Characterization of adhesion and viability of early seeding hematopoietic cells in the host bone marrow in vivo and in situ.
OBJECTIVE
Homing and seeding are essential early events of engraftment that depend on the interaction of hematopoietic cells with the host bone marrow (BM) stroma. We used optical techniques to characterize the adhesion patterns and viability of bone marrow cells (BMC) at the level of recipient BM microenvironment.


METHODS
Donor cells labeled with PKH dyes were tracked in vivo through an optical window placed over the femoral epiphysis of nonconditioned recipients. Adhesion to BM stroma was assessed with laser tweezers, and viability was assayed by fluorescence resonance energy transfer of the pair PKH67-propidium iodide (PI) in freshly excised femurs.


RESULTS
Three hours after intravenous injection, an estimated 30% of the labeled cells in the femur were immobile. The percent of adherent cells increased to 74+/-3% and 97+/-2% on days +1 and +3, respectively, (p<0.001), and similar fractions of cells were viable at these times (p<0.001). The observation that all adherent cells were viable suggested a correlation between these parameters. The day +3 BM-seeded cells rescued secondary myeloablated allogeneic hosts. Seeding in the host BM was accompanied by (4.5-fold) enrichment of cells expressing SCA-1 and was 22-fold higher for lineage-negative Lin(-) cells compared to lineage-positive cells (p<0.001). Pretreatment of Lin(-) cells with anti-VLA-4 antibodies caused a 2.4-fold decrease in homing and a 4.6-fold decrease in seeding (p<0.001).


CONCLUSIONS
The data indicate that adhesion is rate-limiting determinant of homing and early seeding, and a crucial event that preserves the viability of cells toward successful engraftment. The role of VLA-4 is more important for primary seeding than it is for homing to the BM.
Askenasy
Nadir
N
Frankel Laboratory of Bone Marrow Transplantation, Department of Pediatric Hematology and Oncology, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva 49202, Israel. anadir@012.net.il
Yolcu
Esma S
ES
Shirwan
Haval
H
Stein
Jeremiah
J
Yaniv
Isaac
I
Farkas
Daniel L
DL
D000818:Animals; D001853:Bone Marrow; D016026:Bone Marrow Transplantation; D002448:Cell Adhesion; D002465:Cell Movement; D002470:Cell Survival; D005269:Femur; D005456:Fluorescent Dyes; D006085:Graft Survival; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D039041:Integrin alpha4beta1 / Q000502:physiology; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence; D017154:Stromal Cells
D005456:Fluorescent Dyes; D039041:Integrin alpha4beta1