Introduction: Heparin, a negatively-charged glycosaminoglycan (GAG), is known to play an important role in sequestration of positively charged growth factors. To modulate cellular response, the binding and release of growth factors can be modified by reducing the negative charge of heparin via desulfation[1]. In previous work, we have shown that transforming growth factor-β1 (TGF-β1), a protein commonly used to induce chondrogenesis in mesenchymal stem cells (MSCs), remains bioactive and has the ability to signal to surrounding environments when loaded on a novel heparin cell coating[2],[3]. However, the effect of TGF-β1 in culture media on the differentiation of MSCs coated with different heparin species has not been explored. Thus, we believe that sulfated heparin coated MSC aggregates will undergo more chondrogenic differentiation due to a stronger interaction with TGF-β1, compared to desulfated heparin coated aggregates.
Methods and Materials: Human MSCs were coated as single cells in suspension with sulfo-NHS-biotin (4mM), avidin (0.5mg/mL) and biotinylated-heparin (Hep) or biotinylated-desulfated heparin (Hep-) (5mg/mL). Each layer was incubated for 30min in rotary. Once coated, 2000-cell aggregates were formed in 96-well V-bottom plates and cultured in serum-free media with 10ng/mL TGF-β1 in hypoxia. On timepoints, samples were processed for histological analysis (n=12), gene expression analysis (n=4) and immunohistochemical staining (n=12). Gene expression was normalized to β-actin and 18s, and reported as an average fold regulation over the day1 level + standard deviation. Statistical analysis was performed using two-way ANOVA and Tukey’s post hoc test (p<0.05).
Results and Discussion: H&E staining revealed that coating with Hep or Hep- resulted in rounded cell morphology within the aggregate and fewer rounded cells were observed at day21 (Fig1).
Gene expression revealed that in Hep- coated aggregates, the chondrogenic marker and hypertrophic marker of col II and col X, respectively, was upregulated 90-fold and 35-fold, which was significantly higher than levels in noncoated and Hep coated at day21 (Fig 2A&C). Aggrecan was significantly increased by day21 compared to day 1 both Hep and Hep- coating, however they was not significantly different from each other (Fig 2B). Col I expression initially increased at day7, but no significance difference was observed at day21 (Fig 2D). Immunostaining revealed that the ECM components col II, aggrecan and col X were deposited over time throughout the aggregates for all groups, however, only more obvious staining for col X at day21 was observed in Hep- coated aggregates (data not shown).
Conclusions: Contrary to the hypothesis, this study revealed that a desulfated heparin cell coating promotes more chondrogenic differentiation in MSC aggregates than a fully-sulfated heparin coating. Although counterintuitive, the results may be explained by the negatively charged heparin sequestering and retaining TGF-β1, thus preventing it from interacting with cells. While coating with GAGs can be used as a novel technology to modulate cell differentiation within an aggregate, sulfation pattern on these coatings may need to be tailored to enhance the effects of soluble growth factors in vitro. These results present a novel platform for culture of MSC aggregates in serum-free conditions for potential applications in cell-based cartilage regeneration.
This work was supported by the funding source NSF DMR awarded to Johnna S. Temenoff (DMR 1207045).
References:
[1] Miller, T., et al., “Molecular engineering of glycosaminoglycan chemistry for biomolecule delivery,” Acta Biomaterialia.Vol 10(4): p. 1705-19, 2014.
[2] Somoza, R.A., et al., “Chondrogenic Differentiation of Mesenchymal Stem Cells: Challenges and Unfulfilled Expectations,” Tissue Engineering: Part B. Vol 20(6): p. 596-608, 2014.
[3] Lei, J., et al., “Characterization of a multilayer heparin coating for biomolecule presentation to human mesenchymal stem cell spheroid,” Biomaterials Science. Vol 2(5): p. 666-673, 2014.