Impact of Inflammatory Signals on the Regenerative, Anti-inflammatory and Immunomodulatory Potential of Human Mesenchymal Stem Cells and their Secretome

Adult multipotent stem cells can be easily isolated from a variety of adult human tissues and organs including bone marrow and adipose tissue. Especially mesenchymal stromal cells (MSCs) possess immunomodulatory capabilities affecting the majority of immune cells. As of November 2015, more than 540 clinical trials utilising MSCs have been registered in the database clinicaltrials.org.
Notably, the therapeutic benefit of MSC-administration revealed in different proof of concept and clinical studies is nowadays believed to be a consequence of paracrine/endocrine effects rather than driven by the engraftment of MSCs into affected tissues and differentiation towards lost cell types. Related to their proposed paracrine mode of action, several pre-clinical reports and a recent clinical treatment attempt of a Graft-versus-host disease (GvHD) patient provided evidence that MSCs exert their therapeutic functions at least partly via extracellular vesicles (EVs) like exosomes and microvesicles.

After transplantation into damaged/lesioned tissue, MSCs are exposed to inflammatory signals which are mostly sensed through Toll-like receptors (TLRs) and the Tumor Necrosis Facror Receptor I (TNFRI). Notable, in vitro exposure of MSCs to pro-inflammatory signals is known to improve their anti-inflammatory and regenerative potential.

In summary, cell-free approaches including EV-based therapies potentially involving inflammatory priming could be considered as an alternative, more cost effective and safer therapeutic option for numerous degenerative disorders and associated symptoms and complications.


Hypothesis:

We hypothesise that priming with inflammatory signals can increase the anti-inflammatory potential of MSCs and their secretome.
Aims:

- Determination of the most efficient priming protocol
- Investigation of anti-inflammatory and immunomodulatory potential of MSC and and secreted factors before and after inflammatory priming

Planned Research:

Human MSCs will be cultivated and stimulated with pro-inflammatory signals. Unstimulated MSCs and cells exposed to different pro-inflammatory trigger will be compared regarding their anti-inflammatory potential. Here, a NF-kappaB-luciferase based screening system will be used (already established in the PI`s lab). In parallel, the immunomodulatory potential will be assessed in the macrophage cell line THP-1. Briefly, polarisation of the macrophages in M1 and M2 phenotype will be investigated using immunocytochemistry, ELISA and flow cytometry. Finally, the `bystander` effects will be assessed. In addition, we will investigate the influence of inflammatory signals on MSC proliferation, apoptosis, viability, migration, and differentiation into bone, cartilage and fat cells.

Methods:

- Human stem cell culture
- Cell culture of cell lines
- Confocal laser scanning microscopy
- Immunocytochemistry
- Time-lapse microscopy
- Migration assays
- Stem cell differentiation assays
- Viability (MTT) and proliferation assays
- Gene reporter assays
- RT-PCR and qPCR
- Flow cytometry
- Western blotting

Key Words: Stem Cells, Regenerative Medicine, Inflammation

More information:

http://www.wideralab.org