title: Live-cell, temporal gene expression analysis of osteogenic differentiation in adipose-derived stem cells.
abstract: Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3-5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs.
authors.affiliation: 1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University , Providence, Rhode Island.
authors.forename: Hetal V
authors.initials: HV
authors.lastname: Desai
authors.forename: Indu S
authors.initials: IS
authors.lastname: Voruganti
authors.forename: Chathuraka
authors.initials: C
authors.lastname: Jayasuriya
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
authors.forename: Eric M
authors.initials: EM
authors.lastname: Darling
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000293:Adolescent; D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008875:Middle Aged; D015335:Molecular Probes / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics*; D015203:Reproducibility of Results; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D013997:Time Factors; D055815:Young Adult
chemical_list: D015335:Molecular Probes; D000469:Alkaline Phosphatase