Tianxue Fan

Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow.

Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so‐called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti‐PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6‐P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6‐P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis‐supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6‐P cells express a high level of the PA6 molecule, which is reactive with anti‐PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells.

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day‐14 colony‐forming unit‐spleen (CFU‐S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU‐S formation nor the hemopoietic colonies in the recipient liver were observed.

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes.

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra‐bone‐marrow (IBM) route (IBM‐BMT) over the intravenous route (IV‐BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM‐BMT or IV‐BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor‐derived cells in total and of c‐kit+ cells were observed at 2 through 6 days after IBM‐BMT. In parallel, significantly higher numbers of colony‐forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM‐BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM‐BMT over IV‐BMT.

c‐kit<low Pluripotent Hemopoietic Stem Cells Form CFU‐S on Day 16

Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage− (Lin−)/CD71− cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class Ihigh/c‐kitlow and MHC class Ihigh/c‐kit

A novel strategy for organ allografts using sublethal (7 Gy) irradiation followed by injection of donor bone marrow cells via portal vein.

A new strategy for organ allografts that does not require recourse to immunosuppressants is established in mice. The strategy includes sublethal (7 Gy) irradiation followed by the injection of donor bone marrow cells (BMCs) via the portal vein (P.V.) and organ allografts 1 day after irradiation. Irradiation doses (≤7 Gy) are found to allow the recipients to survive without the need to reconstitute the BMCs, as the recipient hematolymphoid cells can gradually recover. One hundred percent of recipients irradiated with 7 Gy followed by either P.V. or i.v. injection of donor BMCs accept organ allografts (the skin, pancreas, and adrenal glands) for more than 1 year. However, organ allograft survival rates decrease when irradiation doses are reduced; the skin graft survival rate of mice treated with 6.5 Gy and P.V. injection of BMCs is 79%, whereas that of mice treated with 6.5 Gy and i.v. injection is 50%, indicating that the P.V. injection of BMCs induces persistent tolerance more effectively than the i.v. injection. H-2 typing reveals that almost all the hematolymphoid cells (>98%) in the peripheral blood and hematolymphoid organs are donor-derived even 1 year after the treatment (7 Gy and P.V.). The T cells are tolerant to both donor-type and host-type MHC determinants. The major mechanism underlying the persistent tolerance induced by this strategy seems to be because of clonal deletion. This simple and safe strategy would be of great advantage for human organ transplantation.

Development of Mouse Dendritic Cells from Lineage-Negative c-kit <low Pluripotent Hemopoietic Stem Cells In Vitro

Dendritic cells (DCs) are essential for the presentation of antigens in the primary immune response. To examine the generation of DCs from hemopoietic stem cells in the bone marrow (BM), lineage‐negative (Lin−)/CD71− bone marrow cells (BMCs) from C57BL/6 mice were separated into major histocompatibility complex (MHC) class Ihigh/ c‐kitlow and MHC class Ihigh/c‐kit80%) generated cells expressed high levels of DC surface markers such as DEC205 and MHC class II, and they were potent stimulators in the primary allogeneic T cell activation. The development of DCs from c‐kit

Enhancement of IL-7 following irradiation of fetal thymus

The effect of ionizing radiation on intra-thymic T cell development was investigated using a fetal thymic organ culture (FTOC) method in vitro. When double-negative (DN) fetal (day 15) thymocytes were co-cultured with an irradiated (25 Gy) fetal (day 15) thymus in the absence of direct contact or mitogenic stimulation, the induction of TCRgammadelta+ T cells was observed. About 50% of the TCRgammadelta+ T cells developed after 4-day-co-culture with the irradiated fetal thymus, whereas only a few TCRgammadelta+ T cells developed after co-culture with the non-irradiated fetal thymus. About 50% of the TCRgammadelta+ T cells were CD8+ cells with alphabeta heterodimeric chains. Cultured supernatants of the irradiated fetal thymi also induced the differentiation from DN thymocytes to CD8+ TCRgammadelta+ T cells after 3-day-culture. To clarify the factor in the cultured supernatants, several neutralizing antibodies (Abs) were used. Only anti-IL-7-Ab inhibited the differentiation from DN thymocytes to CD8+ TCRgammadelta+ T cells. RT-PCR revealed the increased expression of IL-7 mRNA in the fetal thymus 24 hours after radiation. Electron microscope studies demonstrated proliferative epithelial cells in the irradiated fetal thymus. These findings strongly suggest that fetal thymic epithelial cells affected by irradiation proliferate and enhance the production of IL-7, which induces the differentiation of CD8+ TCRgammadelta+ T cells from DN thymocytes.

A New Assay Method for Late CFU‐S Formation and Long‐Term Reconstituting Activity Using a Small Number of Pluripotent Hemopoietic Stem Cells

We have previously reported that Lin−/CD71−/MHC class Ihigh/c‐kit

Treatment of Shwachman Syndrome by Japanese Herbal Medicine (Juzen‐Taiho‐To): Stimulatory Effects of Its Fatty Acids on Hemopoiesis in Patients

Juzen‐taiho‐to (a Japanese herbal medicine) has been traditionally administered to patients with anemia, neutropenia, or wasting syndrome. We previously attempted to isolate and purify the hemopoiesis‐stimulatory components in Juzen‐taiho‐to extracts using an in vitro hemopoietic stem cell (HSC) assay method in which mouse HSCs can proliferate on a stromal cell line (MS‐5). We have found that fatty acids (particularly oleic acid and linolenic acid) actively promote the proliferation of HSCs, and that the effect is mediated by stromal cells, rather than by any direct action on the HSCs.

Lin−CD34− bone marrow cells from adult mice can differentiate into neural-like cells

Numerous studies have shown that some populations of bone marrow cells (BMCs) have the capacity to differentiate into neural cells, which is useful for repairing brain lesions. In this paper, we analyze neural differentiation features of lineage-negative/CD34-negative (Lin(-)CD34(-)) cells in the bone marrow of adult mice. The population of Lin(-)CD34(-) in BMCs was isolated by magnetic bead sorting and fluorescence-activated cell sorter (FACS) using specific lineage (CD4, CD8a, CD11b, CD45R, Gr-1 and TER-119) antibodies and CD34 antibody. First, we cultured Lin(-)CD34(-) BMCs in the presence of RNIF: vitamin A derivative retinoic acid (RA) and neural-inducing factors (platelet-derived growth factor BB (PDGF-BB), epidermal growth factor (EGF) and fibroblast growth factor-basic (FGF-b)). Analyses of RT-PCR and immunocytochemistry indicated that RNIF-treated Lin(-)CD34(-) BMCs expressed neural phenotypes as well as neurogenic transcription factors. When we implanted the Lin(-)CD34(-) BMCs isolated from enhanced green fluorescent protein (eGFP) transgenic mice into the subventricular zone (SVZ) of postnatal mice, eGFP-positive cells survived 3 weeks after the injection in the various brain regions, some of which expressed the neural phenotypes. Our data suggest that certain subsets in the CD34(-) populations of adult bone marrow could have the capacity to differentiate into neural cells in a suitable environment.