Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Abstract

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.

The use of sonication treatment to decellularize aortic tissues for preparation of bioscaffolds

“…49 Previous study has reported that decellularization with various methods including SDS, deoxycholate, and peracetic acid were able to reach up to 99% DNA removal per weight. [50][51][52] Likewise, sonication treatment was also able to obtain maximum efficiency of DNA removal. The efficiency of DNA removal by sonication decellularization system is dependent on sonication intensity.…”

Cell-derived matrices for tissue engineering and regenerative medicine applications

“…When reseeded with mESC, both SPT-EBM and RA-EBM supported greater cell proliferation compared to the proliferation of ESCs cultured as native EBs. However, SPT-EBM increased expression of early differentiation markers Brachyury, FGF-5 and FGF-8 of mESC, whereas the gene expression of ESC cultured on RA-EBM more closely resembled that of native EB 92 . This study demonstrated that EB-derived matrices can support ESC proliferation and differentiation, and that the differentiation protocol used to obtain the EBM can influence the phenotype of reseeded ESCs.…”

“…Similar to the use of MSC-derived matrices to improve somatic stem cell expansion, Goh et al proposed that ESC-derived ECM be used to recapitulate the embryonic microenvironment to improve the efficiency of current ESC lineage-specific differentiation protocols 92 . Two distinct EBM were generated by culturing mESC EBs with or without retinoic acid (RA), an inducer of neural differentiation.…”

“…Contrary to the results from the previous study, in this experiment the different EBM did not appear to have a direct effect on ESC differentiation. The differing outcomes of the two studies are not surprising, as there were fundamental experimental differences, including cell type, EB culture time, media formulations, dosing of RA, decellularization and reseeding methods, and culture of reseeded cells 92, 93 .…”

In vitro organogenesis from pluripotent stem cells

“…15,16 We and others have derived ECMs from various types of PSC aggregates at different developmental stages and demonstrated that the ECMs contain structures and signaling that can bias cell fate decisions. 17,18 The impact of PSC-derived ECMs on cellular differentiation may rely on ECM-bound growth factors such as fibroblast growth factor (FGF)-2, the biochemical composition of ECMs, and the biomechanical properties of ECMs. 19 Endogenous ECMs deposited on Matrigel have been found to support the propagation of hPSCs due to the retention of the paracrine and autocrine factors such as Gremlin and Cerberus, the antagonists of the bone morphogenetic protein (BMP) signaling.…”