Systematic analysis of embryonic stem cell differentiation in hydrodynamic environments with controlled embryoid body size

Abstract

The sensitivity of stem cells to environmental perturbations has prompted many studies which aim to characterize the influence of mechanical factors on stem cell morphogenesis and differentiation. Hydrodynamic cultures, often employed for large scale bioprocessing applications, impart complex fluid shear and transport profiles, and influence cell fate as a result of changes in media mixing conditions. However, previous studies of hydrodynamic cultures have been limited in their ability to distinguish confounding factors that may affect differentiation, including modulation of embryoid body size in response to changes in the hydrodynamic environment. In this study, we demonstrate the ability to control and maintain embryoid body (EB) size using a combination of forced aggregation formation and rotary orbital suspension culture, in order to assess the impact of hydrodynamic cultures on ESC differentiation, independent of EB size. Size-controlled EBs maintained at different rotary orbital speeds exhibited similar morphological features and gene expression profiles, consistent with ESC differentiation. The similar differentiation of ESCs across a range of hydrodynamic conditions suggests that controlling EB formation and resultant size may be important for scalable bioprocessing applications, in order to standardize EB morphogenesis. However, perturbations in the hydrodynamic environment also led to subtle changes in differentiation toward certain lineages, including temporal modulation of gene expression, as well changes in the relative efficiencies of differentiated phenotypes, thereby highlighting important tissue engineering principles that should be considered for implementation in bioreactor design, as well as for directed ESC differentiation.

Fig. 1. Micro-well formation and rotary maintenance of size-controlled EBs

(A) Forced aggregation of ESCs into micro-wells to form size-controlled EBs was accomplished via centrifugation of single cells, followed by 24 h of incubation in wells, and subsequent transfer to rotary orbital suspension (scale bar = 100 μm). (B) After 24 h, the resulting EB populations exhibited narrow size distributions with distinct peaks modulated by ESC seeding density, and (C) relative size differences were maintained over 7 days of differentiation in rotary orbital suspension culture.

Fig. 2. Maintenance of EB populations in different hydrodynamic environments

(A) EBs maintained in rotary orbital suspension exhibited changes in size and shape after 7 days of differentiation. The irregular shape and large size of EBs maintained in static and low rotary orbital speed (25 rpm) conditions indicated agglomeration of individual EBs (scale bar = 400 μm). (B) At 45 and 65 rpm, however, EBs maintained uniform, similarly-sized populations through 14 days (scale bar = 200 μm). (C) Although EB size increased during the course of differentiation, the EB area at 45 and 65 rpm was not significantly different. (D) Additionally, the total number of EBs did not change over time or between conditions, indicating minimal agglomeration of individual pre-formed EBs. * = p < 0.05 (45 rpm) compared to all time points; + = p < 0.05 (65 rpm) compared to all time points; $ = p < 0.05 (65 rpm) compared to all time points except day 14; # = p < 0.05 (65 rpm) compared to all time points except day 7; N.S. = no statistical significance.

Fig. 3. Gene expression profiles of size controlled EBs in hydrodynamic environments

Global gene expression of 84 genes related to ESC pluripotency and differentiation was assessed in EBs from 45 rpm and 65 rpm after 7 and 14 days of differentiation using PCR Arrays. (A) Heatmap visualization of gene expression demonstrated (B) distinct subsets of genes up- and down-regulated compared to ESCs, with (C) several significantly modulated genes being regulated differently between the 45 rpm and 65 rpm conditions. Although several genes exhibited differences in fold change (>1.5 fold, blue; represented as the fold change of 45 rpm/65 rpm), few genes exhibited statistically significant differences (P < 0.05, pink) between the hydrodynamic conditions, (D) including 4 genes increased in 65 rpm, and 1 gene increased in 45 rpm after 7 days of differentiation. (E) Rotary conditions also modulated the temporal expression of genes from the mesoderm (Nkx2.5), endoderm (AFP), and ectoderm (Pax-6) lineages. * P < 0.05.

Fig. 4. Modulation of visceral endoderm and cardiac mesoderm differentiation in response to rotary orbital culture

(A) AFP-GFP transduced ESCs exhibited few GFP+ cells when maintained as an undifferentiated monolayer; however after 14 days of differentiation, EBs from rotary orbital cultures increased expression of GFP (AFP), with (B) EBs cultured at 45 rpm consistently exhibiting increased expression of GFP+ cells after 7, 10 and 14 days of differentiation. (C) Within EBs, the GFP+ cells appeared to cluster in distinct areas, which co-localized with dark regions visualized under phase (overlay) (scale bars = 50 μm, 200 μm). (D) The proportion of EBs exhibiting spontaneous contractile activity was increased following culture 45 rpm compared to 65 rpm after 10, 12, and 14 days of differentiation. * p < 0.05.