(150x) Confirming the Extent and Mechanisms of the Immunosuppressive Enhancement of hMSCs Initiated By Col/Hep Layer By Layer Coatings

Statement of Purpose: There has been an increasing need for highly potent human mesenchymal stromal cells (hMSCs) for the treatment of a number of chronic diseases. Though, for therapies, several millions of cells may be needed for each dose. A current method of reducing dosage and increasing therapeutic potential is to add soluble Interferon Gamma (IFN). Unfortunately, it has been shown that pretreatment with IFN suppresses proliferation of hMSCs. In order to mitigate this negative effect, cells were cultured on unique layer-by-layer (LbL) coatings, composed of collagen and heparin. A side effect found to accompany this approach is an enhancement of immunosuppressive properties. A main concern here is to identify the specific pathways by which the coatings enhance IFN activity. Doing so will allow further evolution of artificial extracellular matrices. First, the hypothesis that the LbL coatings negate the anti-proliferative effect of IFN and enhance IFN’s activity by the activation of the Janus kinase (JAK) pathway, while also immobilizing IFN at proximity to the cell surface, creating a HEP+IFN complex that regulates IFN activity is to be tested.

Methods: The hMSCs were purchased from RoosterBio, sourced from several donors. Coatings (1) used 6.5 bilayers made up by sodium heparin from Celsius Laboratories, Inc., lyphosized type I collagen sponges from Integra Lifesciences Holdings Corp. and Poly(ethylene imine) (PEI) solution from Sigma-Aldrich, each in sodium acetate buffers at pH 5, 4 and 5 respectively. Multiple inhibitors are to be used. As of now, a JAK inhibitor, AG490 from Selleckchem and Sigma Aldrich has been used in the experiments. Upon seeding, the cells were incubated with either normal culture medium or inhibitor incorporated culture medium for 24 hours before being washed with PBS and incubated with either fresh culture medium or IFN incorporated culture medium(50ng/mL), for 3 or 6 days. Preliminary tests were done with PrestoBlue, on a 96 well plate to ascertain the minimum concentration needed for the drug to present as intended and if higher concentrations are toxic to the cells. Several concentrations were tested, ranging from 0.25 to 100μM. Upon settling on 1 μM, experiments began on 24 well plates, using a colorimetric assay for indoleamine-pyrrole 2,3- dioxygenase (IDO) with inhibited and LbL conditions to ascertain if and how much the coatings affect the amount of IDO secreted by the cells and thereby the effect on immunosuppressive properties. The assay works by first incorporating a standard solution to separated supernatant to permit IDO to convert L- tryptophan to N-formyl-kynurenine for 30 mins. This reaction is then stopped by 30% w/v trichloroacetic acid and heated at 58 °C. After cooling, the mixture is combined in a 50/50 ratio with 2% w/v p- dimethylaminobenzaldehyde in acetic acid into a 96 well plate for reading with a spectrometer at 490 nm to quantify the reaction of p-dimethylaminobenzaldehyde with kynurenine. The absorbance results are then normalized by cell count.