019 Mechanism of Stem Cell Migration and Immune Development: Regulation Of

1/3Life Science Open Day | 2006 | Weizmann Institute of Science

972 8 934 2481

Department of

Immunology

972 8 934 4141

Tsvee.Lapidot@weizmann.ac.il

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 (lymphoid and myeloid) hematopoiesis in transplanted immune deficient

NOD/SCID and NOD/SCID/B2mnull mice.

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 chemokinestromal 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. 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.

Published Results:

SDF-1/CXCR4 interactions are involved in stem cell homing and

mobilization

Homing of human stem cells from the circulation to the bone marrow (BM) of

transplanted immune deficient mice, and their subsequent proliferation and differentiationdepend 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

SDF-1/CXCR4 interactions by pretreatment of immature human CD34+ progenitors with

neutralizing CXCR4 Ab blocked their in vivohoming and repopulation, while untreated

cells could home within hours into the BM of recipient mice.

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

Orit Kollet

Alexander Kalinkovich

Polina Goichberg

Eike Buss

Ayelet Dar

Asaf Spiegel

Shoham Shivtiel

Melania Tesio

Yaron Vagima

Amir Shachanovitz

Kfir Lapid

www.weizmann.ac.il/immunology/LapidotPage.html

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

mailto:Tsvee.Lapidot@weizmann.ac.ilhttp://www.weizmann.ac.il/immunology/LapidotPage.htmlhttp://www.weizmann.ac.il/immunology/LapidotPage.htmlhttp://www.weizmann.ac.il/immunology/LapidotPage.htmlhttp://www.weizmann.ac.il/immunology/LapidotPage.htmlmailto:Tsvee.Lapidot@weizmann.ac.il

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In contrast, increasing CXCR4 expression by in vitrocytokine

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 survivaland 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 vitromigration, 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 characteristicof 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 vitroproliferation

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+ progenitorsin 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. HumanCD34+ 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

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

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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 animportant 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, 687694.

Dar & Lapidot et al (2005) Chemokine receptor CXCR4-dependentinternalization and resecretion of functional chemokine SDF-1

by bone marrow endothelial and stromal cells, Nat Immunol, 6,

1038-46.