Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

Abstract

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

Fig. 1.

Arrays of hydrogel microwells for culturing ES cells. (A) Analysis of EBs cultured within microwells for 7 days. Scanning electron microscopy (SEM) images show the formation of uniform arrays of PEG microwells with different diameters (150 μm, 300 μm, and 450 μm) (Top). Phase contrast (Middle) and fluorescent images (Bottom) of EBs cultured within microwells after 7 days. (Scale bar, 100 μm.) Live and dead cells were stained with calcein AM (green) and ethidium homodimer (red). (Scale bar, 200 μm.) (B) The molecular expression of ES cell pluripotency markers after 3 and 7 days. Oct4 expression of ES cells expressing the Oct4/GFP reporter gene (left column for each day). Immunocytochemical staining of SSEA1 (red)/E-cadherin (green) in EBs within microwells (right column for each day). (Scale bar, 100 μm.)

Fig. 2.

EB size-mediated cardiogenic differentiation of ES cells. (A) Morphology and characterization of beating foci (red arrows) in EB outgrowths. Immunocytochemical characterization of cardiomyogenic differentiation and quantification of beating colonies from EB outgrowths that were replated from microwells. Sarcomeric α-actinin expression (red) of EB outgrowths at day 15 of culture. (Scale bar, 100 μm.) (B) Morphology of beating EBs, immunocytochemical characterization of cardiomyogenic differentiation identified by sarcomeric α-actinin and evaluation of beating EBs cultured in microwells. Inset for 150 μm EB figure indicates control stained only with secondary antibody. (Scale bar, 100 μm.) (C) Time course of cardiomyogenic gene expression from EBs within microwells. (I) Gel pictures, (II) GATA4 mRNA expression, (III) Nkx2.5 mRNA expression, and (IV) ANF mRNA expression. Data shown as mean normalized mRNA expression intensity ± SEM (n = 3, * indicates P < 0.05 compared to 150 μm EB).

Fig. 3.

EB size-mediated endothelial cell differentiation of ES cells. (A) Phase contrast and immunocytochemical characterization of endothelial cell differentiation indentified by CD31 (red) and SMA (green) at day 11. EBs retrieved from microwells (Top) and negative control staining only with secondary antibody (Right fluorescent image). ES cells cultured within microwells for 5 days were plated onto Matrigel substrates and were cultured for additional 6 days (Middle) and a fluorescent image of vessel sprouting from EBs on Matrigel (Right fluorescent image). EBs cultured within microwells for 11 days (Bottom) and a fluorescent image of vessel sprouting from EBs on Matrigel (Right fluorescent image). Each inset indicates phase contrast image of corresponding EBs cultured within microwells. (Scale bar, 100 μm.) (B) Characterization of sprouting vessel activity and frequency of EBs cultured within microwells. The sprouting activity and frequency of ES cells cultured within smaller microwells were higher than those within larger microwells. (Scale bar, 100 μm.) (C) Analysis of gene expression of ES cells to identify endothelial cell differentiation. (I) Gel pictures, (II) PECAM mRNA expression, (III) Flk-1 mRNA expression, and (IV) Tie-2 mRNA expression. Data shown as mean normalized mRNA expression intensity ± SEM (n = 3, * indicates P < 0.05 compared to 150 μm EB).

Fig. 4.

The inhibition and activation of WNT5a for directing EB-size mediated cardiogenic and endothelial cell differentiation of ES cells cultured within microwells. (A) Inhibition analysis: Immunocytochemical characterization for WNT5a-siRNA transfection (green) indicates that siRNA is delivered throughout the cell aggregates (Left). Endothelial cell (red, CD31) and cardiogenic (green, sarcomeric α-actinin) differentiation for siRNA transfected and control EBs. (B) Characterization of EB beating and sprouting frequency. (C) Analysis of gene expression of ES cells cultured within microwells (150 μm diameter) to identify endothelial cell and cardiogenic differentiation. (D) Activation analysis: Immunocytochemical characterization of ES cells cultured with the addition of recombinant mouse WNT5a within microwells (450 μm diameter). (Scale bar, 100 μm.) (E and F) Analysis of cardiogenic and endothelial cell differentiation confirmed by EB beating, vessel sprouting frequency, and gene expression. (n = 3, * indicates P < 0.05 compared to controls).