doi:10.1016/j.scr.2008.05.006
Copyright © 2008 Elsevier B.V. All rights reserved.
The effect of controlled growth factor delivery on embryonic stem cell differentiation inside fibrin scaffolds
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Stephanie M. Willertha, Allison Radera and Shelly E. Sakiyama-Elberta, b, 
, 
aDepartment of Biomedical Engineering, Washington University, St. Louis, MO 63130, USA
bCenter for Materials Innovation, Washington University, St. Louis, MO 63130, USA
Received 14 January 2008;
revised 23 May 2008;
accepted 29 May 2008.
Available online 10 June 2008.
Abstract
The goal of this project was to develop 3-D biomaterial scaffolds that present cues to direct the differentiation of embryonic stem (ES) cell-derived neural progenitor cells, seeded inside the scaffolds, into mature neural phenotypes, specifically neurons and oligodendrocytes. Release studies were performed to determine the appropriate conditions for retention of neurotrophin-3 (NT-3), sonic hedgehog, and platelet-derived growth factor (PDGF) by an affinity-based delivery system incorporated into fibrin scaffolds. Embryoid bodies containing neural progenitors were formed from mouse ES cells, using a 4−/4+ retinoic acid treatment protocol, and then seeded inside fibrin scaffolds containing the drug delivery system. This delivery system was used to deliver various growth factor doses and combinations to the cells seeded inside the scaffolds. Controlled delivery of NT-3 and PDGF simultaneously increased the fraction of neural progenitors, neurons, and oligodendrocytes while decreasing the fraction of astrocytes obtained compared to control cultures seeded inside unmodified fibrin scaffolds with no growth factors present in the medium. These results demonstrate that such a strategy can be used to generate an engineered tissue for the potential treatment of spinal cord injury and could be extended to the study of differentiation in other tissues.
Figure 1. The effect of varying the peptide-to-heparin ratio in the affinity-based delivery system on growth factor release over a 14-day time course. (A) Release profile of NT-3 from the delivery system. (B) Release profile of PDGF from the delivery system. (C) Release profile of Shh from the delivery system. #P < 0.05 versus all other groups. * P < 0.05 versus no delivery system. Error bars indicate standard deviation.
Figure 2. The effects of the affinity-based delivery system on ESNPC differentiation and survival inside fibrin scaffolds. (A) Quantitative analysis of the effects of the different delivery system components on ESNPC differentiation inside fibrin scaffolds. (B) Quantitative analysis of the effects of the different delivery system components on ESNPC viability inside fibrin scaffolds. *P < 0.05 compared to 14-day control cultures in unmodified fibrin with no growth factors present in the medium. Error bars indicate standard deviation.
Figure 3. The effects of controlled growth factor delivery on ESNPC differentiation inside fibrin scaffolds. (A) The effects of controlled release of various NT-3 doses on ESNPC differentiation. (B) The effects of controlled release of various PDGF doses on ESNPC differentiation. (C) The effects of controlled release of various Shh doses on ESNPC differentiation. Markers examined included SSEA-1 (undifferentiated ES cells), nestin (neural progenitors), Tuj1 (early neurons), O4 (oligodendrocytes), and GFAP (astrocytes). *P < 0.05 versus cultures in unmodified fibrin with no growth factors present in the medium. #P < 0.05 compared to optimal dose with no delivery system present. Error bars indicate standard deviation.
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Figure 4. The effects of controlled delivery of growth factor combinations on ESNPC differentiation and survival inside fibrin scaffolds. (A) Quantitative analysis of the effects of the various growth factor combinations on ESNPC differentiation after 14 days of culture. Markers examined included SSEA-1 (undifferentiated ES cells), nestin (neural progenitors), Tuj1 (early neurons), O4 (oligodendrocytes), and GFAP (astrocytes). (B) Real-time RT-PCR analysis to confirm FACS results after 7 days of culture inside scaffolds. Markers examined included Sox2 (neural progenitors), microtubule-associated protein-2 (Map2; early neurons), platelet-derived growth factor α receptor (PDGFαR; early oligodendrocytes), and vimentin (astrocytes). (C) Quantitative analysis of the effects of the various growth factor combinations on ESNPC survival after 14 days of culture. *P < 0.05 versus control cultures in unmodified fibrin with no growth factors present in the medium. #P > 0.05 versus same growth factor combination with no delivery system present. +P < 0.05 versus all other groups. Error bars indicate standard deviation.
Figure 5. Diagrams showing the affinity-based delivery system and cell culture process. (A) Schematic of the affinity-based delivery system. The bidomain peptide becomes covalently cross-linked into the fibrin scaffold during polymerization. This peptide contains a heparin-binding domain, allowing heparin to be noncovalently retained inside the scaffold. The heparin in turn noncovalently binds the growth factor, causing it to remain in the scaffold. (B) Schematic of the process for producing embryoid bodies, followed by seeding and culture inside the fibrin scaffolds.
Table 1.
Ratios of the affinity-based delivery system components
a Estimated retention for one wash at equilibrium. Value estimated from dissociation constants.
b Obtained from release studies shown in
Fig. 2.
Corresponding author. Department of Biomedical Engineering, Washington University, St. Louis, MO 63130, USA. Fax: +1 314 935 7448.
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