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.

A Short Interfering Rna Molecular Beacon for the Attenuation of Mycobacterial Infection

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SOX2 and OCT4 mRNA-Expressing Cells, Detected by Molecular Beacons, Localize to the Center of Neurospheres during Differentiation

“…For instance, it is difficult to know how many beacons penetrate each cell during transfection, and also whether each cell takes up an equal amount of probes [44]. Furthermore, the dynamic range of MBs appears inferior to qRT-PCR, since SOX2 -MBs could only detect a 2-fold reduction in SOX2 mRNA expression in monolayers of differentiated LUHMES cells (Figure 3C), while qRT-PCR demonstrated a 20-fold decrease in SOX2 mRNA levels (Figure 5).…”

Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation

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