Characterization of Adherent Umbilical Cord Blood Stromal Cells Regarding Passage, Cell Number, and Nano-biomarking Utilization

Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.

Umbilical cord blood transplantation from unrelated donors in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

There are very few disease-specific studies focusing on outcomes of umbilical cord blood transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia. We report the outcome of 45 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia who underwent myeloablative single unit cord blood transplantation from unrelated donors within the GETH/GITMO cooperative group. Conditioning regimens were based on combinations of thiotepa, busulfan, cyclophospamide or fludarabine, and antithymocyte globulin. At the time of transplantation, 35 patients (78%) were in first complete remission, four (8%) in second complete remission and six (14%) in third or subsequent response. The cumulative incidence of myeloid engraftment was 96% at a median time of 20 days and significantly better for patients receiving higher doses of CD34(+) cells. The incidence of acute grade II-IV graft-versus-host disease was 31%, while that of overall chronic graft-versus-host disease was 53%. Treatment-related mortality was 17% at day +100 and 31% at 5 years. The 5-year relapse, event-free survival and overall survival rates were 31%, 36% and 44%, respectively. Although the event-free and overall survival rates in patients without BCR/ABL transcripts detectable at time of transplant were better than those in whom BCR/ABL transcripts were detected (46% versus 24% and 60% versus 30%, respectively) these differences were not statistically significant in the univariate analysis (P=0.07). These results demonstrate that umbilical cord blood transplantation from unrelated donors can be a curative treatment for a substantial number of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Ultrastructural characterization of bovine umbilical cord blood cells

The umbilical cord blood (UCB) is an important source of pluripotent stem cells, which motivated researches on ontogeny and transplantation. The morphological characterization of umbilical cord cells is the first step to establish subsequent experiments on these areas. Although some information on humans can be found, no data on UCB is available for bovines. Therefore, this work is the first attempt to conduct an ultrastructural characterization of bovine umbilical cord blood. Blood was collected from the umbilical cord of twenty fetuses by punction of the umbilical vein. Samples were processed for whole leucocytes observation by centrifugation and the buffy coat was collected. Cells were washed and pelleted and prepared according to the standard protocol of the transmission electron microscopy. The presence of cells with morphologic characteristics compatible with the precursors from the erythrocytic, neutrophilic, eosinophilic, basophilic, and lymphocytic lineages was observed. Atypical cells with peculiar morphological features, strongly similar to apoptotic cells, were seen. Bovine neutrophils with three types of cytoplasmic granules were also found in the blood. The ultrastructural characteristics of observed bovine UCB cells where similar to those found in other species, suggesting that bovines could possibly constitute an experimental model for approaches on UCB cells research.

Umbilical cord blood and neonatal endothelin-1 levels in preterm newborns with and without respiratory distress syndrome

Increased pulmonary vascular resistance in preterm newborn infants with respiratory distress syndrome is suggested, and endothelin-1 plays an important role in pulmonary vascular reactivity in newborns. We determined umbilical cord blood and neonatal (second sample) levels of endothelin-1 in 18 preterm newborns with respiratory distress syndrome who had no clinical or echocardiographic diagnosis of pulmonary hypertension and 22 without respiratory distress syndrome (gestational ages: 31.4 ± 1.6 and 29.3 ± 2.3 weeks, respectively). Umbilical cord blood and a second blood sample taken 18 to 40 h after birth were used for endothelin-1 determination by enzyme immunoassay. Median umbilical cord blood endothelin-1 levels were similar in both groups (control: 10.9 and respiratory distress syndrome: 11.4 pg/mL) and were significantly higher than in the second sample (control: 1.7 pg/mL and respiratory distress syndrome: 3.5 pg/mL, P < 0.001 for both groups). Median endothelin-1 levels in the second sample were significantly higher in children with respiratory distress syndrome than in control infants (P < 0.001). There were significant positive correlations between second sample endothelin-1 and Score for Neonatal Acute Physiology and Perinatal Extension II (r = 0.36, P = 0.02), and duration of mechanical ventilation (r = 0.64, P = 0.02). A slower decline of endothelin-1 from birth to 40 h of life was observed in newborns with respiratory distress syndrome when compared to controls. A significant correlation between neonatal endothelin-1 levels and some illness-severity signs suggests that endothelin-1 plays a role in the natural course of respiratory distress syndrome in preterm newborns.

Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount™-BD and ISHAGE protocols

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount™ - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount™ (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 ± 19.4 CD34+ cells/µL and with the ProCount™ method we found 36.6 ± 23.2 CD34+ cells/µL. With the ProCount™ method, CD34+ bright cell counts were 9.3 ± 8.2 cells/µL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Human umbilical cord blood cells in infarcted rats

Therapy with bone marrow-derived cells has been used in ischemic patients with reported success. The aim of this study was to determine the therapeutic efficacy of fresh and frozen human umbilical cord blood cells (hUCB) in Wistar rats submitted to permanent occlusion of the left coronary artery. Three hours after myocardial infarction, 2 x 10(7) hUCB cells or vehicle were administered by intramyocardial injection. The animals were divided into five groups: control (N = 10), sham operated (N = 10), infarcted that received vehicle (N = 9), infarcted treated with cryopreserved hUCB (N = 7), and infarcted treated with fresh hUCB (N = 5). Cardiac function was evaluated by electrocardiogram (ECG) and echocardiogram (ECHO) before cell therapy, and by ECG, ECHO, cardiopulmonary test, and left ventricular pressure measurements 3 weeks later. After 3 weeks, both groups treated with hUCB still had Q wave present in L1, âQRS >90° and reduced shortening fraction (less than 50%). In addition, cardiac indexes of left ventricular contractility and relaxation were 5484 ± 875 and -4032 ± 643 mmHg (cryopreserved hUCB) and 4585 ± 955 and -2862 ± 590 mmHg (fresh hUCB), respectively. These values were not statistically different from those of saline-treated animals. Cardiopulmonary exercise test profile was typical of infarcted hearts; exercise time was about 14 min and maximal VO2 was 24.77 ± 5.00 mL·kg-1·min-1. These data show that hUCB therapy did not improve the cardiac function of infarcted animals or prevent cardiac remodeling.

Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10(6) cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6) cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

Analysis of acylcarnitine profiles in umbilical cord blood and during the early neonatal period by electrospray ionization tandem mass spectrometry

Acylcarnitine profiling by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a potent tool for the diagnosis and screening of fatty acid oxidation and organic acid disorders. Few studies have analyzed free carnitine and acylcarnitines in dried blood spots (DBS) of umbilical cord blood (CB) and the postnatal changes in the concentrations of these analytes. We have investigated these metabolites in healthy exclusively breastfed neonates and examined possible effects of birth weight and gestational age. DBS of CB were collected from 162 adequate for gestational age neonates. Paired DBS of heel-prick blood were collected 4-8 days after birth from 106 of these neonates, the majority exclusively breastfed. Methanol extracts of DBS with deuterium-labeled internal standards were derivatized before analysis by ESI-MS/MS. Most of the analytes were measured using a full-scan method. The levels of the major long-chain acylcarnitines, palmitoylcarnitine, stearoylcarnitine, and oleoylcarnitine, increased by 27, 12, and 109%, respectively, in the first week of life. Free carnitine and acetylcarnitine had a modest increase: 8 and 11%, respectively. Propionylcarnitine presented a different behavior, decreasing 9% during the period. The correlations between birth weight or gestational age and the concentrations of the analytes in DBS were weak (r £ 0.20) or nonsignificant. Adaptation to breast milk as the sole source of nutrients can explain the increase of these metabolites along the early neonatal period. Acylcarnitine profiling in CB should have a role in the early detection of metabolic disorders in high-risk neonates.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Umbilical Cord Blood Banking

The umbilical cord, previously considered as waste and discarded at birth, is a source of haematopoietic stem cells. Current therapeutic uses of umbilical cord blood stem cells and the promise of these cells for the treatment of degenerative diseases in the future have led to the establishment of cord blood banks in many parts of the world. Although umbilical cord blood banking raises many ethical and legal issues, this article focuses on the controversy created by the coexistence of public and private cord blood banks in many countries. Policy statements adopted by professional associations and advisory groups indicate that, based on the current state of medical evidence, childbearing women with no current or potential familial need of stem cell transplantation should be encouraged to donate cord blood to public banks.

T lymphocyte subsets of the umbilical cord blood of dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Levels of toxic and essential metals in maternal and umbilical cord blood from selected areas of South Africa-results of a pilot study

This pilot study uses concentrations of metals in maternal and cord blood at delivery, in seven selected geographical areas of South Africa, to determine prenatal environmental exposure to toxic metals. Samples of maternal and cord whole blood were analysed for levels of cadmium, mercury, lead, manganese, cobalt, copper, zinc, arsenic and selenium. Levels of some measured metals differed by site, indicating different environmental pollution levels in the regions selected for the study. Mercury levels were elevated in two coastal populations studied (Atlantic and Indian Ocean sites) with mothers from the Atlantic site having the highest median concentration of 1.78 mu g/L ranging from 0.44 to 8.82 mu g/L, which was found to be highly significant (p < 0.001) when compared to other sites, except the Indian Ocean site. The highest concentration of cadmium was measured in maternal blood from the Atlantic site with a median value of 0.25 mu g/L (range 0.05-0.89 mu g/L), and statistical significance of p < 0.032, when compared to all other sites studied, and p < 0.001 and p < 0.004 when compared to rural and industrial sites respectively, confounding factor for elevated cadmium levels was found to be cigarette smoking. Levels of lead were highest in the urban site, with a median value of 32.9 mu g/L (range 16-81.5 mu g/L), and statistically significant when compared with other sites (p < 0.003). Levels of selenium were highest in the Atlantic site reaching statistical significance (p < 0.001). All analysed metals were detected in umbilical cord blood samples and differed between sites, with mercury being highest in the Atlantic site (p < 0.001), lead being highest in the urban site (p < 0.004) and selenium in the Atlantic site (p < 0.001). To the best of our knowledge this pilot investigation is the first study performed in South Africa that measured multiple metals in delivering mothers and umbilical cord blood samples. These results will inform the selection of the geographical sites requiring further investigation in the main study.

Integrated analysis of halogenated organic pollutants in sub-millilitre volumes of venous and umbilical cord blood sera

A rapid, robust and economical method for the analysis of persistent halogenated organic compounds in small volumes of human serum and umbilical cord blood is described. The pollutants studied cover a broad range of molecules of contemporary epidemiological and legislative concern, including polychlorobiphenyls (PCBs), polychlorobenzenes (CBs), hexachlorocyclohexanes (HCHs), DDTs, polychlorostyrenes (PCSs) and polybromodiphenyl ethers (PBDEs). Extraction and clean-up with n-hexane and concentrated sulphuric acid was followed with analysis by gas chromatography coupled to electron capture (GC-ECD) and GC coupled to negative ion chemical ionisation mass spectrometry (GC-NICI-MS). The advantages of this method rest in the broad range of analytes and its simplicity and robustness, while the use of concentrated sulphuric acid extraction/clean-up destroys viruses that may be present in the samples. Small volumes of reference serum between 50 and 1000 μL were extracted and the limits of detection/quantification and repeatability were determined. Recoveries of spiked compounds for the extraction of small volumes (≥300 μL) of the spiked reference serum were between 90% and 120%. The coefficients of variation of repeatability ranged from 0.1-14%, depending on the compound. Samples of 4-year-old serum and umbilical cord blood (n = 73 and 40, respectively) from a population inhabiting a village near a chloro-alkali plant were screened for the above-mentioned halogenated pollutants using this method and the results are briefly described. © 2010 Springer-Verlag.