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.

Feasibility of trialling cord blood stem cell treatments for cerebral palsy in Australia

Umbilical cord blood may have therapeutic benefit in children with cerebral palsy (CP), but further studies are required. On first appearance it seems that Australia is well placed for such a trial because we have excellence in CP research backed by extensive CP registers, and both public and private cord blood banks. We aimed to examine the possibilities of conducting a trial of autologous umbilical cord blood cells (UCBCs) as a treatment for children with CP in Australia.

Vitamins D and A can be successfully measured by LC-MS/MS in cord blood diluted plasma

OBJECTIVES: In widely used protocols for the collection and isolation of cord blood mononuclear cells, investigators are left with substantial volumes of diluted plasma which could be used for other measurements. The aim of this study was to ascertain the validity of umbilical cord blood (UCB) diluted plasma samples for vitamin D, A and E analysis compared to UCB serum samples. DESIGN & METHODS: Twenty UCB matched samples of diluted plasma and serum were collected. The samples were analysed by two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods on two separate occasions. RESULTS: The results of 25(OH)D3 obtained by the two laboratories demonstrated close agreement with a mean difference of 0.14nmol/L [95% confidence interval (95% CI), -6.8 to 7.1]. Both methods demonstrate close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma; mean difference 2.2nmol/L [95% CI, -9.5 to 13.9] and 4.1nmol/L [95% CI, -14.5 to 6.1] for the results from Lab A and Lab B, respectively. Vitamin A was quantified by Lab A in UCB serum and diluted UCB plasma; mean difference 0.07μmol/L [95% CI, -0.41 to 0.28]. Results of 25(OH)D3 epimer and vitamin E in the diluted UCB plasma were below the limit of quantification, and could not be compared with UCB serum. CONCLUSIONS: Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC-MS/MS. By contrast, quantification of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in analytical sensitivity.

Adiponectin in umbillcal cord blood is inversely related to low-density lipoprotein cholesteril but not ethnicity

Context: Adiponectin is a recognized protective risk marker for cardiovascular disease in adults and is associated with an optimal lipid profile. The role of adiponectin at birth is not well understood, and its relationship with the neonatal lipid profile is unknown. Because ethnic disparities in cardiovascular risk have been attributed to low adiponectin and its associated low high-density lipoprotein cholesterol (HDL-C), investigation at birth may help determine the etiology of these risk patterns.

Objective: Our objective was to investigate the relationship between neonatal adiponectin and lipid profile at birth in two ethnic groups in cord blood.

Design, Setting, and Participants: Seventy-four healthy mothers and their newborns of South Asian and White European origin were studied in this cross-sectional study at St. Mary’s Hospital, Manchester, United Kingdom.

Main Outcome Measures: Serum adiponectin, total cholesterol, HDL-C, low-density lipoprotein cholesterol (LDL-C), and triglyceride levels were measured in umbilical venous blood at birth and in maternal blood collected at 28 wk gestation.

Results: Cord adiponectin was significantly inversely associated with cord LDL-C (r = –0.32; P = 0.005) but not HDL-C. In a multiple regression analysis, cord LDL-C remained the most significant association of cord adiponectin (ß = –0.13; P < 0.001). We did not find any significant ethnic differences in cord adiponectin or lipids with the exception of triglycerides, which were significantly lower in South Asian newborns (P < 0.05).

Conclusion: This is the first report of an inverse relationship between cord adiponectin and LDL-C at birth. In contrast to adult studies, we found no significant association between adiponectin and HDL-C in cord blood. Our results and the strong independent association between adiponectin and HDL-C observed in adult studies suggest a role for adiponectin in lipid metabolism. Ethnic differences in adiponectin may arise after birth.

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.

Cord blood monocyte-derived inflammatory cytokines suppress IL-2 and induce nonclassic "T(H)2-type" immunity associated with development of food allergy

Food allergy is a major health burden in early childhood. Infants who develop food allergy display a proinflammatory immune profile in cord blood, but how this is related to interleukin-4 (IL-4)/T helper 2 (T(H)2)-type immunity characteristic of allergy is unknown. In a general population-derived birth cohort, we found that in infants who developed food allergy, cord blood displayed a higher monocyte to CD4(+) T cell ratio and a lower proportion of natural regulatory T cell (nT(reg)) in relation to duration of labor. CD14(+) monocytes of food-allergic infants secreted higher amounts of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) in response to lipopolysaccharide. In the presence of the mucosal cytokine transforming growth factor-β, these inflammatory cytokines suppressed IL-2 expression by CD4(+) T cells. In the absence of IL-2, inflammatory cytokines decreased the number of activated nT(reg) and diverted the differentiation of both nT(reg) and naïve CD4(+) T cells toward an IL-4-expressing nonclassical TH2 phenotype. These findings provide a mechanistic explanation for susceptibility to food allergy in infants and suggest anti-inflammatory approaches to its prevention.

Identification of novel genes differentially expressed in haemopoietic progenitor cells

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.

Expansion of human hematopoietic stem/progenitor cells on decellularized matrix scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.

Enhanced Ex Vivo expansion of human hematopoietic progenitors on native and spin coated acellular matrices prepared from bone marrow stromal cells

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion.

Framing the discourses of harm and loss: a case study of power relations, mobile phones, and children in Australia

The current penetration of mobile phones in Australia is 92% and it records one of the world’s highest rates of ownership among children under 18. The paper reviews the literature on mobile phones and Australian children and examines the various discourses dominating the public debates; the systematic frames used in these discourses; and whose interests are served in the process. The frames discussed fall under the optimistic (gains); pessimistic (losses, costs or harms); pluralistic (technology per se is neutral but how it is used matters); historical development (skills learnt and the importance of using mobiles); futuristic predictions (promises and dangers for the future); current uses (connectivity, convergence and interactivity); and the techno-realist view (as a mixed blessing) views of technology. Taking the critical perspective and borrowing from Joshua Meyrowitz, the paper illustrates how mobile phones have eroded parental power over how, when, where and with whom their children communicate, surpassing adult supervision, intervention or knowledge, while at the same time, becoming a ‘digital leash’ for parents to re-establish their control an d an ‘umbilical cord’ for their off spring to remain connect! ed with parents, at all times.

Mobile phones and children : an Australian perspective

Mobile phones in Australia record one of the world’s highest rates of ownership among children under 18. This paper examines issues of mobile phones and Australian children and the various discourses (systematic frames) used in discussing their effects. These are the optimistic (gains); pessimistic (losses, costs or harms); pluralistic (technology per se is neutral but how it is used matters); historical development (importance and skills learnt); futuristic predictions (promises and dangers); current uses (connectivity, convergence and interactivity); and techno-realist view (as a mixed blessing). Taking the Justification View of Technology that sees technological adoption as a gamble and borrowing from Joshua Meyrowitz, it examines how mobile phones have eroded parental power over how, when, where and with whom their children communicate, while at the same time, becoming a ‘digital leash’ for parents to re-establish their control and an ‘umbilical cord’ of children to remain connected with parents at all times.

Homocysteine concentration, related B vitamins, and betaine in pregnant women recruited to the Seychelles Child Development Study

Background: Both folate and betaine are important predictors of total homocysteine (tHcy) during pregnancy. However, studies to date have only been undertaken in populations with Western dietary patterns.

Objective: We investigated the predictors of tHcy in pregnant women recruited in the Seychelles, a population where access to fortified foods is limited and where women habitually consume diets rich in fish, eggs, rice, and fruit.

Design: Pregnant women (n = 226) provided blood samples at enrollment, at week 28 of gestation, and at delivery. Cord blood was obtained from a subset of participants (n = 135).

Results: As in other studies, maternal tHcy was lower during pregnancy than at delivery, whereas folate and vitamin B-12 status declined significantly to delivery. Despite low maternal folate status at delivery (median: 9.0 nmol/L), with 35% of women in the deficient range (serum folate: <6.8 nmol/L), cord blood folate status (median: 40.2 nmol/L) was similar to concentrations reported in Western populations. Folate was a significant predictor of tHcy at all time points (P < 0.001). In contrast with previous studies, betaine was only a significant predictor of maternal tHcy (P < 0.001) when the essential amino acid methionine was low.

Conclusions: The current study reports 2 important findings. First, fetal requirements for folate are paramount, such that cord blood folate status is maintained, even when maternal status is low. Second, betaine is a significant predictor of tHcy in pregnant women with low serum folate and low serum methionine concentrations.

An aqueous Chlorella extract inhibits IL-5 production by mast cells in vitro and reduces ovalbumin-induced eosinophil infiltration in the airway in mice in vivo

An aqueous extract of the edible microalga, Chlorella pyrenoidosa (CP) (1), has recently been tested for its immunomodulatory effects in a human clinical trial. Here, the CP extract was dialyzed and fractionated using Sephadex G 100 chromatography. The effects of a dialyzed aqueous CP extract, fraction 2, on mast cell mediator release in vitro and ovalbumin-induced allergic airway inflammation in vivo were examined. In vitro, treatment of mouse bone marrow-derived mast cells with 2 for 18 h significantly inhibited antigen (trinitrophenyl-BSA)-induced IL-5 production. In vivo, treatment of mice with 2 during ovalbumin sensitization and stimulation process significantly reduced eosinophil and neutrophil infiltration in the airways. Moreover, fractions obtained by size exclusion chromatography of 2 inhibited IgE-dependent cytokine GM-CSF production from human cord blood-derived mast cells. Taken together, these results suggest that 2 is composed of biopolymers with anti-allergic potential.

Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor

Osteoclasts are bone resorbing multinucleated cells (MNCs) derived from macrophage progenitors. IL-33 has been reported to drive osteoclastogenesis independently of receptor activator of NFκB ligand (RANKL) but this remains controversial as later studies did not confirm this. We found IL-33 clearly elicited functional dentine-resorbing osteoclast formation from human adult monocytes. However, monocytes from only 3 of 12 donors responded this way, while all responded to RANKL. Human cord blood-derived progenitors and murine bone marrow macrophages lacked an osteoclastogenic response to IL-33. In RAW264.7 cells, IL-33 elicited NFκB and p38 responses but not NFATc1 signals (suggesting poor osteoclastogenic responses) and formed only mononuclear tartrate-resistant acid phosphatase positive (TRAP(+)) cells. Since TGFβ boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFβ co-treatment, which resulted in small numbers of MNCs expressing key osteoclast markers TRAP and calcitonin receptors. Thus, IL-33 possesses weak osteoclastogenic activity suggesting pathological significance and, perhaps, explaining previous conflicting reports.