IntroductionMesenchymal stem cells (MSCs) present in bone marrow (BM) are thought to give rise to cells that constitute the hematopoietic microenvironment (HME). 1 MSCs have been isolated from BM and various tissues from humans and many other species, expanded in culture, and shown to differentiate into osteocytes, chondrocytes, adipocytes, and myoblasts under defined conditions in vitro. 2 In culture, MSCs produce a number of cytokines and extracellular matrix proteins and express cell adhesion molecules, all of which are involved in the regulation of hematopoiesis. 3,4 They also support the development of hematopoietic colonies in vitro. 4 However, in contrast to hematopoietic stem cells (HSCs) that have been prospectively isolated and extensively studied at the singlecell level both in vitro and in vivo, MSCs have only been defined and isolated by physical and functional properties in vitro. Consequently, little is known about their phenotypic and functional characteristics in vivo.Systemic administration of MSCs for facilitation of bone marrow transplantation has been proposed based on the in vitro characteristics of MSCs. 5 In recent studies, cotransplantation of human MSCs and HSCs resulted in increased chimerism or accelerated hematopoietic recovery (or both) in animal models and in humans, 6-9 suggesting a role for MSCs in the engraftment and repopulation of HSCs. Although the existence of donor MSCs has been documented in the BM of recipient animals following MSC infusion, 9,10 the methods used to detect engraftment, such as polymerase chain reaction (PCR) or staining of cytospin samples, could not unambiguously distinguish engraftment from cell survival or nonspecific lodgment on the vascular bed. In addition, Awaya et al examined stromal cells of patients who received BM transplants and confirmed that all donor signals were, in fact, derived from macrophages. 11 To our knowledge, there is no physical evidence that transplanted human MSCs have indeed engrafted in the BM of adult animals and directly participated in the enhanced engraftment of HSCs.To assess the engraftment, spatial distribution, and lineage commitment of MSCs as well as their roles in hematopoiesis in vivo, we transplanted enhanced green fluorescent protein (eGFP)-marked human MSCs into the tibiae of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice by intra-bone marrow transplantation (IBMT), a method previously shown to improve the engraftment of both hematopoietic and nonhematopoietic cells in mice. [12][13][14] We used a dual-color genetic marking strategy 15 along with immunofluorescent staining to distinguish and investigate transplanted cells in situ. We show that transplanted human MSCs integrated into the functional components of the HME and that these MSC-derived cells appeared to be actively involved in the maintenance of human hematopoiesis in murine BM. Materials and methods Isolation of human cord blood CD34 ؉ cellsHuman umbilical cord blood (CB) samples were obtained from full-term deliveries with infor...

To measure the ability of human hematopoietic stem cells (HSCs), the SCIDrepopulating 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 differentia-

No abstract