Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. 75:766770, 2012. (C) 2011 Wiley Periodicals, Inc.

Prediction of the Temperature Distribution of Partially Submersed Umbilical Cables

The objective of this work is to predict the temperature distribution of partially submersed umbilical cables under different operating and environmental conditions. The commercial code Fluent (R) was used to simulate the heat transfer and the air fluid flow of part of a vertical umbilical cable near the air-water interface. A free-convective three-dimensional turbulent flow in open-ended vertical annuli was solved. The influence of parameters such as the heat dissipating rate, wind velocity, air temperature and solar radiation was analyzed. The influence of the presence of a radiation shield consisting of a partially submersed cylindrical steel tube was also considered. The air flow and the buoyancy-driven convective heat transfer in the annular region between the steel tube and the umbilical cable were calculated using the standard k-epsilon turbulence model. The radiative heat transfer between the umbilical external surface and the radiation shield was calculated using the Discrete Ordinates model. The results indicate that the influence of a hot environment and intense solar radiation may affect the umbilical cable performance in its dry portion.

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.

The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: A quantitative morphological and immunohistochemical study

Previous studies have shown that particulate matter (PM) compromise birth weight and placental morphology. We hypothesized that exposing mice to ambient PM would affect umbilical cord (UC) morphology. To test this, mice were kept in paired open-top exposure chambers at the same location and ambient conditions but, in one chamber, the air was filtered (F) and, in the other, it was not (NF). UCs were analysed stereologically and by immunohistochemistry to localize isoprostane and endothelin receptors. The cords of mice from NF chambers were smaller in volume due to loss of mucoid connective tissue and decrease in volume of collagen. These structural changes and in umbilical vessels were associated with greater volumes of regions immunostained for isoprostane, ETAR and ETBR. Findings indicate that the adverse effects of PM on birth weight may be mediated in part by alterations in UC structure or imbalances in the endogenous regulators of vascular tone and oxidative stress. (C) 2012 Elsevier Inc. All rights reserved.

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aims. Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. Methods. MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (alpha-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive (R) system. After a mean of 18 +/- 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. Results. Post-thaw cell recovery: 114 +/- 2.90% (mean +/- SEM). Recovery of viable cells: 93.46 +/- 4.41%, 90.17 +/- 4.55% and 81.03 +/- 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 +/- 1.56%, 72.71 +/- 2.12%, 70.20 +/- 2.39% and 63.02 +/- 2.33% (P<0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 +/- 0.17 X, with a doubling time of 38 +/- 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. Conclusions. A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.

Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.

Prediction of the temperature distribution of partially submersed umbilical cables

The objective of this work is to predict the temperature distribution of partially submersed umbilical cables under different operating and environmental conditions. The commercial code Fluent® was used to simulate the heat transfer and the air fluid flow of part of a vertical umbilical cable near the air-water interface. A free-convective three-dimensional turbulent flow in open-ended vertical annuli was solved. The influence of parameters such as the heat dissipating rate, wind velocity, air temperature and solar radiation was analyzed. The influence of the presence of a radiation shield consisting of a partially submersed cylindrical steel tube was also considered. The air flow and the buoyancydriven convective heat transfer in the annular region between the steel tube and the umbilical cable were calculated using the standard k-ε turbulence model. The radiative heat transfer between the umbilical external surface and the radiation shield was calculated using the Discrete Ordinates model. The results indicate that the influence of a hot environment and intense solar radiation may affect the umbilical cable performance in its dry portion.

[Complex umbilical cord vein pulsations in cases with twin-twin transfusion syndrome: prognostic implications]

Umbilical vein pulsations (UV-P) are due to various etiologies. The aim of the present study was to investigate the incidence and type of UV-P in monochorionic twins with twin-twin transfusion syndrome (TTTS).

Single umbilical artery in twin pregnancies

Our purpose was to evaluate the antenatal incidence of single umbilical artery (SUA) in twin pregnancies according to chorionicity and to assess its relationship with outcome.

Evaluation of the isoflurane-sparing effects of lidocaine infusion during umbilical surgery in calves

OBJECTIVE: To evaluate the isoflurane-sparing effects of lidocaine administered by constant rate infusion (CRI) during umbilical surgery in calves. STUDY DESIGN: Randomized 'blinded' prospective clinical study. ANIMALS: Thirty calves (mean 4.7 +/- SD 2.5 weeks old) undergoing umbilical surgery. METHODS: After premedication with xylazine (0.1 mg kg(-1) , IM), anaesthesia was induced with ketamine (4 mg kg(-1) , IV) and maintained with isoflurane in O(2) administered through a circle breathing system. The calves were assigned randomly to receive a bolus of 2 mg kg(-1) lidocaine IV after induction of anaesthesia, followed by CRI of 50 mug kg(-1) minute(-1) (group L, n=15) or a bolus and CRI of 0.9% sodium chloride (NaCl, group S, n=15). End-tidal isoflurane was adjusted to achieve adequate depth of anaesthesia. Heart rate, direct arterial blood pressure and body temperature were measured intraoperatively. Groups were compared by t- tests, anova or Mann-Whitney rank sum test as appropriate. RESULTS: The end-tidal concentration of isoflurane (median, IQR) was significantly lower in group L [1.0% (0.94-1.1)] compared to group S [1.2% (1.1-1.5)], indicating a 16.7% reduction in anaesthetic requirement during lidocaine CRI. Cardiopulmonary parameters and recovery times did not differ significantly between groups. CONCLUSION AND CLINICAL RELEVANCE: Lidocaine CRI may be used as a supplement to inhalation anaesthesia during umbilical surgery in calves in countries where such a protocol would be within the legal requirements for veterinary use in food animals. This study did not show any measurable benefit to the calves other than a reduction in isoflurane requirement.

Stem Cells From Umbilical Cord Wharton's Jelly From Preterm Birth Have Neuroglial Differentiation Potential

Objective:The aim of the study is to determine the neuroglial differentiation potential of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from preterm birth when compared to term delivery.Study Design:The WJ-MSCs from umbilical cords of preterm birth and term controls were isolated and induced into neural progenitors. The cells were analyzed for neuroglial markers by flow cytometry, real-time polymerase chain reaction, and immunocytochemistry. Results:Independent of gestational age, a subset of WJ-MSC displayed the neural progenitor cell markers Nestin and Musashi-1 and the mature neural markers microtubule-associated protein 2, glial fibrillary acidic protein, and myelin basic protein. Neuroglial induction of WJ-MSCs from term and preterm birth resulted in the enhanced transcription of Nestin and Musashi-1.Conclusions:Undifferentiated WJ-MSCs from preterm birth express neuroglial markers and can be successfully induced into neural progenitors similar to term controls. Their potential use as cellular graft in neuroregenerative therapy for peripartum brain injury in preterm birth has to be tested.

Use of human embryonic stem cells and umbilical cord blood stem cells for research and therapy: a prospective survey among health care professionals and patients in Switzerland

BACKGROUND: Scientific progress in the biology of hematopoietic stem cells (HSCs) provides opportunities for advances in therapy for different diseases. While stem cell sources such as umbilical cord blood (UCB) are unproblematic, other sources such as human embryonic stem cells (hESCs) raise ethical concerns. STUDY DESIGN AND METHODS: In a prospective survey we established the ethical acceptability of collection, research, and therapy with UCB HSCs versus hESCs among health care professionals, pregnant women, patients undergoing in vitro fertilization therapy, parents, and HSC donors and recipients in Switzerland. RESULTS: There was overall agreement about an ethical justification for the collection of UCB for research and therapy in the majority of participants (82%). In contrast, research and therapy with hESCs was acceptable only by a minority (38% of all responders). The collection of hESCs solely created for HSC collection purposes met overall with the lowest approval rates. Hematologists displayed among the participants the highest acceptance rates for the use of hESCs with 55% for collection, 63% for research, and 73% for therapy. CONCLUSIONS: This is the first study assessing the perception of hESCs for research and therapy in comparison with UCB HSCs in different target groups that are exposed directly, indirectly, or not at all to stem cell-based medicine. Our study shows that the debate over the legitimacy of embryo-destructive transplantation medicine is far from over as particularly hESC research continues to present an ethical problem to an overwhelming majority among laypersons and even among health care professionals.