Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial Infarction in Mice

Abstract

Rationale: Stem cell therapy faces several challenges. It is difficult to grow, preserve, and transport stem cells before they are administered to the patient. Synthetic analogs for stem cells represent a new approach to overcome these hurdles and hold the potential to revolutionize regenerative medicine.

Objective: We aim to fabricate synthetic analogs of stem cells and test their therapeutic potential for treatment of acute myocardial infarction in mice.

Methods and results: We packaged secreted factors from human bone marrow-derived mesenchymal stem cells (MSC) into poly(lactic-co-glycolic acid) microparticles and then coated them with MSC membranes. We named these therapeutic particles synthetic MSC (or synMSC). synMSC exhibited a factor release profile and surface antigens similar to those of genuine MSC. synMSC promoted cardiomyocyte functions and displayed cryopreservation and lyophilization stability in vitro and in vivo. In a mouse model of acute myocardial infarction, direct injection of synMSC promoted angiogenesis and mitigated left ventricle remodeling.

Conclusions: We successfully fabricated a synMSC therapeutic particle and demonstrated its regenerative potential in mice with acute myocardial infarction. The synMSC strategy may provide novel insight into tissue engineering for treating multiple diseases.

Keywords: artificial cells; mesenchymal stem cells; myocardial infarction; regeneration; stem cells; tissue engineering.

Figure 1. Fabrication and characterizaiton of synMSC

(A) Schematic illustration of the fabrication process of synMSC. Microparticles (MP) were fabricated by treating mesenchymal stem cells conditioned-media with Poly (lactic-co-glycolic acid) (PLGA). Synthetic mesenchymal stem cells (synMSC) were formed by coating the MP with MSC membranes. After that, we tested the therapeutic effects of synMSC injection in mice with acute myocardial infarction. (B) Scanning electron microscopy images (left) and fluorescent images (right) on the structure of MP and synMSC. MP was labeled with Texas red succinimidyl ester (red), and synMSC as cell membranes labeled with green fluorescent DiO (red particle with green coat). Scale bar: 10 μm. (C, D) Quantitative analyses on the diameter and expressions of MSC markers in the MP, synMSC, and MSC. (E, F and G) Quantitative analyses on the release of vascular endothelia growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), and insulin-like growth factor-1 (IGF-1) from synMSC. n=3 for each group. All data are mean ± SD.

Figure 2. Potency and stability of synMSC in vitro

(A) Fluorescent images of neonatal rat cardiomyocytes (NRCM) stained with alpha sarcomeric actin (green) and co-cultured with MP, synMSC, and MSC (red). Scale bar: 100 μm. (B, C) Quantitative analyses of NRCM numbers and contractility when co-cultured with MP (blue bars), synMSC (red bars), and MSC (green bars). (D) Quantitative analyses on the number of MP and synMSC binding to NRCM. (E) Fluorescent images (above) and white light microscopy images (below) on synMSC morphology and aggregation before and after freeze/thaw. Scale bar: above, 10 μm; below, 100 μm. (F, G) Quantitative analyses on the size and surface antigen expressions of synMSC pre and post freeze/thaw. (H) Representative fluorescent images and illustration showing macrophage (green) attraction after the injection of freeze/thawed MSC and synMSC (red) into a mouse heart. Scale bar: 100μm. (I) Quantitative analyses of the CD68⁺ macrophages in freeze/thawed MSC- or synMSC- injected mouse heart. n=4 for each group. All data are mean ± SD. (B, C)* P < 0.05 when compared to control, (D)* P < 0.05 when compared to MP, (I)* P < 0.05 when compared to synMSC.

Figure 3. Benefits of synMSC injection in mice with myocardial infarction

(A) Representative PET/CT images and SPECT/CT images obtained at baseline and endpoint of mice after MI with or without synMSC treatment. (B) Quantitative analyses on the percentage of altered infarct area and left ventricular volume (endpoint vs baseline) in control and synMSC treated mice. (C) Masson’s trichrome staining images from the base, mid-papillary and apical regions of the infarcted heart two weeks after MI of control, synMSC and MSC treated mice. Quantitative analyses of infarct wall thickness (D) and infarct size (E) of left ventricle in control, synMSC and MSC treated mice. n=8 for each group. All data are mean ± SD. * P < 0.05 when compared to control.

Figure 4. Injection of synMSC promoted endogenous repair in the infarcted heart

(A, B, C) Representative fluorescent images showing c-kit-positive, CD34-positive, and ki67-positive cells in the infarcted heart after control, synMSC, or MSC treatement. Arrows indicate the positively stained cells. Scale bar: (A), (C): 20 μm; (B): 50 μm. (D, E, F) Quantitative analyses on c-kit-positive cells, CD34-positive cells, and ki67-positive cells in the infarcted heart after control, synMSC, or MSC treatment. n=6 for each group. All data are mean ± SD. * P < 0.05 when compared to control.