Engineering human cell spheroids to model embryonic tissue fusion <i>in vitro</i>

<div><p>Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human <i>in vitro</i> assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior <i>in vitro</i> using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.</p></div

2D-DIGE analysis of osteogenic differentiation of HWJSC spheroids at day 7 versus day 1.

<p>HWJSC spheroids were cultured for either 1 or 7 days, and 2D-differential gel electrophoresis and mass spectrometry was performed to identify the differentially expressed proteins that were present on at least one gel (out of a total of 3 gels, representing randomized samples from 3 independent experiments) and identified with at least 2 peptides. Fold change was calculated for each unique spot, with an ANOVA p-value cutoff of 0.055. The protein name, symbol, SwissProt Accession, SameSpots #, and ANOVA p-value are provided in columns for each unique identified protein, and protein expression fold change in day 7 versus day 1 samples is presented as a bar graph centered around a fold change of 1 (representing no difference between day 1 and day 7).</p

Immunofluorescence staining for extracellular matrix proteins in day 1 and day 7 HWJSC spheroids.

<p>HWJSC spheroids were cultured for either 1 day or 7 days (with 6 days of osteogenic differentiation), fixed, and stained for extracellular matrix proteins: collagen I (A) collagen IV (B), laminin (C), and fibronectin (D) and counter-staining with Hoechst. Scale bars represent 100 μm.</p

Generation of co-cultured spheroids consisting of osteogenic HWJSC spheroids seeded with epithelial cells.

<p>A: Schematic of HWJSC staining with CellTracker Orange, spheroid generation and osteogenic differentiation (7 d), washing in OM, and coating with CellTracker Green-stained HPEKp epithelial cells. B: Representative fluorescent maximum intensity projections (MaxIP) of HPEKp seeded on HWJSC spheroids after one day of incubation at epithelial/mesenchymal ratios (E/M) of 0.1 (i), 0.2 (ii), 0.4 (iii), 0.8 (iv), 1.2 (v), and 1.4 (vi). C: Quantified HPEKp/HWJSC co-localization after one day of incubation at E/M ratios from 0.1–1.4, presented as mean ± 95% confidence interval of fractional epithelial coverage represented by 7 confocal z-stacks per condition and a total of 6 independent experiments. Asterisks represent statistical significance with one-way ANOVA and Tukey’s post-hoc test of each condition relative to E/M = 0.1, 0.2, and 0.4. All conditions were statistically higher than E/M = 0.1 and 0.2. Mean epithelial coverage was statistically indistinguishable between E/M = 0.8, 1.2, and 1.4, and E/M = 1.4 was indistinguishable from E/M = 0.4. D: Quantified HPEKp/HWJSC co-localization versus mean HWJSC spheroid diameter, presented as mean fractional epithelial coverage represented by at least one confocal z-stack per data point. E: Quantified HPEKp/HWJSC co-localization versus mean HPEKp seeding density in cells/cm² of HWJSC spheroid surface area, calculated from mean HWJSC spheroid diameter in each image, presented as mean fractional epithelial coverage represented by at least one confocal z-stack per data point. F: Regression analysis, presented with a Pearson correlation coefficient and p-value for each variable, of 6 independent variables versus epithelial coverage: HWJSC spheroid diameter, HPEKp passage #, HWJSC passage #, HPEKp/HWJSC seeding ratio, HPEKp seeding density (cells/cm²), and HPEKp seeding density (cells/cm³).</p

Characterization of HWJSC spheroids and time-course of osteogenic differentiation.

<p>A: Schematic of HWJSC spheroid generation in agarose microwells, generated using AggreWell 800 as a template. B: Data representing the mean spheroid diameter of HWJSC spheroids at three different initial seeding densities (1k, 2k, 3k HWJSCs per spheroid) and in either osteogenic differentiation medium (OM) or growth medium (GM). HWJSCs were seeded in GM on day 0 and were subsequently cultured at day 1 and beyond in either OM or GM for the remainder of the culture (with medium changes every 2–3 days). HWJSC spheroid mean diameter ± 95% confidence interval was calculated for 40 spheroids per condition per time point from one representative experiment. C: Quantified mean intensity of 2k HWJSC spheroids cultured in OM and stained using an alkaline phosphatase substrate kit. Data are presented as the mean fluorescence intensity ± SEM for three independent experiments, normalized to the day 1 time point. Asterisks denote statistical significance calculated on raw mean intensity values at α = 0.05 of each condition relative to the day 1 and day 4 time points (*) or relative to the day 7 and day 14 time points (**) using two-way ANOVA and Tukey’s post-hoc test. D: Fluorescent micrographs representing maximum intensity projections of HWJSC spheroids that were stained for alkaline phosphatase activity at the indicated time point after the initiation of spheroid culture. Scale bar represents 200 μm. E-J: Fold change in gene expression over time of 2k HWJSC spheroids cultured in OM. Data represent fold change relative to GAPDH housekeeping gene and the day 1time point for MYC (E), PPARG (F), CD44 (G), VIM (H), BGLAP (I), and RUNX2 (J). Asterisks denote statistical significance relative to a mean value of ‘1’ using a one-tailed Student’s t-test (α = 0.05) for three independent experiments.</p