Subpopulations Of Bone Marrow Cells Research Articles

Promotion of Chimerism and Tolerance by Flt3 Ligand-Mobilized Facilitating Cells Is Associated with Upregulation of CXCR4 and SDF-1.

Facilitating cells (FC) are a CD8+/TCR− subpopulation of bone marrow (BM) cells that enhance engraftment of hematopoietic stem cells (HSC) in allogeneic recipients without causing graft-versus-host disease. They also significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. Treatment of mice with Flt3 ligand (Flt3-L) induces significant expansion of FC and HSC in the BM and their mobilization into peripheral blood (PB). In the present study, we evaluated the function of Flt3-L expanded FC and potential mechanism of action. Flt3-L treatment resulted in an 8.5-fold increase of FC in BM and a 100-fold increase in PB compared to untreated mice. When FC from the PB and BM of Flt3-L-treated donors were transplanted with HSC into allogeneic recipient mice, BM FC exhibited significantly impaired function while PB FC were potently functional (Fig. a). Strikingly, this correlated with an increase in mRNA for the chemokine SDF-1 and its receptor, CXCR4, which were significantly increased in PB FC (≥10 fold) and decreased in Flt3-L-expanded BM FC as compared to untreated control BM FC (Fig. b). The impaired function for BM FC was restored within 5 days after cessation of Flt3-L-treatment. Taken together, these data suggest that FC can be mobilized efficiently with Flt3-L and their engraftment facilitating function is associated with upregulation of transcripts for SDF-1 and CXCR4, suggesting that FC may chemoattract HSC by secreting SDF-1 and enhance their homing to the new microenvironment after transplantation. The fact that FC express CXCR4 suggests that they may co-migrate with HSC to the hematopoietic microenvironment after transplantation, being chemoattracted by BM-derived SDF-1. In summary, we provide evidence that FC are mobilized with HSC; the SDF-1-CXCR4 axis is involved in FC:HSC interaction; and FC co-migrate with HSC in response to an SDF-1 gradient. [Display omitted]

Gene Expression Profiles for Normal Human Bone Marrow-Derived CD34+ Stem Cells and CD34−/CD33+ Myeloid-Committed Progenitor Cells in Response to Daily Granulocyte-Colony-Stimulating Factor (G-CSF) Treatment.

G-CSF is widely used to accelerate marrow recovery after cancer chemotherapy, to facilitate collection of hematopoietic progenitor cells, and to treat severe chronic neutropenia. Although G-CSF was originally defined as a stimulus for myeloid cell proliferation, it has potent anti-apoptotic properties, affects synthesis of proteins stored in neutrophil granules, and has many other effects on cells of the myeloid lineage. To improve understanding of the molecular and cellular effects of G-CSF, particularly related to its use for the treatment of severe chronic neutropenia, we performed gene expression profile studies using Affymetrix oligonucleotide arrays and purified bone marrow cell sub-populations from normal volunteers treated with daily subcutaneous G-CSF (300 mcg/sc/qd) for five days. Under local anaesthesia, paired marrow aspirates were obtained from the posterior iliac crest before and after 5 daily doses of G-CSF. CD34+ and CD34−/CD33+ cells were purified using Miltenyi immunomagnetic beads. Two rounds of amplification of total RNA isolated from purified CD34+ or CD33+cells was used to obtain sufficient cRNA for hybridization. Expression data from scanned chips were first analyzed using the RMA algorithm. The limma package of the Bioconductor project was used to identify differentially expressed genes. Limma uses an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The statistical analysis of CD33+ cells revealed that 150 of more than 12,000 genes examined were up- or down-regulated >2-fold in response to G-CSF treatment. The top 10 genes with up- or down-regulated level of expression include clusterin, neutrophil elastase, two transcription factors, gelsolin, Grb2, phospholipase D3, protein kinase C, the major vault protein, and serine-threonine kinase. In the myeloid-committed CD34-/CD33+ progenitor cells, genes with altered expression level represent those with gene products involved in the cell cycle, regulation of apoptosis, the cytoskeleton, the inflammatory response, or serine proteases and transcription factors. Most of the genes up-regulated in CD33+ cells (e.g. neutrophil elastase, phospholipase D, protein kinase C) were down-regulated in CD34-positive cells in response to G-CSF. The results of the comparative analyses revealed the normal signature gene expression profiles for CD34+ and CD34−/CD33+ cells and identified genes that may mediate specific G-CSF effects.

Differential promotion of angiogenesis by distinct bone marrow cell subpopulations

Abstract Introduction: Bone marrow mononuclear cells (BMMC) have the capacity to differentiate into endothelial cells and promote neovascularization in hindlimb ischemia. However, the specific bone marrow cell population that promotes revascularization is unknown. Therefore, we determined the ability of unfractionated BMMC, CD34+ and CD34− from bone marrow to promote angiogenesis in an in-vivo murine model. Methods: BMMC were obtained from total bone marrow harvested from C57Bl/6 mice. CD34+ and CD34− subpopulations were isolated using magnetic bead separation. 1cc Matrigel was admixed with either unfractionated BMMC or CD34+ or CD34− cells was injected subcutaneously. Matrigel +/− VEGF (500ng/cc) was injected as positive and negative controls. Matrigel implants were harvested 14 days later and were analyzed by immunohistochemistry with determination of CD31+ and smooth-muscle actin (SMA) positive areas by morphometry. Results: No angiogenesis was observed in plain Matrigel. All implants seeded with bone-marrow-derived cells promoted neovessel formation in a dose-dependent manner. Unfractionated BMMC promoted formation of neovessels containing both CD31+ and SMA + cells. CD31+ area was greatest in implants seeded with CD34+ cells. In contrast, SMA+ area was greatest in implants containing CD34− cells, which promoted development of larger SMA-containing vessels. Conclusions: BMMC are capable of promoting angiogenesis in an in-vivo Matrigel model in a dose-dependent manner. CD34+ bone marrow cells are most capable of promoting endothelial development. In contrast, the CD34− subpopulation promotes development of larger arteries containing vascular SMA. These results have important implications for the development of cell-based therapies for limb ischemia.

Clinical Tolerance: the end of the beginning.

Clinical Tolerance: the end of the beginning.

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as “GFP/CCl 4 model” for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific “niche” made by CCl 4 induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.

Neuronal differentiation of murine bone marrow Thy-1- and Sca-1-positive cells.

Recent evidence suggests that cells from bone marrow can acquire neuroectodermal phenotypes in cell culture or after transplantation in animal models and in the human brain. However, isolation of the bone marrow cell subpopulation with neuronal differentiation potential remains a challenge. To isolate and expand neural progenitors from whole murine bone marrow, bone marrow was obtained from hind limb bone of C57BL6 mice and plated in culture with neuronal medium with basic fibroblast growth factor and epidermal growth factor. After 5-7 days in culture, cellular spheres similar to brain neurospheres appeared either floating or attached to culture dishes. These spheres were collected, dissociated, and expanded. The bone marrow-derived spheres were positive for nestin as assessed by immunocytochemistry and by reverse transcriptase polymerase chain reaction. Thy-1- and Sca-1-positive bone marrow cells selected by magnetic cell sorting resulted in a higher yield of nestin-positive spheres. After exposure to neuronal differentiative medium retinoic acid with and without Sonic hedgehog, cells positive for neuronal markers tubulin III (TuJ-1) and neurofilament (NF) were detected. The mRNA profile of these cells included the expression of TuJ-1, neuronal-specific enolase (NSE), and NF-light chain. To evaluate the in vivo behavior of these cells, spheres derived from bone marrow-derived cells of transgenic green fluorescent protein (GFP) mice were transplanted into newborn mouse brain. Two months later, the mouse neural cortex contained a minor proportion of GFP(+) cells co-expressing neuronal markers (TuJ-1, NF, MAP-2, NeuN). Although cell fusion phenomena with the host cells could not be ruled out, bone marrow-derived neurosphere transplantation could be a strategy for cellular mediated gene therapy.

Telomere length in subpopulations of human hematopoietic cells.

In order to test the hypothesis that the telomere length in human hematopoietic cells correlates with their proliferative potential, we analyzed the telomere length in highly purified subpopulations of bone marrow cells. Cells were sorted on the basis of CD34 and CD38 cell surface markers, and two samples were additionally sorted on the basis of Hoechst 33342 dye efflux allowing isolation of side population (SP) cells. The telomere length in limiting numbers of sorted cells was analyzed using a newly developed fluorescence in situ hybridization (flow-FISH) method in which hybridization of telomere probe in cells of interest is measured relative to control cells in the same tube. In all seven bone marrow samples analyzed, the telomere length in CD34(+)CD38(-) cells was longer than in CD34(+)CD38(+) cells from the same donor (p < 0.02). Results with sorted SP cells were less clear: the telomere fluorescence in these cells was very heterogeneous, and a reproducible difference in telomere length relative to CD34(+)CD38(-) cells could not be observed. We conclude that the telomere length in subpopulations of hematopoietic cells does appear to be correlated with the known proliferative potential of such cells and that further characterization of cells on the basis of telomere length is warranted for enrichment of very rare precursors of hematopoietic and other tissues.

Freshly isolated bone marrow cells induce death of various carcinoma cell lines.

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review. Among many types of organs transplanted using DBMC at this center, it was found that the cadaver renal recipients (CAD) had significantly decreased chronic rejection and higher graft survival when compared to equivalent non-infused controls. DBMC infusion was also associated with a marginal and non-specific immune depression. It was also observed that the number of chimeric donor cells gradually increased in the iliac crest bone marrow compartment with a concomitant decrease in the peripheral blood and that the increase was more rapid in living-related donor (LRD)-kidney/DBMC recipients in spite of a lower number of DBMC infused (<25%) than in the CAD-kidney/DBMC group. In the LRD recipients with residual anti-donor responses, purified chimeric cells of either donor or recipient inhibited recipient immune responses to the donor significantly more strongly than the freshly obtained bone marrow from the specific donor or volunteer suggesting an active regulatory role for chimeric cells. A number of (non-chimeric) subpopulations of bone marrow cells including CD34 + stem cells and the CD34 − early progeny like CD38 +, CD2 +, CD5 + and CD1 + lymphoid cells as well as CD33 + (but CD15 −) myeloid cells down-regulated the MLR and CML responses of allogeneic PBMC stimulated with (autologous) donor spleen cells. These regulatory effects appeared to be refractory to the action of commonly used immunosuppressive drugs and occurred during the early phase of the immune response through cell–cell interactions. Most of these DBMC sub-populations had stimulatory capabilities, albeit markedly lower than donor spleen cells, but only through the indirect antigen presentation pathway. When co-cultured with allogeneic stimulators, purified CD34 + cells were found to give rise both to CD3 − TCR αβ +, as well as CD3 + TCR αβ + cells and, thereby, responded in MLR to allogeneic stimulation (but did not generate cytotoxic effector cells). Also, a number of DBMC subpopulations inhibited the CML and to a lesser extent the MLR, of autologous post-thymic responding T cells stimulated with allogeneic irradiated cells, mediated through soluble factors. Finally, non-chimeric DBMC also inhibited the proliferative and cytotoxic responses of autologous T cells to EBV antigens, inducing T suppressor cells, which in turn could inhibit autologous anti-EBV CTL generation and B cell anti-CMV antibody production. These studies all suggested a strong inhibitory property of a number of DBMC sub-populations in vitro and in vivo with the notion that they promote unresponsiveness.

A Subpopulation of Murine Bone Marrow Cells Fully Differentiates along the Myogenic Pathway and Participates in Muscle Repair in the mdx Dystrophic Mouse

Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and α-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.

Increased bone morphogenetic protein-6 expression in mouse long bones after estrogen administration.

High-dose estrogen administration is known to induce new bone formation in mouse long bones. To study the role of regulatory proteins in this response, we examined associated changes in femoral messenger RNA (mRNA) for candidate factors. 17beta-estradiol (E2) 0.5 mg was administered to intact female mice by weekly injection, and Northern blot analysis was performed 1, 2, 4, 8, 12, and 16 days after the first injection. In contrast to other factors, an increase was observed in mRNA for bone morphogenetic protein-6 (BMP-6), which reached significance at day 8 and subsequent time-points. Estrogen-induced changes in BMP-6 protein expression were assessed by immunocytochemistry in longitudinal femoral sections. In untreated animals, BMP-6 was expressed by a significant proportion of growth plate chondrocytes and a subpopulation of bone marrow cells. In contrast, osteoblasts were consistently BMP-6 negative. From as early as 4 days after starting estrogen, clusters of slightly elongated BMP-6-positive cells were observed within the marrow cavity; the majority were close to active bone formation surfaces. Double immunolabeling studies revealed that only approximately 10% of BMP-6-positive bone marrow cells co-expressed the osteoblast transcription factor Cbfa1 suggesting that they are largely distinct from the osteoblast precursor population generated concurrently. BMP-6-positive cells expressed neither leukocyte nor erythroid markers (CD45 and TER-119, respectively), consistent with a stromal origin. We conclude that estrogen-induced osteogenesis in female mice is associated with increased levels of BMP-6 mRNA in mouse femurs, which seems to reflect the emergence of clusters of BMP-6 positive stromal cells adjacent to active bone formation surfaces. These findings raise the possibility that BMP-6 serves as a paracrine mediator of estrogen's osteogenic action in mice.

Expression of Leukemia-Associated Antigen, JL1, in Bone Marrow and Thymus

The identification of immunophenotypic markers with restricted expression has long been a critical issue in diagnostic and therapeutic advances for acute leukemias. We previously developed a monoclonal antibody against a new thymocyte surface antigen, JL1, and showed that JL1 is expressed in the majority of acute leukemia cases. In this study, using multiparameter flow cytometric analyses, we found that JL1 was uniquely expressed in subpopulations of normal bone marrow (BM) cells, implying the association of JL1 with the differentiation and maturation process. Although CD34(+) CD10(+) lymphoid precursors and some of maturing myeloid cells express JL1, neither CD34(+) CD38(-/lo) nor CD34(+) AC133(+) noncommitted pluripotent stem cells do. As for the myeloid precursors, CD34(+) CD33(+) cells do not express JL1. During lymphopoiesis, JL1 on the earliest lymphoid precursors disappear in the CD20(+) sIgM(+) stage of B-cell development or after CD1a down-regulation in thymocytes. Despite the highly restricted expression of JL1 in normal BM cells, most of the leukemias express JL1 irrespective of their immunophenotypes. These results indicate that JL1 is not only a novel differentiation antigen of hematopoietic cells, but also a leukemia-associated antigen. Therefore, we suggest that JL1 be a candidate molecule in acute leukemia for the diagnosis and immunotherapy that spares the normal BM stem cells.

New blood cells help an overburdened brain

Neurodegenerative disorders are all too common and devastating for those affected and their family and friends. Studies of patients and animal models not only provides information necessary for developing preventative and therapeutic approaches, but can also reveal novel basic principles of neuroscience. Such is the case with studies of glycolipid storage disorders that result in progressive neuronal dysfunction and degeneration. These disorders, which include Tay–Sachs, Sandhoff and Gaucher's diseases, are caused by genetic defects in lysosomal enzymes that are necessary for the degradation of glycosphingolipids, thus resulting in excessive accumulation of glycolipids in lysosomes. In the nervous system of these patients, the intracellular accumulation of glycolipids can trigger damage and death of neurons and oligodendrocytes. Because the glycolipids accumulate in the cells that degenerate, it had been assumed that the cells ‘self-destruct’. Surprisingly, Wada and co-workers 1xMicroglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation. Wada, R. et al. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10954–10959Crossref | PubMedSee all References1 now provide compelling evidence that neurons that accumulate the ganglioside GM2 in Sandhoff disease do not commit suicide, but instead are killed by enraged microglia.Wada and co-workers now provide compelling evidence that neurons that accumulate the ganglioside GM2 in Sandhoff disease do not commit suicide...Using a mouse model in which disruption of the β-hexosaminidase gene causes a neurological phenotype similar to Sandhoff disease in humans, Wada et al. show, using cDNA microarray analysis, that inflammation-like changes in gene expression occur in the brains of the Sandhoff mice. They further show that activated microglia are present in vulnerable brain regions before evidence of neuronal death. Remarkably, when bone marrow from normal mice was transplanted into Sandhoff mice depleted of bone marrow cells by treatment with γ-radiation, the activation of microglia and neuronal death were suppressed. However, the amelioration of neuronal damage was not associated with a decrease in ganglioside accumulation in the neurons, suggesting that it is not the lack of degradation of glycolipid that is responsible for the demise of neurons in Sandhoff disease. For more than 15 years it had been known that bone marrow transplantation can halt the progression of the disease symptoms outside of the nervous system in animals with lysosomal storage disorders 2xCorrection of feline arylsulphatase B deficiency (mucopolysaccharidosis VI) by bone marrow transplantation. Gasper, P.W. et al. Nature. 1984; 312: 467–469Crossref | PubMed | Scopus (61)See all References2 as a result of repopulation of organs, such as the spleen and liver, with macrophages. However, it was not known if, and how, the neurodegenerative process might be affected by bone marrow transplantation. The work of Wada et al. strongly suggests that β-hexosaminidase deficiency in microglia causes them to become hyper-reactive and attack neurons in the brainstem and spinal cord. Replacing the enraged microglia with less-reactive macrophages from normal mice results in a suppression of the inflammatory cascade that probably kills the neurons in Sandhoff patients.These new findings also reveal an important link between blood cells, brain function and disease. Whereas the involvement of inflammatory processes, in which microglia play a central role, is now well-established in neurodegenerative disorders, it had been assumed that the microglia were resident cells rather than blood-derived macrophages. However, the microglial population can turnover much more rapidly than previously appreciated, particularly in the settings of injury and disease. This raises the intriguing possibility that other subpopulations of bone marrow cells, some of which might be capable of differentiating into neurons and oligodendrocytes, might circulate and enter the brain. Findings from recent studies have shown that stem cells in the brain are responsive to environmental stimuli, ranging from mental and physical exercise to dietary restriction 3xDietary restriction increases survival of newly-generated neural cells and induces BDNF expression in the dentate gyrus of rats. Lee et al. J. Mol. Neurosci. 2000; 15: 105–113See all References3. Clearly, it will be of considerable interest to determine the effects of such stimuli on blood cells that take up residency in the brain and to see whether such alterations in blood–brain cell transfer play a role in neurodegenerative conditions other than lysosomal storage disorders.

Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses.

The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. Compared with spleen cells as controls, un-irradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports). The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34+ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38+, CD2+, CD5+, and CD1+ lymphoid cells (all were depleted of CD3+ cells) as well as CD33+ (but CD15 negative) myeloid precursors. However, positively selected CD19+ B-cells and CD15+ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3+ TcR-alphabeta+ CD4+ and CD28+ cells. A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.

CD44v10 in Hematopoiesis and Stem Cell Mobilization

Transplantation of hematopoietic progenitor cells provides in many instances of malignant tumors an ultimate chance of curative therapy, whereby the transfer of peripheral blood stem cells (PBSC) may even be advantageous as compared to bone marrow cells. Yet, the transfer of PBSC requires mobilization of stem cells into the periphery, which is mostly achieved via hematopoietic growth factors like G-CSF. Although G-CSF has been found to efficiently mobilize stem cells in most instances, some patients do not or insufficiently respond to G-CSF treatment In addition, G-CSF treatment may by accompanied by maturation of the most primitive progenitors and this may have an impact on stem cell homing and recovery of hemopoiesis. Therefore, additional approaches for stem cell mobilization have been searched for, in particular mobilization via a blockade of an adhesion molecule expressed by CD34-positive cells, like VLA-4 (CD49d) and the hematopoietic isoform of CD44 (CD44s).We recently described that in the mouse one of the CD44 variant isoforms, CD44v10, is expressed on a subpopulation of bone marrow cells, whereas a CD44v10 receptor-globulin only binds to stromal elements. These features appeared promising for anti-CD44v10 as a means of stem cell mobilization. Indeed, treatment with anti-CD44v10 revealed promising results concerning the recovery of multilineage colony forming units in the spleen and the peripheral blood. We here summarize features of expression and function of CD44 in hematopoiesis an provide further evidence for anti-CD44v10 as a means to mobilize hematopoietic progenitor cells.

Donor bone marrow and transplantation tolerance: historical perspectives, molecular mechanisms and future directions (review).

The induction of transplantation tolerance, defined as the indefinite existence of an intact, functioning allograft, has been a goal of transplantation immunologists for decades because of the morbid effects of immunosuppressive drugs required to maintain graft function. One promising method of tolerance induction involves the peritransplant administration of cytoablative drugs followed by administration of a low dose of donor bone marrow cells. A subpopulation of donor bone marrow cells bearing the CD8 accessory molecule cause the functional deletion of graft reactive T cells via a CD95 dependent mechanism that also involves the cytokine TGFbeta. This interaction is bi-directional in which the bone marrow cell deletes the graft reactive T cell that recognizes it. Therefore, only T cells that recognize the donor antigens are deleted, leaving the immune response to other antigens intact. This protocol has been successfully used in rodents and an outbred large animal model. Clinical trials of donor bone marrow administration are now underway and initially indicate that administration of donor bone marrow is clinically safe.

Involvement of CD44 variant isoform v10 in progenitor cell adhesion and maturation

CD44 has been described repeatedly to be involved in hematopoiesis. Here, we addressed the question of functional activity of CD44 variant isoform v10 (CD44v10) in progenitor cell maturation by in vivo and in vitro blocking studies with a monoclonal antibody and a receptor globulin. We became interested in this question by the observation that CD44v10 is expressed, although at a low level, on a subpopulation of bone marrow cells. Flow cytometry revealed that 15%–20% of hematopoietic cells in the fetal liver and 25%–35% of bone marrow cells in adult mice were CD44v10 positive. The majority of CD44v10 + cells was HSA +/J11d + and CD43 +. CD44v10 was not detected on CD4 +, CD8 +, lgM +, or lgD + cells. A CD44v10 receptor globulin did not bind to hematopoietic progenitor cells, but to stromal elements. The CD44v10–CD44v10 ligand interaction had a major impact on the adhesion of progenitor cells to stromal elements. When healthy animals received repeated injections of either anti CD44v10 or the CD44v10 receptor globulin, committed progenitors were mobilized and significantly augmented numbers were recovered in the spleen and the peripheral blood. Furthermore, the CD44v10–CD44v10 ligand interaction, which had no impact on progenitor expansion, influenced progenitor maturation, particularly of the B-cell lineage. Although the nature of the CD44v10 ligand remains to be explored, the supportive role of CD44v10 in progenitor maturation and, importantly, the efficient mobilization of progenitor cells by anti-CD44v10 and a CD44v10 receptor globulin could be of clinical benefit in peripheral blood stem cell transplantation.

Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL.

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5×10 6 cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1–3×10 8 immature and mature DC per mouse at 90–95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10–12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10–12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-α for the last 24 h, where 50–70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.

Involvement of CD44 exon v10 in B-cell activation.

The family of CD44 glycoproteins has been suggested to be involved in lymphocyte homing, maturation and activation. Using in vitro blocking studies with a monoclonal antibody, we here addressed the question of functional activity of CD44 variant exon v10 (CD44v10) in B-cell activation. We became interested in this question by the observation that CD44v10O was transiently expressed on activated T cells, B cells and monocytes as well as on a subpopulation of bone marrow cells. A potential ligand, as revealed by staining with a CD44v10 receptor globulin, was only detected on monocytes. Anti-CD44v10 had no major impact on T-cell activation and no influence on primed B cells, but interfered with the mounting of a primary B-cell response to T-independent and T-dependent antigens. Addition of anti-CD44v10 at different stages during the activation process revealed that CD44v10 was not engaged in B-cell-T-cell interactions. The antibody exerted some effect on monocyte activation as defined by a slight decrease in IL-1 production, but most efficiently inhibited antigen-specific as well as mitogen-induced B-cell activation when present during the coculture of virgin B cells with monocytes. These findings, together with the observation that a CD44v10 ligand was only detected on monocytes but not on lymphocytes, point towards a requirement for CD44v10 in a B-cell-monocyte interaction. Furthermore, since activation of B cells by engagement both of the B-cell receptor and of mitogen receptors was inhibited by anti-CD44v10, the data suggest that a costimulatory function of CD44v10 proceeds independent of the B-cell receptor.