Human Umbilical Cord Blood Samples Research Articles

Preexisting maternal immunity to AAV but not Cas9 impairs in utero gene editing in mice.

In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.

Novel Angiogenesis Role of GLP-1(32-36) to Rescue Diabetic Ischemic Lower Limbs via GLP-1R-Dependent Glycolysis in Mice.

Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism. In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated Glp1r-/- mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment. We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway. Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease. URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.

Permutation-based significance analysis reduces the type 1 error rate in bisulphite sequencing data analysis of human umbilical cord blood samples

ABSTRACT DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.

Error-Corrected Sequencing of Cord Bloods Identifies Pediatric AML-Associated Clonal Hematopoiesis

Background:Recently, several studies have used next-generation sequencing (NGS) to characterize an age-specific increase in the variant allele frequencies (VAF) and number of somatic mutations in AML-associated genes in adults without hematological malignancies. Termed Clonal Hematopoiesis of Indeterminate Significance (CHIP), this phenomenon is defined as being >2% VAF and associated with an increased risk of hematologic malignancy later in life. The threshold of 2% VAF is arbitrarily defined because mutations below the threshold could not be reliably called due to the inherent error rate of sequencing. In light of this, the lab has previously developed error-corrected sequencing (ECS) to identify point mutations and small indels from adult blood and marrow samples at VAF as low as 0.01%. Moving on to pediatric samples, the most commonly mutated genes in pediatric AML vary substantially from adult AML, and the prevalence of clonal hematopoiesis in pediatric AML-specific genes in early childhood is unknown. Using ECS, we investigated the prevalence of clonal hematopoiesis in 94 adult and pediatric AML-associated genes in 31 random human umbilical cord blood (UCB) samples. We hypothesized clonal mutations in cord blood would be rare because of the age-related linear acquisition of mutations observed in older individuals, but the genes in question had not been previously assessed.Methods:Random cord blood samples were obtained from the St. Louis Cord Blood Bank (http://www.slcbb.org/). ECS libraries from extracted genomic DNA were prepared according to established protocol. A total of 1602 amplicons were targeted (total queried genome space ~270kb) using an Illumina panel designed to enrich 94 genes frequently mutated in both adult and pediatric AML. The libraries were sequenced on the Illumina HiSeq 3000 platform and raw sequence data was analyzed using a validated bioinformatic pipeline established in the lab. A subset of called mutations was secondarily validated using the QX200 droplet digital PCR platform (Bio-Rad) in the Druley lab.Results and Conclusions:We found that 18.2% of cord blood samples harbor validated somatic mutations with VAFs ranging from 0.002 - 0.006. With ECS, we showed that the prevalence of clonal hematopoiesis in younger population is much higher than previously expected. These results suggest that clonal hematopoiesis is likely a stochastic process that happens in everyone as early as fetal development, yet the incidence of pediatric leukemia is estimated to be 7.7 cases per million children.Future work:In order to rule out the possibility of maternal blood contamination of the UCB, we are flow sorting CD34+ cells from the cord blood samples and screening for the mutations. DisclosuresNo relevant conflicts of interest to declare.

Sesn2 attenuates the damage of endothelial progenitor cells induced by angiotensin II through regulating the Keap1/Nrf2 signal pathway.

Endothelial progenitor cell (EPC) dysfunction is an important physiopathological mechanism in the dynamics of the formation of atherosclerosis. It has been reported that angiotensin II (Ang-II) damages the function of EPCs in atherosclerotic plaque through induction of oxidative stress. Sestrin 2 (Sesn2) serves as an antioxidant role in oxidative stress, however, the exact mechanisms underlying the dynamics of how Sesn2 may factor into EPCs after Ang-II treatments needs to be illustrated. We isolated EPCs from human umbilical cord blood samples and treated with Ang-II. Western blotting, qRT-PCR, transwell assays, immunofluorescence and so on were used to investigate the mechanisms underlying the roles of Sesn2 in EPCs treated with Ang-II. Ang-II was found to promote the apoptosis of EPCs as well as inhibited the mRNA and protein expression of Sesn2. Upregulation of Sesn2 attenuated the negative effect of Ang-II. Sesn2 increased the protein expression of Nrf2 by enhancing P62-dependent autophagy. Silencing of Nrf2 enhanced the degree of apoptosis of EPCs as well as resulted in the impairment of EPC functions through inducing the promotion of (reactive oxygen species) ROS production. Our study results indicated that Sesn2 facilitated the viability of EPCs After treatment with Ang-II, as well as provided a potential therapeutic target to alleviate the progression of atherosclerosis.

Fabrication of blood-derived elastogenic vascular grafts using electrospun fibrinogen and polycaprolactone composite scaffolds for paediatric applications.

The development of tissue-engineered vascular grafts (TEVGs) for paediatric applications must consider unique factors associated with this patient cohort. Although the increased elastogenic potential of neonatal cells offers an opportunity to overcome the long-standing challenge of in vitro elastogenesis, neonatal patients have a lower tolerance for autologous tissue harvest and require grafts that exhibit growth potential. The purpose of this study was to apply a multipronged strategy to promote elastogenesis in conjunction with umbilical cord-derived materials in the production of a functional paediatric TEVG. An initial proof-of-concept study was performed to extract fibrinogen from human umbilical cord blood samples and, through electrospinning, to produce a nanofibrous fibrinogen scaffold. This scaffold was seeded with human umbilical artery-derived smooth muscle cells (hUASMCs), and neotissue formation within the scaffold was examined using immunofluorescence microscopy. Subsequently, a polycaprolactone-reinforced porcine blood-derived fibrinogen scaffold (isolated using the same protocol as cord blood fibrinogen) was used to develop a rolled-sheet graft that employed topographical and biochemical guidance cues to promote elastogenesis and cellular orientation. This approach resulted in a TEVG with robust mechanical properties and biomimetic arrangement of extracellular matrix (ECM) with rich expression of elastic fibre-related proteins. The results of this study hold promise for further development of paediatric TEVGs and the exploration of the effects of scaffold microstructure and nanostructure on vascular cell function and ECM production.

Serological and molecular detection of Neospora caninum and Toxoplasma gondii in human umbilical cord blood and placental tissue samples

Neosporosis primarily affects cattle and dogs and is not currently considered a zoonotic disease. Toxoplasmosis is a zoonosis with a worldwide distribution that is asymptomatic in most cases, but when acquired during pregnancy, it can have serious consequences. The seropositivity rates determined by the indirect fluorescent antibody test for Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii) were 24.3% (49 samples) and 26.8% (54 samples), respectively. PCR positivity for N. caninum was observed in two samples of cord blood (1%) using the Nc5 and ITS1 gene, positivity for T. gondii was observed in 16 samples using the primer for the B1 gene (5.5% positivity in cord blood and 2.5% positivity in placental tissue). None of the samples showed structures characteristic of tissue cysts or inflammatory infiltrate on histopathology. Significant associations were observed only between N. caninum seropositivity and the presence of domestic animals (p = 0.039) and presence of dogs (p = 0.038) and between T. gondii seropositivity and basic sanitation (p = 0.04). This study obtained important findings regarding the seroprevalence and molecular detection of N. caninum and T. gondii in pregnant women; however, more studies are necessary to establish a correlation between risk factors and infection.

Contamination Rates by Delivery Method of Human Umbilical Cord Blood Samples in the United Arab Emirates and Gulf Cooperation Council Countries

The use of umbilical cord blood, which is recognized as a rich source of hematopoietic stem cells, has become an alternative source to bone marrow for transplantation. Cord blood units used for transplant might be rejected due to positive bacterial microbiology. According to common cell therapy standards a microbiology bacterial identification is required to know the type of bacterial, and to determine if the bacteria is considered critical or non-critical prior to the transplant determination.

BCR/ABL preleukemic fusion gene in subpopulations of hematopoietic stem and progenitor cells from human UCB.

The BCR/ABL preleukemic fusion gene (PFG) is one of the most frequent fusion genes in acute lymphoblastic leukemia (ALL) and was also detected in hematopoietic cells from umbilical cord blood (UCB) of healthy newborns. Since hematopoietic stem/progenitor cells (HSPC) are considered to be a critical cellular target for origination of leukemia, we have studied the presence of BCR/ABL PFG in expanded subpopulations of HSPC and differentiated cells from UCB of those healthy newborns, who have previously been tested positive for BCR/ABL by screening of their UCB mononuclear cells using RT-qPCR and FISH methods. We isolated cells from human UCB samples positive for BCR/ABL and negative controls. The isolated cells were sorted into 5 hematopoietic and progenitor cell subpopulations. We analyzed BCR/ABL in sorted and expanded subpopulations of UCB using FISH and RT-qPCR. We found that the number of BCR/ABL positive cells was similar in each studied subpopulation and the same as in differentiated lymphocytes. Our data showed that there is no specific subpopulation of hematopoietic and progenitor stem cells with an increased leukemogenic potential due to the presence of higher copies of BCR/ABL.

Insulin-Like Peptide 3 (INSL3) Serum Concentration During Human Male Fetal Life.

Context: Insulin-like peptide 3 (INSL3), a protein hormone produced by Leydig cells, may play a crucial role in testicular descent as male INSL3 knockout mice have bilateral cryptorchidism. Previous studies have measured human fetal INSL3 levels in amniotic fluid only.Objective: To measure INSL3 serum levels and mRNA in fetal umbilical cord blood and fetal testes, respectively.Design: INSL3 concentrations were assayed on 50 μl of serum from male human fetal umbilical cord blood by a non-commercial highly sensitive and specific radioimmunoassay. For secondary confirmation, quantitative real-time PCR was used to measure INSL3 relative mRNA expression in 7 age-matched human fetal testes.Setting: UT Southwestern Medical Center, Dallas, TX and Medical University of South Carolina, Charleston, SC.Patients or other Participants: Twelve human male umbilical cord blood samples and 7 human male testes were obtained from fetuses 14–21 weeks gestation. Male sex was verified by leukocyte genomic DNA SRY PCR.Interventions: None.Main Outcome Measures: Human male fetal INSL3 cord blood serum concentrations and testicular relative mRNA expression.Results: INSL3 serum concentrations during human male gestational weeks 15–20 were 2–4 times higher than published prepubertal male levels and were 5–100 times higher than previous reports of INSL3 concentrations obtained from amniotic fluid. Testicular fetal INSL3 mRNA relative expression was low from weeks 14–16, rose significantly weeks 17 and 18, and returned to low levels at week 21.Conclusions: These findings further support the role of INSL3 in human testicular descent and could prove relevant in uncovering the pathophysiology of cryptorchidism.

Detection of BPDE-DNA adducts in human umbilical cord blood by LC-MS/MS analysis

Benzo [a]pyrene (BaP) is a model compound for the study of polycyclic aromatic hydrocarbon (PAH) carcinogenesis. Upon metabolism, BaP is metabolized to the ultimate metabolite, BaP trans-7,8-diol-anti-9,10-epoxide (BPDE), that reacts with cellular DNA to form BPDE-dG adducts responsible for BaP-induced mutagenicity, carcinogenicity, and teratogenicity. In this study, we employed our developed LC-MS/MS method to detect and quantity BPDE-dG adducts present in 42 normal human umbilical cord blood samples and 42 birth defect cases. We determined that there is no significant difference in the level of BPDE-dG formation between the normal and birth defect groups. This represents the first time to use an LC-MS/MS method to quantify BPDE-dG in human umbilical blood samples. The results indicated that under experimental conditions, BPDE-dG adducts were detected in all the human umbilical cord blood samples from the normal and birth defect groups.

A Novel Predictor of Response to Gemtuzumab Ozogamicin Therapy in AML Provides Strategies for Sensitization of Leukemia Stem Cells in Individual Patients

Acute myeloid leukemia (AML) patients with normal cytogenetics, NPM1 mutation, and no FLT3-ITD are considered to be at low molecular risk (LMR). We previously reported that most LMR patients have a low LSC17 score; these patients derive benefit from the addition of low fractionated doses of gemtuzumab ozogamicin (GO) to standard treatment and have favorable survival outcomes compared to patients with high LSC17 scores (Ng, Nature 2016). We recently developed a 13-gene sub-score (LMR13) that can be calculated from the LSC17 assay; a high LMR13 score identifies not only patients with molecularly-defined LMR disease, but also patients with LMR-like gene expression (GE), treatment response, and survival outcome. Similar to LMR cases, LMR-like patients gain a significant overall, event-free, and relapse-free survival (OS, EFS, RFS) benefit from the addition of GO treatment as observed in the ALFA-0701 trial cohort (OS: P=0.05; EFS: P=0.009; RFS: P=0.02). To gain mechanistic insight into the molecular determinants of GO response in LMR and LMR-like patients, we modelled the pathway through which GO traverses upon targeting a cell using a curated list of n=245 genes that includes the GO binding receptor CD33, lysosomal markers, ATP-binding cassette (ABC) transporters, DNA damage response/repair machinery, and pro/anti apoptotic factors. Sparse statistical regression was applied to a training dataset of n=495 AML patients (GSE6891) to identify the minimal subset of pathway components that were most associated with high LMR13 scores as a surrogate for GO responsiveness. This process selected n=16 genes which were then used to construct a random forest classifier to predict GO response. The final decision-tree based model, termed GO12, retained n=12 of the 16 pathway genes, and was able to accurately identify LMR and LMR-like patients across n=5 independent AML cohorts totaling n=1188 patients (AUC≈80.8%). As expected, ALFA-0701 patients who were predicted to be LMR-like with >50% certainty achieved significantly better survival when GO was added to their induction regimen (OS: P=0.03; EFS: P To determine if there is a therapeutic window of GO effects on normal versus leukemic hematopoietic stem cells, we applied the GO12 model to GE data derived from hematopoietic stem and progenitor cell (HSPC)-enriched human umbilical cord blood (hUCB) samples (GSE29105, GSE42414, GSE58299). The baseline predicted probability of GO sensitivity of these populations was at most 17%. Simulated GE knockdown (KD) of all possible combinations of the key resistance factors up to 3 standard deviations revealed a maximal model-predicted probability of GO response of 52%, consistent with robust inherent resistance of hUCB-HSPC to GO-mediated cytotoxicity. In contrast, applying the same analysis to GE data derived from AML patient samples predicted that LSC-containing populations can be sensitized to GO with up to 75% probability through double or triple KD of key GO resistance factors. Furthermore, analysis of chromatin accessibility (ATAC-Seq) data revealed that GO toxin (calicheamicin) DNA-binding motifs are situated within genomic loci where chromatin is significantly more open in LSC-enriched than in LSC-depleted or hUCB-derived HSPC/mature cell fractions (P Disclosures Bullinger:Amgen: Honoraria, Speakers Bureau; Bayer Oncology: Research Funding; Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau. Dohner:Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Amgen: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Dombret:Seattle Genetics: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Menarini: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Ambit (Daiichi Sankyo): Consultancy, Honoraria; Sunesis: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Jazz Pharma: Consultancy, Honoraria, Research Funding; Ariad (Incyte): Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau; Roche/Genentech: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau; Cellectis: Consultancy, Honoraria, Other: Travel expenses; Celgene: Consultancy, Honoraria, Other: Travel expenses, Speakers Bureau; Immunogen: Consultancy, Honoraria; Shire-Baxalta: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Otsuka: Consultancy, Honoraria.

Contamination Rates by Delivery Method of Human Umbilical Cord Blood Samples in the United Arab Emirates and Gulf Cooperation Council Countries

Contamination Rates by Delivery Method of Human Umbilical Cord Blood Samples in the United Arab Emirates and Gulf Cooperation Council Countries

MicroRNA Microarray Profiling during Megakaryocyte Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells.

Objective In order to clarify the role of microRNAs (miRNA) in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133+ cells. Materials and Methods In this experimental study, human CD133+ hematopoietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis. Results A total of 10 and 14 miRNAs were upregulated (e.g. miR-1246 and miR-148-a) and down-regulated (e.g. miR- 551b and miR-10a) respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis. ConclusionWe conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment.

Identification of CD146 as a novel molecular actor involved in systemic sclerosis

We highlight for the first time that CD146/sCD146 is involved in fibrotic process during SSc. sCD146 could thus constitute a new biomarker to assess disease activity, and potentially a new target for therapeutic applications.

Ex Vivo Expansion and Characterization of Human Umbilical Cord Blood CD34+ Stem Cells

Limited CD34+ cell number in human umbilical cord blood (hUCB) samples stimulated the development of in vitro HSC expansion. hUCB CD34+ cells cultured with cytokine cocktails were increased about 7.3 folds after 4 days' culture in vitro. For in vivo study, CD34+ cells obtained directly from hUCB or from in vitro expansion were transplanted into NOD/SCID mice (n=4 for each group). Four groups of mice were injected with saline, 0.4 million unexpanded CD34+ cells, 0.4 million in vitro expanded CD34+ cells, and 2.9 million in vitro expanded CD34+ cells, respectively. Peripheral blood (PB) was collected and multi‐lineage differentiation was assessed at 1, 3, and 8 weeks post transplantation. CD34+ cells were decreased below 1% in group 2 and 3 but not in group 4 after 3 weeks. On the other hand, CD45+, CD41+, CD71+and CD15+ populations were increased in groups 2‐4 compared with those of 1 week. 8 weeks post transplantation, human CD34+ cells were hardly detected in PB from any groups. However, PB and bone marrow cells expressing human cell surface markers of various hematopoietic lineages were detectable in groups 2‐4 [Figure 1]. These results indicated that CD34+ cells from hUCB either before or after expansion in vitro are capable of differentiation into hematopoietic cells of various lineages in vivo. Our study also indicates that in vitro expanded CD34+ cells are successfully long term engrafted to bone marrow of mice [Figure 2]. An efficient experimental method has been developed for the expansion of hUCB CD34+ cells in vitro and these cells can be potentially explored for various applications in the clinic.

Fibroblast-endothelial partners for vascularization strategies in tissue engineering.

Cell-based approaches have emerged as a promising therapy to achieve successful vascularization in tissue engineering. Since fibroblasts activation and migration is required for physiological events relying on angiogenesis, we hypothesize herein that different fibroblasts exhibit distinct capacity to promote capillary-like structures assembly, by mature and progenitor endothelial cells (ECs). Outgrowth endothelial cells (OECs) were isolated from human umbilical cord blood samples and characterized by immunofluorescence and imaging flow cytometry for endothelial markers. Coculture systems were established using either human umbilical vein ECs (HUVECs) or OECs with fibroblasts, being evaluated at 7, 14, and 21 days of culture. Two types of human dermal fibroblasts (HDF) were used, namely neonatal human foreskin fibroblasts-1 (HFF-1) and juvenile HDF. OECs expressed EC markers and formed capillary-like structures. HFF-1 exhibited higher expression of transglutaminase-2, while HDF exhibited a higher expression of α-smooth muscle actin (α-SMA) and podoplanin, which were not observed for HFF-1. Formation of capillary-like structures was only observed in cocultures with HDF and not with HFF-1. No significant differences were found between HDF and OECs or HUVECs cocultures. These findings suggest that HDF is a preferential cell source for promoting vascularization, either using mature or progenitor ECs, probably due to their higher expression of α-SMA and podoplanin, and increased synthesis of extracellular matrix. This work opens new research possibilities regarding the use of specific fibroblast populations cocultured with ECs, as efficient partners for vascular development in regenerative medicine strategies.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.

OS079. Fetal deglycosylated apolipoprotein C-III (Apo C-III0) concentration is altered in intrauterine growth restriction

We recently demonstrated that serum lipid levels are altered in growth restricted fetuses (IUGR) [1]. We now aimed to analyse the proteome profile of umbilical cord blood in order to gain a greater understanding about metabolic changes in IUGR fetuses. umbilical cord blood serum samples of IUGR (n=15) and of gestational age matched controls (CN; n=15) were subjected to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were forwarded to MALDI-TOF mass spectrometric profiling. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis. Concentrations of proteins of interest were additionally measured by ELISA. Statistical estimations were performed by Student's t-test and calculation of Spearman's correlation coefficient. MALDI mass spectra showed on average more than 60 protein ion signals between m/z 4000 and 25,000. The six best differentiating ion signals were found at m/z 8205, m/z 8766, m/z 13,945, m/z 15,129, m/z 15,308, and m/z 16,001. One of the constituent of this proteome signature is the deglycosylated form of apolipoprotein C-III, apo C-III0 (8766 m/z) that is known to prevent triglycerides from catabolism. While total Apo CIII concentration tended to be decreased (IUGR 22.54μg/mL SD 10.25. CN 29.9μg/mL SD 15.46. p=0.1355) calculated Apo C-III0 concentration levels has been found to be more abundant in the IUGR cord blood serum samples (IUGR 1.99μg/mL SD 0.85. CN 1.15μg/mL SD 0.55. p<0.0001). Moreover, fetal triglycerid levels were significantly increased in IUGR (IUGR 16.7mg/dL SD 7.58. CN 56.5mg/dL SD 49.92. p-value after log transformation =0.0008)and apo C-III0 was highly correlated to fetal triglyceride levels (rho=0.694). Using mass spectrometric approaches we successfully developed an IUGR specific proteome signature derived from human umbilical cord blood samples. Most interesting the deglycosylated form of the apolipoprotein C-III (apoC-III0) was found to be significantly increased in IUGR and thus might lead to reduced triglyceride catabolism. This observation is in agreement with the known observation of triglyceride levels being increased in IUGR fetuses. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.