The binding of boronated peptides to low affinity mammalian saccharides.

A 54-member library of boronated octapeptides, with all but the boronated residue being proteinogenic, was tested for affinity to a set of saccharides commonly found on the terminus of mammalian glycans. After experimentation with a high-throughput dye-displacement assay, attention was focused on isothermal titration calorimetry as a tool to provide reliable affinity data, including enthalpy and entropy of binding. A small number of boronated peptides showed higher affinity and significant selectivity for N-acetylneuraminic acid over methyl-α-d-galactopyranoside, methyl-α/ß-l-fucopyranoside and N-acetyl-d-glucosamine. Thermodynamic data showed that for most of the boronated peptides studied, saccharide binding was associated with a significant increase in entropy, presumably resulting from the displacement of semiordered water molecules from around the sugar and/or peptide.

Progress in bio-manufacture of platelets for transfusion.

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Formation and characterisation of a modifiable soft macro-porous hyaluronic acid cryogel platform.

A facile method for the synthesis of cell supportive, highly macro-porous hyaluronic acid (HA) hydrogels via cryogelation is presented. Unmodified HA was chemically cross-linked via EDC/NHS zero-length cross-linking at sub-zero temperatures to yield cryogels with high porosity and high pore interconnectivity. The physical properties of the HA cryogels including porosity, average pore size, elasticity and swelling properties were characterised as a function of cryogelation conditions and composition of the precursor solution. The HA cryogels swell extensively in water, with the average porosities observed being ~90% under all conditions explored. The morphology of the cryogels can be controlled, allowing scaffolds with an average pore size ranging from 18 ± 2 to 87 ± 5 µm to be formed. By varying the cross-linking degree and HA concentration, a wide range of bulk elastic properties can be achieved, ranging from ~1 kPa to above 10 kPa. Preliminary cell culture experiments, with NIH 3T3 and HEK 293 cell lines, performed on biochemically modified and unmodified gels show the cryogels support cell proliferation and cell interactions, illustrating the biomedical potential of the platform.

Brief Report: Factors Released by Megakaryocytes Thrombin Cleave Osteopontin to Negatively Regulate Hematopoietic Stem Cells.

Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4 ß1 and α9 ß1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl(-/-) mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl(-/-) mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN.

Predictions for optimal mitigation of paracrine inhibitory signalling in haemopoietic stem cell cultures.

INTRODUCTION: Recent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Stimulatory signals can enhance proliferation and promote differentiation, whilst inhibitory signals can significantly limit culture output. METHODS: Numerical models of various mitigation strategies are presented and applied to determine effectiveness of these strategies toward mitigation of paracrine inhibitory signalling inherent in these culture systems. The strategies assessed include mixing, media-exchange, fed-batch and perfusion. RESULTS: The models predict that significant spatial concentration gradients exist in typical cell cultures, with important consequences for subsequent cell expansion. Media exchange is shown to be the most effective mitigation strategy, but remains labour intensive and difficult to scale-up for large culture systems. The fed-batch strategy is only effective at very small Peclet number, and its effect is diminished as the cell culture volume grows. Conversely, mixing is effective at high Peclet number, and ineffective at low Peclet number. The models predict that cell expansion in fed-batch cultures becomes independent of increasing dilution rate, consistent with experimental results previously reported in the literature. In contrast, the models predict that increasing the flow rate in perfused cultures will lead to increased cell expansion, indicating the suitability of perfusion for use as an automated, tunable strategy. The effect of initial cell seeding density is also investigated, with the model showing that perfusion outperforms dilution for all densities considered. CONCLUSIONS: The models predict that the impact of inhibitory signalling in HSC cultures can be mitigated against using media manipulation strategies, with the optimal strategy dependent upon the protein diffusion time-scale relative to the media manipulation time-scale. The key messages from this study can be applied to any complex cell culture scenario where cell-cell interactions and paracrine signalling networks impact upon cell fate and cell expansion.

FOXN1 (GFP/w) reporter hESCs enable identification of integrin-ß4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1(+) thymic epithelial progenitors.

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.

Design, synthesis and binding properties of a fluorescent α9ß1/α4ß1 integrin antagonist and its application as an in vivo probe for bone marrow haemopoietic stem cells.

The α9ß1 and α4ß1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4ß1 integrin have been thoroughly investigated with respect to HSC function, the role of α9ß1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9ß1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9ß1 integrin with potent cross-reactivity against α4ß1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9ß1 and α4ß1 integrins, which showed faster binding to α4ß1 integrin relative to α9ß1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9ß1 and α4ß1 integrins; (2) investigating the biochemical properties of α9ß1 and α4ß1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.

Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation.

We report transplanted hemopoietic stem cells (HSC) preferentially lodge within two cells of mature megakaryocytes (MM). With both populations comprising ~0.2% of bone marrow cells, this strongly suggests a key functional interaction. HSC isolated from the endosteum (eLSKSLAM) showed significantly increased hemopoietic cell proliferation while in co-culture with MM. Furthermore, eLSKSLAM progeny retained HSC potential, maintaining long-term multi-lineage reconstitution capacity in lethally ablated recipients. Increased hemopoietic cell proliferation was not MM contact dependent and could be recapitulated with media supplemented with two factors identified in MM-conditioned media: insulin-like growth factor binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1). We demonstrate that HSC express the receptor for IGF-1 and that IGF-1/IGFBP-3 induced increased hemopoietic cell proliferation can be blocked by an anti-IGF-1 neutralising antibody. However, co-cultures of 8N, 16N or 32N MM with eLSKSLAM showed that MM of individual ploidy did not significantly increase hemopoietic cell proliferation. Our data suggests that MM are an important component of the HSC niche and regulate hemopoietic cell proliferation through cytokine release.

Potent agonists of a hematopoietic stem cell cytokine receptor, c-Mpl.

Several growth factors feature prominently in the control of hematopoiesis. Thrombopoietin, a class I hematopoietic cytokine, plays critical roles in regulating hematopoietic stem cell numbers and also stimulates the production and differentiation of megakaryocytes, the bone marrow cells that ultimately produce platelets. Thrombopoietin interacts with the c-Mpl cell-surface receptor. Recently, several peptide and small-molecule agonists and antagonists of c-Mpl have been reported. We conducted a bioinformatics and molecular modeling study aimed at understanding the agonist activities of peptides that bind to c-Mpl, and developed new potent peptide agonists with low nanomolar activity. These agonists also show very high activity in human CD34(+) primary cell cultures, and doubled the mean blood platelet counts when injected into mice.

Cryogels for biomedical applications.

The use of hydrogels as support materials for the growth and proliferation of mammalian cells has been well documented as they closely mimic the gel-like properties - and in some cases also the chemical properties - of the extracellular matrix (ECM), which naturally surrounds the cells of any biological tissue. Macro-porous hydrogels set below the freezing point of the solvent, so-called 'cryogels', have recently gained significant interest in the fields of tissue engineering and in vitro cell culture, thanks to their inherent interconnected macro-porous structure and ease of formation in comparison to other macro-pore forming techniques. This review highlights recent advances in cryogelation techniques and starting materials that can be utilised to synthesise biocompatible and biologically relevant cryogels as well as discussing physicochemical characterisation techniques for these materials. Lastly, emerging trends in the application of cryogels, particularly as three-dimensional ECM mimicking scaffolds for cell culture and tissue engineering, are discussed.

Granulocyte colony stimulating factor expands hematopoietic stem cells within the central but not endosteal bone marrow region.

Granulocyte colony stimulating factor (G-CSF) is clinically well established for the mobilization of hematopoietic stem cells (HSC). Extensive data on the underlying mechanism of G-CSF induced mobilization is available; however, little is known regarding the functional effect of G-CSF on HSC within the bone marrow (BM). In this study we analyzed the proportion and number of murine HSC in the endosteal and central bone marrow regions after 4 days of G-CSF administration. We demonstrate that the number of HSC, defined as CD150(+)CD48(-)LSK cells (LSKSLAM cells), increased within the central BM region in response to G-CSF, but not within the endosteal BM region. In addition the level of CD150 and CD48 expression also increased on cells isolated from both regions. We further showed that G-CSF mobilized proportionally fewer LSKSLAM compared to LSK cells, mobilized LSKSLAM had colony forming potential and the presence of these cells can be used as a measure for mobilization efficiency. Together we provide evidence that HSC in the BM respond differently to G-CSF and this is dependent on their location. These findings will be valuable in developing new agents which specifically mobilize HSC from the endosteal BM region, which we have previously demonstrated to have significantly greater hematopoietic potential compared to their phenotypically identical counterparts located in other regions of the BM.

Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

Phenotypically identical hemopoietic stem cells isolated from different regions of bone marrow have different biologic potential.

Hemopoietic stem cells (HSCs) reside within a specified area of the bone marrow (BM) cavity called a "niche" that modulates HSC quiescence, proliferation, differentiation, and migration. Our previous studies have identified the endosteal BM region as the site for the HSC niche and demonstrated that hemopoietic stem and progenitor populations (HSPCs, LSK) isolated from different BM regions exhibit significantly different hemopoietic potential. In this study, we have analyzed subpopulations of LSK cells isolated from different regions of the BM and showed that CD150(+)CD48(-)LSK HSCs within the endosteal BM region have superior proliferative capacity and homing efficiency compared with CD150(+)CD48(-)LSK HSCs isolated from the central BM. Furthermore, we show, for the first time, that a subset of CD150(+)CD48(+)LSK progenitor cells, previously defined as B-lymphoid primed hemopoietic cells, are capable of multilineage reconstitution, however, only when isolated from the endosteal region. In addition, we provide evidence for an unrecognized role of CD48 in HSC homing. Together, our data provide strong evidence that highly purified HSCs show functional differences depending on their origin within the BM and that the most primitive HSCs reside within the endosteal BM region.

The effect of bovine endosteum-derived particles on the proliferation of human mesenchymal stem cells.

There is a large biomanufacturing and clinical need for cost-effective and simple techniques to expand mesenchymal stem cells whilst retaining their multipotency. Endosteum-derived particles were prepared, characterised and examined as a biomaterial to facilitate the in vitro expansion of human mesenchymal stem cells. Bovine endosteum-derived particles are composed of chondroitin sulphate glycosaminoglycans with 4- and 6-sulphation and N-sulphated heparan sulphate glycosaminoglycans. The particles were positive for perlecan, laminin and fibronectin by immunohistochemistry and alpha-mannose, alpha-glucose, terminal N-acetyl-alpha-D-glucosamine, N-acetyl-alpha-galactosamine and alpha-fucose, using lectin binding. Human mesenchymal stem cells showed greater than 96% attachment to the particles after one day in spinner culture. After 7 days, the stem cells on decalcified particles were viable and had a 5-fold higher growth than the stem cells grown on Cytodex-2 beads. Significantly more stem cells were recovered from decalcified particles compared with mineralised particles (P < 0.05). Differentiation to chondrogenic, osteogenic and adipogenic lineages was maintained after culturing stem cells on the demineralised particles. We conclude that bovine endosteum-derived particles can be extracted from bone marrow to retain sulphated proteoglycans and glycosylated proteins. These particles are a suitable biomaterial for supporting the growth and retaining the multipotency of human mesenchymal stem cells.

Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML.

Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the betac subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene betac Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34(+)CD38(-)CD123(+) leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins.

Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.

The use of experimental murine models to assess novel agents of hematopoietic stem and progenitor cell mobilization.

The recent explosion in the understanding of the cellular and molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) mobilization has facilitated development of novel therapeutic agents, targeted at improving mobilization kinetics as well as HSPC yield. With the development of new agents comes the challenge of choosing efficient and relevant preclinical studies for the testing of the HSPC mobilization efficacy of these agents. This article reviews the use of the mouse as a convenient small animal model of HSPC mobilization and transplantation, and outlines the range of murine assays that can be applied to assess novel HSPC mobilizing agents. Techniques to demonstrate murine HSPC mobilization are discussed, as well as the role of murine assays to confirm human HSPC mobilization, and techniques to investigate the biologic phenotype of HSPC mobilized by these novel agents. Technical aspects regarding mobilization regimens and control arms, and choice of experimental animals are also discussed.

Detection and quantification of functionally defined hematopoietic progenitor cells and tissue specific mRNA within the peripheral blood of myeloma patients after administration of granulocyte colony-stimulating factor and erythropoietin.

OBJECTIVE: Hematopoietic progenitor cells (HPC) as well as tissue committed stem cells expressing mRNA specific to various somatic tissues are thought to be part of the CD34+ bone marrow compartment. In this study, we explore and quantify their mobilization in patients with multiple myeloma undergoing chemotherapy upon administration of granulocyte colony-stimulating factor (G-CSF) plus/minus erythropoietin (EPO). PATIENTS AND METHODS: HPC were quantified by flow cytometry and functional assays within the blood of healthy donors and myeloma patients before and after chemotherapy followed by G-CSF or G-CSF + EPO given subcutaneously. The mRNA expression was studied by quantitative polymerase chain reaction (PCR). Cytokines and peripheral blood protease levels were measured by an enzyme-linked immunosorbent assay. RESULTS: EPO did not significantly alter the number of HPC mobilized by G-CSF alone, and mRNA specific for liver, brain, muscle and kidney was detected in both treatment groups. Quantitative PCR analysis revealed a 2.7-fold increased expression of glial fibrillary acidic protein after G-CSF + EPO administration compared to G-CSF alone (P = 0.003). The concentration of G-CSF rose from 62 +/- 22 pg/mL and 48 +/- 10 pg/mL to 28 +/- 9 ng/mL and 85 +/- 10 ng/mL after 10 d of treatment with G-CSF and G-CSF + EPO, respectively. The concentration of neutrophil elastase (NE) rose only in the G-CSF group by a factor 1.5. CONCLUSION: The alteration of G-CSF and NE levels as well as the expression of tissue committed RNA after the administration of EPO in addition to G-CSF indicate that different growth factors mobilize different stem cells that might potentially be used for the support of tissue repair in future treatment protocols.

Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues.

Previous studies revealed that mAb BB9 reacts with a subset of CD34(+) human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map BB9 expression throughout hematopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and surrounding endothelial cells are BB9(+). Thereafter, BB9 is expressed by primitive hematopoietic cells in fetal liver and in umbilical cord blood (UCB). BB9(+)CD34(+) UCB cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice contribute 10-fold higher numbers of multilineage blood cells than their CD34(+)BB9(-) counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34(+) population. Protein microsequencing of the 160-kDa band corresponding to the BB9 protein established its identity as that of somatic angiotensin-converting enzyme (ACE). Although the role of ACE on human HSCs remains to be determined, these studies designate ACE as a hitherto unrecognized marker of human HSCs throughout hematopoietic ontogeny and adulthood.