Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

Abstract

Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes.

Figure 1. Suspension culture of progenitor cardiac spheres and simulated microgravity increase cell viability and CM yield.

(A) Experimental design. hPSCs were induced to differentiate into CMs at day 0 using growth factors or small molecules. At day 4 or 5, cells were dissociated and forced to aggregate into progenitor cardiac spheres. The progenitor cardiac spheres were cultured under simulated microgravity at days 5–8 or 6–9 and maintained under standard gravity until day 20 (designated as 3D-MG). Parallel 2D and 3D cultures were maintained under standard gravity throughout, designated as 2D-SG and 3D-SG, respectively. At day 20, cells were analyzed for cell viability, CM purity, yield and density. (B) Representative flow cytometry analysis. Cell viability was analyzed by EMA staining and EMA negative cells were identified as live cells. Purity of CMs was analyzed by intracellular staining of α-actinin, a CM-associated marker. (C) Summary of IMR90-iPSC differentiated cell viability, CM purity, cell yield and density. (D) Summary of H7 hESC differentiated cell viability, CM purity, cell yield and density. (E) Summary of H9 hESC differentiated cell viability, CM purity, CM yield and density. The CM yield was defined as the number of viable CMs generated from one undifferentiated stem cell. Data are presented as mean ± SD of 3–6 biological samples for each culture condition. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.

Figure 2. Cellular and molecular features of CMs derived from progenitor cardiac spheres expanded under standard gravity and simulated microgravity.

(A) Immunocytochemistry analysis of the cardiac structural proteins α-actinin and cTnI and the adhesion molecule cadherin in cells harvested at day 20. Nuclei were stained with DAPI. Scale bars = 50 μm. (B,C) Expression of genes encoding structural proteins and calcium handling proteins in these cells determined by qRT-PCR. Data are presented as mean ± SD of 4 biological replicates x 3 reactions/sample. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. Structural and functional characterization of hPSC-CMs cultured in 2D-SG, 3D-SG and 3D-MG conditions.

(A) Structural analysis of hPSC-CMs. Cells were dissociated, replated and stained for sarcomeric α-actinin (green) and DAPI (blue). Overall appearance of myofibrillar structure was categorized into 3 different levels: Score 1 cells are α-actinin^pos but without clear sarcomeric striations; Score 2 cells have diffuse punctate staining pattern and some patterned striations in partial cell area; and Score 3 cells have highly organized and well-defined myofibrillar structure with distinct paralleled bands of z-discs distributed throughout the cell area. Percentage of the cells by the scores was generated by counting 102 and 136 cells for 3D-SG and 3D-MG, respectively. Note that 3D-MG produced CMs with higher levels structural maturation. Scale bar = 25 μm. (B) Calcium transients of hPSC-CMs cultured in 3D-SG and 3D-MG conditions. Representative traces of hPSC-CMs field-stimulated at 1 Hz were acquired by optical fluorescence imaging. Measurements of calcium transients are presented as mean ± SD of n = 37 and 40 line scans for 3D-SG and 3D-MG culture conditions, respectively. **P < 0.01; ****P < 0.0001. (C) Representative patch clamp recording and summary of action potential parameters. Electrophysiological parameters measured: N, cell number; dV/dtmax, maximum action potential upstroke velocity; MDP, maximum diastolic potential; APA, action potential amplitude; APD50, action potential duration at 50% of repolarization; APD90, action potential duration at 90% of repolarization. Data are presented as mean ± SD.

Figure 4. MEA recordings of hPSC-CMs cultured in 2D-SG, 3D-SG and 3D-MG conditions.

(A) A representative image of MEA chamber seeded with cardiospheres. Extracellular recordings from differentiated culture harvested at day 20 without treatment (baseline) and after the treatment with indicated pharmacological agents. Scale bars = 100 μm. (B) Representative MEA recordings of cells without treatment (baseline), of cells treated alone with isoproterenol as indicated, and of cells first treated with isoproterenol alone followed by co-treatment of carbamylcholine (isoproterenol + carbamylcholine). (C,D) Response of cell beating to the indicated pharmacological treatments. n = 4 biological samples. (E) Representative MEA recording before and after cells were treated with nifedipine at final concentrations of 1 and 10 μM. (F) Normalized mean cFPD of cells before and after the nifedipine treatment. n = 3–4 biological samples. All data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Simulated microgravity and 3D culture promote the induction of cardiac progenitors and CM differentiation.

Flow cytometry analysis of cells exposed to microgravity (3D-MG) and standard gravity (2D-SG and 3D-SG) at various time points for their expression of markers associated with (1) cardiac mesoderm, (A) KDR/PDGFRα and (B) CD13; (2) cardiac progenitors, (C) ISL1; and (3) cardiomyocytes, (D) SIRPA and (E) cTnT/α-actinin. Data are presented as representative flow cytometry analysis and summary based on mean ± SD of 3 biological replicates.

Figure 6. Simulated microgravity and 3D culture increase the proliferation of cardiac progenitors.

(A) Representative images, flow cytometry analysis and summary of cells at differentiation day 10 that were co-stained with EdU and an antibody against NKX2-5, a marker for cardiac progenitors. Scale bars = 100 μm. The quantitative summary was based on flow cytometry analysis. Data are presented as mean ± SD of 3 biological replicates. Scale bar = 100 μm. (B) Similar analyses to (A) except that Ki-67 instead of EdU was co-stained with NKX2-5. (C) Similar analyses to (A) except that IAK1 instead of EdU was co-stained with NKX2-5. (D) Relative expression levels of genes associated with proliferation and cell cycle in cells at differentiation day 8 according to qRT-PCR. Data are presented as mean ± SD of 8 biological replicates x 3 reactions/sample. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 7. Simulated microgravity and 3D culture increase cell viability of cardiac progenitors.

(A) Representative flow cytometry plots of viability of the cells at day 8 and assessed by Annexin V and PI. The nature of the cells in each quadrant is noted in the far-right diagram. (B) Summary of the viability, apoptotic and dead cells at day 8. Data are presented as mean ± SD of 8 biological replicates. (C) Summary of the cell yield and density. Yield was calculated based on the number of viable cells at day 8 generated from each input undifferentiated stem cell. Data are presented as mean ± SD of 8 biological replicates. (D) Relative expression levels of genes associated with pro-survival in cells at differentiation day 8 according to qRT-PCR. Data are presented as mean ± SD of 8 biological replicates x 3 reactions/sample. (E) Representative flow cytometry analysis and summary of cells at differentiation day 8 that were co-stained with p-AKT and NKX2-5. Data are presented as mean ± SD of 3 biological replicates *P < 0.05; **P < 0.01; ***P < 0.001.