using-microfluidic-devices-to-measure-lifespan-cellular-phenotypes

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Peking University

Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.

Human feeder cells support establishment and definitive endoderm differentiation of human embryonic stem cells.

CCAAT enhancer binding protein-α (C/EBP-α) is a transcript factor that regulates adipocyte differentiation and induces apoptosis in hepatic stellate cells (HSCs) in vivo and in vitro. However, the effect of C/EBP-α on hepatocytes in vivo remains unknown. This study investigated whether C/EBP-α exerts different apoptotic effects on hepatocytes and HSCs in vitro and in vivo. An adenovirus vector-expressing C/EBP-α gene was constructed, and a rat hepatic stellate cell lines (HSC-T6) and hepatocytes were transfected. A CCl(4)-induced liver fibrosis model in mice was also utilized. C/EBP-α induced apoptosis in hepatocytes and HSCs, but a significant difference between these cell types was observed in vitro. The mitochondrial pathway was involved in the apoptotic process and was predominant in HSC-T6 apoptosis. In the CCl(4)-induced mice liver fibrosis model, the administration of Ad-C/EBP-α decreased extracellular matrix deposition, including collagen and hydroxyproline content, and γ-GT levels, a marker of liver damage, were reduced significantly. Immunohistochemistry and TUNEL assay results showed an increase of apoptosis in HSCs, but hepatocytes were less affected. C/EBP-α induced differential apoptotic effects in hepatocytes and HSCs in vitro and in vivo. This differential effect could be a potential target for the treatment of hepatic fibrosis with little hepatic toxicity.

C/EBP-α ameliorates CCl(4)-induced liver fibrosis in mice through promoting apoptosis of hepatic stellate cells with little apoptotic effect on hepatocytes in vitro and in vivo.

Qi Ou-yang

Peking University

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells