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. 2013 Apr 18;8(4):e61856.
doi: 10.1371/journal.pone.0061856. Print 2013.

Extracellular matrix aggregates from differentiating embryoid bodies as a scaffold to support ESC proliferation and differentiation

Saik-Kia Goh  1 , Phillip Olsen, Ipsita Banerjee
Affiliations

Extracellular matrix aggregates from differentiating embryoid bodies as a scaffold to support ESC proliferation and differentiation

Saik-Kia Goh et al. PLoS One. .

Erratum in

  • PLoS One. 2013;8(10). doi:10.1371/annotation/201f73d1-bf44-4528-b71c-b537aad520f3

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interesting exist.

Figures

Figure 1
Figure 1. EB preparation, morphology and gene analysis.
(A) Preparation scheme for EB derived ECM scaffold. (B–C) Generation of EB via rotary suspension culture resulted in homogenous-sized EBs. Histology showing both groups of native EB – (D) SPT EB and (E) RA treated EB. Arrow heads indicate numerous cavities within the SPT EBs more than RA treated EB. (F–H) Quantification of gene expression via qRT-PCR shows that RA induces neural differentiation of EBs. (F) Nestin, (G) Pax6, and (H) Brachyury. Relative expression is normalized to SPT EB. H&E staining of EB sections shows presence of neural rosettes (dotted lines) in the (K) RA treated EBs confirming neuroepithelial tissue formation in contrast to (I) SPT EB. Immunohistochemical analysis of consecutive sections demonstrated positive for anti-Nestin staining in (J) RA treated EB but minimally expressed in (L) SPT EB. All values are mean ± SD, p<0.01(**), n = 3, represents pooled of EBs from 3 experimental repeats.
Figure 2
Figure 2. ECM characterization of native EBs resulting from different treatment - SPT vs. RA.
Immunofluorescence images of both groups of EBs – (A) SPT and (B) RA composing ECM molecules. Arrow heads indicate more cavities were found within the SPT EBs than the RA EBs. (C) Metamorph image analysis showing the quantification of the mean fluorescence intensity (MFI) of ECM markers stained in both group of EBs. Bar = 100 µm. All values are mean ± SD, p<0.05 (*), n = 6, represents pooled of EBs from 6 experimental repeats.
Figure 3
Figure 3. Decellularization of SPT and RA treated embryoid bodies.
(A) Panel images depict the decellularization process of EB. (B) Histological analysis of decellularized EB by H&E staining to show cell removal efficiency with three different detergents. (C) H&E staining of both groups of decellularized EB scaffolds showing absence of intact nuclei. (D) SEM images after decellularization process shows dense particulate material without distinct individual cell in both groups of EBs.
Figure 4
Figure 4. Immunofluorescence images of ECM biomolecules preserved after decellularization treatment in both groups of EBs.
(A) dSPT EB and (B) dRA EB. Both groups of decellularized EB preserved Collagen I, Collagen IV, Fibronectin, and Laminin after decellularization treatment.
Figure 5
Figure 5. Analysis of seeding efficiency of EB scaffolds by three different seeding methods.
(A) Schematic illustration to demonstrate three different methods of cell seeding. (B) Whole mount fluorescence images of seeded EB scaffolds demonstrate three different seeding strategies on decellularized EB scaffolds examined at 6 hours after initial seeding. (C) Alamar Blue assay depicts higher seeding efficiency in both the orbital shaker and hanging drop seeding method than the static method. Bar = 450 µm. All values are mean ± SD, p<0.01 (**), n = 3, represents individual seeded EBs in 3 experimental repeats.
Figure 6
Figure 6. Examination of viability, engraftment location and proliferation kinetics of the seeded EB constructs.
(A) Whole mount fluorescence images of seeded EB scaffolds at day 2 depict more ESR1 cells attach and grow on dSPT than dRA EB scaffolds. (B) Alamar blue assay depicts higher proliferation of ESR1 seeded on both decellularized EB scaffolds compared to native ESR1 EB over the course of 6 days. (C) Representative live/dead staining images show the survival of the engrafted ESR1 cells on both dSPT and dRA EB scaffolds after 2 days of culture. (D) Image analysis of live/dead assay to depict the mean percentage of live cells. (E) XY single-plane-two-photon imaging of cell-seeded constructs was done at depths of 33, 64 and 94 µm – this demonstrates that engrafted cells attached on the surface of the scaffolds with minimal infiltration into the ECM scaffold. All values are mean ± SD, n = 3, represents individual seeded EBs in 3 experimental repeats.
Figure 7
Figure 7. Gene and protein expression of the seeded EB constructs after 6 days of culture.
(A) qRT-PCR result at day 6 to depict gene expressions related to early gastrulation. The expression level is normalized to native ESR1 EB at day 6. Higher early differentiation markers –Brachyury, FGF5 and FGF8 were found on ESR1 seeded on dSPT EB scaffold. VE gene – AFP and pluripotency gene – Nanog are downregulated on both seeded constructs compared to native EB. Neuroectoderm marker – Nestin was upregulated on seeded dSPT EB scaffold but downregulated in seeded dRA EB scaffold. (B) Brachyury protein expression was investigated and confirmed by IHC – representative images showing higher cell numbers found positive for Brachyury in ESR1 seeded on dSPT EB scaffold compared to dRA EB scaffold. (C) Cell number area positive for Brachyury were measured and compared among ESR1 seeded on dSPT, dRA EB scaffolds and native ESR1 EB and average Brachyury immunoreactive area was significantly greater on ESR1 seeded on dSPT scaffold. All values are mean ± SD; p<0.05 (*), P<0.01 (**), n = 3, represents individual seeded EBs in 3 experimental repeats.
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References

    1. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Journal of Embryology and Experimental Morphology 87: 27–&. - PubMed
    1. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, et al. (2000) Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Molecular Medicine 6: 88–95. - PMC - PubMed
    1. Daley WP, Peters SB, Larsen M (2008) Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 121: 255–264. - PubMed
    1. Page-McCaw A, Ewald AJ, Werb Z (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8: 221–233. - PMC - PubMed
    1. Shukla S, Nair R, Rolle MW, Braun KR, Chan CK, et al. (2010) Synthesis and organization of hyaluronan and versican by embryonic stem cells undergoing embryoid body differentiation. J Histochem Cytochem 58: 345–358. - PMC - PubMed

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