Abstract 2257: Cardiac Tissue Explant Culturing: A Novel Method to Study Human Pediatric Injury-Related Cytokine Signaling
Background. Paracrine functions of the human heart have been postulated to provide important signaling for injury-related regenerative responses associated with cell migration and proliferation. Reported herein is a novel method to study the poorly characterized human cytokine responses to injury.
Methods. Freshly discarded samples of right atrial appendage from pediatric patients with end-stage congestive heart failure (n=4; CHF) and without CHF (n=1; Control) were minced and cultured in Dulbecco’s MEM + 20% bovine calf serum. Media were sampled before and after addition of tissue and after 3, 7, and 14 days in culture. The relative abundance of 120 cytokines was determined using monoclonal antibody array with chemi-luminescent detection (RayBio, USA). Cytokine-specific antibody binding ≥ 30% on an arbitrary scale between 0 (negative control) to 100% (positive control) was defined as newly expressed.
Results. In this unique culture method model, cell migration from the tissues was first observed by day 7 in culture. Cell counts were determined for positively selected out-trafficking c-kit+ cells using monoclonal antibody and magnetic nanoparticle separation technique (StemCell Technologies, Can.) The majority (~90%) of nucleated cells were c-kit+ with yields averaging 6000 cells/0.1 gm tissue. By day 14, the number of c-kit+ cells had doubled. The number of expressed cytokines within the first 7 days in the Control and CHF groups was 14 and 8, respectively, with 7 common to both. Fewer cytokine responses and 1 unique cytokine, GRO-α, were detected from tissues derived from patients with CHF. The 7 unique cytokines in the Control and not expressed in the CHF group were: angiopoietin-2, ENA-78, GCSF, HGF, MIF, TIMP-2 and NAP-2.
Conclusions. Explant culturing is a reproducible method to discover human heart signaling responses to injury/hypoxia that are temporally associated with observed cell migration and proliferation of cardiac tissue-derived stem/progenitor cells independent of confounding contributions from circulating cellular and soluble factors.
This research has received full or partial funding support from the American Heart Association, Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).