ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells

Using differential display polymerase chain reaction, a gene was identified in CD34+-enriched populations that had with low or absent expression in CD34- populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34+ cells than in CD34- cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34+38- phenotype and was down-regulated with the proliferation and differentiation of CD34+ cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Identification and characterization of a lymphocytic Rho-GTPase effector: rhotekin-2

Rhotekin belongs to the group of proteins containing a Rho-binding domain that are target peptides (effectors) for the Rho-GTPases. We previously identified a novel cDNA with homology to human rhotekin and in this study we cloned and characterized the coding region of this novel 12-exon gene. The ORF encodes a 609 amino-acid protein comprising a Class I Rho-binding domain and pleckstrin homology (PH) domain. Cellular cDNA expression of this new protein, designated Rhotekin-2 (RTKN2), was shown in the cytosol and nucleus of CHO cells. Using bioinformatics and RTPCR we identified three major splice variants, which vary in both the Rho-binding and PH domains. Real-time PCR studies showed exclusive RTKN2 expression in pooled lymphocytes and further purification indicated sole expression in CD4^_pos T-cells and bone marrow-derived B-cells. Gene expression was increased in quiescent T-cells but negligible in activated proliferating cells. In malignant samples expression was absent in myeloid leukaemias, low in most B-cell malignancies and CD8^pos T-cell malignancies, but very high in CD4^pos/CD8^pos T-lymphoblastic lymphoma. As the Rho family is critical in lymphocyte development and function, RTKN2 may play an important role in lymphopoiesis.

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34+ hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Prevention of suicide : police powers, parliamentary intent and judicial interpretation

While in most countries suicide is no longer a crime, it is also acknowledged that the state has an interest in the preservation of human life, prevention of suicide, and protection of vulnerable persons from harming themselves. In a civil, secular and democratic society, however, the public law principle of state protective powers has to be balanced against the private law principle of personal autonomy (personal self-determination). Under the doctrine of autonomy, competent adults of sound mind can make legally binding voluntary choices, including the so-called ‘death-choice’ (refusal of life-sustaining or life-prolonging treatment as well as suicide). To add to the complexity, whereas the powers of the state in relation to suicide and its prevention have been codified, the concepts of personal autonomy and personal liberty are grounded in common law. Stuart v Kirkland–Veenstra [2008] VSCA 32, which is at present being considered by the High Court of Australia, exemplifies tensions that arise in the suicide-prevention area of jurisprudence. This article explores powers and duties of police officers in relation to suicide prevention and the notion of mental illness by reference to the Kirkland–Veenstra case, the relevant statutory framework and the common law.

RTKN2 induces NF-KappaB dependent resistance to intrinsic apoptosis in HEK cells and REgulates BCL-2 genes in human CD4+ lymphocytes

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4+ T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNFα. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4+ T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4+ T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.

Mechanisms of resistance to the cytotoxic effects of oxysterols in human leukemic cells

We have developed hematopoietic cells resistant to the cytotoxic effects of oxysterols. Oxysterol-resistant HL60 cells were generated by continuous exposure to three different oxysterols—25-hydroxycholesterol (25-OHC), 7-beta-hydroxycholesterol (7β-OHC) and 7-keto-cholesterol (7κ-C). We investigated the effects of 25-OHC, 7β-OHC, 7κ-C and the apoptotic agent staurosporine on these cells. The effect of the calcium channel blocker nifedipine on oxysterol cytotoxicity was also investigated. Differential display and real-time PCR were used to quantitate gene expression of oxysterol-sensitive and -resistant cells. Our results demonstrate that resistance to the cytotoxic effects of oxysterols is relatively specific to the type of oxysterol, and that the cytotoxicity of 25-OHC but not that of 7β-OHC and 7κ-C, appears to occur by a calcium dependent mechanism. Oxysterol-resistant cells demonstrated no significant difference in the expression of several genes previously implicated in oxysterol resistance, but expressed the bcl-2 gene at significantly lower levels than those observed in control cells. We identified three novel genes differentially expressed in resistant cells when compared to HL60 control cells. Taken together, the results of this study reveal potentially novel mechanisms of oxysterol cytotoxicity and resistance, and indicate that cytotoxicity of 25-OHC, 7β-OHC and 7κ-C occur by independent, yet overlapping mechanisms.

Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen

Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1–6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21^Cip1 gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for exvivo expansion of these cells.

Three-dimensional tissue scaffolds from interbonded poly(e-caprolactone) fibrous matrices with controlled porosity

In this article, we report on the preparation and cell culture performance of a novel fibrous matrix that has an interbonded fiber architecture, excellent pore interconnectivity, and controlled pore size and porosity. The fibrous matrices were prepared by combining melt-bonding of short synthetic fibers with a template leaching technique. The microcomputed tomography and scanning electron microscopy imaging verified that the fibers in the matrix were highly bonded, forming unique isotropic pore architectures. The average pore size and porosity of the fibrous matrices were controlled by the fiber/template ratio. The matrices having the average pore size of 120, 207, 813, and 994 mm, with the respective porosity of 73%, 88%, 96%, and 97%, were investigated. The applicability of the matrix as a three-dimensional (3D) tissue scaffold for cell culture was demonstrated with two cell lines, rat skin fibroblast and Chinese hamster ovary, and the influences of the matrix porosity and surface area on the cell culture performance were examined. Both cell lines grew successfully in the matrices, but they showed different preferences in pore size and porosity. Compared with two-dimensional tissue culture plates, the cell number on 3D fibrous matrices was increased by 97.27% for the Chinese hamster ovary cells and 49.46% for the fibroblasts after 21 days of culture. The fibroblasts in the matrices not only grew along the fiber surface but also bridged among the fibers, which was much different from those on two-dimensional scaffolds. Such an interbonded fibrous matrix may be useful for developing new fiber-based 3D tissue scaffolds for various cell culture applications.

Three dimensional scaffolds from multilobal fibres for enhanced cell growth

Multilobal fibres are irregularly shaped fibres with several surface channels or grooves. Scaffolds created from these fibres have high surface area which may enhance cell density. This study compared the cell growth of dermal fibroblasts and osteoblast-like SaOS2 cells on multilobal fibre nonwoven scaffolds to the conventional fibre scaffolds. Cells were cultured on round nylon, trilobal nylon, round polyethylene terephthalate (PET) and multilobal PET scaffolds for 14 days. There were more cells growing on trilobal nylon and PET multilobal scaffolds than their round counterparts. The preference to the type of multilobal scaffolds was cell dependent. The density of fibroblast increased 37% on trilobal nylon compared to round nylon scaffolds after 14 days of culture. SaOS2 cells preferred the multilobal PET scaffolds, exhibiting a 66% increase in cell number after 14 days of culture. Scaffolds manufactured from multilobal fibres have the ability to accommodate a high number of cells, demonstrating a great potential in tissue engineering applications.

Biofunctionalization of 3D nylon 6,6 scaffolds using a two-step surface modification

Nylon is a relatively inert polymer. The ability to easily functionalize nylon with biomolecules will improve the utilization of nylon in biological systems. A potential use of the biofunctionalized nylon scaffolds is in devices for cell therapeutics that can specifically select cells present in small numbers, such as hematopoietic stem cells. This study developed a versatile and simple two-step technique combining oxygen plasma treatment with wet silanization to graft biomolecules onto nylon 6,6 3D porous scaffolds. Scaffolds that were exposed to oxygen plasma exhibited up to 13-fold increase in silane attachment ((3-mercaptopropyl)trimethoxysilane/(3-aminopropyl)trimethoxysilane) compared to untreated scaffolds. To address the limitation of nondestructive characterization of the surface chemistry of 3D scaffolds, fluorescent CdSe/ZnS nanoparticles were used as a reporting tool for -NH(2) functionalized surfaces. Scaffolds that were covalently bound with neutravidin protein remained stable in phosphate buffered saline up to four months. Functionality of the neutravidin-grafted scaffolds was demonstrated by the specific binding of CD4 cells to the scaffold via CD4-specific antibody. Ultimately, these neutravidin-functionalized 3D nylon scaffolds could be easily customized on demand utilizing a plethora of biotinylated biomolecules (antibodies, enzymes and proteins) to select for specific cell of interest. This technique can be extended to other applications, including the enhancement of cell-scaffold interactions.

Enhanced cell growth using non-woven scaffolds of multilobal fibres

Multilobal fibres contain several grooves and have higher surface area than round fibres. Cell density can be enhanced when cultured on scaffolds manufactured with multilobal fibres. This study compared the cell growth of dermal fibroblasts and osteoblast-like SaOS2 cells on polymeric scaffolds produced from multilobal fibres to the conventional round-fibred scaffolds. Cells were cultured on round nylon 6,6, trilobal nylon 6,6, round polyethylene terephthalate (PET) and multilobal PET scaffolds for 14 days. There were more cells cultured on trilobal nylon 6,6 and PET multilobal scaffolds than their round counterparts. Preference to the type of multilobal scaffolds was cell dependent. Fibroblasts increased by 21.8 ± 1.9 fold to 6.3 × 105 cells (p < 0.001) when cultured on trilobal nylon 6,6 scaffolds while SaOS2 cells exhibited a 16.7 ± 2.8 fold increase (2.9 × 105 cells, p < 0.001) on the multilobal PET scaffolds after 14 days of culture. The ability of multilobal fibres to accommodate large quantities of cells presents an excellent alternative to round fibres as scaffolds for tissue engineering.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bonemarrowfailure and immunodeficiencies. The currentmethods forHSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell- free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellularmatrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and itmay also provide insights into the constitution of the niche in which these cells reside in the bone marrow.

Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34+ hematopoietic progenitor cells

Background aims. Cord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.

Methods. This is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34 hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.

Results. Optimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.

Conclusions. This study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.