Murine Bone Marrow Stem Cells Research Articles

Short-Term Storage of Mobilized Peripheral Blood Stem Cells in a Closed System Changes the Microenvironment and May Affect the Quantity of CD34+ and CD34+CD38-CD45RA-CD90+ Cells

Hypoxic conditions preserve the multipotency and self-renewing capacity of murine bone marrow and human cord blood stem cells. Blood samples stored in sealed blood gas tubes become hypoxic as leukocytes metabolize and consume oxygen. Taken together, these observations suggest that peripheral blood stem cell (PBSC) samples stored under airtight conditions become hypoxic, and that the stem cells contained may undergo qualitative or quantitative changes. This study aimed to determine the effect of storage for 8 hours in a sealed system on PBSC samples. Granulocyte colony-stimulating factor-mobilized PBSC samples were collected prospectively from 9 patients with myeloma or amyloidosis prior to apheresis, followed by measurement of CO2, O2, hydrogen ion (pH), lactate, and glucose concentrations in the blood and immunophenotyping of stem cell and multipotent progenitor cell populations before and after 8 hours of storage in sealed blood collection tubes. Blood concentrations of O2 and glucose and pH measurements were significantly decreased, whereas concentrations of CO2 and lactate were significantly increased after storage. Significantly higher concentrations of CD34+ cells (552 ± 84 cells/106total nucleated cells [TNCs] versus 985 ± 143 cells/106TNCs; P=.03), CD34+CD38- cells (98 ± 32 cells/106TNCs versus 158 ± 52 cells/106TNCs; P=.03), CD34+CD38+ cells (444 ± 92 cells/106TNCs versus 789 ± 153 cells/106TNCs; P=.03), and CD34+CD38-CD45RA-CD90+ cells (55 ± 17 cells/106TNCs versus 89 ± 25 cells/106TNCs; P=.02) were detected after 8 hours of storage. The changes in concentrations of CD34+CD38+ cells and CD34+ cells were inversely associated with the change in glucose concentration (P=.003 and P < .001, respectively) and positively associated with the change in lactate concentration (P=.01 and P <.001, respectively) after 8 hours of airtight storage. Storage of PBSC samples in a sealed, airtight environment is associated with microenvironmental changes consistent with hypoxia and increased concentrations of immunophenotypically defined stem cells. These results may have clinical implications with regard to the collection and processing of stem cell products and warrant confirmation with functional and mechanistic studies.

Modulation of Host Defense in Chronic Granulomatous Disease (CGD) Via Adoptive Transfer of Neutrophil Progenitor Cells

Ectopic expression of transcription factor HoxB8 in murine bone marrow stem cells programs cells to stably expand exponentially in culture. When HoxB8 expression is regulated through a conditional promoter, cells are capable of expansion through HoxB8 induction or differentiation into neutrophil-like cells through HoxB8 withdrawal. We have previously demonstrated that some derivations of HoxB8 progenitor cells are capable of engraftment in an unconditioned murine host leading to rapid formation of a transient population of donor neutrophils in the periphery. Donor derived neutrophils (ddN) function comparably to host wild-type (WT) neutrophils ex vivo. Based on these findings, we hypothesize that conditionally immortalized HoxB8 progenitor cells have the potential to serve as a novel neutrophil driven cellular therapy. To investigate the use of HoxB8 progenitor cells as a cellular therapy for disease, we utilized a well characterized murine model of the congenital disorder CGD. Individuals with CGD are particularly susceptible to infections with Staphylococcus aureus due to loss of reactive oxygen species (ROS) production via the NADPH oxidase. Impaired neutrophil function leads to increased inflammation and decreased microbial killing. We found that prophylactic treatment of CGD mice with an adoptive transfer (AT) of WT HoxB8 progenitor cells protected them from an otherwise lethal pulmonary infection (Figure 1). Mice which received an AT of progenitor cells derived from a CGD animal did not have improved outcomes. This suggests the effector functions of the WT ddN were responsible for the change in survival. To better understand the mechanism of protection derived from the AT, we developed a novel fluorescent killing assay. This assay demonstrated the positive influence of the WT ddN on the host CGD neutrophils intracellular killing capacity in vivo. In addition to prophylactic treatment, post infectious treatment with the progenitor cells improved survival to >50% (Figure 2). Interestingly, twelve hours post adoptive transfer, donor derived cells represent a small population of the BAL cellular environment (<1%). Evaluation of the BAL and serum cytokine environment at this time-point provide insight to the immuno-modulatory role of the donor derived cells. Results from this study demonstrate the promise of a neutrophil progenitor based cellular therapy. This approach is unique from those currently employed as progenitor cells engraft without conditioning, expand and differentiate in vivo unlike granulocyte transfusions, and may be genetically modified in culture to potentially address the challenges of microbial resistance genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhanced osteogenic differentiation of mesenchymal stem cells by surface lithium modification in a sandblasted/acid-etched titanium implant.

This study investigated the osteogenesis-related cell functions of osteoprogenitor cells modulated by surface chemistry modification using lithium (Li) ions in a current clinical oral implant surface in order to gain insights into the future development of titanium (Ti) implants with enhanced osteogenic capacity. Wet chemical treatment was performed to modify a sandblasted/acid-etched (SLA) Ti implant surface using Li ions. The osteogenesis-related cell response to the surface Li ion-modified SLA sample was evaluated using two kinds of murine bone marrow stem cells, bipotent ST2 cells and primary multipotent mesenchymal stem cells (MSCs). The modified surface exhibited the formation of an Li-containing Ti oxide layer with plate-like nanostructures. The Li-incorporated surface enhanced early cellular events, including spreading, focal adhesion formation and integrin mRNA expression (α2, α5, αv and β3), and accelerated osteogenic differentiation of bipotent ST2 cells compared with unmodified SLA surface. Surface Li modification significantly increased GSK-3β phosphorylation and suppressed β-catenin phosphorylation, and promoted the subsequent osteogenic differentiation of primary MSCs. These results indicate that surface chemistry modification of SLA implants by wet chemical treatment with Li ions induces a more favorable osseointegration outcome through the promotion of the osteogenic differentiation of bone marrow MSCs via the positive regulation of GSK-3β and β-catenin activity.

HoxB8‐conditional Neutrophil Progenitor Transplant Provides a Therapeutic Benefit for S. aureusPulmonary Infection in a Murine Model of Chronic Granulomatous Disease (CGD)

CGD is a congenital disease marked by neutrophil dysfunction leading to increased infectious susceptibility. Despite prophylactic treatment with antimicrobial agents, outcomes for CGD patients are troubling with a typical lifespan of 30‐40 years due to breakthrough infections. As we strive to stay ahead of the impacts of drug‐resistant microbes, it is reasonable to consider cellular therapeutics which replace immune cells to treat CGD and other immunodeficiencies. We hypothesize that a conditionally‐immortalized neutrophil progenitor cell line has a translational application as a therapy for immune compromised hosts. This cell line was first generated by Wang, et al. through inducing ectopic expression of the transcription factor HoxB8 in murine bone marrow stem cells. Induction of HoxB8 leads to exponential growth of cells, while withdrawal leads to differentiation into neutrophil‐like cells. We have found that unconditioned transplant of HoxB8 progenitors lead to robust engraftment in the bone marrow. Engrafted progenitor cells differentiate in vivointo donor‐derived neutrophils found circulating in the peripheral blood. Donor neutrophils are also recruited to the lungs of mice infected with Staphylococcus aureus. Using a novel fluorescent killing assay, we demonstrate that donor‐derived neutrophils can kill pulmonary S. aureus both in vitro and in vivo with comparable efficiency to wild type host neutrophils. Strikingly, our results find that treatment with HoxB8‐conditional progenitors improves survival of CGD animals infected with S. aureus from 0% to &gt;50%. This work demonstrates the potential of conditionally‐immortalized neutrophil progenitors as a novel cellular therapy in the context of neutrophil dysfunction.

OSTEOGENIC POTENTIAL OF NANOSTRUCTURED CARBONATED HYDROXIAPATITE MICROSPHERES ASSOCIATED WITH MESENCHYMAL STEM CELL IN VITRO AND IN VIVO

<h3>Background</h3> Bone tissue is capable of self-healing in the event of injuries. However, situations such as the patient‘s age or size of the damage can compromise natural healing, bringing to the fore the need for interventions to treat bone injuries. Tissue engineering presents treatment alternatives involving the use of cells and associated biomaterials. In this context, studies using biomaterials claim that carbonated hydroxyapatite (CHA) is more resorbable than hydroxyapatite (HA) and has biological similarity to bone hydroxyapatite. <h3>Methods</h3> Parameters of indirect contact (cytotoxicity, proliferation, and migration) of the extract obtained from spheres of carbonated hydroxyapatite associated with murine bone marrow stem cells (BMSCs) in vitro were evaluated. Then the osteogenic potential of CHA extract in murine BMSCs in vitro was assessed by quantification of calcium matrix, alkaline phosphatase levels and gene expression Col1A1 and Bglap. Non-critical size bone defects were performed in rat femur. After 14 days of healing, bone blocks were obtained for analysis from treated animals and compared to non-treated animals by histological and histomorphometric evaluation. <h3>Results</h3> The CHA extract showed osteoinductive properties in vitro without the need to add osteogenic inducing medium. The ability to induce osteogenic differentiation in undifferentiated cells was confirmed by increased activity of alkaline phosphatase, formation of mineralized nodules and increased expression of the Col1A1 and Bglap genes after 7 and 14 days of differentiation. All groups presented bone neoformation at the injury site 14 days after the operation. Histomorphometric analysis showed no statistical difference between groups in the parameters of bone formation, biomaterial and connective tissue. <h3>Conclusion</h3> The CHA spheres have in vitro osteoinductive properties and, when loaded with murine BMSCs, have the potential for bone repair and regeneration in vivo, presenting biocompatibility and osteoconductive, making it a promising alternative for bone tissue engineering applications.

Linker histone H1.2 and H1.4 affect the neutrophil lineage determination.

Neutrophils are important innate immune cells that tackle invading pathogens with different effector mechanisms. They acquire this antimicrobial potential during their maturation in the bone marrow, where they differentiate from hematopoietic stem cells in a process called granulopoiesis. Mature neutrophils are terminally differentiated and short-lived with a high turnover rate. Here, we show a critical role for linker histone H1 on the differentiation and function of neutrophils using a genome-wide CRISPR/Cas9 screen in the human cell line PLB-985. We systematically disrupted expression of somatic H1 subtypes to show that individual H1 subtypes affect PLB-985 maturation in opposite ways. Loss of H1.2 and H1.4 induced an eosinophil-like transcriptional program, thereby negatively regulating the differentiation into the neutrophil lineage. Importantly, H1 subtypes also affect neutrophil differentiation and the eosinophil-directed bias of murine bone marrow stem cells, demonstrating an unexpected subtype-specific role for H1 in granulopoiesis.

Characterization of a Novel JAK1 Pseudokinase Mutation in the First Case of Trisomy 21-Independent GATA1-Mutated Transient Abnormal Myelopoiesis

Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.

AGE-ASSOCIATED INCREASE IN KYNURENINE SUPPRESSES AUTOPHAGY AND PROMOTES APOPTOSIS IN MESENCHYMAL STEM CELLS

The age-related increase of the tryptophan metabolite, kynurenine (KYN), has been associated with osteoporosis progression. Increased activity of by Indoleamine-(2,3)-dioxygenase(IDO), are responsible for the elevation of KYN levels in bone tissue. IDO activity is elevated with age and could be a promising therapeutic target forosteopenia and osteoporosis. Previously, our group has shown that the serum level of KYN is elevated with age and correlates with bone loss in vivo. Kynurenine suppress the expression and activity of chemokine CXCL12 essential for osteogenesis, bone marrow stem cells homing. Bone Marrow Stem Cells (BMSC) cultured in 1% FBS were treated with CXCL12 (100ng/ml) in the presence of saline control or the autophagic flux-inhibition agent chloroquine (CQ). CXCL12 treatment increased autophagy by upregulating the degree of LC3B-II by 20%. CXCL12 treatment also significantly increased co-localization of LC3B and LAMP-2 in serum starved cells. In the present study, we tested the theory that kynurenine plays an opposite role to CXCL12 by suppressing the autophagy cell survival pathway and by inducing apoptosis. Treatment of nutrient-deprived murine BMSCs with 10 or 100 µM of KYN suppresses autophagy in a dose dependent fashion while increasing cellular apoptosis. Treatment of BMSCs with KYN downregulated autophagic flux in BMSC preventing CQ-induced CL3B/LAMP-2 colocalization. KYN treatment prevented conversion of LC3B-I to LC3B-II in CQ-treated cells by 30 percent. At the same time, KYN treatment induces apoptosis, by increasing TUNEL-positive cells number by more than 50 percent. Additionally, KYN treatment significantly increased the levels of cleaved isoforms of PARP and caspase-3.

Long-Term Effect of Mesenchymal Stromal Cell Derived Extracellular Vesicles on the Restoration of Engraftment of Stem Cells in Radiation Exposed Mice

Extracellular vesicles (EVs) including exosomes and microvesicles, have been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that pretreated irradiated murine bone marrow stem cells with human or murine MSC-EV in vitro, could significantly improve the engraftment capacity of radiation-damaged stem cells up to 9 months post-transplantation. Interestingly, the restoration of engraftment was not significantly observed within the first month of post-transplant, the predominant reversal effect occurred on later period of post-transplant from 3 months up to 9 months. This is indicating a long-term effect of MSC-EVs on reversal of radiation damage of stem cell engraftment capacity. To confirm this hypothesis, in our current study, the effect of human MSC-EVs on reversal of engraftment capacity of bone marrow stem cells post-radiation was investigated by an in vivo study. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-EVs or vehicle were then injected intravenously 24, 48 and 72 hours after irradiation. The whole bone marrow were harvested at 6, 12, 26 and 53 weeks post EV-injection and then transplanted into 950 cGy exposed B6.SJL mice and engraftment evaluated at 1 and 3 months post-transplantation. In those transplanted mice at 6 weeks post-EV injection, there was slight increase in the restoration of engraftment rate (the percent of irradiated mice with EV/Vehicle treatment engraftment rate compared to healthy non-irradiated mice engraftment rate) in EV treated mice (17.58±2.32% compared to untreated mice (13.80±1.41%) after 1 month post-transplantation. However, for those mice transplanted at 12, 26, and 53 weeks post-EV injection, there were the significant higher restorations of engraftment rate in EV treated mice (40.48±6.03%, 33.93±3.76%, and 56.62±3.635) compared to untreated mice (12.39±1.30%, 15.14±2.21%, 36.21±3.63%) after 1 month transplantation respectively. The similar restorations of engraftment were also seen in 3 months post-transplantation. Our study also showed that there was a significant inhibition of stem cell engraftment at 53 weeks post 500cGy whole body radiation mice which was 36.21±3.63% of engraftment rate from healthy mice. Thus our data suggest that there is a long-term effect of MSC-EVs on the restoration of engraftment of stem cells in radiation-exposed mice. DisclosuresNo relevant conflicts of interest to declare.

Cyclosporine May Inhibit the Effect of Extra-Physiologic Oxygen Shock/Stress on Peripheral Blood Stem Cells

Introduction: Adequate numbers of stem cells with preserved multi-potency and self-renewing capabilities are necessary for successful hematopoietic reconstitution after bone marrow transplantation. Although hematopoietic stem cells (HSC) reside in the bone marrow under a hypoxic microenvironment (1-4% O2), human HSC are collected and processed in ambient air (21% O2) (Spencer et al., Nature 508:269-73). Exposure of murine bone marrow and human cord blood to ambient air for as little as 30 minutes triggered stem cell differentiation from quiescent pluripotent long term stem cells into activated multipotent progenitors (MPPs), a phenomenon called extra-physiologic oxygen shock / stress (EPHOSS)(Mantel et al., Cell 161:1553-65). The effect of EPHOSS on HSCs is mediated through reactive oxygen species (ROS) which open the mitochondrial permeability transition pore (MPTP) and trigger stem cell differentiation. Cyclosporine (CSA) inhibits the MPTP regulator cyclophilin D and prevents MPTP opening (Kroemer et al., Physiol Rev 87:99-163). Currently, mobilized peripheral blood stem cells (PBSCs) are the major source of grafts for hematopoietic cell transplantation. We hypothesized that EPHOSS is detrimental to human PBSCs similar to murine bone marrow and human cord blood stem cells and CSA will protect human PBSCs from the effects of EPHOSS and inhibit their differentiation from pluripotent long term HSC into short term MPPs.Study design and methods. This is a proof-of-concept, prospective, non-interventional study. We obtained G-CSF mobilized PBSCs from healthy related donors under an IRB approved protocol. All donors provided written informed consent. Blood containing HSCs was collected from each donor with minimal exposure to ambient air (<60 seconds). The sample was immediately split and incubated for 60 minutes with and without CSA (50 µg/mL). CSA treated and untreated PBSCs were immunophenotyped by multi-parameter flow cytometry for HSCs (CD34+CD38-CD90+CD45RA-), MPP (CD34+CD38-CD90-CD45RA-) and common lymphoid progenitors (CD34+CD38+CD127+). We used a CD34 ISHAGE-based gating strategy to accurately enumerate HSC and MPP.Results. In four separate experiments, CSA treated PBSCs had a higher median (range) number of HSCs/106 total nucleated cells (TNC) compared to untreated PBSCs ((297 (51 - 512) versus 185 (47 -392), respectively). CSA treated PBSCs also had a higher median (range) HSC:MPP ratio compared to untreated PBSCs ((0.56 (0.40 - 0.70) versus 0.43 (0.30 - 0.50), respectively) suggesting that the differentiation of HSC to MPP upon exposure to air was decreased in the CSA treated PBSCs.Conclusions / future directions. This is the first report describing the effect of EPHOSS in human PBSCs. Our preliminary data suggest that EPHOSS promotes the differentiation of human PBSCs from HSC to MPP and that CSA may inhibit this process. Further confirmatory and mechanistic studies exploring the contribution of MPTP and ROS to EPHOSS are ongoing. The results from this research may potentially change the current practice of collecting PBSCs in ambient air. DisclosuresMcCarthy:Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Chen:Bellicum Pharmaceuticals: Research Funding.

Introducing copper and collagen (via poly(DOPA)) coating to activate inert ceramic scaffolds for excellent angiogenic and osteogenic capacity.

Traditional calcium polyphosphate (CPP) scaffolds have attracted wide attention for repairing bone defects owing to their low cytotoxicity and controllable degradation. However, because of poor mechanical strength, significant brittleness and suboptimal osteoinductivity and osteoconductivity, their further clinical applications are restricted. To overcome these limitations, collagen (Col) coated Cu(ii) ion-doped calcium polyphosphate (CCPP) scaffolds were employed and dopamine (DOPA) was used as a linkage (CCPP/D/Col) to ensure their stable and tight structure. Controllable Cu(ii) ion continuously released from scaffolds together with collagen coating could simultaneously enhance the cytocompatibility, compressive strength and ductility, bone-related gene expression and new bone regeneration. In comparison with the initial CPP specimens, these multifunctional CCPP/D/Col composite scaffolds' crystal grains of CCPP were arranged regularly and well-ordered, and the size and rugosity were more suitable for cell spreading and attachment. Murine bone marrow stem cells (BMSCs) seeded on CCPP/D/Col scaffolds possessed better proliferation and migration, rapid attachment and enhanced expression of osteogenic-related genes, which indicated better bone regeneration. The potential mechanism of this process was further elucidated. Both copper doping and collagen coating could effectively stabilize hypoxia-inducible factor-1α (HIF-1α) that thus stimulates the expression of vascular endothelial growth factor (VEGF). In addition, they could also promote the osteogenic differentiation of cells through stimulating bone-related gene expression. The concept of introducing active ions and biological macromolecules to modify inert ceramics may offer a new strategy to construct a multifunctional composite scaffold for bone tissue regeneration.

Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of curative treatment strategies for hematologic malignancies. The homing process ends with selective access and anchorage of HSCs to their specialized niches within the BM. Adhesion molecules are targets to either enhance homing in cases of stem cell transplantation or reduce BM retention to harvest mobilized HSCs from the blood of matched donors. A major adhesion protein which is functionally expressed on HSCs and is involved in their homing and retention is the integrin alpha4beta1 (Very late antigen-4; VLA4). In this protocol we introduce an adhesion assay optimized for VLA4 expressing murine bone marrow stem cells. This assay quantifies adherent HSCs by flow cytometry with HSC enriching cell surface markers subsequent to the isolation of VLA4 expressing adherent cells.

The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Background. Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237).Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs.Materials and Methods. Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression.Results. We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression.Conclusions. Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Short Lef-1 Isoform, Lacking The β-Catenin Binding Domain, Is Not Acting As a Dominant Negative Lef-1 Variant, But Is a Hematopoietic Active Protein With Unique DNA Binding Properties

Studies in the hematopoietic system of human and mice have demonstrated that the Wnt signaling pathway is essential for maintenance, activation and proliferation of normal hematopoietic stem cells. Lef-1 is a member of the Lef-1/T-cell-factor (Tcf) family of transcription factors regulated by the canonical Wnt signaling pathway and generally directly interacts with β-catenin in transcriptional complexes to induce expression of target genes, including the cell cycle regulators cyclin D1 and c-myc. We have previously shown that aberrant expression of Lef-1 perturbs normal hematopoietic stem cell (HSC) and progenitor function and induces acute myeloid leukemia in the murine bone marrow transplantation model and that it is a novel prognostic independent factor in patients with normal karyotype AML (Petropoulos et al. JEM, 2008; Metzeler et al., Blood 2012). Furthermore, we could recently demonstrate that shRNA mediated depletion of Lef-1 drastically compromises the function of long-term repopulating stem cells (Edmaier et al., Leukemia, in press), indicating a vital function of Lef-1 at the level of the most primitive stem cell compartment. So far, all these Lef-1 functions are ascribed to the full-length transcript of Lef-1, which acts as transcriptional mediator of Wnt signaling via its β-catenin binding domain, whereas the shorter Lef-1 isoform, deriving from an intronic promoter is thought to function as a dominant negative variant. To dissect the role of the ‘dominant negative' Lef-1 isoform we first retrovirally engineered primary murine bone marrow cells to express the N-terminal deleted Lef-1 (Lef-1d56), lacking the β-catenin binding domain in comparison to the full-length Lef-1 (Lef-1WT) and the empty retroviral EGFP control. Loss of binding to β-catenin was validated by Co-IP for Lef-1d56. Of note, Lef-1d56 induced a 2,05fold and significant increase at the level of clonogenic cells in vitro compared to the control (n=3; p<0,05) and did not differ substantially from the activity of Lef-1 WT. β-catenin binding did not impact Lef-1 activity at the level of the short-term repopulating stem cells as documented in the CFU-S assay with almost comparable CFU-S frequencies between the wild-type and the mutant Lef-1 (d56) (73 CFU-S/1x105 and 64 CFU-S/1x105 , respectively, compared to 38 CFU-S/1x105 for the EGFP control; p<0.05). In clear contrast, loss of β-catenin interaction reduced the CRU frequency dramatically (1:597.197(Lef-1 WT) and 1:1.675.238 (Lef-1 d56), respectively and 1: 1.233.152 for the EGFP control; p=0,066), indicating the necessity of Lef-1 to collaborate with β-catenin at the level of the long-term repopulating stem cell. Retroviral expression of Lef-1 d56 in murine bone marrow stem and progenitor cells for 48h induced a distinct gene expression profile and deregulated more genes than Lef-1WT compared to the EGFP control. Differentially expressed genes between Lef-1d56 and Lef-1WT comprised known hematopoietic factors such as like Gata2, Ets1 and genes associated to the G-protein coupled receptor protein signaling. ChIP-Seq in hematopoietic murine cells revealed unique binding sites for Lef-1d56 compared to Lef-1WT with binding of the Lef-1d56 to promoters of Wnt5a and CD81. Taken together, our data clearly indicate that loss of the β-catenin binding site does not convert Lef-1 into an overall dominant negative variant, but creates a ‘neomorphic' isoform with distinct biological and DNA binding properties. Analyses are ongoing which quantify the expression of LEF-1WT and the β-catenin domain lacking LEF-1 isoform in over 100 patients with normal karyotype AML in comparison to normal human stem and progenitor cells to test potential differences between normal and leukemic cells as well as associations of the two LEF-1 transcripts with the mutational status, gene expression and treatment outcome in this AML patient group. Disclosures:No relevant conflicts of interest to declare.

Rapid screening of potential metallic glasses for biomedical applications

This paper presents a rapid screening process to select potential titanium and zirconium based metallic glasses (MGs) for bio-material applications. Electrochemical activity of 7 MGs including 6 bulk metallic glasses and 1 thin-film deposited MG in simulation body and human serum is first inspected. A low-voltage potential state test is also developed to simulate the cell membrane potential that the implant MGs will suffer. Results show that the MGs composed of Ti65Si15Ta10Zr10 and Ta57Zr23Cu12Ti8 exhibit excellent electrochemical stability in both simulation body fluid and human serum. In addition, the copper content in the MGs plays an important role on the electrochemical activity. MGs with the copper content higher than 17.5% show significant electrochemical responses. The cytotoxicity of the solid MG samples and the corrosion released ions are also evaluated by an in-vitro MTT test utilizing the murine bone marrow stem cells. Results indicate that all the solid MG samples show no acute cytotoxicity yet the corrosion released ions show significant toxicity for murine bone marrow stem cells. The rapid screening process developed in the present study suggests that the Ti65Si15Ta10Zr10 metallic glass has high potential for biomedical applications due to its good electrochemical stability and very low cytotoxicity.

Characterization of ion currents of murine CD117pos stem cells in vitro and their modulation under AT2R stimulation

Hematopoietic stem cells, especially CD117(pos) cells, have been found to possess a regenerative potential in various tissues, in particular cardiac muscle. However, the characterization of the relevant ion currents of stem cells prior to implantation lacks documentation. Activation of angiotensin II type 2 receptor (AT2 R) can lead to further cell differentiation and receptor auto-expression and might thus influence electrophysiological properties of CD117(pos) stem cells. This study was designed to functionally characterize membrane currents of CD117(pos) cells under normal and AT2 R-stimulated conditions. CD117(pos) murine bone marrow stem cells were isolated with MACS technique and stimulated for the AT2 R with angiotensin II and losartan for 3-5days prior to patch-clamp measurements. RT-PCR was used to determine channel expression. Endothelial properties were analysed with immunocytochemistry and acLDL uptake assay. A well-expressed inward rectifying current (IKir ) was identified in cultured CD117(pos) cells. Furthermore, a ZD 7288 (HCN channel blocker)-sensitive current component was isolated. Voltage-dependent potassium currents and chloride currents were less expressed. A small fraction of cells demonstrated voltage- and time-dependent inward currents. In AT2 R-stimulated cells inward rectifying the hyperpolarization-induced inward currents were slightly attenuated on the translational level but showed increased mRNA expression. Cultured CD117(pos) cells express CD31 and VEGFR-2 and significantly increased the uptake of acLDL. CD117(pos) cells do not have properties of action potential-generating cells and moderately change their excitability during AT2 R stimulation. Electrophysiological and molecular properties of control and AT2 R-stimulated cells point to a differentiation to vascular endothelial cells. This could increase beneficial vascularization in injured tissues.

Histamine receptor H4 regulates mast cell degranulation and IgE induced FcεRI upregulation in murine bone marrow-derived mast cells

There is increasing evidence that histamine regulates the immune system via histamine H4 receptors, therefore we sought to investigate the functions of the H4 receptor on mast cells. Mast cells were differentiated from murine bone marrow stem cells, and the expression of mast cell surface markers FcεRI and CD117 were measured using flow cytometry. Real-time qRT-PCR was used to determine the expression of mH4R; as a measure of antigen-dependent degranulation, β-hexosaminidase release assay was carried out using IgE sensitized mast cells. We determined that the expression kinetics of FcεRI and mH4R can be described with a function that has one maximum value in the time range of the culture’s differentiation. Antigen-dependent degranulation of murine bone marrow-derived mast cells could be inhibited by a selective H4 antagonist/inverse agonist only when it was present during the IgE sensitization phase of degranulation. In addition, flow cytometric analysis revealed that the H4 antagonist/inverse agonist also inhibited IgE induced FcεRI upregulation. The inhibition percentage of H4 antagonist on IgE induced FcεRI upregulation was determined to be dependent upon the maturity of the mast cell cultures, and this time-dependency was consistent with the expression kinetics of both mH4R and FcεRI. These results imply that H4R has regulatory roles in FcεRI expression and FcεRI mediated functions in mast cells. In conclusion the present study shows that H4 receptors potentially play a role in IgE induced FcεRI upregulation and in the sensitization phase but not the effector phase of mast cell degranulation.

Enhanced in vitro biocompatibility/bioactivity of biodegradable Mg–Zn–Zr alloy by micro-arc oxidation coating contained Mg2SiO4

There is a great attraction to apply magnesium alloy as an orthopedic implant material due to its biodegradable feature and suitable mechanical properties. In order to further improve the biocompatibility of magnesium alloy, a Mg2SiO4-containing micro-arc oxidation (MAO) coating was generated on ZK60 magnesium alloy, in this study. The surface characterization by SEM, XRD and EDS showed that a rough and porous reaction layer with major composition of O, Mg and Si was formed on the surface of ZK60 alloy. The result of polarization test showed that the MAO coating significantly enhanced the corrosion resistance of biodegradable ZK60 alloy. In vitro biocompatibility/bioactivity evaluation of the MAO-coated alloy compared with the naked alloy was performed using murine bone marrow stem cells (BMSCs). The results revealed that the extract of MAO-coated alloy had no obvious cytotoxicity during the 5days culturing, and the surface of MAO coating exhibited a better cell adhesion and affinity. Alkaline phosphatase (ALP) activity assay demonstrated that the ALP level of the BMSCs in the extract of MAO-coated alloy was much higher than that of the naked alloy, indicating that the release of Mg and Si ions from the coating was beneficial for the differentiation of BMSCs. Result of blood hemolysis test showed that the MAO coating group had a drastically decreased hemolytic ratio (1.04%) compared with the naked alloy group (28.78%) (p<0.01), meaning a great improvement of hemocompatibility. In conclusion, the results of this study suggested that the Mg2SiO4-containing MAO coating is an effective way to improve the biocompatibility/bioactivity of magnesium alloys.

Detection and isolation of human disseminated tumor cells in the murine bone marrow stem cell niche.

The presence of disseminated tumor cells (DTCs) in the bone marrow is associated with poor prognosis of cancer patients. However, little is known about the biology of DTCs due to lack of relevant animal models. Here, we describe the methods for detecting and isolating human DTCs from the murine bone marrow niche by PCR using human Alu sequences and by fluorescence-activated cell sorting and immunohistochemistry using anti-HLA antibody. These strategies could be useful for exploring the biology of DTCs.

Abstract 5167: Notch1 is required for endothelial cell lineage differentiation from adult bone marrow stem cells in vitro

Abstract Background and Objectives. Adult bone-marrow stem cells (BMSCs) are able to differentiate into endothelial progenitor cells (EPCs) and subsequently endothelial cells (ECs) under appropriate conditions. BMSCs-derived EPCs/ECs contribute to post-natal neovascularization and are important tools for regenerative medicine. However, the molecular mechanism determining specific differentiation of endothelial lineage remains unknown. The Notch signaling pathway plays an integral role in the differentiation of a variety of cell lineages. We hypothesize that Notch signaling is involved in controlling endothelial lineage differentiation from BMSCs and examined the role of Notch1 signaling in this process. Methods and Results. An in vitro method for differentiation of endothelial cell lineage from murine BMSCs was developed. Mononuclear cells were isolated from mouse whole BM cells by light density separation using Ficoll-Paque® Plus, and cultured in the differentiation medium to differentiate endothelial cells. The endothelial phenotype of differentiated cells was characterized by immunostaining and flow cytometry analyses. Those fractions of cells with VEGFR2+/CD31+ or Tie2+/CD31+ were isolated and tested. VEGFR2+/CD31+ cells could form networks on the MatrigelTM, indicating that these cells are ECs. mRNA expression of Notch pathway components in VEGFR2+/CD31+ cells was examined by real-time RT-PCR. Multiple components of the Notch signaling pathway were expressed in the differentiated ECs. The effect of loss-of-function in Notch1 on the differentiation of endothelial cell lineage was examined using BMSCs from Notch1Flox/Flox mice. Cells expressing VEGFR2 were significantly decreased when Notch1 is deleted. Conclusions. A novel and reliable method has been developed to differentiate murine adult bone marrow stem cells into endothelial cell lineage in vitro. The role of Notch1 in determining endothelial cell differentiation was tested on this platform. Our results implicated that Notch1 is required for the differentiation of endothelial cell lineage from BMSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5167.