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

Abstract

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.

Figure 1. Bioinspired approach for preparing pluripotent stem cells to implant by injectable delivery.

(a) A schematic illustration of the multi-step procedure to prepare the totipotent-pluripotent stem cells for implantation in the uterus wall, including proliferation to form a microscale cell aggregate (that is, morula) in zona pellucida, pre-differentiation of morula into trophoblast cells and inner cell mass in the zona pellucida, hatching out of the zona pellucida and re-encapsulation in the trophoblast before implantation during early embryo development in the female reproductive system. (b) A schematic illustration of the bioinspired procedure for producing 3D microscale constructs of murine embryonic stem cells (mESCs) together with real images, showing the analogy between the bioinspired approach and the aforementioned natural procedure. The bioinspired approach mimics the natural procedure phenomenologically rather than mechanistically. Scale bar, 100 μm

Figure 2. Pre-differentiation of the mESC aggregates into the early cardiac stage.

(a) Microarray data showing significantly increased expression of mesoderm and cardiac marker genes and significantly decreased expression of pluripotency marker genes in the aggregated cells after pre-differentiation. (b) Flow cytometry data showing successful pre-differentiation of the mESC aggregates with diminished expression of pluripotency protein makers (OCT-4 and NANOG). (c) Flow cytometry data showing early cardiac pre-differentiation with significantly increased expression of cardiac specific protein marker (cTnT) and the early cardiac protein marker (NKX2.5).

Figure 3. Characterization of the aggregates pre-differentiated to the early cardiac stage.

(a) SEM images showing successful encapsulation of the pre-differentiated cell aggregates with the alginate-chitosan micromatrix (ACM) both outside (first two columns) and inside (third column) the aggregates. Scale bars, 5 μm. (b) Confocal fluorescence micrographs of the middle plane of the ACM-A showing micromatrix inside the aggregates indicated by labelling alginate in the ACM with FITC to show up with green fluorescence. Scale bar, 50 μm. (c) The elastic modulus of the pre-differentiated aggregates is significantly increased after ACM encapsulation. The modulus was determined by atomic force microscopy (AFM) nanoindentation. Error bars represent standard deviation (s.d., n=3). *P<0.05 (Student two-tailed t-test).

Figure 4. Therapy of myocardial infarction by injecting ACM-encapsulated pre-differentiated aggregates.

(a) A schematic illustration of surgical ligation (X) of the LAD at its proximal location to create large-area myocardial infarction (MI) and implantation of samples by intramyocardial injection at three different locations. (b) Typical gross images of a heart with no MI and MI hearts with five different treatments showing granulomas in single cell (Single) and Bare-A treated mice (arrows). Scale bar, 3 mm. (c) Quantitative data of cumulative granuloma occurrence in both wild-type (WT) and Card9 knockout (KO) mice, showing treatments with single, Bare-A, Bare-A-KO have significantly higher occurrence of granuloma than the other treatments including ACM-A. The animal number (n) was 10, 31, 29, 38, 31, 24 and 9 for No MI, Saline, Single, Bare-A, ACM-A, ACM and Bare-A-KO, respectively. *P<0.05 (Chi-square test). (d) Typical micrograph of granuloma with immunofluorescence staining of F4/80 (for macrophage, red) and CD3 (for T cells, red) showing many immune cells within a loose matrix in the granuloma collected at 28 days after injected with Bare-A. The nuclei are stained blue. Scale bar, 20 μm. (e) Data of implanted cells (expressing green fluorescence protein, GFP) retained and survived in the heart after 28 days showing a significantly higher cell survival with the ACM-A treatment than all the other treatments. The cell survival was quantified by counting cells with green fluorescence in the heart from the apex to the point of ligation. Error bars represent s.d. (n=3). *P<0.05 (one-way ANOVA). (f) Survival of WT MI mice at 28 days after injection, showing the ACM-A treatment can maintain a significantly higher animal survival than all the other treatments. The animal number (n) was 31, 29, 38, 31 and 24 for Saline, Single, Bare-A, ACM-A and ACM, respectively. *P<0.05 (one-way ANOVA). (g) Ejection fraction measured by PV loops showing the ACM-A treatment significantly improves the heart function after MI, compared with saline, Bare-A and ACM treatments. Error bars represent s.d. (n=4 for Single and n=3 for other groups). *P<0.05 (one-way ANOVA).

Figure 5. Cardiac regeneration in situ with the ACM-encapsulated pre-differentiated aggregates.

(a) Low-magnification sagittal micrographs of Masson's trichrome stained tissue sections (top row) and zoom-in views of the left ventricular wall (bottom row) showing extensive fibrosis in the MI hearts treated with saline, materials alone (that is, ACM), single cells and Bare-A while it is minimal with the ACM-A treatment. Scale bar: 2 mm (top row) and 100 μm (bottom row). (b) Quantitative analysis showing the ACM-A treatment significantly reduces fibrosis in the MI heart. The n=3 and *P<0.05 (one-way ANOVA). (c) Micrographs of sectioned and H&E stained tissue (Morphology) in the MI zone of heart treated with the ACM-A showing green fluorescent CM-like cells (GFP) and their seamless integration with the neighbouring non-fluorescent native host tissue (Merged). Scale bar, 100 μm. (d) Immunohistochemically stained tissue from the MI zone of hearts treated with the ACM-A showing positive staining of CM (cTnI, connexin 43, and α-actinin, in red) markers co-localized with injected GFP cells. The striated pattern of the cardiac tissue with green fluorescence is evident. Scale bar, 10 μm. All tissues were harvested on day 28 after intramyocardial injection.