Introduction

Cell types associated with the nervous system, namely neurons, astrocytes and oligodendrocytes, all originate from a single class of progenitor cells, called neural stem cells (NSCs). NSCs have been isolated from both the developing central nervous system (CNS) and peripheral nervous system (PNS) 1, 2, 3. During CNS development NSCs line the neural tube and sequentially give rise to populations of neurons, astrocytes and finally oligodendrocytes 4. In the developing PNS, cells delaminating from the dorsal most region of the neural tube, maintain the ability to self renew and give rise not only to cells of neural lineages, but in addition can differentiate to mesenchymal derivatives 5. NSCs have also been isolated from the adult CNS, dispelling the notion that the adult brain was incapable of plastic change at a cellular level 6, 7.

Much interest has been focused on how the extrinsic environment influences NSC fate choice. In vitro NSCs, derived from the CNS, have been shown to respond to the addition of single extrinsic determinants and differentiate to specific neural and mesenchymal lineages (Figure 1). Though this has helped elucidating many of the cell's intrinsic mechanisms involved in NSC differentiation, including both signaling and epigenetic pathways, it fails to reflect the broad spectrum of signaling factors known to converge on these cells in vivo. Recently the use of biomaterials has begun to reveal new ways to investigate what controls NSC fate choice 8, 9. This is an important first step, as it gets researchers closer to the goal of integrating NSCs into the broad range of envisioned stem cell therapies. In this review we will focus on recent advances in this area and discuss how material science is pointing the way forward in our understanding of NSC state and fate, and how biomaterials may be of potential use in implementing NSCs into future stem cell therapy.

Figure 1
figure 1

NSCs isolated from the developing forebrain can differentiate to different cell fates in vitro upon use of single factors supplemented in the growth media. Stem cell characteristics such as self-renewal and differentiation potential of NSCs are maintained by fibroblast growth factor (FGF). Neuronal and astrocytic differentiation can be induced by platelet derived growth factor (PDGF) and cardiotrophin-1 (CT-1) / cilliary neurotrophic factor (CNTF) respectively 46, 47, 48. Oligodendrocytic differentiation can be induced by thyroid hormone (T3), and in addition NSCs can also differentiate to mesenchymal cell fates by stimulation with bone morphogenic protein-4 (BMP4) 47, 49.

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Neural stem cells as multipurpose tools in cell therapy

The potential for controlled differentiation into all neural tissues makes NSCs very attractive for regeneration therapy in the nervous system. Additional, and perhaps unexpected, properties of NSCs expand their potential use as therapeutic tools. These include the capacity to home in on injury or inflammation sites and to provide trophic support to remaining healthy tissues.

Using exogenous NSCs in cell replacement therapy

Neural stem cells can potentially be used to replace tissue loss due to trauma or neurodegenerative diseases. Ideally, the precise architecture of the tissue should be reconstructed and integrated with surrounding tissues. Importantly, several neural cell types need to be replaced in this scenario. NSCs derived in vitro from human embryonic stem cells 10 or isolated from human fetal brain 11, when transplanted into the adult rodent brain, differentiated into neurons, astrocytes and oligodendrocytes. Notably, NSCs integrated into the neurogenic niche of the subventricular zone and contributed to neurogenesis in the olfactory bulb. In the hippocampus, transplanted neural progenitors were observed to differentiate into region-specific neuronal subtypes and functionally integrate into the local circuitry 12.

NSC transplantation into disease or injury rodent models has largely presented promising results. Transplanted NSCs have been reported to survive, migrate primarily towards the pathological site, and differentiate. Adult mouse NSCs transplanted into a mouse model of multiple sclerosis caused an increase in the numbers of oligodendrocyte progenitor and mature cells that are of both the host and transplant origin, resulting in a remarkable recovery from the disease 13. In a rat model of brain ischemia, transplanted human NSCs migrated to the injury and differentiated mostly into neurons 14. NSCs transplanted intravenously in a model of intracerebral hemorrhage differentiated into neurons and astrocytes at the lesion site, resulting in functional recovery 15. Neuronal differentiation and functional recovery were reported following transplantation of NSCs derived from the rat embryonic spinal cord into a model of contusion spinal cord injury (SCI) 16. Improved motor function was also reported in a model of weight-drop SCI after transplantation of NSCs retrieved from adult rats 17. NSCs differentiated mostly into astrocytes and oligodendrocytes but these animals showed aberrant axonal sprouting at the injury site and allodynia-like hypersensitivity of forepaws. Allodynia was also observed after transplantation of neural progenitor-like C17.2 cells into the injured spinal cord 18.

Injury-induced recruitment of endogenous NSCs

The presence of stem cell niches in multiple regions of the CNS in the adult has raised the hypothesis that NSCs may be naturally called into action during pathological states. Tracing of nestin positive neural progenitors resident in the adult spinal cord has revealed that these cells proliferate and differentiate into neurons at the lesion area following dorsal compression injury 19. Focal ischemia was shown to increase the normal rate of neurogenesis in the cortex and hippocampus 20, 21, and the results could be enhanced by exogenous delivery of growth factors 21. Eventual endogenous stem cell repair responses are obviously unsatisfactory in pathology. Engineering improvements on these responses can potentially be used for therapeutic purposes.

NSCs as vehicles for trophic support

Some of the beneficial effects observed post-NSC transplantation have been attributed to trophic or anti-inflammatory support provided by NSCs. Adult NSCs transplanted into a model of chronic CNS inflammation accumulate in perivascular areas and cause apoptosis of encephalitogenic T cells 22. C17.2 cells were shown to naturally secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) 23. Axonal growth in a SCI site following transplantation was ascribed to this effect.

NSCs as vehicles for gene delivery

Neural stem cells transplanted into the adult brain demonstrate a remarkable capacity to target sites of disease or injury 24. This characteristic, dubbed “pathotropism”, has been explored to convert NSCs into gene and drug delivery devices. GDNF-overexpressing NSCs prevented degeneration of dopaminergic neurons when transplanted into a Parkinson's disease model 25 and improved cognitive function after traumatic brain injury 25, 26, 27. This strategy may be particularly beneficial in the targeted delivery of tumor suppressing drugs to highly invasive CNS tumors. Significant decrease in medulloblastoma tumor mass in nude mice was achieved by transplantation of a human NSC line engineered to secrete the prodrug activating enzyme cytosine deaminase 28. Interleukin-12-secreting NSCs were shown to target glioma cells, recruit T cells and elicit anti-tumor immunity 29. Tumor targeting NSCs in this study were later identified as astrocyte progenitors expressing chemokine receptor 4 30.

Biomaterials for neural stem cell delivery

Stem cells are highly responsive to the chemical and physical state of their microenvironment. Precise engineering of microenvironmental cues using artificial materials (Table 1) can potentially control the spatial and temporal progression of regenerative processes initiated by NSC transplantation.

Table 1 A summary of some biomaterials used for transplantation of NSCs in vivo and/or controlling the characteristics of NSCs in vivo and in vitro
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Biomaterials and neural stem cell therapy - in vivo studies

Artificial materials can, at a first level, simply provide a physical support for the introduction of NSCs into a lesion site where a cavity has formed, such as in stroke or trauma. Polyglycolic acid (PGA), a degradable hydrogel, was used as a scaffold in the transplantation of the neural stem-like cell line C17.2 into an infarction cavity formed by the ligation of the carotid artery 31. In this model of severe cerebral palsy, transplanted cells differentiated into neurons and astrocytes and appeared to form connections with host neurons. An implant of NSCs embedded in a blend of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of PLGA and polylysine was used in a rat hemisection model of SCI 32. The animals exhibited motor recovery and diminished glial scar formation. Interestingly, the beneficial effects of the implant, especially regarding glial scar formation, were attributed in part to the implant material itself.

Additional levels of environmental control can be achieved by modifying the material to secrete active compounds that provide trophic support to the NSCs or to other cell types. As described above, NSCs in vitro respond strongly to single soluble factors by choosing to differentiate into predominantly one of the possible cell lineages. Biomaterial driven differentiation can be envisaged where the cell types of interest in a particular disease or injury situation can be produced, possibly in the desirable temporal sequence. NSCs were grown in vitro in PLGA microparticles encapsulating NGF and coated with polylysine 33. The microparticle/NSC suspensions were then injected into the caudate/putamen of adult rats and caused elevation of NGF levels and increased NGF driven choline acetyltransferase activity in surrounding tissues. The spatial and temporal concentration profiles of NGF encapsulated in ethylene vinyl acetate (EVAc) matrix slabs in turn placed inside a collagen gel embedded with NSC or PC12 cells were described in mathematical detail 34. Artificial extracellular matrices were generated by fusing an elastin backbone to Notch ligands, a signaling pathway heavily involved in the control of NSC state and fate 35.

The mechanical and geometrical properties of the biomaterial can be tailored to drive neurons to bridge the ends of an injury. This strategy is of particular interest in SCI therapy, where there is a clear preferred direction for axonal growth. Alginate hydrogels engineered to form parallel fibers were seeded with adult spinal cord NSCs and implanted into spinal cord lesions 36, resulting in NSCs elongating along the biomaterial fibers. Retinal precursor cells (RPCs) were seeded onto PLGA/PLLA (poly(L-lactic acid) polymers containing pores oriented normal to the plane of the scaffold 37. RPCs delivered by the polymer/progenitor cell composite graft differentiated and cell delivery was much improved compared to injection of RPCs alone.

Biomaterials and neural stem cell therapy - in vitro studies

Polymer-based materials offer a high degree of flexibility in chemical and physical properties. Microarray technologies can be instrumental in screening for optimal materials for NSC delivery. Arrays of blends of biodegradable polyesters were tested for human mesenchymal stem cell/biomaterial interactions 38. Cell attachment and glial differentiation of C17.2 cells were also tested in smaller arrays in the same study. Human NSC differentiation was tested in printed arrays of extracellular matrix molecules and signaling factors 8. The extracellular matrix molecules laminin and fibronectin and the artificial polymers ProNectin and Polyethyleneimine (PEI) were immobilized onto arrays in combination with cytokines 39. A comprehensive analysis was carried out to examine the fates of NSCs cultured onto the different polymer/growth factor combinations.

In vitro studies of single biomaterials for supporting NSC maintenance and differentiation have focused mostly on hydrogels. Aligned fibers of PLLA were shown to align embedded NSCs along the direction of the fibers 40. The width of the fibers did not affect the alignment response but NSCs proliferated more on nano- than on micro-scale fibers. Polyethylene glycol (PEG) alone 41 or in combination with polylysine 42 also supported the culture of NSCs. NSC progression from proliferation to neuronal differentiation and formation of functional synapses was reported for NSCs interdispersed in Type I collagen 43. A particularly innovative approach relies on the ability of certain peptides such as IKVAV 44 or RADA 16-I peptides 45 to self-assemble into nanofibers. Strikingly, IKVAV nanofibers promoted neuronal differentiation of NSCs and decreased astrocytic differentiation relative to laminin, an extracellular matrix protein containing IKVAV sequences.

Conclusions

Cell therapy using NSCs is a promising approach and the use of polymers and biomaterials can improve the outcome after transplantation in vivo as well as improve differentiation efficiency and survival in vitro. Yet there is an urgent need for the development of novel approaches based on biodegradable hydrogels, not only containing and administering “nurturing” factors but also exerting control over unwanted and disastrous outcomes, such as overgrowth and cell death. Such “second generation” polymers can very well be a crucial factor for successful implementation of neural stem cell therapy in the future.