Gelatin Based Polymer Cell Coating Improves Bone Marrow-Derived Cell Retention in the Heart after Myocardial Infarction

Abstract

Background: Acute myocardial infarction (AMI) and the ensuing ischemic heart disease are approaching an epidemic state. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Polymer based cell coating is biocompatible and has been shown to be safe. Here, we assessed the therapeutic utility of gelatin-based biodegradable cell coatings on bone marrow derived cell retention in ischemic heart.

Methods: Gelatin based cell coatings were formed from the surface-mediated photopolymerization of 3% gelatin methacrylamide and 1% PEG diacrylate. Cell coating was confirmed using a multimodality approach including flow cytometry, imaging flow cytometry (ImageStream System) and immunohistochemistry. Biocompatibility of cell coating, metabolic activity of coated cells, and the effect of cell coating on the susceptibility of cells for engulfment were assessed using in vitro models. Following myocardial infarction and GFP+ BM-derived mesenchymal stem cell transplantation, flow cytometric and immunohistochemical assessment of retained cells was performed.

Results: Coated cells are viable and metabolically active with coating degrading within 72 h in vitro. Importantly, cell coating does not predispose bone marrow cells to aggregation or increase their susceptibility to phagocytosis. In vitro and in vivo studies demonstrated no evidence of heightened immune response or increased phagocytosis of coated cells. Cell transplantation studies following myocardial infarction proved the improved retention of coated bone marrow cells compared to uncoated cells.

Conclusion: Gelation based polymer cell coating is biologically safe and biodegradable. Therapies employing these strategies may represent an attractive target for improving outcomes of cardiac regenerative therapies in human studies.

Keywords: Bone marrow mesenchymal stem cells; Cell coating; Myocardial infarction; Photo-polymerization; Polymer.

Figure 1.. BM-derived MSCs remained viable after coating with polymer.

Coated BM-MSCs were visualized by imaging flow cytometer-ImageStream System. Coated and uncoated BM-MSCs were fixed, stained with antibodies against collagen I antibody (AF-647) for the presence of cell surface gelatin coating, and acquired on an ImageStream flow cytometer. (Panel A) Representative FACS plots and images demonstrating nucleated coated GFP+ BM-MSCs with Hoechst33342 and Alexa-Fluor 647 positive staining. (Panel B) Coated cells remained alive after coating process as stained double positively with picosirius red (red for gelatin coating) and calcein (green for viability) as assessed by microscopy (upper panel) and flow cytometry (lower panel).

Figure 2.. Coating did not predispose cells to aggregate.

Coated and uncoated control BM-MSCs were analyzed for cell-cell aggregates using flow cytometry by examining the forward scatter area vs. height (FSC) plots. Events falling outside the diagonal of forward scatter area versus height were considered to be aggregates. On forward scatter plots, where the area under the curve is plotted against the maximum height of the curve for each event, single cells fall along the diagonal, indicated by the drawn gate. We did not observe changes in cell aggregates with our coating strategy.

Figure 3.. Cell coating began to degrade in vitro within 24 hr.

Coated and uncoated control A549 lung carcinoma cells were cultured for up to three days. Coated cells were identified with Picosirius red. The fluorescence in the FL-1 channel of the coated cells was due to the presence of eosin, which was used to initiate polymerization. The plots demonstrate the progressive loss of coating on cultured cells over time.

Figure 4.. Coated BM-MSCs are metabolically active.

Coated and control uncoated cells were assessed for up to five days in vitro. Each day, cultures of cells were examined for metabolic activity by the MTT assay. After an initial lag phase, the metabolic activity of coated cells was comparable to that of the control uncoated cells.

Figure 5.. Coating does not predispose coated cells to be engulfed by macrophages.

All A549 target cells were labeled with Deep Red while coated cells were identified by Picosirius Red. Target cells were cultured with unlabeled RAW264.7 macrophage effector cells for 15 minutes. Effector cells that had engulfed target cells were found in the lower left region (Panel A). Fewer professional phagocytes were present when coated A549 cells were co-cultured (right plot) than uncoated cells (left plot). The brightness of deep red, an indication of engulfment, was plotted for cultures indicating that coating may protect target cells from engulfment as detailed in the quantitative assessment of plots (Panel B).

Figure 6.. The coating on the cells did not induce a localized inflammatory response.

Paraffin imbedded sections were stained for CD68 and quantified as number of cells per power field were quantified. Quantitative analysis did not show significant difference between mice treated with coated or uncoated cells (N = 3 mice/group).

Figure 7.. Coated cells were more efficiently retained in the heart.

Flow cytometry analyses of digested heart tissue demonstrate higher percentage of GFP+ cells in mice treated with coated cells comparted to mice transplanted with uncoated cells. Panel A shows the gating strategy for GFP+ cells in the heart as assessed 7 days after MI and transplantation. Panel B illustrates a quantitative analysis of GFP+ cells in digested heart tissue of mice treated with coated and uncoated bone marrow cells showing higher percentage of GFP+ cells and trend towards higher GFP mean fluorescence intensity in mice treated with coated MSCs. Immunohistochemical analysis demonstrated higher numbers of retained GFP+ bone marrow cells in mice treated with coated cells (Panel C). Quantitatively, mice treated with coated cells exhibited higher numbers for GFP+ cells per high power field as compared to mice treated with uncoated cells (Panel D) (N = 4 mice/group; *P<0.05, **P<0.01).

Figure 7.. Coated cells were more efficiently retained in the heart.

Flow cytometry analyses of digested heart tissue demonstrate higher percentage of GFP+ cells in mice treated with coated cells comparted to mice transplanted with uncoated cells. Panel A shows the gating strategy for GFP+ cells in the heart as assessed 7 days after MI and transplantation. Panel B illustrates a quantitative analysis of GFP+ cells in digested heart tissue of mice treated with coated and uncoated bone marrow cells showing higher percentage of GFP+ cells and trend towards higher GFP mean fluorescence intensity in mice treated with coated MSCs. Immunohistochemical analysis demonstrated higher numbers of retained GFP+ bone marrow cells in mice treated with coated cells (Panel C). Quantitatively, mice treated with coated cells exhibited higher numbers for GFP+ cells per high power field as compared to mice treated with uncoated cells (Panel D) (N = 4 mice/group; *P<0.05, **P<0.01).