Tsvee Lapidot

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells

[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stemand progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the marrow have not been completely elucidated and are still an object of intense study. One important aspect is the modification of expression and affinity of adhesion molecules by stem and progenitor cells which are required both for stem cell retention, migration and development. Adhesion is regulated by expression of the adhesion molecules, their affinity and avidity. Affinity regulation is related to the molecular binding recognition and bond strength. Here, we describe the in vitro FACS assay used in our research to explore the expression, affinity and function of the integrin α4β1 (also termed VLA-4) for murine bone marrow retained EPCR+ long term repopulation HSC (LT-HSC) (Gur-Cohen et al., 2015).

Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells

[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a nonmotile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of curative treatment strategies for hematologic malignancies. The homing process ends with selective access and anchorage of HSCs to their specialized niches within the BM. Adhesion molecules are targets to either enhance homing in cases of stem cell transplantation or reduce BM retention to harvest mobilized HSCs from the blood of matched donors. A major adhesion protein which is functionally expressed on HSCs and is involved in their homing and retention is the integrin alpha4beta1 (Very late antigen-4; VLA4). In this protocol we introduce an adhesion assay optimized for VLA4 expressing murine bone marrow stem cells. This assay quantifies adherent HSCs by flow cytometry with HSC enriching cell surface markers subsequent to the isolation of VLA4 expressing adherent cells.