title: A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow.
abstract: To measure the ability of human hematopoietic stem cells (HSCs), the SCID-repopulating cell (SRC) assay has been widely used. Conventionally, human HSCs are transplanted into a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse via a tail vein. However, those cells must go through various obstacles until they reach the mouse marrow environment, which could explain the generally low homing efficiency in this system. Thus, the capability of HSCs may not be studied accurately by this intravenous transplantation method. In our attempt to reveal actual SRC potential, ie, self-renewal and multilineage differentiation in recipient bone marrow, we introduced cells into mouse marrow directly (intrabone marrow [iBM]) to minimize the effect of factors that may interfere with the homing of HSCs and compared the results obtained by intravenous and iBM methods. When cord blood CD34(+)CD38(-) cells were transplanted in NOD/SCID mice by iBM, a 15-fold higher frequency of SRC, 1 in 44 CD34(+)CD38(-) cells, was achieved compared with 1 in 660 by the intravenous method. Furthermore, the iBM transplant showed high levels of engraftment in the secondary transplantation. Pretreatment of CD34(+) cells with antibodies that block either very late antigen 4 (VLA-4) or VLA-5 reduced engraftment partially, whereas blockage of both molecules resulted in complete inhibition of engraftment, which suggests that VLA-4 and VLA-5 are involved in different processes in engraftment or have complementary roles. Our results indicate that the iBM injection strategy is a more sensitive and direct way to measure the capability of human SRCs and is useful to investigate the interaction of HSCs and marrow environment in vivo.
authors.affiliation: Division of Hematopoiesis, Research Center for Regenerative Medicine, Department of Hematology, and Institute of Medical Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yahata
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ando
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Sato
authors.forename: Hiroko
authors.initials: H
authors.lastname: Miyatake
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Yukari
authors.initials: Y
authors.lastname: Muguruma
authors.forename: Shunichi
authors.initials: S
authors.lastname: Kato
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Hotta
mesh_terms: D000818:Animals; D001853:Bone Marrow; D019070:Cell Lineage; D002465:Cell Movement; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D017930:Genes, Reporter; D006085:Graft Survival; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007231:Infant, Newborn; D007267:Injections; D007275:Injections, Intravenous; D039041:Integrin alpha4beta1 / Q000502:physiology; D039081:Integrin alpha5beta1 / Q000502:physiology; D008164:Luminescent Proteins / Q000032:analysis; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009928:Organ Specificity; D019718:Receptors, CXCR4 / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013623:Tail / Q000098:blood supply; D018183:Transplantation Chimera; D014183:Transplantation, Heterologous / Q000379:methods*
chemical_list: D039041:Integrin alpha4beta1; D039081:Integrin alpha5beta1; D008164:Luminescent Proteins; D019718:Receptors, CXCR4; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins