Evaluation of Endothelial Cells Differentiated from Amniotic Fluid-Derived Stem Cells

Amniotic fluid holds great promise as a stem cell source, especially in neonatal applications where autologous cells can be isolated and used. This study examined chemical-mediated differentiation of amniotic fluid-derived stem cells (AFSC) into endothelial cells and verified the function of AFSC-derived endothelial cells (AFSC-EC). AFSC were isolated from amniotic fluid obtained from second trimester amnioreduction as part of therapeutic intervention from pregnancies affected with twin-twin transfusion syndrome. Undifferentiated AFSC were of normal karyotype with a subpopulation of cells positive for the embryonic stem cell marker SSEA4, hematopoietic stem cell marker c-kit, and mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105. Additionally, these cells were negative for the endothelial marker CD31 and hematopoietic differentiation marker CD45. AFSC were cultured in endothelial growth media with concentrations of vascular endothelial growth factor (VEGF) ranging from 1 to 100 ng/mL. After 2 weeks, AFSC-EC expressed von Willebrand factor, endothelial nitric oxide synthase, CD31, VE-cadherin, and VEGF receptor 2. Additionally, the percentage of cells expressing CD31 was positively correlated with VEGF concentration up to 50 ng/mL, with no increase at higher concentrations. AFSC-EC showed a decrease in stem cells markers c-kit and SSEA4 and were morphologically similar to human umbilical vein endothelial cells (HUVEC). In functional assays, AFSC-EC formed networks and metabolized acetylated low-density lipoprotein, also characteristic of HUVEC. Nitrate levels for AFSC-EC, an indirect measure of nitric oxide synthesis, were significantly higher than undifferentiated controls and significantly lower than HUVEC. These results indicate that AFSC can differentiate into functional endothelial-like cells and may have the potential to provide vascularization for constructs used in regenerative medicine strategies

Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes

Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC

Antibiotic Prophylaxis for Presumptive Group B Streptococcal Infection in Preterm Premature Rupture of the Membranes: Effect on Neonatal and Maternal Infectious Morbidity

Objective: The purpose of this study was to determine if the prevalence of neonatal and maternal infectious morbidity in patients with preterm premature rupture of membranes (PROM) who received ampicillin prophylaxis for presemptive group B streptococcal colonization is increased compared to those who received no prophylaxis

Antepartum management of rhesus alloimmunisation. A literature review

Introducción: la aloinmunización Rh es una enfer-medad frecuente en Colombia pese al uso de inmuno-globulina anti-D (Rho) en los embarazos de alto riesgo. Asimismo, es una condición que requiere la identificación temprana de los factores de riesgo, así como el adecuado tamizaje con el fin de lograr una remisión oportuna a una unidad materno-fetal con experiencia para disminuir las complicaciones fetales y brindar la terapia indicada en caso de ser necesario.Objetivo: revisar la exactitud de los métodos de tamizaje y de diagnóstico temprano, así como también la efectividad de los métodos no invasivos e invasivos de tratamiento.Metodología: se realizó una revisión de la lite-ratura existente de acuerdo con las bases de datos PubMed, EBSCO, Ovid y ProQuest desde el año 2000 hasta el 2008, la cual incluyó artículos de revisión e investigaciones originales. Resultados: la titulación de anticuerpos y el pico de velocidad máximo sistólico de la arteria cerebral media son las herramientas que permiten realizar la evaluación y la identificación de las pacientes en riesgo. El tratamiento incluye transfusión intrauterina y parto oportuno mientras que la prevención con la inmunoglobulina anti-D (Rho) continúa indicada.Conclusión: la aloinmunización Rh aún es una pato-logía de interés en el control de las pacientes obstétricas de bajo y alto riesgo. El conocimiento que se obtenga de la enfermedad permitirá realizar el diagnóstico opor-tuno y, de esta manera, identificar los fetos en riesgo que son susceptibles de terapia intrauterina.Introduction: rhesus alloimmunisation remains a common disease in Colombia in spite of universal immunisation having been implemented with immunoglobulin anti-D (Rho) for all susceptible pregnancies. Rh alloimmunisation is a condition requiring risk factors to be identified, all pregnancies to be suitably screened and timely referral to a maternal foetal medicine unit ensured to minimise foetal complications and provide foetal intervention as necessary. Objective: this review was aimed at summarising the available data to provide the reader with tools helping to improve medical care by reviewing the exactitude of screening methods and early diagnosis and the effectiveness of non-invasive and invasive methods of treatment.Methodology: the literature in PubMed, EBSCO, Ovid and ProQuest databases was reviewed. Original papers, reviews, guidelines and bulletins published between 2000 and 2008 were included. Results: antibody titres and middle cerebral artery Doppler were seen to be the screening tools usually used for identifying haemolytic anaemia in the foetus and neonates in Rh alloimmunisation. Rh alloimmunization treatment included close follow-up, intrauterine transfusion and timely delivery and prevention of Rh alloimmunisation by immunoglobulin anti- D (Rho). Conclusion: knowledge of the disease will lead to early recognition of the risk factors and early diagnosis for identifying foetuses at risk which are susceptible to intrauterine therapy

Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC tandem mass spectrometry

Using the bottom-up approach and liquid chromatography (LC) in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-β-amyloid (1 17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high-performance LC tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10 antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyroglutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig VL genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core-fucosylated, biantennary, complex-type structures containing zero to two galactose residues

Serial Intrauterine Transfusions for a Hydropic Fetus with Severe Anemia and Thrombocytopenia Caused by Parvovirus: Lessons Learned

Abstract Introduction Fetal exsanguination is a rare complication of cordocentesis. Successful correction of fetal thrombocytopenia is essential for the reduction of risks. Case Report A 25-year-old, gravida 3, P2-0-0-0-2, was referred at 27 weeks of gestation for evaluation of newly diagnosed nonimmune hydrops secondary to parvovirus infection. Despite the use of ancillary platelet transfusions to correct the severe fetal thrombocytopenia, prolonged bleeding from the cord puncture site still occurred, necessitating five intrauterine transfusions to ultimately correct the fetal anemia. Conclusions The use of a smaller-diameter procedure needle, correction of the fetal thrombocytopenia early in the procedure, and external cord compression with the ultrasound transducer were ultimately successful measures in allowing for minimal loss of transfused red cells from the intravascular compartment