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Focuses on the prospective isolation of stromal progenitor cells from mouse bone marrow (BM). Discussion on the stromal tissue of the BM; Prospects and limitations of cell therapies based on the use of marrow stromal tissue; Major barriers to the isolation of stromal progenitors from mouse BM.

Accurately defining hierarchical relationships between human stem cells and their progeny, and using this knowledge for new cellular therapies, will undoubtedly lead to further successful treatments for life threatening and chronic diseases, which represent substantial burdens on patient quality of life and to healthcare systems globally. Clinical translation relies in part on appropriate biomarker, in vitro manipulation and transplantation strategies. CD164 has recently been cited as an important biomarker for enriching both human haematopoietic and skeletal stem cells, yet a thorough description of extant human CD164 monoclonal antibody (Mab) characteristics, which are critical for identifying and purifying these stem cells, was not discussed in these articles. Here, we highlight earlier but crucial research describing these relevant characteristics, including the differing human CD164 Mab avidities and their binding sites on the human CD164 sialomucin, which importantly may affect subsequent stem cell function and fate.

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The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, co-expressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

The fetal liver is the site of a major expansion of the hematopoietic stem cell (HSC) pool and is also a privileged organ to study megakaryocyte progenitor differentiation. We identified in the mouse fetal liver at day 13.5 a discrete stromal cell population harboring a CD45 - TER119 - CD31 - CD51 + VCAM-1 + PDGFRα - (V + P - ) phenotype that lacked colony-forming unit fibroblast activity and harbored an hepatocyte progenitor signature. This previously undescribed V + P - population efficiently supported megakaryocyte production from mouse bone marrow HSC and human peripheral blood HSC-myeloid progenitors cultured in the presence of limited cytokine concentrations. Megakaryocytes obtained in V + P - cocultures were polyploid, positive for CD41/CD42c, and efficiently produced proplatelets. Megakaryocyte production appeared to be mediated by an expansion of the progenitor compartment through HSC-stromal cell contact. In conclusion, the fetal liver contains a unique cellular microenvironment that could represent a platform for the discovery of regulators of megakaryopoiesis., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Background Aims: Despite ethnic diversity and ready availability of cryopreserved, human leukocyte antigen-typed cord blood (CB), delayed engraftment remains a significant hurdle to successful CB transplantation. Suboptimal homing of CB hematopoietic stem and progenitor cells (HSPCs) to the hematopoietic microenvironment (HM) is thought to be responsible and due to low levels of HSPC fucosylation. Fucosylation (decoration with sialyl-Lewis X ) may improve HSPC homing to HM by increasing the strength of HSPC/E-selectin interactions, where E-selectin is constitutively expressed by HM microvasculature. Enforced fucosylation of CB HSPCs using fucosyltransferases, increases the rate and magnitude of engraftment in xenogeneic transplant models. However, it is unclear whether endogenously fucosylated and non-fucosylated CB HSPC are qualitatively identical or whether endogenous fucosylation marks a qualitative difference between CB HSPC. If qualitatively identical, non-fucosylated CB HSPCs represent a good target for enforced fucosylation with improved engraftment conferred on an increased number of otherwise qualitatively identical HSPC. If qualitatively different, then conferring engraftment upon a majority, possibly lower "quality," non-fucosylated HSPCs by enforced fucosylation might inadvertently compromise engraftment., Methods: Functional (xenogeneic engraftment, colony-forming unit and selectin-binding assays) and phenotypic analyses of fluorescence-activated cell sorting-isolated, endogenously fucosylated and non-fucosylated CB CD34 + cells were performed., Results: Endogenous fucosylation of CB HSPCs exists as a continuum. Endogenously fucosylated HSPCs engrafted more efficiently in a xenogeneic transplantation model than non-fucosylated HSPCs. Outside of the differences in endogenous fucosylation, no other qualitative (functional and/or phenotypic) differences were identified., Discussion: The majority of endogenously non-fucosylated CB HSPCs represent a good target for enforced fucosylation with the goal of improving engraftment following CB transplantation., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

In this study, we show that matrix dense cortical bone is the more potent compartment of bone than bone marrow as a stromal source for mesenchymal stem cells as isolated from adult rats. Lineage-depleted cortical bone-mesenchymal stem cells demonstrated >150-fold enrichment of colony forming unit-fibroblasts per cell incidence. compared to lineage-depleted bone marrow-mesenchymal stem cells, corresponding to a 70-fold increase in absolute recovered colony forming unit-fibroblasts. The composite phenotype Lin(-)/CD45(-)/CD31(-)/VLA-1(+)/Thy-1(+) enriched for clonogenic mesenchymal stem cells solely from cortical bone-derived cells from which 70% of clones spontaneously differentiated into all lineages of bone, cartilage, and adipose. Both populations generated vascularized bone tissue within subcutaneous implanted collagen scaffolds; however, cortical bone-derived cells formed significantly more osteoid than bone marrow counterparts, quantified by histology. The data demonstrate that our isolation protocol identifies and validates mesenchymal stem cells with superior clonal, proliferative, and developmental potential from cortical bone compared to the bone marrow niche although marrow persists as the typical source for mesenchymal stem cells both in the literature and current pre-clinical therapies., Competing Interests: Declaration of conflicting interest: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

CD34(+) cell dose provides a measure of hematopoietic tissue that predicts the rate of engraftment upon transplant. It is positively correlated with multiple measures of hematopoietic recovery, including platelet engraftment. Here we identify a subpopulation of CD34(+) cells that coexpress a surface antigen--MA6, which is more positively correlated with platelet engraftment in a clinical setting than CD34(+) alone. The specific identity and function of MA6 remain to be determined, however, it is expressed by primitive megakaryocyte (MK) progenitors, but is lost with differentiation and is not expressed by platelets. Commitment of CD34(+)MA6(+) cells to the MK lineage was confirmed by in vitro assays and their significance in hematopoietic transplantation explored by flow cytometric analysis of cryopreserved samples of granulocyte colony stimulating factor-mobilized peripheral blood progenitor cell (PBPC) products along with a retrospective analysis of platelet engraftment data. Platelet engraftment by day 21 was predicted by receipt of ≥ 6 × 10(6) CD34(+) cells/kg or ≥ 0.3 × 10(6) CD34(+)MA6(+) cells/kg. Subsequent analysis of cord blood (CB) CD34(+) cells revealed <0.2% coexpressed MA6(+), compared to 8% of PBPC CD34(+) cells. This low proportion of CD34(+)MA6(+) cells may be responsible, at least in part, for the delayed platelet engraftment associated with CB transplantation. However, platelet engraftment is markedly improved in recipients of ex vivo-expanded CB. This may be a consequence of an increased proportion of CD34(+)MA6(+) cells present in the ex vivo-expanded product and also suggests that optimizing ex vivo culture conditions to generate CD34(+)MA6(+) cells might further improve platelet engraftment in CB recipients.

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.

Background Aims: Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo., Methods: CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment., Results: Both FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes., Conclusions: Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

This study assessed the potential of highly purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) in combination with the anti-scarring protein decorin to repair the injured spinal cord (SC). Donor hMPCs isolated from spinal cord injury (SCI) patients were transplanted into athymic rats as a suspension graft, alone or after previous treatment with, core (decorin(core)) and proteoglycan (decorin(pro)) isoforms of purified human recombinant decorin. Decorin was delivered via mini-osmotic pumps for 14 days following sub-acute (7 day) or chronic (1 month) SCI. hMPCs were delivered to the spinal cord at 3 weeks or 6 weeks after the initial injury at T9 level. Behavioral and anatomical analysis in this study showed statistically significant improvement in functional recovery, tissue sparing and cyst volume reduction following hMPC therapy. The combination of decorin infusion followed by hMPC therapy did not improve these measured outcomes over the use of cell therapy alone, in either sub-acute or chronic SCI regimes. However, decorin infusion did improve tissue sparing, reduce spinal tissue cavitation and increase transplanted cell survivability as compared to controls. Immunohistochemical analysis of spinal cord sections revealed differences in glial, neuronal and extracellular matrix molecule expression within each experimental group. hMPC transplanted spinal cords showed the increased presence of serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted hMPCs for up to 2 months; however, no evidence of hMPC transdifferentiation into neuronal or glial phenotypes. The number of hMPCs was dramatically reduced overall, and no transplanted cells were detected at 8 weeks post-injection using lentiviral GFP labeling and human nuclear antigen antibody labeling. The presence of recombinant decorin in the cell transplantation regimes delayed in part the loss of donor cells, with small numbers remaining at 2 months after transplantation. In vitro co-culture experiments with embryonic dorsal root ganglion explants revealed the growth promoting properties of hMPCs. Decorin did not increase axonal outgrowth from that achieved by hMPCs. We provide evidence for the first time that (Stro-1(+)) hMPCs provide: i) an advantageous source of allografts for stem cell transplantation for sub-acute and chronic spinal cord therapy, and (ii) a positive host microenvironment that promotes tissue sparing/repair that subsequently improves behavioral outcomes after SCI. This was not measurably improved by recombinant decorin treatment, but does provide important information for the future development and potential use of decorin in contusive SCI therapy., (© 2013 Elsevier Inc. All rights reserved.)

Liposarcomas are tumors arising in white adipose tissue (WAT) with avidity for local recurrence. Aggressive dedifferentiated liposarcomas (DDLS) may arise from well-differentiated subtypes (WDLS) upon disease progression, however, this key issue is unresolved due in large part to knowledge gaps about liposarcoma cellular composition. Here, we wished to improve insights into liposarcoma cellular hierarchy. Tumor section analysis indicated that the populations, distinguishable based on the expression of CD34 (a marker of adipocyte progenitors) and CD36 (a marker of adipocyte differentiation), occupy distinct intra-tumoral locations in both WDLS and DDLS. Taking advantage of these markers, we separated cells from a panel of fresh human surgical specimens by fluorescence-activated cell sorting (FACS). Based on chromosome analysis and the culture phenotypes of the composing populations, we demonstrate that malignant cells comprise four mesenchymal populations distinguished by the expression of CD34 and CD36, while vascular (CD31+) and hematopoietic (CD45+) components are non-neoplastic. Finally, we show that mouse xenografts are derivable from both CD36-negative and CD36-positive DDLS cells, and that each population recreates the heterogeneity of CD36 expression in vivo. Combined, our results show that malignant cells in WDLS and DDLS can be classified according to distinct stages of adipogenesis and indicate immunophenotypic plasticity of malignant liposarcoma cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Insect larvae clearly react to visual stimuli, but the ability of any visual neuron in a newly hatched insect to respond selectively to particular stimuli has not been directly tested. We characterised a pair of neurons in locust larvae that have been extensively studied in adults, where they are known to respond selectively to objects approaching on a collision course: the lobula giant motion detector (LGMD) and its postsynaptic partner, the descending contralateral motion detector (DCMD). Our physiological recordings of DCMD axon spikes reveal that at the time of hatching, the neurons already respond selectively to objects approaching the locust and they discriminate between stimulus approach speeds with differences in spike frequency. For a particular approaching stimulus, both the number and peak frequency of spikes increase with instar. In contrast, the number of spikes in responses to receding stimuli decreases with instar, so performance in discriminating approaching from receding stimuli improves as the locust goes through successive moults. In all instars, visual movement over one part of the visual field suppresses a response to movement over another part. Electron microscopy demonstrates that the anatomical substrate for the selective response to approaching stimuli is present in all larval instars: small neuronal processes carrying information from the eye make synapses both onto LGMD dendrites and with each other, providing pathways for lateral inhibition that shape selectivity for approaching objects.

Mesenchymal stem cells (MSCs) are the focus of intensive efforts worldwide directed not only at elucidating their nature and unique properties but also developing cell-based therapies for a diverse range of diseases. More than three decades have passed since the original formulation of the concept, revolutionary at the time, that multiple connective tissues could emanate from a common progenitor or stem cell retained in the postnatal bone marrow. Despite the many important advances made since that time, substantial ambiguities still plague the field regarding the nature, identity, function, mode of isolation and experimental handling of MSCs. These uncertainties have a major impact on their envisioned therapeutic use.

This study aimed to determine the potential of purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) to repair the injured spinal cord (SC) after transplantation into T-cell-deficient athymic RNU nude rats following acute moderate contusive spinal cord injury (SCI). hMPCs were isolated from the bone marrow (BM) stroma of SCI patients and transplanted as a suspension graft in medium [with or without immunosuppression using cyclosporin A (CsA)]. Extensive anatomical analysis shows statistically significant improvement in functional recovery, tissue sparing, and cyst reduction. We provide quantitative assessment of supraspinal projections in combination with functional outcomes. hMPC-transplanted animals consistently achieved mean BBB scores of 15 at 8 weeks post injury. Quantitative histological staining revealed that graft-recipient animals possessed more intact spinal tissue and reduced cyst formation than controls. Fluorogold (FG) retrograde tracing revealed sparing/regeneration of supraspinal and local propriospinal axonal pathways, but no statistical differences were observed compared to controls. Immunohistochemical analysis revealed increased serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted donor hMPCs 2 weeks posttransplantation, but no evidence of hMPC transdifferentiation was seen. Although hMPCs initially survive at 2 weeks posttransplantation, their numbers were dramatically reduced and no cells were detected at 8 weeks posttransplantation using retroviral/lentiviral GFP labeling and a human nuclear antigen (HNA) antibody. Additional immunosuppression with CsA did not improve hMPC survival or their ability to promote tissue sparing or functional recovery. We propose Stro-1(+)-selected hMPCs provide (i) a reproducible source for stem cell transplantation for SC therapy and (ii) a positive host microenvironment resulting in the promotion of tissue sparing/repair that subsequently improves behavioral outcomes after SCI. Our results provide a new candidate for consideration as a stem cell therapy for the repair of traumatic CNS injury.

Background: Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells., Methods: We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood., Results: Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001)., Conclusions: Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.

In a synaptic active zone, vesicles aggregate around a densely staining structure called the presynaptic density. We focus on its three-dimensional architecture and a major molecular component in the locust. We used electron tomography to study the presynaptic density in synapses made in the brain by identified second-order neuron of the ocelli. Here, vesicles close to the active zone are organized in two rows on either side of the presynaptic density, a level of organization not previously reported in insect central synapses. The row of vesicles that is closest to the density's base includes vesicles docked with the presynaptic membrane and thus presumably ready for release, whereas the outer row of vesicles does not include any that are docked. We show that a locust ortholog of the Drosophila protein Bruchpilot is localized to the presynaptic density, both in the ocellar pathway and compound eye visual neurons. An antibody recognizing the C-terminus of the Bruchpilot ortholog selectively labels filamentous extensions of the presynaptic density that reach out toward vesicles. Previous studies on Bruchpilot have focused on its role in neuromuscular junctions in Drosophila, and our study shows it is also a major functional component of presynaptic densities in the central nervous system of an evolutionarily distant insect. Our study thus reveals Bruchpilot executes similar functions in synapses that can sustain transmission of small graded potentials as well as those relaying large, spike-evoked signals., (Copyright © 2011 Wiley-Liss, Inc.)

Many arthropods possess escape-triggering neural mechanisms that help them evade predators. These mechanisms are important neuroethological models, but they are rarely investigated using predator-like stimuli because there is often insufficient information on real predator attacks. Locusts possess uniquely identifiable visual neurons (the descending contralateral movement detectors, DCMDs) that are well-studied looming motion detectors. The DCMDs trigger 'glides' in flying locusts, which are hypothesised to be appropriate last-ditch responses to the looms of avian predators. To date it has not been possible to study glides in response to stimuli simulating bird attacks because such attacks have not been characterised. We analyse video of wild black kites attacking flying locusts, and estimate kite attack speeds of 10.8±1.4 m/s. We estimate that the loom of a kite's thorax towards a locust at these speeds should be characterised by a relatively low ratio of half size to speed (l/|v|) in the range 4-17 ms. Peak DCMD spike rate and gliding response occurrence are known to increase as l/|v| decreases for simple looming shapes. Using simulated looming discs, we investigate these trends and show that both DCMD and behavioural responses are strong to stimuli with kite-like l/|v| ratios. Adding wings to looming discs to produce a more realistic stimulus shape did not disrupt the overall relationships of DCMD and gliding occurrence to stimulus l/|v|. However, adding wings to looming discs did slightly reduce high frequency DCMD spike rates in the final stages of object approach, and slightly delay glide initiation. Looming discs with or without wings triggered glides closer to the time of collision as l/|v| declined, and relatively infrequently before collision at very low l/|v|. However, the performance of this system is in line with expectations for a last-ditch escape response.

In Schistocerca gregaria ocellar pathways, large second-order L-neurons use graded potentials to communicate signals from the ocellar retina to third-order neurons in the protocerebrum. A third-order neuron, DNI, converts graded potentials into axonal spikes that have been shown in experiments at room temperature to be sparse and precisely timed. I investigated effects of temperature changes that a locust normally experiences on these signals. With increased temperature, response latency decreases and frequency responses of the neurons increase. Both the graded potential responses in the two types of neuron and the spikes in DNI report greater detail about a fluctuating light stimulus. Over a rise from 22 to 35°C the power spectrum of the L-neuron response encompasses higher frequencies and its information capacity increases from about 600 to 1,700 bits/s. DNI generates spikes more often during a repeated stimulus but at all temperatures it reports rapid decreases in light rather than providing a continual measure of light intensity. Information rate carried by spike trains increases from about 50 to 185 bits/s. At warmer temperatures, increased performance by ocellar interneurons may contribute to improved aerobatic performance by delivering spikes earlier and in response to smaller, faster light stimuli.

Obesity complicates a number of diseases through mechanisms that are poorly defined. Mobilization and recruitment of progenitor cells to pathological sites is an important factor in disease progression. Here, we analyzed the influence of obesity on the systemic circulation of CD34(+) cell populations and correlated frequencies of cells displaying previously established cell marker signatures with the BMI. Comparative analysis of peripheral blood mononuclear cells (PBMC) from 12 nonobese (BMI <30 kg/m(2)) and 14 obese (BMI >30 kg/m(2)) disease-free donors by flow cytometry revealed that obesity is associated with a fivefold increased frequency of circulating progenitor cells (CPC), a population consisting of hematopoietic and endothelial precursors. Our data also indicate that obesity is associated with increased frequency of circulating mesenchymal stromal progenitor cells (MSC). In contrast, the frequencies of mature endothelial cells (EC) and CD34-bright leukocytes are unaffected by obesity. Combined, our results indicate that obesity promotes mobilization of progenitor cells, which may have clinical relevance.

Adipose stromal cells (ASCs) serve as mesenchymal progenitors in white adipose tissue (WAT). Intercellular interactions involving ASCs have remained obscure. By merging phage display technology with fluorescence-activated cell sorting (FACS), we screened a combinatorial library for peptides that target mouse ASCs in vivo. We isolated peptide CSWKYWFGEC that specifically homes to ASCs, used it as bait to purify the corresponding ASC surface receptor, and identified it as a previously unreported cleavage product of decorin (DCN) lacking the glycanation site (termed ΔDCN). We demonstrate that ΔDCN is differentially expressed on ASC surface. In a screen for ΔDCN-binding proteins, we identified resistin, an adipokine for which the receptor has been unknown. Expression of ΔDCN in 3T3-L1 cells promoted proliferation and migration but suppressed lipid accumulation upon adipogenesis induction, which was resistin dependent. We conclude that ΔDCN serves as a functional receptor of resistin in adipocyte progenitors and may regulate WAT expansion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A novel bioactive sponge was created with a composite of type I collagen sponges or porous poly(e-caprolactone) (PCL) scaffolds, platelet-rich plasma (PRP), BMP2-loaded nanoporous silicon enclosure (NSE) microparticles, mineralizing peptide amphiphiles (PA), and mesenchymal stem cells (MSC). Primary MSC from cortical bone (CB)  tissue proved to form more and larger colony units, as well as produce more mineral matrix under osteogenic differentiation, than MSC from bone marrow (BM). Coating pre-treatments were optimized for maximum cell adhesion and mineralization, while a PRP-based gel carrier was created to efficiently deliver and retain MSC and  microparticles within a porous scaffold while simultaneously promoting cell recruitment, proliferation, and angiogenesis. Components and composite sponges were evaluated for osteogenic differentiation in vitro. Osteogenic sponges were loaded with MSC, PRP, PA, and NSE and implanted subcutaneously in rats to evaluate the formation of bone tissue and angiogenesis in vivo. It was found that the combination of a collagen sponge with CB MSC, PRP, PA, and the BMP2-releasing NSE formed the most bone and was most vascularized by four weeks compared to analogous composites featuring BM MSC or PCL or lacking PRP, PA, and NSE. This study indicates that CB MSC should be considered as an alternative to marrow as a source of stem cells, while the PRP-PA cell and microparticle delivery system may be utilized for diverse tissue engineering applications.

Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34(+) cell doses transplanted. Here, current strategies directed at improving TNC and CD34(+) cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo co-culture expansion system., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Here, we have prospectively isolated and characterized, for the first time, clonogenic cells with self-renewal capacities from mantle cell lymphoma (MCL), a particularly deadly form of non-Hodgkin's lymphoma (NHL). Self-renewal and tumorigenic activities were enriched in MCL cell fractions that lacked expression of the prototypic B-cell surface marker, CD19. CD45+CD19- cells represented a relatively small fraction of the total MCL tumor cells; however, they recapitulated the heterogeneity of original patient tumors on transplantation into immunodeficient mice. As few as 100 of these cells displayed self-renewal capacities in secondary and tertiary recipient mice by in vivo limiting dilution assays. Similar to leukemic stem cells, CD45+CD19- MCL cells also displayed a quiescent status as determined by dye efflux assays. In summary, this study is the first to isolate subpopulations of MCL cells that have self-renewal and tumorigenic capacities. Identification and characterization of MCL-ICs are important first steps toward understanding how self-renewal and tumorigenicity are regulated in MCL and designing targeted therapies against MCL-ICs will ultimately lead to improved outcomes for MCL patients., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Abstract We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.

We investigate coding in a locust brain neuron, DNI, which transforms graded synaptic input from ocellar L-neurons into axonal spikes that travel to excite particular thoracic flight neurons. Ocellar neurons are naturally stimulated by fluctuations in light collected from a wide field of view, for example when the visual horizon moves up and down. We used two types of stimuli: fluctuating light from a light-emitting diode (LED), and a visual horizon displayed on an electrostatic monitor. In response to randomly fluctuating light stimuli delivered from the LED, individual spikes in DNI occur sparsely but are timed to sub-millisecond precision, carrying substantial information: 4.5-7 bits per spike in our experiments. In response to these light stimuli, the graded potential signal in DNI carries considerably less information than in presynaptic L-neurons. DNI is excited in phase with either sinusoidal light from an LED or a visual horizon oscillating up and down at 20 Hz, and changes in mean light level or mean horizon level alter the timing of excitation for each cycle. DNI is a multimodal interneuron, but its ability to time spikes precisely in response to ocellar stimulation is not degraded by additional excitation. We suggest that DNI is part of an optical proprioceptor system, responding to the optical signal induced in the ocelli by nodding movements of the locust head during each wing-beat.

Locusts respond to the images of approaching (looming) objects with responses that include gliding while in flight and jumping while standing. For both of these responses there is good evidence that the DCMD neuron (descending contralateral movement detector), which carries spike trains from the brain to the thoracic ganglia, is involved. Sudden glides during flight, which cause a rapid loss of height, are last-chance manoeuvres without prior preparation. Jumps from standing require preparation over several tens of milliseconds because of the need to store muscle-derived energy in a catapult-like mechanism. Locusts' DCMD neurons respond selectively to looming stimuli, and make connections with some motor neurons and interneurons known to be involved in flying and jumping. For glides, a burst of high-frequency DCMD spikes is a key trigger. For jumping, a similar burst can influence timing, but neither the DCMD nor any other single interneuron has been shown to be essential for triggering any stage in preparation or take-off. Responses by the DCMD to looming stimuli can alter in different behavioural contexts: in a flying locust, arousal ensures a high level of both DCMD responsiveness and glide occurrence; and there are significant differences in DCMD activity between locusts in the gregarious and the solitarious phase., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

During the course of studies to investigate whether MPC circulate in response to G-CSF, the agent most frequently used to induce mobilization of hematopoietic progenitors, we observed that while G-CSF failed to increase the number of MPC in circulation (assayed in vitro as fibroblast colony-forming cells, CFU-F), G-CSF administration nevertheless resulted in a time-dependent increase in the absolute number of CFU-F within the BM, peaking at Day 7. Treatment of BM cells from G-CSF-treated mice with hydroxyurea did not alter CFU-F numbers, suggesting that the increase in their numbers in response to G-CSF administration is not due to proliferation of existing CFU-F. Given previous studies demonstrating that G-CSF potently induces bone turnover in mice, we hypothesized that the increase in CFU-F may be triggered by the bone resorption that occurs following G-CSF administration. In accord with this hypothesis, administration of an inhibitor of osteoclast differentiation, osteoprotegerin (OPG), prevented the increase of CFU-F numbers induced by G-CSF. In conclusion, these data indicate that the cytokine treatment routinely used to mobilize hematopoietic stem cells could provide a readily applicable method to induce in vivo expansion of MPC for clinical applications., (Copyright 2010 Elsevier B.V. All rights reserved.)

Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle in the use of CB is its low cell dose, which is largely responsible for an elevated risk of graft failure and a significantly delayed neutrophil and platelet engraftment. As a positive correlation has been shown between the total nucleated cell (TNC) and CD34(+) cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with MSC in a cytokine cocktail. We show that this strategy can be further improved if CD3(+) and/or CD14(+) cells are first depleted from the CB MNC before ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible as clinical-grade devices and reagents are available. Ultimately, the aim of improving TNC and CD34(+) transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.

The use of umbilical cord blood (UCB) grafts for hematopoietic stem cell transplantation (HSCT) is a promising technique that permits a degree of human leukocyte antigen mismatch between the graft and the host without the concomitant higher rate of graft-versus-host disease that would be observed between an adult marrow graft and a mismatched host. A disadvantage to the use of UCB for HSCT is that immune reconstitution may be significantly delayed because of the low stem cell dose available in the graft. Ex vivo expansion of UCB CD34 cells would provide a greater number of stem cells; however, there are persistent concerns that ex vivo-expanded CD34 cells may lose pluripotency and the ability to contribute meaningfully to long-term engraftment. To address this issue, we transduced CD34-selected UCB cells with a lentiviral construct expressing luciferase, and determined homing and engraftment patterns in vivo by noninvasive bioluminescent imaging in sublethally irradiated NOD/SCID/IL-2Rgamma(-/-) (NSG) mice. Graft contribution to multilineage commitment was also confirmed by analysis of primary and secondary transplants by flow cytometry and immunohistochemistry. Our results demonstrate that, other than a mild delay at the onset of engraftment, there were no significant differences in lineage repopulation or in long-term or secondary engraftment between culture-expanded and unexpanded UCB CD34-selected cells. The results suggest that multipotent stem cells can be expanded ex vivo and can contribute meaningfully to long-term hematopoietic engraftment.

The connection between obesity and accelerated cancer progression has been established, but the mediating mechanisms are not well understood. We have shown that stromal cells from white adipose tissue (WAT) cooperate with the endothelium to promote blood vessel formation through the secretion of soluble trophic factors. Here, we hypothesize that WAT directly mediates cancer progression by serving as a source of cells that migrate to tumors and promote neovascularization. To test this hypothesis, we have evaluated the recruitment of WAT-derived cells by tumors and the effect of their engraftment on tumor growth by integrating a transgenic mouse strain engineered for expansion of traceable cells with established allograft and xenograft cancer models. Our studies show that entry of adipose stromal and endothelial cells into systemic circulation leads to their homing to and engraftment into tumor stroma and vasculature, respectively. We show that recruitment of adipose stromal cells by tumors is sufficient to promote tumor growth. Finally, we show that migration of stromal and vascular progenitor cells from WAT grafts to tumors is also associated with acceleration of cancer progression. These results provide a biological insight for the clinical association between obesity and cancer, thus outlining potential avenues for preventive and therapeutic strategies.

Objective: The effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on their osteogenic differentiation., Methods: Murine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)(+) CD31(-) CD45(-) population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1beta and TNFalpha. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR)., Results: We show that osteogenic differentiation from the MSC population is suppressed by IL-1beta and TNFalpha. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, alpha1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFalpha inhibited osteonectin and osteopontin mRNA expression and only IL-1beta reduced cell proliferation., Conclusions: The convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1beta and TNFalpha, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC.

Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed beta-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased beta-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.