Bioengineering of injectable encapsulated aggregates of pluripotent stem cells for therapy of myocardial infarction

It is difficult to achieve minimally invasive injectable cell delivery while maintaining high cell retention and animal survival for in vivo stem cell therapy of myocardial infarction. Here we show that pluripotent stem cell aggregates pre-differentiated into the early cardiac lineage and encapsulated in a biocompatible and biodegradable micromatrix, are suitable for injectable delivery. This method significantly improves the survival of the injected cells by more than six-fold compared with the conventional practice of injecting single cells, and effectively prevents teratoma formation. Moreover, this method significantly enhances cardiac function and survival of animals after myocardial infarction, as a result of a localized immunosuppression effect of the micromatrix and the in situ cardiac regeneration by the injected cells.

Staining done over intact aggregates before and after pre-differentiation. Scale bar: 100 µm. b, Staining done on single cells disassociated from their aggregates before and after the pre-differentiation. Scale bar: 20 µm. The data show successful pre-differentiation of the aggregated mESCs into the early cardiac stage. All the fluorescence images shown are confocal, which means the views are cross-sections through the aggregates or single cells. Although the NKX2.5 protein functions in the nuclei, we also observed its stain in the cytoplasm, probably because the protein is synthesized in the cytosol. The striated pattern is difficult to discern in the pre-differentiated cells because the cells were pre-differentiated into the early cardiac stage rather than mature cardiomyocytes. Figure 4. Biodegradation of the micromatrix of alginate and chitosan in vitro. a, Quantitative data showing alginate-chitosan micromatrix (ACM) encapsulation slows down the attachment of the predifferentiated cells from their aggregates when cultured in tissue culture petri dishes. Error bars represent s.d. (n=3). **, p < 0.01 (Student's two-tailed t test). Typical differential interference contrast (DIC) and fluorescence images showing the morphology and high cell viability of bare (b) and ACM-encapsulated (c) pre-differentiated aggregates cultured in petri dishes for 15 min, 1 day, and 3 days. Scale bar: 100 µm. Cells gradually detached from ACM-encapsulated aggregates and attached on petri dish as a result of the gradual degradation of the oxidized alginate in the ACM. Bare-A: Bare pre-differentiated aggregates. ACM-A: ACM-encapsulated, pre-differentiated aggregates.

Supplementary Figure 5. Biodegradation of the micromatrix of alginate and chitosan in vivo. a, IVIS images
showing successful labeling non-oxidized (NonOxi) alginate and oxidized (Oxi) alginate (by default and used for making the alginate-chitosan micromatrix or ACM in this study) with indocyanine green (ICG). b, ICG fluorescence in the MI heart of mice injected with Bare-A, NonOxi-ACM-A-ICG made of ICG labeled non-oxidized alginate, and ACM-A-ICG made of ICG labeled oxidized alginate. No fluorescence was observed in the hearts treated with Bare-A on days 0 (I h), 1, and 3. The fluorescence in the hearts treated with ACM-A-ICG reduced over time and disappeared on day 3, indicating the ACM gradually degraded over three days in vivo in the heart. The degradation of NonOxi-ACM-A-ICG is slower than that of ACM-A-ICG and the fluorescence was still observable on day 3 in the heart. c, ICG fluorescence in mice subcutaneously injected with Bare-A, NonOxi-ACM-A-ICG, and ACM-A-ICG. The trend of degradation for the subcutaneously injected NonOxi-ACM-A-ICG and ACM-A-ICG is similar to the intramyocardially injected NonOxi-ACM-A-ICG and ACM-A-ICG. The former degrades slower than the latter. Figure 6. The surgical procedure of permanent ligation to simulate myocardial infarction. a, A schematic illustration of permanent ligation of the left anterior descending artery (LAD) at its proximal location. Ligation at the proximal location results in large-area myocardial infarction (MI). b, A picture of the mouse heart collected after surgery, showing the location of ligation as compared to the sketch in panel a. c, A picture of the heart in situ before ligation showing fresh red on the outer surface of the anterior wall of the left ventricle. d, A picture of the heart in situ after ligation of the LAD at the proximal location, showing the pale appearance of the anterior wall of the left ventricle due to the lack of blood supply indicating successful ligation. Of note, the long suture is to be cut before closing the chest. e, Representative photographs of the cross-sectional sections of a heart from base to apex stained with tetrazolium chloride and blue at 24 h after ligation, showing nonischemic/normal regions (blue), area at risk (red), and area with infarction (white). The infarct (the total white area in all the cross-sections) was quantified to be 65.0 ± 1.6% (mean ± s.d., n=3) of the total area of all the cross-sections, which is considered as large-area infarct. Scale bar: 3 mm The tissues were stained by hematoxylin and eosin (H&E). In the MI heart treated with saline, single cells (Single), Bare-A, and ACM, there are macrophages (solid arrows) and fibroblasts in the fibrotic area, while fibrosis is negligible for the No MI control and minimal in the ACM-A treated heart. There are some degenerated cardiomyocytes with multiple nuclei (dashed arrow) in the ACM-A treated hearts, but no massive macrophages. In addition, the granulomas from single cells and bare-A treated mice exhibit strong inflammation indicated by the presence of massive macrophages (arrows). Scale bar: 50 µm

Supplementary Figure 9. Retention of implanted cells in the left ventricular wall.
Low magnification phase and fluorescence micrographs and their merged views of tissue sections from the left ventricle of MI heart of wild-type mice at 28 days after treated with saline, single cell (Single), Bare-A, ACM-A, and ACM. The implanted cells with green fluorescence are visible for all the groups with cells (Single, Bare-A, and ACM-A) and the ACM-A treatment leads to the highest cell retention. No green fluorescence was observed in the treatment groups without cells (Saline and ACM), indicating the green fluorescence is due to the implanted cells with green fluorescent protein (GFP) rather than auto fluorescence of scar tissue that is must abundant in the saline and ACM treated heart. Scale bar: 1 mm. The images were produced automatically using Zeiss MosaiX for tiling and imaging stitching. All mice were under anesthesia during the echocardiography experiments. By four weeks, the MI mice in all treatment groups show major changes in heart structure while the change is the least for mice with the ACM-A treatment. The ventricular contraction amplitude for the ACM-A treatment is improved compared to that for the other four treatments.
Supplementary Figure 16. Heart rate measured by echocardiography before and after surgery. It shows no significant difference among the different treatment groups before surgery (baseline) and at 4 weeks after the surgery (endpoint). Error bars represent s.d. The n=4,9,8,5,8,and 4 for No MI, Saline, Single, Bare-A, ACM-A, and ACM, respectively. No significance was detected between the different groups (one way ANOVA).

Heart rate, bpm
Before the surgery 4 weeks after the surgery Treatments