Stem Cell Research (2008) 1, 205–218

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The effect of controlled delivery on embryonic stem cell differentiation inside scaffolds Stephanie M. Willerth a, Allison Rader a, Shelly E. Sakiyama-Elbert a,b,* a Department of Biomedical Engineering, Washington University, St. Louis, MO 63130, USA b Center for Materials Innovation, Washington University, St. Louis, MO 63130, USA

Received 14 January 2008; received in revised form 23 May 2008; accepted 29 May 2008

Abstract The goal of this project was to develop 3-D 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 and oligodendrocytes. Release studies were performed to determine the appropriate conditions for retention of neurotrophin-3 (NT-3), sonic hedgehog, and -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 a4−/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 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 injury and could be extended to the study of differentiation in other tissues. © 2008 Elsevier B.V. All rights reserved.

Introduction surrounding tissue. One of the most commonly used methods for incorporating growth factors or other cues involves cova- Combining bioactive scaffolds with embryonic stem (ES) cells lently tethering them to the scaffold material (Beckstead et provides a desirable alternative for replacement of damaged al., 2006; Fan et al., 2007; Gomez et al., 2007; Gomez and tissues (Ferreira et al., 2007; Levenberg et al., 2003, 2005; Schmidt, 2007; Ho et al., 2007; Kuhl and Griffith-Cima, 1996; Willerth et al., 2006, 2007a). For such engineered tissues to Taqvi et al., 2006; Yim and Leong, 2005). These methods function and integrate effectively in vivo, the cues necessary have been previously used to promote ES cell differentiation for promoting optimal ES cell differentiation should be re- and survival (Beckstead et al., 2006; Ho et al., 2007; Taqvi et tained inside such scaffolds, allowing them to be accessible al., 2006; Yim and Leong, 2005). While such strategies are to the cells and preventing loss due to diffusion into the highly effective at retaining such cues inside scaffolds, cova- lent linkages can impede internalization and, in turn, affect the activity and function of growth factors. Affinity-based * Corresponding author. Department of Biomedical Engineering, delivery systems have been explored as alternative methods Washington University, St. Louis, MO 63130, USA. Fax: +1 314 935 7448. of retaining growth factors inside scaffolds via noncovalent E-mail address: [email protected] (S.E. Sakiyama-Elbert). interactions (Benoit and Anseth, 2005; Benoit et al., 2007;

1873-5061/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scr.2008.05.006 206 S.M. Willerth et al.

Edelman et al., 1991; Sakiyama-Elbert and Hubbell, 2000a,b; oligodendrocytes while helping to improve the microenvir- Taylor et al., 2004, 2006; Taylor and Sakiyama-Elbert, 2006; onment at the injury site, allowing for regeneration by the Wissink et al., 2001). These systems utilize the interaction spared host cells. between and a target drug to retain the drug inside a The goal of this work was to generate an optimized scaffold, mimicking the way the scaffold for repair of SCI that contained an affinity-based sequesters growth factors in vivo. These systems improve delivery system, growth factors, and ESNPCs. To achieve this the retention of growth factor activity and allow growth goal, delivery conditions for retention of NT-3, PDGF-AA, and factor internalization. Such biomimetic delivery systems Shh were determined and then the ability of these factors to have shown promise as a potential treatment for spinal cord direct the differentiation of ESNPCs seeded inside scaffolds injury (SCI), but these scaffolds alone are not sufficient to when presented by the delivery system was determined. produce functional recovery (Taylor et al., 2004, 2006; Taylor Once optimal doses for each growth factor were found, these and Sakiyama-Elbert, 2006). doses were tested in combination to determine their effec- The use of progenitor cells derived from ES cells as a means tiveness at promoting differentiation of the ESNPCs into of restoring cellular populations lost because of SCI has also neurons and oligodendrocytes to serve as a replacement for been investigated with some promising results (Keirstead et the tissue lost to SCI. Specifically, the goal was to determine al., 2005; Liu et al., 2000; McDonald et al., 1999; Nistor et al., the doses of growth factor that needed to be presented by 2005). In a study by McDonald et al., mouse ES cells were the delivery system to replicate the differentiation observed induced to form embryonic stem cell-derived neural lineage in the previous soluble growth factor studies (Willerth et al., precursor cells (ESNPCs) using the 4−/4+ retinoic acid treat- 2007a) that would allow presentation of similar cues follow- ment protocol (Bain et al., 1995) and then injected into a rat ing cell transplantation. Additionally, control experiments model of SCI as treatment (McDonald et al., 1999). These cells were performed to confirm that the effects observed were promoted a modest increase in functional recovery (∼3on due to the enhanced retention of growth factors by the the Basso, Beattie, and Bresnahan scale). However, few cells delivery system. These scaffolds could also be used to repli- (∼5k) differentiated into neurons and the viability of the cate the microenvironment present during the different transplanted cells was low (∼10k). Similar results have been stages of development and may have further applications for obtained by other groups when stem cells were transplanted studying the cues necessary to direct stem cell differentia- directly in the injured spinal cord (Cao et al., 2001, 2002). tion for other lineages. The glial scar promotes differentiation of stem cells into and discourages cell migration into the injury site. Implan- Results table biomimetic scaffolds could provide a permissive microenvironment for transplanted stem cells and cues to promote survival and differentiation into neurons and Determining delivery system conditions for oligodendrocytes at the injury site. maximum growth factor retention inside fibrin Toward this goal, previous work from our lab has iden- scaffolds tified the appropriate conditions for culturing mouse ESNPCs inside three-dimensional fibrin scaffolds and soluble growth To determine the delivery system conditions that would ge- factors that promote the differentiation of ESNPCs into nerate the slowest release of the individual growth factors to neurons and oligodendrocytes (Willerth et al., 2006, 2007a). ESNPCs, two peptide-to-heparin ratios were tested in the The later study found that combining neurotrophin-3 (NT-3) context of the delivery system by changing the concentration with platelet-derived growth factor (PDGF) and NT-3 with of heparin present. Changing the heparin concentration also sonic hedgehog (Shh) increased the fraction of ESNPCs that alters the ratio of heparin to growth factor and all of these differentiated into neurons and oligodendrocytes compared ratios are listed in Table 1. The two chosen heparin con- to control cultures in unmodified fibrin with no growth fac- centrations (62.5 and 6.25 μM) resulted in either a 4:1 or a tors present in the medium, making them worthy of further 40:1 heparin to peptide ratio, respectively. These ratios investigation for use with our affinity-based delivery system. were selected based on previous work (Taylor et al., 2004; Additionally, these growth factors have shown beneficial Wood and Sakiyama-Elbert, 2008) and had been shown to effects when tested in the context of SCI. Delivery of NT-3 provide sustained release of growth factors that possess an has been shown to enhance neural fiber sprouting into the affinity for heparin. The concentration of heparin changes injury site (Taylor et al. 2004, 2006) and promote regenera- the number of binding sites available to the growth factor tion of the corticospinal tract (Schnell et al., 1994; Taylor et present in the scaffold and thus affects the rate of growth al., 2004, 2006; Tuszynski et al., 2003). Treatment with all factor release from the scaffolds. The goal of this release three PDGF isoforms (PDGF-AA, PDGF-AB, and PDGF-BB) pro- study was to determine which ratio resulted in the slowest motes angiogenesis to the injury site, which may help growth factor release from the scaffolds and retained the produce a more hospitable environment for regeneration to maximum amount of growth factor. Release was evaluated occur (Hiraizumi et al., 1992, 1993, 1996). Shh induces over a 2-week period, which corresponds roughly to the de- proliferation of neural progenitors when applied after SCI, gradation time of the fibrin scaffolds in vivo. Over the first and when used in combination with transplanted oligoden- 24 h, the scaffolds were washed extensively (five times) to drocyte progenitor cells, it has been shown to improve reco- remove unincorporated components, followed by one wash a very and increase the amount of white matter spared from day for the remaining 13 days of the study. injury (Bambakidis and Miller, 2004; Bambakidis et al., 2003). Figure 1A shows the results of the 14-day release study to Thus, these growth factors serve two purposes in vivo. They determine the optimal peptide-to-heparin ratio for NT-3 can promote the differentiation of ESNPCs into neurons and delivery. When no heparin was present, virtually all of the Stem cell differentiation inside fibrin scaffolds 207

Table 1 Ratios of the affinity-based delivery system components Growth factor Peptide-to- Heparin-to-growth Estimated fraction Fraction of growth Fraction of growth heparin ratio factor ratio of growth factor factor retained in factor retained in bound a scaffold after 24 h b scaffold after 14 days b Neurotrophin-3 4:1 1700:1 0.89 0.61 0.39 40:1 170:1 0.74 0.44 0.25 Platelet-derived 4:1 1600:1 0.98 0.53 0.43 growth factor 40:1 160:1 0.82 0.37 0.26 Sonic hedgehog 4:1 1300:1 0.74 0.19 0.10 40:1 130:1 0.64 0.02 0.01 a Estimated retention for one wash at equilibrium. Value estimated from dissociation constants. b Obtained from release studies shown in Fig. 2.

NT-3 (∼99k) was released from the scaffold by the end of 14 components on differentiation and cell viability. For in days. In contrast, both the 4:1 and the 40:1 ratios provided vitro differentiation studies, scaffolds were polymerized sustained NT-3 delivery over the same time period. The 4:1 containing the different components at the indicated peptide-to-heparin ratio retained the most NT-3 (∼40k)in concentrations and all scaffolds were washed five times the scaffold at the end of the study, which was significantly over 24 h before cell seeding to remove unincorporated more than the amount retained when the 40:1 ratio was components and to simulate the conditions of an in vivo used. The different ratios produced two different rates of setting in which the unincorporated components would be NT-3 release, with the 4:1 ratio being selected for delivery of washed away. The effects of different delivery system NT-3 to the ESNPCs seeded inside the scaffolds because it components on differentiation and viability after 14 days of generated the slowest release and maximized growth factor culture can be seen in Figs. 2A and 2B. When peptide alone or retention. peptide and heparin were incorporated into the scaffolds, a Similar results were obtained for the release studies decrease in astrocytes was observed. When heparin alone involving delivery of PDGF (Fig. 1B). Three different rates of was added to the scaffolds, a decrease in nestin (neural release were observed, depending on the amount of heparin progenitor) staining was observed in addition to the decrease present in the delivery system. When no heparin was present, in the percentage of astrocytes. However,combining peptide ∼10k of the PDGF remained in the scaffold at the end of with heparin restored the nestin to levels similar to those of the study. When a 4:1 ratio was used, ∼42k of the PDGF was the cultures in unmodified fibrin with no growth factors retained in the scaffold, while the 40:1 ratio resulted in present in the medium. The incorporation of the delivery ∼26k retention. Thus the 4:1 peptide-to-heparin ratio was system components into the fibrin scaffold did not have any selected for the in vitro differentiation studies because it effect on cell viability compared to the unmodified fibrin retained significantly more PDGF than the 40:1 ratio. scaffolds. Release of Shh from the delivery system was also char- A series of various growth factor doses was delivered to acterized for the different heparin-to-peptide ratios (Fig. ESNPCs seeded inside fibrin scaffolds. Doses were selected 1C). The 4:1 ratio generated the slowest rate of release, but based on the previous studies conducted with growth factors it retained only ∼10k of Shh at the end of the 14-day study. present in the culture medium (Willerth et al., 2007a) and the However, when no heparin was present or the 40:1 ratio was results of the release studies. The purpose of this study was to used, Shh was released from the system much more rapidly, determine the optimal dose for promoting differentiation of with 99k of the Shh released by day 2 of the 14-day study. neurons and oligodendrocytes from ESNPCs seeded inside This release rate was much faster than the rate observed for fibrin scaffolds containing the delivery system. As seen in Fig. the scaffolds containing NT-3 or PDGF with no heparin pre- 3A, all doses of NT-3 delivered resulted in a reduction of the sent. For Shh, only the 4:1 ratio produced a release rate that fraction of cells differentiating into astrocytes. Only the 125- was significantly different from that of the scaffolds con- ng (417 ng/ml) dose produced an increase in the fraction of taining growth factor without heparin. Overall, the 4:1 neurons and neural progenitors obtained compared to the peptide-to-heparin ratio resulted in maximum growth factor group cultured in unmodified fibrin with no growth factors retention inside the scaffolds for all growth factors tested. present in the medium. Additionally, at this dose, an increase in the fraction of cells differentiating into oligodendrocytes was observed compared to the same dose of NT-3 when no Determining optimal growth factor doses from the delivery system was present. Accordingly, the 125-ng dose of delivery system for promoting ESNPC differentiation NT-3 was selected for use in the combination growth factor into neurons and oligodendrocytes studies since it increased the fraction of cells that differ- entiated into both neurons and oligodendrocytes. Before the effects of controlled growth factor delivery on the Based on previous work, various PDGF doses ranging from resulting cell phenotypes of the ESNPCs seeded inside were 10 to 40 ng were delivered to ESNPCs seeded into fibrin examined, a set of control experiments was performed to scaffolds, as seen in Fig. 3B. The 20-ng dose (66 ng/ml) of determine the effects of each of the delivery system PDGF promoted the highest level of differentiation of ESNPCs 208

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. #Pb0.05 versus all other groups.*Pb0.05 versus no ..Wlet tal. et Willerth S.M. delivery system. Error bars indicate standard deviation. Stem cell differentiation inside fibrin scaffolds 209

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. *Pb0.05 compared to 14-day control cultures in unmodified fibrin with no growth factors present in the medium. Error bars indicate standard deviation. into oligodendrocytes while reducing the fraction of cells drocytes produced while reducing the fraction of cells dif- differentiating into astrocytes compared to the group cul- ferentiating into astrocytes compared to the cultures in tured in unmodified fibrin with no growth factors present in unmodified fibrin with no growth factors present in the the medium. For a 10-ng dose of PDGF (33 ng/ml), no diffe- medium (Fig. 3C). The lowest Shh dose (50 ng total) produced rences were observed compared to controls cultured in un- an increase only in the fraction of cells differentiating into modified fibrin with no growth factors present in the medium neurons while decreasing the fraction of cells differentiating and for the higher dose, only a decrease in the fraction of into astrocytes. However, no difference in the fraction of cells staining positive for glial fibrillary acidic protein (GFAP) cells differentiating into oligodendrocytes was observed was observed. When the 20-ng dose of PDGF was polymerized compared to the cultures in unmodified fibrin with no growth into scaffolds with no delivery system present, only a de- factors present in the medium. A decrease in the fraction of crease in the fraction of cells differentiating into astrocytes cells differentiating into astrocytes was the only difference was observed compared to the group cultured in unmodified observed for both the highest dose of Shh delivered (150 ng) fibrin with no growth factors present in the medium. and the 100-ng Shh dose with no delivery system present. When Shh was delivered from scaffolds containing the Based on these results, three different growth factor delivery system, the 100-ng dose resulted in increases in the combinations, NT-3 and PDGF, NT-3 and Shh, and NT-3, PDGF, fraction of cells differentiating into neurons and oligoden- and Shh, were released from fibrin scaffolds both with and 210 S.M. Willerth et al.

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). *Pb0.05 versus cultures in unmodified fibrin with no growth factors present in the medium. #Pb0.05 compared to optimal dose with no delivery system present. Error bars indicate standard deviation. without the delivery system present, and the resulting diffe- All three growth factor combinations had significant rentiation and viability were quantified as seen in Figs. 4A effects on the differentiation of ESNPCs compared to both and 4B. The optimal dose of each growth factor for pro- the group with no growth factor present and the scaffolds moting ESNPC differentiation into neurons and oligodendro- containing the same doses of growth factors without a cytes that had been determined from the individual studies delivery system. These three combinations all produced an was used in these combination studies. As seen in Fig. 4A, increase in the fraction of cells staining positive for nestin no differences in the fraction of cells staining positive for and O4 compared to both the control group cultured in SSEA-1 was observed for any of the experimental groups, but unmodified fibrin scaffolds with no growth factor present all experimental groups did exhibit a decrease in the fraction and the control group consisting of the same growth factor of cells staining positive for GFAP compared to the group doses with no delivery system. As mentioned before, the cultured in unmodified fibrin scaffolds with no growth factor control cultures without a delivery system are important to present. show that the delivery system is retaining more growth Stem cell differentiation inside fibrin scaffolds 211 factor as well as presenting it in a biologically active manner. Release studies determined the appropriate delivery Controlled delivery of certain growth factor combinations system conditions needed to retain the maximum amount (the dual combination of NT-3 and PDGF or the triple of growth factor using the affinity-based delivery system. combination of NT-3, PDGF, and Shh) increased the fraction The release studies were performed in the absence of cells to of cells differentiating into neurons compared to both the quantify the amount of growth factor passively released over group with no growth factor present and the group consisting time. In the in vitro culture system and in vivo, the remaining of the same growth factor doses with no delivery system. growth factor is actively released from the scaffolds when Controlled delivery of NT-3 and Shh increased only the cells activate plasmin, which enzymatically degrades the fraction of cells differentiating into neurons compared to scaffold. In the in vitro culture system, the growth factors the group with no growth factor present. Additionally, can act upon the ESNPCs seeded inside the scaffolds, and in controlled delivery of NT-3 and PDGF also produced an vivo, the growth factors can be released by both the ESNPCs increase in the fraction of cells differentiating into neurons and the surviving cells that remained after the injury. compared to the controlled delivery of the other two growth Two different heparin concentrations were tested in the factor combinations tested, making this combination (NT-3 context of the delivery system and the 4:1 ratio resulted in and PDGF) most effective for achieving the desired goal of maximum growth factor retention for all growth factors promoting differentiation of ESNPCs into neurons and tested. These results suggest that the increase in heparin oligodendrocytes. concentration provided more binding sites to retain the To confirm the results observed using fluorescence-acti- growth factors inside the scaffold. For the 4:1 peptide-to- vated cell sorting (FACS), real-time reverse transcriptase- heparin ratio, there is an ∼1300-1700 times excess of binding polymerase chain reaction (RT-PCR) was used to examine the sites (heparin molecules) per growth factor, while the 40:1 expression levels of four different markers for these cell ratio yields only ∼130-170 times excess of binding sites phenotypes, including Sox2 (neural progenitors), microtubule- (Table 1). associated protein-2 (Map2; early neurons), platelet-derived Table 1 shows the estimated fraction of growth factor growth factor α receptor (PDGFαR; early oligodendrocytes), bound at equilibrium calculated from the dissociation and vimentin (astrocytes). Figure 4B shows the results of constants for each growth factor. The release rates of the the real-time RT-PCR performed to confirm the effects of growth factors correlate with the estimated bound fraction controlled delivery of NT-3 and PDGF compared to unmodi- at equilibrium. The affinity-based delivery system was pre- fied fibrin scaffolds and scaffolds containing NT-3 and PDGF dicted to retain more PDGF and NT-3 than Shh at equilibrium. with no delivery system present. For the Sox2 marker, no This trend was observed in the release studies with the 4:1 statistical differences were observed between groups. ratio, which was confirmed by the 14-day release studies. Increases in the expression levels of Map2 and PDGFαR For NT-3 and PDGF,the delivery system retained different compared to the unmodified fibrin scaffolds were observed amounts of growth factor depending on the peptide-to- only for the group cultured in the scaffolds containing the heparin ratio. The pattern of growth factor release was delivery system with NT-3 and PDGF. Both groups treated consistent with previously published release studies of NT-3 with NT-3 and PDGF, both in the presence and in the absence and PDGF-BB, a different PDGF isoform (Taylor et al., 2004; of delivery system, showed a decrease in the expression of Thomopoulos et al., 2007). Only the 4:1 ratio retained Shh vimentin compared to the group cultured in unmodified inside the scaffolds. Shh had never been tested previously fibrin with no growth factors present in the medium. Figure with the affinity-based delivery system, but it has been 4C shows the effects of the various growth factor combina- reported to have a high affinity for heparin (Rubin et al., tions on cell viability. All growth factor combinations that 2002; Zhang et al., 2007). The quick release of Shh could contained PDGF (both in presence and in the absence of the potentially be due to repulsive interactions between Shh and delivery system) promoted an increase in cell viability com- the scaffold itself. pared to the group with no growth factor present. An initial burst of growth factor release was observed over the first 24 h. In an in vivo setting, this unbound growth factor would be washed away from the scaffold by cere- Discussion brospinal fluid present at the injury site. To account for this effect, all scaffolds were washed five times before cell To produce an engineered tissue as a treatment for SCI, seeding and the addition of the second scaffold layer. Diffe- delivery conditions and appropriate growth factor dosing for rent effects on ESNPC differentiation were observed when promoting ESNPC differentiation into neurons and oligo- the same growth factor dose was polymerized inside the dendrocytes were determined, as both of these cell types scaffolds in the presence of delivery system compared to could potentially contribute to functional recovery after SCI. when no delivery system was present. Neurons could help to restore the circuitry disrupted after It is also important to consider the effects of the delivery injury, allowing for reinnervation (Lee et al., 2007). Oli- system on the differentiation of ESNPCs, as bioactive pep- godendrocytes could restore the myelination of the spared tides have been previously used to promote differentiation axons present at and around the injury site and such oligo- of neural progenitors seeded on nanofibers (Silva et al., dendrocyte replacement strategies have been shown to pro- 2004). The presence of peptide and heparin, both individu- mote functional recovery in animal models of SCI (Keirstead ally and in combination, reduced the fraction of ESNPCs that et al., 2005; Liu et al., 2000; Nistor et al., 2005). Addi- differentiated into astrocytes. The reduction in the fraction tionally, minimizing production was important, as of cells that differentiated into astrocytes was observed for they could contribute to the glial scar and inhibit regenera- all of the delivery system groups except when it was used to tion (Fawcett and Asher, 1999). delivery 10 ng total of PDGF, suggesting that the presence of 212 S.M. Willerth et al. Stem cell differentiation inside fibrin scaffolds 213 growth factor does not interfere with this beneficial effect. delivery system required polymerizing high doses of Shh into The presence of heparin alone decreased the fraction of the scaffolds to promote differentiation, and more effective neural progenitors present, but this decrease was not ob- retention of Shh by an affinity-based delivery system might served when both peptide and heparin were present, be obtained by using different scaffold material. suggesting that the peptide may help maintain nestin ex- Controlled delivery of all three growth factor combina- pression. When looking at the effects of the growth factors tions produced significant changes compared to both unmo- on ESNPC differentiation, it is important to view the results dified fibrin scaffolds with no growth factors present in the in the context of these control experiments. For other tissue- medium and scaffolds containing the same growth factor engineering applications, it will be important to run similar combinations with no delivery system. The main difference control experiments to ensure these components do not between the three combinations tested was in the fraction of interfere with the desired stem differentiation. cells differentiating into neurons. The combination of NT-3 For each of the growth factors tested, a range of con- and PDGF produced the largest fraction of cells differentiat- centrations was selected by combining the results of the ing into neurons compared to all the groups tested, making it release studies and previous studies that looked at the the optimal combination for further testing in the context of effects of growth factors in the culture medium on ESNPC an in vivo model of SCI. This result was also confirmed using differentiation inside fibrin scaffolds (Willerth et al., 2007a). real-time RT-PCR analysis with increases in the expression Controlled delivery of a 125-ng dose (417 ng/ml) from the levels of Map2 and PDGFaR and a decrease in vimentin ex- affinity-based delivery system produced and oligo- pression levels compared to the cultures in unmodified fibrin dendrocyte differentiation comparable to 25 ng/ml NT-3 in scaffolds with no growth factors present. the media. Soluble NT-3 has also been shown to promote the These results are consistent with other literature that has differentiation of human ES cells into neurons in three- shown different situations in which combining NT-3 with dimensional settings, suggesting these scaffolds might also PDGF produces additive effects. Previous studies have shown be useful for human ES cell lines (Levenberg et al., 2005). NT- that this combination promotes the survival of Schwann cell 3 also promotes the survival of oligodendrocyte precursors precursors and remyelination in rodent models of multiple (Kumar et al., 1998), which could potentially explain the sclerosis, suggesting that their signaling pathways may share increase in O4 staining observed with controlled delivery of an intermediate that produces these additional benefits the optimal dose (100 ng NT-3) with the delivery system. (Fressinaud, 2005; Lobsiger et al., 2000). Also, the inclusion Controlled delivery of NT-3 also produced an increase in the of PDGF in combination with other growth factors produced percentage of cells staining positive for nestin, which could an increase in cell viability. This increase in viability may potentially be due to the delivery system, as this effect increase the survival rate of the transplanted cells in vivo. was not observed in the previous studies with NT-3 in the This study has produced an engineered neural tissue medium. consisting of a fibrin scaffold containing an affinity-based From our previous work, the controlled release dose of delivery system, growth factors, and ESNPCs ready for in vivo PDGF (20 ng total) corresponds to between 2 and 10 ng/ml testing as a potential treatment for SCI. The growth factor soluble PDGF and replicates the benefits of both doses, delivery system and concentration were optimized to pro- which included an increase in the fraction of cells differ- mote the differentiation of the cells seeded within these entiating into oligodendrocytes and a decrease in the frac- scaffolds into neurons and oligodendrocytes and to increase tion of cells differentiating into astrocytes. These results are cell viability. The concentrations found in this study for consistent with other studies that have demonstrated PDGF's promoting the differentiation of ESNPCs into these cell phe- role in promoting oligodendrocyte development (Calver et notypes can be easily translated to other materials with al., 1998). known release properties for in vivo SCI applications. Such a Controlled delivery of 100 ng of Shh produced similar strategy can be extended to generate replacements for other ESNPC differentiation to both 10 and 25 ng/ml Shh in the types of damaged tissues. Finally, this affinity-based delivery medium. The increase in neuronal differentiation was ex- system could also be used to study signaling events during pected, as many studies have demonstrated that Shh com- development. Its ability to mimic the extracellular matrix bined with retinoic acid promotes the differentiation of ES and present growth factors in a controlled manner allows for cells into motor neurons (Dutton et al., 1999; Lee et al., the reproduction of signals present during development in an 2007; Li et al., 2005; Miles et al., 2004; Soundararajan et al., in vitro setting. Thus, this delivery system allows for 2006, 2007). Shh also plays important roles in promoting generation of tissue-engineered scaffolds as well as provid- oligodendrocyte differentiation (Danesin et al., 2006; Oh et ing an in vitro system for studying the development biology al., 2005). The poor retention of Shh by the affinity-based of stem cells.

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. *Pb0.05 versus control cultures in unmodified fibrin with no growth factors present in the medium. #PN0.05 versus same growth factor combination with no delivery system present. +Pb0.05 versus all other groups. Error bars indicate standard deviation. 214 S.M. Willerth et al. Stem cell differentiation inside fibrin scaffolds 215

Materials and methods peptide-to-heparin ratios produce sustained growth factor release from fibrin scaffolds (Taylor et al., 2004; Wood and All materials were purchased from Sigma-Aldrich unless Sakiyama-Elbert, 2008). otherwise noted. To produce the components needed for the delivery sys- tem, the bidomain peptide ( sequence: (AcG) NQEQVSPK(βA)FAKLAARLYRKA, substrate Preparation of fibrin scaffolds containing the indicated by italics) was synthesized using standard Fmoc biomimetic delivery system chemistry (amino acids from EMD Biosciences and peptide synthesis solvents from Applied Biosystems) and purified Plasminogen-free (Calbiochem) obtained from using reverse-phase C18 chromatography, with the peptide human plasma was dissolved in Tris-buffered saline (TBS; pH sequence being confirmed using matrix-assisted laser deso- 7.4) for 2 h at 37 °C. Sterile fibrinogen solutions were pre- rption ionization mass spectroscopy as previously described pared as previously described (Willerth et al., 2007b). Fibrin (Willerth et al., 2007b; Wood and Sakiyama-Elbert, 2008). scaffolds (10 mg/ml) were polymerized under conditions Sterile porcine heparin solutions (average molecular weight previously determined be optimal for ES cell culture (Will- 18 kDa; concentrations 6.25 and 62.5 μM) were prepared and erth et al., 2006). The affinity-based delivery system (Fig. 5) the following growth factors were delivered using this consists of three components: a bidomain peptide, heparin, system: NT-3 (Peprotech), PDGF-AA (R and D Systems), and and growth factor. The peptide contains a Factor XIIIa Shh (R and D Systems). These components were added at the substrate derived from α -plasmin inhibitor (Ichinose et al., 2 indicated concentrations during the fibrin polymerization 1983), allowing it to be covalently cross-linked into the fibrin process to ensure incorporation into the scaffolds. scaffold, and a heparin-binding domain derived from anti- III (Tyler-Cross et al., 1994), which binds heparin noncovalently and retains it inside the fibrin scaffold. The Characterization of controlled growth factor heparin can in turn bind growth factors with an affinity for release from fibrin scaffolds heparin and retain them inside the scaffold. Thus these two reversible reactions (peptide binding to heparin and heparin Fibrin scaffolds (400 μl) were polymerized to contain 0.25 binding to growth factor) allow the delivery system to retain mM bidomain peptide, 100 ng/ml of growth factor (40 ng growth factors noncovalently inside the fibrin scaffolds. At total), and varying concentrations of heparin. Control scaf- equilibrium, five different species are present inside the folds contained no heparin, while the experimental scaffolds scaffold, including unbound heparin, unbound growth factor, contained either 6.25 or 62.5 μM heparin, corresponding heparin-growth factor complexes, heparin bound to the to 40:1 and 4:1 peptide-to-heparin ratios. Two different immobilized peptide, and heparin-growth factor complexes peptide-to-heparin ratios were tested to determine which that are bound to the peptide. ratio retains the maximum amount of growth factor inside The goal of using this delivery system is to maximize the the scaffold. The peptide-to-heparin ratios and the heparin- fraction of heparin-growth factor complexes that are bound to-growth factor ratios tested are listed in Table 1. to the peptide inside the fibrin scaffold. For promoting These acellular scaffolds were washed five times over the differentiation of ESNPCs, it is important to maximize the first 24 h with 1 ml of TBS containing 2k bovine serum amount of growth factor retained inside the scaffold to albumin (BSA) followed by once a day for the next 13 days, ensure retention in an in vivo setting. Changing the resulting in a 14-day time course. Each wash was collected in concentration of heparin present affects the amount of a silanized tube and stored at −20 °C. After the end of the growth factor retained inside the scaffold by shifting the study, the remaining growth factor was released from the chemical equilibrium of the system. In the reversible scaffold using extraction buffer (phosphate-buffered saline reaction, when heparin and growth factor bind to form a containing 10 mg/ml heparin, 2 M NaCl, 1k BSA, 0.01k complex, adding excess unbound heparin should cause Triton-X, pH 7.4) as previously described (Willerth et al., increased formation of heparin-growth factor complexes at 2007b; Wood and Sakiyama-Elbert, 2008). The amount of equilibrium according to LeChatlier's principle. These com- NT-3 and Shh present in the washes and scaffolds was plexes can in turn bind noncovalently to the immobilized quantified using ELISA kits (R and D Systems). To quantify the peptide, becoming immobilized inside the scaffold. Differ- amount of PDGF present, a monoclonal α-PDGF-AA antibody ent heparin concentrations will result in different amounts (clone MAB1055; R and D Systems) was used as the capture of heparin-growth factor complexes being formed, affecting antibody and a biotinylated α-PDGF-AA antibody (clone the amount of heparin-growth factor complexes that can BAF1055) served as the detection antibody. Absorbance bind to the immobilized peptide at equilibrium. In this readings at 450 nm were measured using a 96-well plate manner, the amount of heparin present affects the rate at reader (Multiskan RC Labsystems). The experimental wells which growth factor is released from the scaffolds. Previous were compared to a concentration curve of known standards work from our lab has demonstrated that 4:1 and 40:1 to determine the quantity of growth factor present.

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. 216 S.M. Willerth et al.

Embryoid body formation and culture inside fibrin 1-h incubations with primary and secondary antibody. For scaffolds intracellular markers (nestin, Tuj1, and GFAP), cells were fixed with 1k paraformaldehyde, permeabilized with 0.5k RW4 mouse ES cells were cultured on gelatin-coated plates saponin solution, and blocked with 5k NGS solution. These using complete medium (Dulbecco's modified Eagle medium cells were then incubated with primary and secondary containing 10k fetal bovine serum, 10k newborn calf serum, antibodies for 30 min each. Cells stained with secondary and 0.3 M nucleoside mix, all from Invitrogen except the antibody only were used to eliminate nonspecific background nucleosides, which were from Sigma) containing 1000 U/ml secondary antibody staining. A more stringent control would leukemia inhibitory factor (LIF) and 10-4 M β-mercaptoethanol have been to use an isotype control primary antibody to (BME) as described previously (Willerth et al., 2006, 2007a). ensure the specificity of the staining. For quantitative ana- Undifferentiated ES cells were induced to form embryoid lysis of cell viability, cells were stained using the Live/Dead bodies (EBs) containing ESNPCs using the 4−/4+ retinoic acid Viability/Cytotoxicity kit (Invitrogen) as previously described treatment protocol before being seeded onto the scaffolds (Willerth et al., 2007a). The cells were incubated with PBS (Bain et al., 1995). The cells were cultured in complete me- solutions containing 0.1 μM calcein AM and 16 μM ethidium dium without LIF or BME for the first 4 days followed by an homodimer for 30 min. Cellular fluorescence was detected additional 4 days of culture in complete medium containing using a FACSCalibur flow cytometer (Becton-Dickinson) and 100 nM retinoic acid. The medium was changed every other analysis was performed using CellQuest software (Becton- day during this process. The 4−/4+ retinoic acid treatment Dickinson). Representative plots showing the fluorescence protocol produces a cell population consisting of immature for the different control and experimental groups are shown neural progenitors (Willerth et al., 2007a). Of these cells, for the cultures seeded inside unmodified fibrin scaffolds 61F6k stain positive for the SSEA-1 marker, which is with no growth factors present and the cultures seeded inside expressed by undifferentiated mouse ES cells, and 71F6k scaffolds containing the delivery system along with PDGF and of these cells stain positive for nestin, which is a neural NT-3 in Supplementary Figs. 1 and 2. progenitor marker. These cells did not stain positive for any of the mature neural cell markers, including β-tubulin III (early Quantitative real-time RT-PCR neurons), O4 (oligodendrocytes), and GFAP (astrocytes). For studying the effects of controlled growth factor To confirm the results observed using FACS, quantitative release, 300 μl of scaffold containing delivery system and real-time RT-PCR was used to examine the expression levels growth factors was added into each well of a 24-well plate of markers for different cell phenotypes for three different (Corning) and allowed to polymerize for 1 h at 37 °C. These groups, including EBs cultured in unmodified fibrin, in scaffolds were then washed five times with 1 ml of TBS over scaffolds containing NT-3 and PDGF with no delivery system the next 24 h, with each wash being applied for at least 2 h present, and in scaffolds containing NT-3 and PDGF with a to remove unincorporated delivery system components. delivery system. Real-time RT-PCR was used to examine the Following aspiration of the final wash, an individual EB expression of the following four markers: Sox2 (neural was placed on each scaffold and a second layer of scaffold progenitors), Map2 (early neurons), PDGFαR (early oligoden- (100 μl) without delivery system was then added to drocytes), and vimentin (astrocytes). surround each EB. After an additional hour of incubation To obtain RNA for real-time RT-PCR analysis, the cells were at 37 °C, 1 ml of complete medium was added and the cells cultured inside scaffolds for 7 days. An earlier time point was were cultured in scaffold for 14 days, with the medium chosen because mRNA upregulation often occurs before being changed to neural basal medium containing B27 changes in protein expression are observed. Cells were then supplement (both from Invitrogen) on day 3 as previously isolated from the scaffolds using trypsin as described above described (Willerth et al., 2006, 2007a). A schematic of this and the resulting cells were lysed using Buffer RLT (Qiagen). process is shown in Fig. 5B. These lysates were homogenized using a Qiashredder kit and RNA was then isolated using an RNEasy kit (both from Qiagen). Fluorescence-activated cell sorting From the isolated RNA, cDNA was synthesized using the Quantitech reverse transcription kit (Qiagen) according to After 14 days of culture, the medium was aspirated from each the manufacturer's instructions. Using the Quantitech SYBR scaffold, followed by a wash with phosphate-buffered saline Green PCR Mastermix (Qiagen) combined with gene-specific (PBS; pH 7.4). Cells were dissociated using trypsinization and QuantiTect primer assays, quantitative real-time PCR was manually removed from the scaffolds and then resuspended performed using an Applied Biosystems 7000 real-time PCR in PBS. The cells were then stained and sorted as previously system using the following PCR cycle: 50 °C for 2 min, 95 °C described (Willerth et al., 2007a). For cell phenotype ana- for 10 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 lysis, cells were stained for the following markers: stage- s, 72 °C for 30 s, with fluorescent signal detected at 72 °C. specific embryonic antigen 1 marker (SSEA-1; undifferen- Target genes were normalized to β-actin expression to tiated mouse ES cells; 1:25; Chemicon), nestin (neural pre- account for variations in cDNA concentration between cursors; 1:100; Chemicon), β-tubulin III (Tuj1; early neurons; samples. The differences in gene expression levels were 1:1000; Covance), O4 marker (1:100; oligodendrocytes; calculated using the comparative DCt method, which allows Chemicon), and GFAP (astrocytes; 1:40; Immunostar). Spe- for determining relative expression of RNA between two cies-specific secondary antibodies (AlexaFluor 488; Invitro- samples. In this method, the crossover threshold (Ct) refers to gen) were used to detect the primary antibodies. For cell- the cycle number when the fluorescence released by the PCR surface markers (SSEA-1 and O4), live cells were blocked reaction begins to increase exponentially. The difference using PBS containing 5k normal goat serum (NGS) followed by between relative Ct values for each sample was used to Stem cell differentiation inside fibrin scaffolds 217 estimate the fold difference in expression between samples Cao, Q.L., Howard, R.M., Dennison, J.B., Whittemore, S.R., 2002. compared to unmodified fibrin scaffolds. Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord. Exp. Neurol. 177, 349–359. Statistical analysis Cao, Q.L., Zhang, Y.P., Howard, R.M., Walters, W.M., Tsoulfas, P., Whittemore, S.R., 2001. Pluripotent stem cells engrafted into For the release studies, four independent release experiments the normal or lesioned adult rat spinal cord are restricted to a (one scaffold per experiment) were analyzed for each set of glial lineage. Exp. Neurol. 167, 48–58. delivery system conditions. For cell phenotype analysis, three Danesin, C., Agius, E., Escalas, N., Ai, X., Emerson, C., Cochard, P., independent experiments consisting of one 24-well plate Soula, C., 2006. Ventral neural progenitors switch toward an containing fibrin scaffolds with EB cultures per experiment oligodendroglial fate in response to increased Sonic hedgehog were performed for each condition tested. For cell viability (Shh) activity: involvement of sulfatase 1 in modulating Shh – analysis, three independent experiments analyzing 6 wells signaling in the ventral spinal cord. J. 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