Synthesis and organization of hyaluronan and versican by embryonic stem cells undergoing embryoid body differentiation

Abstract

Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial-mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and -5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) -1, -2, and -3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenvironments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Figure 1

Hyaluronan accumulation during embryoid body (EB) formation. Sections (5 μm) from day 4, 7, and 10 EBs were stained with hyaluronan binding protein (dark red color); hematoxylin-stained nuclei are shown in blue. Hyaluronan was increasingly prominent with differentiation time (A–C). Localized pockets of hyaluronan staining can be seen by day 7 (B,E), and higher magnification (G–I, magnified view of boxes in D–F) shows hyaluronan surrounding individual cells. Scale bars at left apply to each row.

Figure 2

Versican accumulation during EB formation. Sections (5 μm) from day 4, 7, and 10 EBs were stained with anti-versican antibody (dark red color); hematoxylin-stained nuclei are shown in blue. Some versican expression can be seen by day 4 (A,D), with increased quantities observed by days 7 (B,E) and 10 (C,F). Versican (V0 and V1 isoforms) appears to localize in areas of lower cell density, while higher magnification images (G–I, magnified view of boxes in D–F) reveal versican staining within acellular spaces. Scale bars at left apply to each row.

Figure 3

Quantification of hyaluronan synthesis by EBs. A quantitative hyaluronan (HA)-binding protein-based kit was used to measure hyaluronan retained within embryonic stem cells (ESCs) and day 4, 7, and 10 EBs (A) as well as hyaluronan secreted in medium conditioned by ESCs and day 2, 4, 6, 8, and 10 EBs (B). Hyaluronan content in ESCs, EBs, and conditioned medium samples progressively increased over the course of differentiation. Day 10 EBs and day 8- and 10-conditioned media samples contained significantly greater amounts of hyaluronan than samples collected from all other time points examined. (A) # indicates significant differences from ESCs, day 4, and day 7 (p<0.01); (B) ‡ indicates significant differences from ESCs and days 2–6 (p<0.01) (ANOVA with post hoc Tukey test). Error bars represent standard deviations; n=3 per group.

Figure 4

Analysis of versican synthesis and proteolysis in differentiating EBs. Purified, chondroitinase-treated protein lysates from triplicate samples of ESCs and day 4, 7, and 10 EBs were probed with rabbit anti-mouse versican β-GAG domain antibody (A). An increase in proteins identified by the β-GAG antibody (versican core proteins V0 and V1, approximately 450 and 350 kDa, respectively) were apparent after 7 and 10 days of differentiation in triplicate samples and were not detected in ESCs and EBs after 4 days of differentiation. Protein lysates were also examined for the versican cleavage product, known as the DPEAAE fragment, generated by ADAMTS-1 over the course of EB culture (B). The 70-kDa DPEAAE fragment was present at day 4 and appeared to increase with differentiation time through day 10. β-Actin was used as a loading control. ADAMTS-1 and -5, known versicanases, were examined via SuperArray RT-PCR arrays, and expression of each gene was found to increase significantly by day 10 compared with day 4 of differentiation, while ADAMTS-8, a protease unrelated to versican degradation, remained unchanged (C). ADAMTS-5 immunofluorescent staining detected protease expression with day 7 (D) and day 10 (E) EBs. † indicates significant differences from day 4 EBs (p<0.05) (ANOVA with post hoc Tukey test). Error bars represent standard deviations; n=3 per group.

Figure 5

Quantification of hyaluronan synthase (HAS) and versican gene expression. qRT-PCR was used to compare expression levels of HAS isoforms 1, 2, and 3 and versican splice variants V0, V1, V2, and V3 during the course of EB differentiation. HAS-2 increased with time and was consistently expressed at significantly higher levels than either HAS-1 or HAS-3. Isoforms V0, V1, and V3 increased by 10 days of differentiation, while V2 was consistently expressed at relatively low levels. (A,B) # indicates significant differences from ESCs, day 4, and day 7 (p<0.02); solid line indicates a p value of <0.01; dashed line indicates p<0.02; and dotted line indicates p<0.05 (ANOVA with post hoc Tukey test). Error bars represent standard deviations; n=6 per group.

Figure 6

Hyaluronan and versican spatial distributions. EBs were stained for both hyaluronan (green; A–C) and versican isoforms V0 and V1 (red; D–F); merged images show localization of the two molecules in yellow (G–I) and at higher magnifications (J–L). Hyaluronan and versican appear to localize in areas of lower cell density, and deposition of both molecules increases over the course of differentiation. Bar in A applies to A–I; bar in J applies to J–L.

Figure 7

Cellular expression of epithelial–mesenchymal phenotype markers relative to versican localization. Adjacent 5-μm sections from day 10 EBs were fluorescently stained with pan-cytokeratin or N-cadherin primary antibody (FITC-labeled secondary, green) and versican (isoforms V0 and V1; TRITC-conjugated secondary antibody) (red); nuclei were counterstained with Hoechst dye (blue). Cytokeratin localized primarily in the periphery of the EBs, with some evidence of invasion into the EB interiors, while versican was localized exclusively in cytokeratin-negative areas (A,B). In contrast, N-cadherin localized to areas occupied by versican (C,D). Staining was performed on adjacent sections of the same EBs, which were matched based on morphology and location on each slide. Bar = 100 μm.