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

Respiratory dysfunction in unsedated dogs with golden retriever muscular dystrophy

Golden retriever muscular dystrophy (GRMD) is a well-established model of Duchenne muscular dystrophy. The value of this model would be greatly enhanced with practical tools to monitor progression of respiratory dysfunction during treatment trials. Arterial blood gas analysis, tidal breathing spirometry, and respiratory inductance plethysmography (RIP) were performed to determine if quantifiable abnormalities could be identified in unsedated, untrained, GRMD dogs. Results from 11 dogs with a mild phenotype of GRMD and 11 age-matched carriers were compared. Arterial blood gas analysis was successfully performed in all dogs, spirometry in 21 of 22 (95%) dogs, and RIP in 18 of 20 (90%) dogs. Partial pressure of carbon dioxide and bicarbonate concentration were higher in GRMD dogs. Tidal breathing peak expiratory flows were markedly higher in GRMD dogs. Abnormal abdominal motion was present in 7 of 10 (70%) GRMD dogs. Each technique provided objective, quantifiable measures that will be useful for monitoring respiratory function in GRMD dogs during clinical trials while avoiding the influence of sedation on results. Increased expiratory flows and the pattern of abdominal breathing are novel findings, not reported in people with Duchenne muscular dystrophy, and might be a consequence of hyperinflation