The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells

Abstract

The differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs) using embryoid bodies (EBs) is relatively inefficient and highly variable. Formation of EBs using standard enzymatic disaggregation techniques results in a wide range of sizes and geometries of EBs. Use of a 3-D cuboidal microwell system to culture hESCs in colonies of defined dimensions, 100-500 microm in lateral dimensions and 120 microm in depth, enabled formation of more uniform-sized EBs. The 300 microm microwells produced highest percentage of contracting EBs, but flow cytometry for myosin light chain 2A (MLC2a) expressing cells revealed a similar percentage (approximately 3%) of cardiomyocytes formed in EBs from 100 microm to 300 microm microwells. These data, and immunolabeling with anti-MF20 and MLC2a, suggest that the smaller EBs are less likely to form contracting EBs, but those contracting EBs are relatively enriched in cardiomyocytes compared to larger EB sizes where CMs make up a proportionately smaller fraction of the total cells. We conclude that microwell-engineered EB size regulates cardiogenesis and can be used for more efficient and reproducible formation of hESC-CMs needed for research and therapeutic applications.

Figure 1. Schematic of microwell controlled EB formation and differentiation

Cuboidal microwells were molded using polyurethane as described in the Methods, and hESCs seeded to the Matrigel coated bottom of the wells. hESC colonies from the microwells were used to form EBs which were cultured and characterized as schematically indicated.

Figure 2. hESC EBs formed from microwells yield homogeneous size distributions with predictable number of cells per EB

EBs formed following one day of suspension culture from hESCs cultured in microwells of 100 μm (A), 200 μm (B), 300 μm (C), 400 μm (D) and 500 μm (E) lateral dimensions and 120 μm depth compared to EBs from standard hESC culture on Matrigel (F). Between 68 and 185 EBs were evaluated for each sample set done in parallel in each experiment. Scale bar is 300 μm. (G) EBs derived from microwells 120 μm deep x 100–500 μm lateral dimensions were isolated and trypsinized after 1 day suspension culture, and the average number of cells per EB was counted for a total of 3 independent experiments. Assuming the volume of the starting microwell determined cell number, the data were fit to a simple polynomial equation, cell number=A(X-Y dimension)² where A is a constant. The data were well fit to the model (R²= 0.99) with A=0.0163±0.0004. (H) Histogram of the volume-weighted distribution of EB size relative to EB diameter for each starting microwell size. The microwell EBs sizes could well be described by Gaussian distributions (dashed curves) centered at 105 μm (100), 181 μm (200), 276 μm (300), 345 μm (400), 384 μm (500). In contrast, the EBs derived from unconstrained culture were more broadly size distributed and not well described by a simple Gaussian distribution. (I) Histogram of EB size distribution after 5 days in suspension culture showed differential growth of EBs from different sized microwells. The microwell EB sizes could well be described by Gaussian distributions centered at 162 μm (100), 237 μm (200), 291 μm (300), 301 μm (400), and 323 μm (500). A range of 189 to 536 EBs were counted for each sample set.

Figure 3. Time course of development of spontaneously contracting EBs comparing microwell and control EBs

EBs formed from microwells of 100, 200, 300, 400 and 500 μm lateral dimensions and from unconstrained standard culture were plated and observed for development of spontaneous contractions. The number of EBs contracting was scored every three days starting at day 9 after EB formation until day 30 as a percentage of attached EBs. Average contracting percentages were obtained by pooling EB counts from three independent experiments done in triplicate using at least 474 total EBs for each sample set. Error bars represent SEM. Data were compared using one-way ANOVA and Tukey post tests. At day 9, 12 and 15, there were no significant differences between any of the microwell EBs and control EBs. On day 18, the 300 and 400 μm EBs were significantly different from the control EBs (P < 0.05), and from day 21 to day 30, the 300 and 400 μm EBs were significantly different from the control EBs (P < 0.05), and the 300 μm EBs were also significantly different from the 100 μm EBs (P < 0.05).

Figure 4. Expression of cardiac genes NKX2-5, TNNT2, and MYL7 in microwell and control EBs

Total RNA was isolated from each size of microwell EB culture and unconstrained control EBs. Real-time RT-PCR was performed using Taqman gene expression assays, and the fold change in expression for each gene is plotted for the tested microwell size relative to the control. Error bars indicate fold change range calculated by the comparative Ct method for triplicates from three independent experiments. Data were compared using one-way ANOVA and Tukey post tests with * indicating the gene expression in microwell EBs is significantly different from that of the control EBs, P < 0.05.

Figure 5. Flow cytometric analysis of cells expressing MLC2a, a marker for cardiomyocytes, in EBs from microwell and control EBs

A) Flow cytometric analysis of cells expressing MLC2a in the microwell EBs and the control EBs. Cells were harvested from microwell EBs of 100, 300, and 500 μm lateral dimensions compared to EBs from standard unconstrained culture following 30 days of culture. B) Average number MLC2a positive cells comparing different sized microwell EBs to control EBs from 3 independent experiments, each experiment had 3 wells of EB culture in 6-well plate for flow cytometry. Error bars indicate SEM. Data were compared using blocked one-way ANOVA and Tukey post tests.

Figure 6. Immunolocalization of cardiomyocytes in EBs formed from different sized microwells

Panels A – C show immunolabeling with MF20 antibody (red) which recognizes sarcomeric myosin present in cardiomyocytes and nuclei are stained with DAPI (blue), in 30 days old EBs formed from 100 μm, 300 μm and 500 μm microwells , respectively. Panels D - F show immunolabeling with anti-MLC2a antibody (red), which recognizes a myofilament protein expressed in all early embryonic cardiomyocytes, and nuclei are stained with DAPI (blue), in 30 day old EBs formed from 100 μm, 300 μm and 500 μm microwells, respectively. Scale bar is 300 μm.