Lentiviral Arrays for Live-cell Dynamic Monitoring of Gene and Pathway Activity During Stem Cell Differentiation

Abstract

Uncovering the complexity of mesenchymal stem cell (MSC) differentiation requires novel methods to capture the dynamics of the process in a quantitative and high-throughput manner. To this end, we developed a lentiviral array (LVA) of reporters to capture the dynamics of gene and pathway activity during MSC differentiation into adipogenic, chondrogenic, and osteogenic lineages. Our results identified signature promoters and pathways with unique activation profile for each MSC lineage. In combination with chemical inhibitors, lineage-specific reporters predicted the effects of signaling pathway perturbations on MSC differentiation. Interestingly, some pathways were critical for differentiation into all lineages, while others had differential effects on each lineage. Our study suggests that when combined with large chemical or siRNA libraries, the reporter LVA can be used to uncover novel genes and signaling pathways affecting complex biological processes such as stem cell differentiation or reprogramming.

NANOG Reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF-Dependent Gene Expression

“…F, G). In addition, we subcloned the SRF DNA binding site (transcription motif, CArG‐RE) upstream of the ZsGreen fluorescent reporter into the LVDP vector that was developed in our laboratory and used it to introduce the CArG‐RE sequence into LP and LP NANOG MSC. Indeed, NANOG expression induced a 2.2 ± 0.2‐fold increase ( p < .05) in the activity of CArG‐RE (Fig.…”

“…To quantify the effect of NANOG on CArG‐response element (RE) activity, image analysis was performed as described before . Briefly cells were transduced with lentivirus dual promoter (LVDP) vector carrying the CArG‐RE.…”

CDH2 and CDH11 act as regulators of stem cell fate decisions

“…[159] In addition, the envelope of lentiviral particles was engineered to bind covalently to fibrin hydrogels during polymerization [160, 161], thereby enabling generation of lentiviral arrays (LVA) that were employed to measure the activity of several Pr/RE participating in the inflammatory response[162]. More recently, we generated a library of Pr/RE to monitor MSC differentiation towards adipogenic, osteogenic, chondrogenic or myogenic lineages and used it to identify novel pathways that may be involved in lineage specification[163, 164]. Potentially, this technology may be combined with novel microfabrication methods to determine how the extent of intercellular adhesion influences stem cell specification decisions of adult stem cells, cancer stem cells or hiPSC and potentially also the pluripotency networks that are critical for cellular reprogramming.…”

Simultaneous Pathway Activity Inference and Gene Expression Analysis Using RNA Sequencing

“…Reporter assays provide a quantitative assessment of signal transduction pathway activation by inferring the activity of pathway-specific transcription factors in terms of protein activity (Gorman et al, 1982; Bronstein et al, 1994). However, this approach is not amenable to the analysis of multiple signaling pathways in a single population of cells due to a paucity of orthogonal protein activity readouts (Bellis et al, 2011; Padmashali et al, 2014). In contrast, global gene expression profiling offers a more information-rich, unbiased approach to identify key mediators of signal transduction and gene regulation (Tian et al, 2005).…”