MS-5 Cells Research Articles

CXCR7 Expression Restricts CXCR4/SDF-1 Mediated Hematopoietic-Supporting Activity of Stromal Cells by Decreasing Extracellular SDF-1 Availability.

Abstract 1452Poster Board I-475Stromal cell-derived factor 1 (SDF-1)/CXCR4 axis plays a major role in regulating the interactions between hematopoietic stem and progenitor cells (HPSC) and their stromal microenvironment within the bone marrow. A second SDF-1/CXCL12 receptor, CXCR7 binds SDF-1 with high affinity but little is known about its function in hematopoiesis. In the present study, we demonstrate that the activity of CXCR7 is crucial for proper maintenance of hematopoietic activity on stromal layers. Using quantitative reverse transcription-PCR analysis, we demonstrate that CXCR7 is highly expressed in stromal cells in contrast to hematopoietic cells showing that it functions primarily in the stromal microenvironment compartment. CXCR7 stable expression in UT7 hematopoietic cells fails to support SDF-1 induced migration and signalling but inhibits migration of CXCR4 expressing cells in paracrine manner. Overexpression of CXCR7 in MS-5 stromal cells lead to a reduction of SDF-1 concentration in the supernatants. In addition, supernatants from these cells had substantially lower efficiency in promoting integrin alpha-4 beta-1–mediated adhesion and migration of Mo7e cells to vascular cell adhesion molecule-1 (VCAM-1) and CS-1/fibronectin than their control GFP counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1–dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to VCAM-1 when supernatants from CXCR7 expressing MS-5 cells were used compared with supernatants from GFP expressing cells. Finally, phenotypical primitive murine cells displayed reduced hematopoietic activity when cultured on CXCR7 expressing MS-5 cells. This reduced hematopoietic activity is partly reverted when recombinant SDF-1 was added to CXCR7 overexpressing MS-5 cells. Taken together, our results indicate that CXCR7-controlled disponibility of SDF-1 expression influences BM cell migration, adhesion and hematopoietic activity and thus behaves as a decoy receptor regulating the SDF-1 level. DisclosuresBouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.

Osteoblast-Stem Cell Contact Is Required for B-Lymphopoietic Proliferation, Not Differentiation.

Abstract 3635Poster Board III-571Nonparenchymal stromal cells form the cellular constituents of tissue-specific stem cell niches. One key question in stem cell biology is the nature of signals provided by stromal cells in general, and via stem cell-stromal cell contact in particular, that direct the survival, proliferation and differentiation of tissue stem cells. We and others have previously reported that osteoblasts dictate the differentiation of hematopoietic stem cells (HSC) to B lymphopoiesis, supporting the acquisition of E2A, EBF1 and Pax5, as well as cell surface B220, CD18 and sIgM, with Flt3-ligand (FL) and interleukin 7 (IL-7) required for these activities. In this context, a fundamental unanswered question is whether, and if so why, B lymphopoiesis requires direct cell-cell contact between HSCs and osteoblasts. In the present study, we asked whether HSC contact with osteoblasts plays its principle role in B lymphopoiesis by supporting B differentiation, by supported proliferation, or both. Magnetically purified mouse Lin- Kit+ (LK) bone marrow cells were cultured for 11 days in the presence of 50 ng/ml FL plus 100 ng/ml IL-7, in direct contact with OP-9 or MS-5 osteoblastoid cells, or in proximity to OP-9/MS-5 cells but separated by a Transwell filter. Following co-culture, the hematopoietic cells were counted, the presence of B lineage-specific cell surface markers measured by flow cytometry, and the expression levels of B-lineage transcription factors E2A, EBF and Pax5 measured by quantitative PCR. LK cultured in FL + IL-7 acquired B220 as well as low levels of E2A expression, but did not express Pax5 expression nor acquire cell surface CD19 or IgM, suggesting differentiation to Pre-Pro B cells but not beyond; in contrast, LK cultured in direct contact with OP-9 differentiated fully to mature B cells, expressing Pax5 and cell surface CD19 and IgM. LK cultured above OP9 cells but with cell-cell contact prevented by an interposed Transwell also fully acquired the transcription factor and cell surface antigenic profiles of mature B cells, but the proliferation that accompanies OB-contact driven differentiation was blocked:Cells/mL Day 0Cells/mL Day 11Percent of InputOP-9 Direct Contact2.3 × 1057.8 × 1063400%MS-5 Direct Contact2.3 × 1051.6 × 106670%OP-9 Transwell2.3 × 1051.3 × 1045%MS-5 Transwell2.3 × 1052.0 × 1049%These data indicate that all the signals required to induce HSC differentiation to mature B cells are contained within the combination of cytokines and other molecules secreted by OP-9 cells, but that direct contact with OP-9 cells provides a proliferative stimulus not achieved by OP-9 secreted molecules, either via a direct receptor-ligand interaction or indirectly, by contact-induced secretion of specific soluble proliferative factors from osteoblasts. Experiments to distinguish these alternatives, and to identify whether such signals trigger nutrient influx, direct activation of Akt, MAPK or other key proliferative pathways, are now in progress. The proliferative role of stromal cell-parenchymal cell contact within the stem cell niche likely may have general implications to normal and neoplastic cell physiology. Disclosures:No relevant conflicts of interest to declare.

Neurotrophism of Bone Marrow Stromal Cells to Embryonic Stem Cells: Noncontact Induction and Transplantation to a Mouse Ischemic Stroke Model

Embryonic stem (ES) cell-derived cell products may serve as a source of cells for regenerative medicine. Currently available technologies for the induction of ES cells into neural lineage cells require extended culturing in vitro and complex procedural manipulations, with variable yields of heterogeneous cells, which have hindered the prospective use of cell derivatives for treatment of ischemic stroke. We established a simple and efficient method to derive mouse ES cells into neural lineage cells using an 8-day coculture with the bone marrow stromal cells MS5, followed by a 6-day propagation culture and a 4-day selection culture. The protocol generated a relatively high yield of neural lineage cells without any mesodermal and endodermal lineage commitment. In in vivo study, these derived cells could improve the cognitive function of ischemic stroke mice. Three weeks after transplantation, migration of implanted cells to lesioned areas was noted. It was also evident of a normalization of pyramidal neuron density and morphology in hippocampal CA1 region. One (1/17) episode of teratoma development was noted. Data suggested that MS5 cells may exert a neurotrophic effect to enhance neural differentiation of ES cells and MS5-induced ES cell-derived cells appeared to be applicable to cell therapy for ischemic stroke.

Up Regulation of transendothelial migration on Hematopoietic Progenitor Cells (HPC) and neutrophils by Leukotreine B4 (LTB4) via elevated Endothelial Permeability

Backgrounds : The mechanisms on HPC mobilization by Hematopoietic growth factors seem to be multifactorial processes. Though the roles of protease (ie. NE, MMPs) and cell adhesion molecules (CAMs) seem to be important, the precise mechanisms on HPC mobilization are still unknown. LTB4 is lipid mediator derived from membrane phospholipids during the process of inflammation, and have many roles (ie; inducer of chemotaxis, the production of nitric oxide, transepithelial migration of neutrophil). The major activities of LTB4 include the recruitment and activation of leukocytes, suggesting that it has considerable functional overlap with the chemokine family of chemoattractant peptides, which also direct the recruitment of leukocytes. We previously had reported that LTB4 was able to mobilize HPC of 8.5-fold baseline within only 4 hours without significant side effects in the murine models (Blood 2005, 106;11(2):401b-402b) and LTB4 receptor and reactive oxygen species (ROS) were involved in the pathway of mobilization induced by LTB4 or G-CSF using C57BL/6 mice (Blood 2006, 108;11(2):395b, Blood 2007, 110;11(2):303B). In this study, we investigated the cellular mechanisms of HPC mobilization which was induced by LTB4.Methods : We studied the expression of LTB4 receptors (BLT1 and BLT2) on the surface of murine HPC using RT-PCR. To elucidate the effects of LTB4 on the cells which compose the bone marrow environment for the HPC mobilization, we studied the influence of LTB4 on proliferation, transmigration, permeability, and expression of CAMs on HPC and neutrophils from C57BL/6 mice and MS5 cells, murine stromal cell line cells, and bEnd3 cells as well.Results : Proliferations of b.End3 cell and MS-5 cell were not affected by treatment of LTB4. But, permeability of b.End3 cell monolayer were increased 1.3-fold by LTB4 treatment (100 nM) compared with control. After LTB4 treatment, transendothelial migrations of total nucleated cells (TNC) (22.2% vs 9.4%), HPC (2.7% vs 0%) and neutrophils (75.6% vs 13.6%) from the C57BL/6 mice bone marrow were increased compared with control group respectively. These effects were reversed by LTB4 receptor antagonists (ie; LTB4APA and U75302). Transendothelial migration of neutrophil was increased quickly compared with those of HPCs and total nucleated cells from the bone marrow. However, LTB4 didn't affect on the expression of ICAM-1 and VCAM-1 of b.End3 cell, murine microvascular endothelial cell.Conclusions : Through our data, it is suggested that transendothelial migration of HPCs and neutrophils were upregulated by LTB4 via elevated endothelial permeability. Further studies are necessary to find the molecular mechanisms on the mobilization by LTB4. Prompt mobilization by LTB4, which takes only 4 hours may be very useful in the clinic if safty profiles are completed in human.

GSK-3β Inhibition Activates WNT, Delays Division and Preserves the Function in Hematopoietic Stem Cell

Wingless (Wnt) signaling appears to play an integral role in expansion and quiescence of stem and progenitor hematopoietic cell populations. To better understand this dichotomous role and aid in the translation of therapeutic goals including cellular therapies, greater understanding of the molecular mechanisms that regulate Wnt/β-catenin in the bone marrow niche are needed. We explored the impact of Wnt signalling in the context of the human HSC niche by using co-culture of human cord blood or bone marrow derived HSC with mesenchymal stem cells (MSCs) together with pharmacological inhibition of GSK-3β (iGSK-3β). We have previously shown that activation of the Wnt/β-catenin pathway using pharmacological inhibition of GSK-3β (iGSK-3β) in human cord blood derived HSCs acts to preserve their primitive status, often lost during the ex-vivo expansion process (1). Here we show using cell division tracking that iGSK-3β delays stem cell divisions induced by cytokines in co-culture with MSCs. Functional analysis of iGSK-3β-treated cells revealed an increased proportion of primitive cells producing mixed Colony Forming Units (CFU-GEMMs) formed in the methylcellulose cultures and Cobblestone Area Forming Cells identified in long-term co-cultures with the bone marrow stroma MS5 cells. In addition, iGSK-3β-treated cells exhibited a higher frequency of SCID Repopulating Cells. We also show that the iGSK-3β effect, at least in part, is mediated through VLA4 integrin engagement by fibronectin. Importantly, iGSK-3β also induced niche-independent delay in stem cell divisions seen in cytokine supported suspension cultures. Up-regulation of HoxB4 and p21Waf1 and down-regulation of cyclin D1 was observed in HSCs treated with iGSK-3β. These findings confirm that iGSK-3β– regulated Wnt/β-catenin activity is crucial for the maintenance of HSC quiescence, both in the MSC niche and in a niche-independent environment and that iGSK-3β can circumvent the requirement of exogenous Wnt ligand for the induction of Wnt/β-catenin signalling in MSC niche. Our results are consistent recent data showing that blocking of canonical Wnt signaling, by over-expression of dickkopf1 (Dkk1) increases the number of proliferating HSCs in the murine marrow microenvironment but reduces the ability to reconstitute the hematopoietic system of irradiated recipient transgenic mice (2). These findings contribute to a better understanding of the mechanisms regulating the balance between self renewal and quiescence and help to facilitate progress towards clinical application of Wnts as mediators of ex-vivo stem cell expansion or interruption of the Wnt pathway as potential anti-leukemic therapeutic strategy.

Perivascular/Mesenchymal Stem Cells from Multiple Human Tissues Support Hematopoiesis in Vitro.

Mesenchymal stem cells (MSC) can be derived selectively in culture from multiple organs, an omnipresence we have recently suggested to be explained by the perivascular location of native MSC ancestors within intact tissues (Crisan et al. 2008, in press). We have now analyzed the ability of MSC extracted pro- or retrospectively from different human tissues to support hematopoiesis. MSC were either classically derived in primary cultures of umbilical cord blood (UCB) lineage-depleted mononuclear cells (n=3) or enzymatically dissociated adult adipose tissue (n=3), or grown as CD146+ NG2+ CD34-CD56- CD45- pericytes (n=2) purified by flow cytometry from fetal skeletal muscle and cultured over the long term. In both settings, identical MSC were obtained that maintained a stable CD146+ CD90+ CD73+ CD105+ CD34- CD45- surface phenotype and could differentiate into skeletal muscle, fat, bone and cartilage. CD34+ hematopoietic progenitors (n=3) immunoselected from term UCB were seeded (5×10e3cells/cm2 in triplicate) onto confluent irradiated layers of MSC derived from UCB, adipose tissue or fetal muscle pericytes (MSCu, MSCa and MSCmp, respectively) or, as a control, MS5 bone marrow stromal cells that allow the proliferation of very primitive human progenitor cells. All studies were approved by the relevant institutional regulatory board. The cells were cocultured for 5 weeks in a classical long-term culture-initiating cell assay in a complete medium (MyeloCult H5100, Stem Cell Technologies) containing hydrocortisone but no added cytokine. Wells were scored daily for the presence of cobblestone areas (CA) and half of the medium was replaced every week. Eventually, trypsinized cells from each well were characterized by flow cytometry for the expression of hematopoietic cell markers and assayed for CFC potential. After 14 days of incubation, colonies grown in semi-solid medium were scored as derived from colony forming units (CFU)-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) and as high-proliferative-potential colony precursors (HPPC), the most primitive hematopoietic cell so far identified in a clonogenic assay in vitro. Within the CD45+ gate, all trypsinized cultures contained comparable percentages of CD34+lin- cells (MSCu: 51±9%; MSCa: 58±14%; MSCmp: 61±19%; MS5: 59±18%), the most immature hematopoietic cell compartment maintained during the long-term coculture. MSCu and MSCmp supported a similar cell proliferation during the whole culture while on MSCa, CA formed very rapidly and consistently but eventually decreased over the long-term culture. Interestingly, MSCu and MSCmp supported the development of the highest numbers of HPPC and of CFU giving rise to the largest GEMM colonies, as compared to MSCa that gave the same results as the control MS5 cell line. In summary, all MSCs tested were able to support hematopoiesis and CA formation, albeit with differences in growth kinetics and morphology of the colonies. Herein we show for the first time that purified human perivascular cells exhibit robust hematopoiesis support in vitro, in addition to multilineage mesodermal developmental potential. In conclusion, we demonstrate that MSC from novel sources distinct from the bone marrow are able to support hematopoiesis. These results further sustain the identity, beyond acronyms, between marrow stromal cells, long known for their support of hematopoiesis, and mesenchymal stem cells that gained more recent credit in the field of regenerative medicine because of their multilineage differentiation potential.

The Hypoxic Microenvironment in Acute Myelogenous Leukemia: Critical Role of CXCR4 in the Induction of HIF-1α.

The bone marrow microenvironment provides functional and structural support for both normal and leukemic hematopoietic stem cells. Importantly, the marrow microenvironment is known to be hypoxic. Given the many critical functions of HIF-1α, we investigated HIF-1α levels in leukemic cell lines and primary AML blasts. Surprisingly, HIF-1α expression was non-detectable in AML cells grown in suspension cultures under normoxic (21% O2) or hypoxic (1% O2) conditions. Normoxic co-cultures with bone marrow-derived mesenchymal stromal (MSC) or MS5 cells likewise did not induce HIF-1α, but hypoxic co-culture conditions induced HIF-1α protein without changes in HIF-1α mRNA levels, suggesting post-transcriptional regulation. Functionality of HIF-1α was confirmed by concomitant increase in the levels of glucose transporter glut-1, the HIF-1α downstream target. Inhibition of stroma-leukemia cell interactions with the small molecule CXCR4 inhibitor AMD3465 (Genzyme/Anormed) at 100 nM completely abrogated the induction of HIF-1α in HL-60 and MOLM13 AML cells. While SDF-1 was unable to induced HIF-1α under normoxic conditions, it did so under both physical (1% O2) and chemical (COCl2) hypoxic conditions, in two different cell lines. Inhibition of P13K (with LY294002) or MEK/ERK signaling (with CI-1040) abrogated HIF-1α induction under hypoxic conditions. Immunohistochemical staining of bone marrow samples from primary AML confirmed the presence of HIF-1α in leukemic cells localized adjacent to bone-lining stromal elements. Results suggest that1.the bone marrow microenvironment of AML is hypoxic in vivo;2.in leukemia cells HIF-1α induction under low oxygen tension depends on the presence of stromal cells;3.HIF-1α induction is dependent on SDF-1/CXCR4 and is mediated by activation of P13K and MAPK signaling.Altogether these findings suggest that SDF-1α/CXCR4 interactions contribute to the survival of leukemic cells via specific induction of HIF-1α signaling by the bone marrow microenvironment. Disruption of these interactions via CXCR4 inhibition strategies may suppress multiple pro-survival HIF-1α targets in leukemic cells.

Interleukin-7 contributes to human pro–B-cell development in a mouse stromal cell–dependent culture system

The role of interleukin (IL)-7 in human B lymphopoiesis is still controversial. We used an in vitro culture system to verify involvement of IL-7 in development of human pro-B cells from hematopoietic stem cells. Human CD34(+) bone marrow cells were cultured for 4 weeks on MS-5 mouse stromal cells to induce pro-B cells. Expression of IL-7 receptor alpha or other B-cell differentiation marker genes on cultured human CD34(+)bone marrow cells was investigated by reverse transcription polymerase chain reaction (RT-PCR). Colony assay of human CD34(+) bone marrow cells was also performed to determine the effect of IL-7 on colony-forming ability. Neutralizing antibody or reagent that eliminates the effect of IL-7 was added to the culture system, and the number of pro-B cells induced was estimated by flow cytometry. RT-PCR analysis revealed mRNA expression of IL-7 receptor alpha as well as B-cell differentiation marker genes in not only CD19(+) pro-B cells but also CD19(-) CD33(-) cells induced from CD34(+) bone marrow cells after cultivation for 4 weeks on MS-5 cells. Addition of anti-mouse IL-7 antibody, anti-human IL-7 receptor alpha antibody, or JAK3 kinase inhibitor reduced the number of pro-B cells induced, demonstrating that elimination of IL-7 reduces pro-B-cell development. Addition of anti-mouse IL-7 antibody emphasized the colony-forming ability of burst-forming unit erythroid cells. IL-7 produced by MS-5 cells is required for human pro-B-cell development from CD34(+)bone marrow cells in our culture system, and IL-7 appears to play a certain role in early human B lymphopoiesis.

Tumor necrosis factor promotes T-cell at the expense of B-cell lymphoid development from cultured human CD34 + cord blood cells

Human CD34+ cord blood (CB) cells are hematopoietic progenitors useful for stem cell transplantation, even after ex vivo expansion. We investigated the effect of tumor necrosis factor (TNF) on lymphoid development from cultured CD34+ CB cells. Human CD34+ CB cells were cultured in cytokine mixes with or without TNF. Preculture during 60 hours was followed by in vitro differentiation assays, including fetal thymus organ culture and coculture on murine stromal MS-5 cells. In a next step, experiments were extended to CD34+CD38- and CD34+CD38+ CB cells and prolonged preculture. Preculture in the presence of TNF improved differentiation into T cells and diminished the ability to generate B cells, while NK potential and myeloid development were unaffected. Sorted CD34+CD38- CB cells were more potent T-cell precursors after preculture in TNF, compared to CD34+CD38+ CB cells. In precultured CD34+CD38- CB cells, TNF increased GATA3 but decreased EBF1 expression, in line with the skewed lymphoid differentiation induced by TNF. However, when preculture in the presence of TNF was extended to 1 week, T-cell precursors were lost. After short-term culture of CD34+ CB cells in the presence of TNF, T-cell generation is stimulated at the expense of B-cell generation. T-cell progenitors are enriched in the CD34+CD38- fraction. These results have implications on the culture conditions to be used for CB CD34+ cells prior to transplantation.

Expansion of normal and leukemic human hematopoietic stem/progenitor cells requires Rac-mediated interaction with stromal cells

To determine the involvement of Rac signaling in self-renewal and expansion on bone marrow stroma of normal CD34+ cells vs leukemic CD34+ cells from acute myeloid leukemia (AML) patients. Rac signaling was modulated by retroviral introduction of Racl-N17, Racl-V12, or by using the Rac inhibitor NSC23766. In long-term MS5 cocultures (leukemic) expansion, migration, adhesion, and presence of stem/progenitor cells were monitored in both normal as well as leukemic CD34+ cells. Inhibition of Rac signaling impaired migration and adhesion of cord blood (CB) CD34+ cells on MS5 stroma. Long-term inhibition of Rac during a 5-week coculture period on stroma prevented association of hematopoietic progenitors with the bone marrow stromal cells and resulted in a dramatic decrease in the primitive stem cell frequency (long-term culture-initiating cell) in a dose-dependent manner. Many of these phenotypes were reversed in the presence of activated Racl-V12, including improved migration toward, and association with, MS5 cells. CD34+ AML cells were characterized by elevated levels of Rac activity (five of seven patients) and enhanced migration and adhesion to MS5 bone marrow stroma as compared to CB CD34+ cells. A dramatic decrease was observed in the formation of leukemic cobblestone area-forming cells as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. Our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.

Effect of Fibroblast Growth Factor 2 on Stromal Cell-Derived Factor 1 Production by Bone Marrow Stromal Cells and Hematopoiesis

Reduction of intramedullary hematopoiesis and the development of myelofibrosis and splenic hematopoiesis are frequent complications of clonal myeloid disorders that cause severe morbidity and death and present a therapeutic challenge. However, the pathogenesis of these complications is still unknown. We evaluated the effect of fibroblast growth factor 2 (FGF-2), the level of which is elevated in patients with clonal myeloid disorders, on bone marrow stromal cell expression of stromal cell-derived factor 1 (SDF-1), a chemokine that is essential for normal hematopoiesis. Reverse transcription-polymerase chain reaction analysis, immunoblot analysis, and enzyme-linked immunosorbent assays were used to examine effects of human recombinant FGF-2 exposure on SDF-1 expression in mouse stromal MS-5 and S-17 cells. Cocultures of human CD34-positive peripheral blood stem cells or mouse pre-B DW34 cells with mouse stromal cells were used to characterize the functional relevance of the effects of FGF-2 on SDF-1 expression. The in vivo hematologic effects of FGF-2 were determined by systemic administration to mice (n = 10). All statistical tests were two-sided. FGF-2 reduced constitutive SDF-1 mRNA expression and secretion in stromal cells (SDF-1 levels in supernatants: MS-5 cells cultured for 3 days in medium only versus in medium with FGF-2, 95.4 ng/mL versus 22.2 ng/mL, difference = 73.2 ng/mL, 95% confidence interval [CI] = 60.52 to 85.87 ng/mL; P = .002, two-sided Student's t test; S-17 cultured in medium only versus in medium with FGF-2, 203.53 ng/mL versus 32.36 ng/mL, difference = 171.17 ng/mL, 95% CI = 161.8 to 180.6 ng/mL; P<.001). These effects of FGF-2 were reversible. FGF-2 compromised stromal cell support of the growth and survival of pre-B DW34 and myeloid lineage cells, and these effects were reversed in part by exogenous recombinant SDF-1alpha (rSDF-1alpha) (DW34 pre-B cells recovery on S-17 stromal cells, expressed as a percentage of DW34 cells recovered from medium only: with FGF-2 versus without FGF-2, 27.6% versus 100%, difference = 72.4%, 95% CI = 45.34% to 99.51%, P = .008; with FGF-2 plus rSDF1 versus with FGF-2 only, 60.3% versus 27.6%, difference = 32.7%, 95% CI = 9.35% to 56.08%, P = .034; fold increase in number of myeloid lineage cells after culture on S-17 stromal cells: with FGF-2 versus without FGF-2, 0.25-fold versus 3.8-fold, difference = 3.55-fold, 95% CI = 2.66- to 4.44-fold, P<.001; recovery of myeloid cells on S-17 stromal cells, expressed as a percentage of myeloid cells recovered from medium only: FGF-2 plus rSDF-1alpha versus FGF-2 only, 76.5% versus 32.4%, difference = 44.1%, 95% CI = 32.58% to 55.68%, P<.001). Administration of FGF-2 to mice reversibly reduced bone marrow levels of SDF-1 and cellularity and induced immature myeloid cell mobilization, extramedullary hematopoiesis, and splenomegaly. Systemic administration of FGF-2 in mice disrupts normal bone marrow hematopoiesis in part through reduced expression of SDF-1. Thus, endogenous FGF-2 may represent a potential therapeutic target in clonal myeloid disorders characterized by bone marrow failure.

Glycogen Synthase Kinase-3β Regulates Ex-Vivo Expansion and Engraftment of Umbilical Cord Blood Hematopoietic Stem Cells through the Activation of Wnt Signaling.

Rapid availability of an HLA matched marrow donor for allogeneic hematopoietic stem cell transplantation remains a challenge in the clinical setting and has resulted in increased use of alternate stem cell sources, particularly unrelated umbilical cord blood (UCB). Although UCB transplantation has been used to treat a multiplicity of malignant and non-malignant hematological diseases, success has been limited particularly in adult recipients, because of reduced numbers of stem/progenitor cells in a cord unit resulting in higher graft failure rates, delayed hematopoietic reconstitution and increased transplant related mortality when compared to BM or PBSCs. This has prompted investigation of alternate strategies including multi-unit infusions and ex-vivo expansion systems. Clinical trials incorporating cytokine-based HSC ex-vivo expansion have failed to demonstrate significant shortening of hematologic recovery despite substantial increases in cell dose, suggesting loss of HSC function. Activation of Wnt signaling has previously been shown to maintain HSC function; as such we hypothesized that use of a specific inhibitor of glycogen synthase kinase (GSK-3β) to activate Wnt signaling in combination with cytokine (Flt3L, SCF, TPO) based ex-vivo HSC expansion would result in expansion, maintenance of HSC function and ultimately improve hematological recovery. UCB CD34+ cells were treated with a potent GSK-3β inhibitor; 6-bromoindirubin-3′-oxime (BIO), a synthetic cell-permeable derivative of a natural product, indirubin-3′-oxime, isolated from the molusk Tyrian purple. UCB CD 34+ cells treated with BIO exhibited increased expression of β-catenin that re-located from the cytoplasm to the nuclei to activate transcription of Wnt targets including cyclin D1, c-myc, and HoxB4. When compared to control cells (cytokine only); BIO treated cells demonstrated an increase in the absolute number of nucleated, CD34+ and clonogenic cell numbers in 5–7 day expansion cultures with the best expansion observed at 0.1 μM of BIO. Increased cell cycling, reduced apoptosis and up-regulation of the cell cycle inhibitor p21Waf1 previously shown to maintain HSC function in-vivo was demonstrated in BIO treated cells. Furthermore, BIO significantly improved the hematopoietic supporting ability of murine bone marrow stroma MS5 cells. Increased expansion of myeloid, megakaryocytic and B-cell progenitor cells were all observed. Using a NOD/SCID mouse model we have demonstrated both preservation of function and expansion with increased numbers of engrafting BIO treated human CD34+ UCB (120-fold expansion) compared to control cells (45-fold expansion). The increased expansion of human NOD/SCID repopulating cells suggests improved maintenance of stem cell function during ex-vivo expansion of UCB in the presence of BIO. Activation of the Wnt pathway, through GSK-3β inhibition, is a promising strategy to facilitate expansion and maintain function of UCB HSCs and may overcome current limitations of UCB and allow for greater application of this stem cell source.

The GEF Lsc Regulates Hematopoietic Stem Cell Motility, Mobilization and Recruitment.

Lsc is a Rho GTPase guanine nucleotide exchange factor (RhoGEF) that physically and functionally links G-protein coupled receptors (GPCR) to the monomeric GTPase RhoA in mature hematopoietic and other cells. Lsc−/− (LscKO) mice have a peripheral leukocytosis, abnormal neutrophil and B cell motility, and immune response deficiencies. Although Lsc is required for neutrophil homeostasis, its role in hematopoietic stem and progenitor cells is unknown. In this study, we have used LscKO mice to determine if Lsc is required for normal stem cell motility and mobilization. Initially, we used immunofluorescence labeling to demonstrate that hematopoietic stem and progenitor cells express Lsc. This suggested that Lsc may be required for normal hematopoietic stem and progenitor cell migration. Stromal-cell derived factor-1 (SDF-1) is a potent chemokine for hematopoietic stem cells and activates the CXCR4 GPCR. It has been reported that Lsc is not required for SDF-1-stimulated migration of mature murine T and B cells. However, using a bare-filter transwell assay, we found that while LscKO Sca-1+ cells and Sca-1+Lin- cells have normal spontaneous migration, they have significantly increased SDF-1-stimulated migration compared to their wild-type (WT) counterparts, 1.4 and 2.3 fold, respectively. We then demonstrated that adhesion of LscKO Sca-1+ cells to bone marrow (BM) stromal MS-5 cells was normal, indicating that impaired adhesion was not responsible for the abnormal SDF-1-stimulated migration. Using colony assay, we demonstrated that LscKO mice have a normal number of circulating peripheral stem and progenitor cells. Strikingly, after 5 days of G-CSF administration, LscKO mice have 1.6 fold and 2.3 fold the number of peripheral mature WBC and stem and progenitor cells (colony forming units), respectively, compared to WT mice. Recruitment of BM CXCR4+ pro-angiogenic stem and progenitor cells has been linked to enhanced tumor angiogenesis. Because LscKO BM cells had abnormal SDF-1-stimulated migration and mobilization, we hypothesized that Lsc might regulate tumor angiogenesis as well. To this end, we assessed tumor growth in LscKO mice by injecting congenic Lewis lung carcinoma cells subcutaneously into LscKO mice and WT controls. Preliminary experiments revealed that tumors were 3.3 times larger in the LscKO mice as compared to WT mice. Quantification of the tumor vessels with anti-CD31 staining demonstrated that the tumors in LscKO mice were 1.4 fold more vascularized than controls. In summary, our results demonstrate that the Rho GEF Lsc is essential for normal hematopoietic stem cell migration and mobilization. In addition, we propose that absence of Lsc facilitates tumor growth by promoting BM stem and progenitor cell recruitment to the neo-angiogenic vessels, possibly augmenting tumor vascularization.

Involvement of insulin-like growth factor-I and insulin-like growth factor binding proteins in pro–B-cell development

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of proteins thought to modulate IGF function. By employing an in vitro culture system of human hematopoietic stem cells cocultured with murine bone marrow stromal cells, we examined the effects of IGF-I and IGFBPs on early B-cell development. Human CD34(+) bone marrow cells were cocultured with murine stromal MS-5 cells for 4 weeks, and pro-B-cell number was analyzed by flow cytometry. After administration of reagents that are supposed to modulate IGF-I or IGFBP function to the culture, the effect on pro-B-cell development was examined. After cultivation for 4 weeks, effective induction of pro-B-cell proliferation was observed. Experiments using several distinct factors, all of which neutralize IGF-I function, revealed that impairment of IGF-I function results in a significant reduction in pro-B-cell development from CD34(+) cells. In addition, when the effect of recombinant proteins of IGFBPs and antibodies against IGFBPs were tested, IGFBP-3 was found to inhibit pro-B-cell development, while IGFBP-6 was required for pro-B-cell development. IGF-I is essential for development of bone marrow CD34(+) cells into pro-B cells. Moreover, IGFBPs are likely involved in regulation of pro-B-cell development.

Murine serum obtained from bone marrow-transplanted mice promotes the proliferation of hematopoietic stem cells by co-culture with MS-5 murine stromal cells

To examine whether serum obtained from bone marrow-transplanted mice can selectively expand hematopoietic stem cells (HSCs) among whole bone marrow cells in vitro, whole bone marrow cells were cultured with or without MS-5 murine stromal cells in the presence of serum obtained from transplanted mice on day 3 (day 3 serum) or serum from normal mice for 7 days. When whole bone marrow cells and MS-5 cells were co-cultured in day 3 serum for 7 days, the c-kit-positive, Sca-1-positive, lineage marker-negative cells (KSL cells) expanded approximately 25 times; however, when they were co-cultured in normal serum for 7 days, the KSL cells expanded approximately 1.3 times. Direct contact between the whole bone marrow cells and MS-5 cells was essential for expansion of KSL cells in the co-culture, and it upregulated the expression of some cytokines in MS-5. Above all, the day 3 serum specifically upregulated the expression of SCF, SDF-1 alpha, G-CSF, IL-11 and IL-6 in MS-5. The level of testosterone in the day 3 serum was higher than normal serum and the addition of the testosterone in the culture expanded the KSL cells among whole bone marrow cells on MS-5 cells and also upregulated the expression of SDF-1 alpha, IL-11 and IL-6 in MS-5. These data indicates that the serum of bone marrow-transplanted mice contains a factor(s) that induced changes in the expression levels of various cytokines in MS-5 stromal cells and enabled the MS-5 cells to expand the KSL cells among whole bone marrow cells.

Properties of MSC Derived Stromal Cell Lines Developed from Bone Marrow of TNF Knockout Mice.

Mesenchymal stem cells (MSC) forming adherent stromal cell lines have been revealed in suspension fraction (SF) of 70+ weeks old long-term bone marrow culture (LTBMC) of TNF knockout mice.The proportion of CD45+ cells in such old cultures decreased significantly while cellularity of SF increased, accompanied by the first successful generation of cell lines. More than 100 cell lines were established.Almost all cell lines were Sca1 positive, expressed SDF1, had fibroblast-like morphology and expressed collagens type II and IV. Most of cell lines could maintain hematopoiesis (CFU-S, CFU-GM and CAFC production) for 10 weeks. Some of the cell lines were able to maintain growth without differentiation of murine ES cells and some expressed LIF or LIF-receptor, VEGF and FGF1. All cell lines produced hematopoietic growth factors - their supernatants stimulated growth of CFU-GM in semisolid media. The expression of SCF was revealed in all cell lines tested for growth factors production.Quantitative RT-PCR analysis revealed that the expression of hematopoietic growth factors - M-CSF, G-CSF and TPO increased sometimes up to 6 times in comparison with the expression of these factors in adherent cell layer (ACL) of LTBMC. The expression of N-cadherin - one of the main regulators of hematopoietic stem cells (HSC) in niche - increased 7–32 fold in 7 tested cell lines regarding the expression of this RNA in ACL of LTBMC. Whereas the expression level of N-cadherin was lower in wild type bone marrow, NIH3T3 fibroblasts and MS5 cells, maintaining the hematopoiesis and CAFC growth, showed the expression on the higher level than in ACL. The expression of ICAM 1, the molecule also regulating HSC, was reduced in all cell lines. The expression level of genes participating in activating of hematopoietic stem cells in niche such as Jagged, Dll 1 and Ang1 more than halved in 5 out of 7 tested lines. The production of Wnt 5a RNA fluctuated from 0,5 to 12 fold and did not correlate with any other changes in gene expression and the ability of cell lines to maintain hematopoiesis. The level of Ihh, Shh was downregulated in cell lines.The cell line 26dD3 developed in the media containing dexamethasone demonstrated 70 fold increased expression of Kirre - gene supporting HSC but there was no difference in maintaining hematopoiesis and ES cells. High expression of human homolog of this gene (NEPH2) was known to be detected in brain only. At the same time this cell line expressed tubulin b3. These data support a model of lineage specification in which unilineage commitment is prefaced by a “promiscuous” phase of multilineage locus activation. Thus the developed stromal cell lines are not terminally differentiated and use different pathways to maintain hematopoiesis. The TNF deficiency leads to dramatic alterations in regulation of MSC. Mechanisms of this regulation should be scrutinized.

Expansion of Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells Requires Rac-Mediated Interaction with Stromal Cells.

We have studied Rac signal transduction in human cord-blood (CB) and acute myeloid leukemia (AML) CD34+ cells and determined that Rac proteins are critically involved in the interaction between human stem/progenitor cells and stroma. Constitutive activation of Rac signaling was achieved by retroviral introduction of Rac1-V12 into CB-derived CD34+ cells, while inhibition of Rac signaling was established by retroviral introduction of dominant negative Rac1-N17 or by utilizing the Rac inhibitor NSC23766 . Inhibition of Rac signaling resulted in a proliferative disadvantage of CB-CD34+ cells when cultured on MS5 stromal cells. Cells were severely disturbed in their migration towards and direct association with MS5 stroma when Rac signaling was inhibited. The Long Term Culture-Initiating Cells (LTC-ICs) migrated underneath the stromal MS5 layer within 24 hrs after plating, and similar results were obtained for about 50% of the Colony Forming Cells (CFCs). However, transient inhibition of Rac signaling for 24–72 hrs resulted in a shift of LTC-ICs and CFCs to the suspension phase as determined by colony assays and CAFC week 5 enumeration. When Rac signaling signal transduction was inhibited during the 5 week coculture period a dramatic decrease in LTC-IC frequency from 0.6% to 0.15% was observed. Many of these phenotypes were reversed in the presence of activated Rac1-V12, including improved migration towards and association with MS5 cells and elevated LTC-IC frequencies. Importantly, in CAFC assays using AML cells (n=8) that were enriched for leukemic stem cells on the basis of a CD34+/CD38low phenotype we observed a dramatic decrease in leukemic CAFC formation as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766 . Taken together, our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.

Dexmethasone Down-Regulates Stromal Cell-Derived Factor-1 Production in Bone Marrow Stromal Cells: Implications in Myeloma Cell Biology.

Stromal cell-derived factor-1 (SDF-1) plays a role in the homing of myeloma cells to bone marrow. In addition, SDF-1 modestly enhances the proliferation of myeloma cells and inhibits Dexmethasone (Dex)-induced apoptosis of the cells. Dex is currently used to treat multiple myeloma, based on its apoptic effects. In this study, we investigated the regulatory effects of Dex on SDF-1 production in bone marrow stromal cells (BMSCs) and on CXCR4 expression in myeloma cells. As previously reported, it was evident that primary myeloma cells (CD138+ cells obtained from patients with multiple myeloma) and Dex-resistant myeloma cell line RPMI8226 expressed CXCR4 and responded to SDF-1, resulting in chemotaxis. SDF-1 modestly stimulated the proliferation of primary myeloma cells and RPMI8226 cells and protected the cells from Dex-induced apoptosis. Human umbilical vein endothelial cells transduced with the SDF-1 gene using adenoviral vectors better supported the formation of cobblestone areas of primary myeloma cells and RPMI8226 cells in co-culture, similar to hematopoietic progenitor cells; this was blocked by pretreating the myeloma cells with pertussis toxin, indicating that SDF-1 plays a critical role not only in migration of the cells underneath the SDF-1-producing stromal cells but also in proliferation of the cells in contact. Dex up-regulated CXCR4 expression in RPMI8226 cells; however, its regulatory effects on CXCR4 in primary myeloma cells differed among patients. RT-PCR and Northern blot analyses revealed that Dex down-regulated SDF-1 mRNA expression in both primary BMSCs and murine stromal MS-5 cells in a dose-dependent manner. Western blot analysis and ELISA assay confirmed that Dex inhibited SDF-1 production in BMSCs. Furthermore, Dex inhibited cobblestone area formation of RPMI8226 cells in co-culture with MS-5. Interestingly, Dex up-regulated CXCR4 mRNA expression and cytoplasmic CXCR4 in BMSCs. These results indicate that Dexamethasone induces the down-regulation of SDF-1 production in BMSCs, which might mediate, at least in part, its anti-myeloma effects in vivo.

Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.

Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.

Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic Stem Cells and the Granulopoietic Differentiation of More Mature Progenitors

The study of the human hematopoietic system would be facilitated by availability of a relevant animal model. Because the medullar microenvironment is made of different types of cells, interactions between hematopoietic cells and stromal cells are difficult to analyze in detail. As an approach for establishing an in vivo model to dissect these interactions, we grafted murine bone marrow fibroblastic cells (MS-5 cell line) with hematopoietic cells into the kidney capsule of syngenic mice. To identify the origin of cells present in the graft, we used green fluorescent protein-stable transfected MS-5 cells for the transplantation. To analyze the evolution of stromal cells and identify hematopoietic cells able to develop in these conditions, we performed morphology, histochemistry, and immunohistology on tissue sections at different times after transplantation. When injected alone, MS-5 cells differentiate into adipocytes. When injected with a bone marrow suspension or with isolated CD45+ cells (leukocytes), the stromal cells keep their fibroblastic morphology and their alkaline phosphatase expression and sustain granulopoiesis. When injected with hematopoietic stem cells called c-kit+ Sca-1+ Lin- suspension, clusters of hematopoietic cells are also observed: They do not present any granulopoietic activity and do not belong to B or T population nor to erythroid lineage. They are quiescent, induce bone marrow recovery and survival of lethally irradiated recipients, are able to form macroscopic colonies in the spleen, and are able to form very few colonies in vitro, suggesting that they are hematopoietic stem cells. In conclusion, our results show that reticular fibroblastic stromal cells MS-5 sustain the survival of stem cells and are not able to induce their differentiation. However, they can control differentiation, proliferation, and/or survival of hematopoietic cells engaged in myeloid lineage.