Lentiviral arrays for high-throughput, live monitoring gene and pathway activation during stem cell differentiation

Roshan Padmashali, Panagiotis Mistriotis1, Maoshih Liang1, Stelios T. Andreadis1, 2
1. Bioengineering Laboratory, 908 Furnas Hall, Department of Chemical and Biological Engineering, University at
Buffalo, State University of New York, Amherst, NY 14260-4200, USA
2. Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203, USA

Introduction: Mesenchymal stem cells (MSCs) are abundant and multipotent cells with great potential for regenerative medicine and tissue engineering. The conventional tools for investigating gene expression such as qPCR are laborious, cost intensive and they cannot provide functional information on the pathways involved in biological processes. On the other hand, LentiViral microArrays (LVA) provide live-cell, high-throughput monitoring of activation of biological pathways during biological processes such as MSC differentiation. In this paper, we employed LVA with a library of lentiviral vectors to monitor pathway activation during MSC differentiation into bone, fat or cartilage. In addition, we hypothesized that use of LVA in combination with a
small molecular chemical library might identify pathways that control MSC fate commitment.
Materials and Methods: To address this hypothesis, first we engineered a dual-promoter lentiviral vectorâ??LVDP containing a sp(ae)cific transcriptional regulatory element encoding for green fluorescence protein for monitoring gene regulation. Additionally this vector contains a constitutive promoter driving red fluorescence protein providing an internal normalization strategy, thereby allowing measurements of gene or pathway dynamics independent of the gene transfer efficiency. We developed a library of lineage specific reporters containing promoters such as adiponectin, SOX9 and Runx2 for adipogenesis, chondrogenesis and osteogenesis, respectively as well as transcription factor response motifs e.g. Vitamin D, SMAD, C/EBP etc. By

Figure 1 (a) In the experiment setup, MSCs were transduced with various LVDP viruses and the

dynamics of lineage-specific reporters were

monitored real-time with fluoresunce microscopy during differentiation. (b) Heat map summarizing the effects of chemical inhibitors on lineage-specific reporters during differentiation.

employing automated fluorescence microscopy, we determined the kinetics of lineage-specific pathway activation in MSC derived from two different anatomic locations i.e. hair follicle and bone marrow, (Fig 1(a)). Furthermore, we screened a small array of chemical inhibitors to monitor the pathways that may control
differentiation into one or more lineages.

(b)

Results and Discussion: Our results demonstrate differential

reporter activation between the two MSC sources, suggesting that the regulatory pathways controlling differentiation may be dependent on the source of MSC. To identify key molecules affecting MSC differentiation, several inhibitors were selected to illustrate the effects on lineage specific reporters. We found that inhibition of TGF-β receptor promoted adipogenesis as evidenced by decreased activity of the adiponectin promoter. Similarly, inhibition of p38 increased the activity of both chondrogenic (SMAD2/3) and osteogenic (OCBOX) reporters, while inhibition of TGF-β or FGF receptors down-regulated chondrogenic or osteogenic reporters, respectively. Inhibition of the PI3 kinase and glucocorticoids readily suppressed the activity of adipogenic pathways. Also, inhibition of JNK and ERK blocked the expression of osteo-specific reporters such as Vitamin-D and OCBOX. Finally, we identified novel pathways differentially
regulating MSC differentiation along different lineages. The complete results were summarized in Fig 1(b).

Conclusion: We developed a LVA system that enabled efficient monitoring of gene and pathway activation in MSC differentiation. This approach also allowed us to identify differences in the kinetics of pathway activation between different MSC lines, despite their similar differentiation potential. Notably, the LVA enabled screening of small molecule libraries that identified pathways that were necessary for differentiation along particular lineages. Our results suggest that the LVA is a tool with great potential to enhance understanding of stem cell fate specification as well as pathway regulation and drug discovery.