The potential of stem cells to create engineered tissues as a replacement for those lost to injury has been well documented [1, 2]. Due to their pluripotency, mouse embryonic stem (ES) cells have the capacity to produce the cell types found in all three germ layers, including those found in the central nervous system (CNS) [3, 4]. Thus, ES cells provide a potential means of repopulating cells lost due to spinal cord injury (SCI). Culturing mouse ES cells in suspension as embryoid bodies (EBs) using the 4−/4+ retinoic acid treatment protocol developed by Bain et al. produces embryonic stem cell-derived neural progenitor cells (ESNPCs). These cells can differentiate to resemble normal neurons both morphologically and physiologically after additional culture [5, 6]. Cells formed using the 4−/4+ protocol were injected into a chronic SCI model and demonstrated the ability to promote a modest increase in functional recovery . However, the percentage of cells that survived was low (∼10%), and few of the surviving cells differentiated into neurons.
To address these issues, three-dimensional (3D) biomaterial scaffolds could be used to increase the viability of transplanted cells by providing a permissive environment for growth and proliferation as well as to promote the differentiation of ES cells into specific cell types based on the properties of the scaffold [8, , , , , –14]. Fibrin scaffolds have been characterized for use in tissue engineering applications involving stem cells because they promote cell adhesion and migration and are approved for clinical use as surgical sealants [11, 15, , , , –20]. Recent work has determined the optimal conditions for seeding ESNPCs into 3D fibrin scaffolds . Additionally, fibrin scaffolds containing controlled release systems for growth factor delivery show promise as a potential treatment for SCI and could potentially be used as scaffolds for cell transplantation [22, , , –26].
Many different growth factors can be used to influence ESNPCs to differentiate into one of three mature neural phenotypes of the CNS (neurons, oligodendrocytes, and astrocytes). For this particular study, growth factors were selected for study based on two criteria: their ability to promote ES cell differentiation and survival and their potential as a therapeutic for SCI. Neurotrophins such as neurotrophin-3 (NT-3) have been shown to increase ES cell survival and promote differentiation into neural structures in 3D culture [10, 27, 28]. NT-3 delivery from biomaterial scaffolds promotes neural fiber sprouting and, in some cases, increases in functional recovery after SCI [22, 23, 29]. Other growth factors, such as platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), and sonic hedgehog (Shh), have been shown to play important roles in stem cell survival and differentiation into specific neural lineages [28, 30, , , –34]. PDGF plays an important role in oligodendrocyte precursor proliferation and promotes differentiation of human ES cells into oligodendrocytes [35, 36]. Treatment of SCI with PDGF results in increased angiogenesis to the wound site, which can be beneficial [37, –39]. CNTF promotes differentiation of ES cells into astrocytes as well as the survival of mature neurons [28, 34, 40, 41]. The experimental data on the efficacy of CNTF as a treatment for SCI have been conflicting, with some studies suggesting that it can promote increased migration of neurons and astrocytes into the injury site and another study suggesting that neutralization of CNTF results in a lessening of the glial scar [42, 43]. Shh can stimulate differentiation of ES cells into motor neurons when used in conjunction with retinoic acid [32, 33, 44]. Injection of Shh into the site of SCI has been shown to promote proliferation of neural precursors and an increase in oligodendrocyte progenitors cells . Further work showed that implanting oligodendrocyte precursor cells along with Shh into a contusion model of SCI resulted in the sparing of white matter along with functional recovery . Basic fibroblast growth factor (bFGF) can promote ES and neural stem cell proliferation [46, –48]. Additionally, bFGF plays many roles in the injured spinal cord, including promoting neural progenitor proliferation, neuronal survival, and enhancing functional recovery [49, , –52]. Other growth factors, such as epidermal growth factor (EGF) and bone morphogenetic protein (BMP), were considered due to their ability to affect stem cell differentiation [53, , –56]. However, when used as a treatment for SCI, EGF did not promote functional recovery and was not included in the present study [57, 58]. BMP promoted astrocyte formation in vivo when used as a treatment for SCI, making it undesirable for further study .
This work investigated the response of mouse ESNPCs seeded inside of fibrin scaffolds to these five different growth factors: NT-3, bFGF, CNTF, PDGF, and Shh. The influence of these growth factors was studied over a range of concentrations to determine an appropriate dose for each growth factor, and certain growth factors were tested in combination to determine the effect on ESNPC differentiation. For treatment of SCI, it is important to generate neurons and oligodendrocytes from ESNPCs to restore those cells lost to injury while minimizing the amount of astrocytes, which can contribute to the glial scar. The effect of growth factors on cell viability was also assessed to determine which growth factors could help increase cell viability after implantation. This study provides insight into which growth factor combinations promote the differentiation of ESNPCs into neural tissue consisting of neurons and oligodendrocytes to be used as treatment for SCI.