In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3®

Introduction. Primary cells in vitro culture scale-up is a crucial issue in cell-based tissue and organ regeneration therapy. Reducing costs and space occupied by the cells cultured in vitro has been an important target. Cells cultured in vitro with the use of bioreactor with dextran microcarriers (Cytodex®) have potentially a chance to meet many of the cell therapy requirements. Material and methods. We used collagen-coated carriers (Cytodex3®) and a spinner flask bioreactor to develop environment suitable for human myoblast proliferation. In parallel, standard adherent in vitro culture conditions for myoblasts propagation (T-flask) were conducted. Cell cycle characterization, senescence, myogenic gene ex­pression and cell apoptosis were evaluated in order to find differences between two culture systems under study. Results. The number of cells obtained in bioreactor per 106 of starting cells population was approximately ten times lower in comparison with T-flask culture system. The microcarriers cultured adult myoblasts in compari­son with the regular T-flask culture showed faster and more advanced replicative aging and lower proliferative potential. Moreover, the percentage of the cells that entailed an irreversible cell arrest (G0 phase) was also significantly (p < 0.0001) increased. Conclusions. Our results suggest that population of primary human myoblasts obtained from adult individuals and propagated on dextran microcarriers did not meet the requirements of the regenerative medicine regarding quantity and quality of the cells obtained. Nonetheless, further optimization of the cell scaling up process including both microcarriers and/or bioreactor program is still an important option

Hypoglycemia and the Origin of Hypoxia-Induced Reduction in Human Fetal Growth

The most well known reproductive consequence of residence at high altitude (HA >2700 m) is reduction in fetal growth. Reduced fetoplacental oxygenation is an underlying cause of pregnancy pathologies, including intrauterine growth restriction and preeclampsia, which are more common at HA. Therefore, altitude is a natural experimental model to study the etiology of pregnancy pathophysiologies. We have shown that the proximate cause of decreased fetal growth is not reduced oxygen availability, delivery, or consumption. We therefore asked whether glucose, the primary substrate for fetal growth, might be decreased and/or whether altered fetoplacental glucose metabolism might account for reduced fetal growth at HA.Doppler and ultrasound were used to measure maternal uterine and fetal umbilical blood flows in 69 and 58 residents of 400 vs 3600 m. Arterial and venous blood samples from mother and fetus were collected at elective cesarean delivery and analyzed for glucose, lactate and insulin. Maternal delivery and fetal uptakes for oxygen and glucose were calculated.The maternal arterial – venous glucose concentration difference was greater at HA. However, umbilical venous and arterial glucose concentrations were markedly decreased, resulting in lower glucose delivery at 3600 m. Fetal glucose consumption was reduced by >28%, but strongly correlated with glucose delivery, highlighting the relevance of glucose concentration to fetal uptake. At altitude, fetal lactate levels were increased, insulin concentrations decreased, and the expression of GLUT1 glucose transporter protein in the placental basal membrane was reduced.Our results support that preferential anaerobic consumption of glucose by the placenta at high altitude spares oxygen for fetal use, but limits glucose availability for fetal growth. Thus reduced fetal growth at high altitude is associated with fetal hypoglycemia, hypoinsulinemia and a trend towards lactacidemia. Our data support that placentally-mediated reduction in glucose transport is an initiating factor for reduced fetal growth under conditions of chronic hypoxemia

The 12p13.33/RAD52 locus and genetic susceptibility to squamous cell cancers of upper aerodigestive tract

Acknowledgments: The authors thank all of the participants who took part in this research and the funders and support and technical staff who made this study possible. We also acknowledge and thank The Cancer Genome Atlas initiative whose data contributed heavily to this study. Funding: Funding for study coordination, genotyping of replication studies and statistical analysis was provided by the US National Institutes of Health (R01 CA092039 05/05S1) and the National Institute of Dental and Craniofacial Research (1R03DE020116). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Post-ischemic hypothermia attenuates loss of the vascular basement membrane proteins, agrin and SPARC, and the blood\u2013brain barrier disruption after global cerebral ischemia

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague\u2013Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42\u201345 mm Hg). In a separate group of animals, a mild (32 \ub0C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from 3c500 brain vessels (20\u2013100 \u3bcm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using \ub3H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p<0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCMcaptured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h postischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for \ub3H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.Peer reviewed: YesNRC publication: Ye

Dynamic analysis of the blood-brain barrier disruption in experimental stroke using time domain in vivo fluorescence imaging

The blood-brain barrier (BBB) disruption following cerebral ischemia can be exploited to deliver imaging agents and therapeutics into the brain. The aim of this study was (a) to establish novel in vivo optical imaging methods for longitudinal assessment of the BBB disruption and (b) to assess size selectivity and temporal patterns of the BBB disruption after a transient focal ischemia. The BBB permeability was assessed using in vivo time domain near-infrared optical imaging after contrast enhancement with either free Cy5.5 (1 kDa) or Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa) in mice subjected to either 60- or 20-minute transient middle cerebral artery occlusion (MCAO) and various times of reperfusion (up to 14 days). In vivo imaging observations were corroborated by ex vivo brain imaging and microscopic analyses of fluorescent tracer extravasation. The in vivo optical contrast enhancement with Cy5.5 was spatially larger than that observed with BSA-Cy5.5. Longitudinal studies after a transient 20-minute MCAO suggested a bilateral BBB disruption, more pronounced in the ipsilateral hemisphere, peaking at day 7 and resolving at day 14 after ischemia. The area differential between the BBB disruption for small and large molecules could potentially be useful as a surrogate imaging marker for assessing perinfarct tissues to which neuroprotective therapies of appropriate sizes could be deliveredNRC publication: Ye

Postischemic hypothermia protects against loss of agrin and SPARC from the vascular basement membrane in global cerebral ischemia

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins and subsequent endothelial cell activation and proliferation 1. However, the fate and role of BM proteoglycans and non-structural BM constituents, including SPARC (BM-40, osteonectin), in ischemic blood-brain barrier (BBB) pathophysiology remains unknown.NRC publication: Ye