Fetal Spinal Cord Transplantation after Spinal Cord Injury

C H A P T E R

23 Fetal Spinal Cord Transplantation after Spinal Cord Injury: Around and Back Again Lyn B. Jakeman1, Paul J. Reier2 1Department

of Physiology and Cell Biology, Center for Brain and Spinal Cord Repair, The Ohio State University College of Medicine, Columbus, OH, USA; 2Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL, USA

1.  INTRODUCTION AND HISTORY Those living with a spinal cord injury (SCI) are in urgent need of effective treatments to promote functional recovery, thus many exciting therapeutic approaches are currently under active investigation. While a longstanding emphasis of SCI research has been focused on regeneration and reconstruction of white matter tracts, it is increasingly clear that gray matter interneuronal circuits and local propriospinal relays are key elements that are both essential and sufficient for a wide range of spinal cord functions. A repair strategy of considerable interest is the use intraspinal grafts of neural lineage cells, which are likely to become an integral component of future combinatorial approaches [1]. As a precursor to the current emphasis on stem cells, the use of donor embryonic central nervous system (CNS) or fetal spinal cord (FSC) tissue has been especially intriguing. Intraspinal transplantation of fetal neural tissue is a valuable research tool that can directly address several objectives, including: replacement of both neurons and glia, physiological delivery of trophic factors and neurotransmitters, and provision of a developmentally conducive matrix for axonal growth [1,2]. The earliest FSC tissue transplantation studies were performed in rodents and focused on graft survival, differentiation, and integration with the host spinal cord, with the hopes of achieving these primary objectives. These studies revealed the great potential for replacing damaged spinal cord gray matter cells, through the expansion and differentiation of a relatively small number of surviving neural precursor or progenitor cells. Despite the advances gained from this approach to date, true reconstruction and functional

Neural Regeneration http://dx.doi.org/10.1016/B978-0-12-801732-6.00023-9

integration of gray matter circuitry remains a daunting challenge. The following sections are intended to provide some perspectives for neural repair in SCI that have been gained from intraspinal FSC tissue grafting models and insights gained from studies exploring other cellular grafting strategies. The importance of cell survival, differentiation, and integration remain highly relevant today in light of current understanding of the extensive neuroplastic reserve of the injured spinal cord and the potential role of interneurons and relay circuits in optimizing functional recovery [3–8].

1.1  Embryonic Transplants Challenged Neural Regeneration Dogma The idea of transplanting CNS tissues from one subject to another has long had a place in the science fiction genre, yet the early history of brain tissue-to-brain grafts in animal models provided some key principles that have shaped the spinal cord transplantation field to this day. These early studies and the history of grafting are described in detail by other authors and are only briefly summarized here (see [9–11]). Experimentalists of the late nineteenth century first reported results of transplanting adult brain tissues from donor to host brains. Walter Thompson described the survival of some cells from grafts of cat brain tissue to host dog brain, but later analyses suggest that few neurons would have survived this procedure for more than a few weeks, especially in the absence of immunosuppression. An established landmark paper by Elizabeth Dunn in 1917 described placement of 9–10-day-old mouse cerebral cortex tissues into the cortex of littermates and reported viable grafts

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in four of 44 subjects, with some semblance of normal neural cytoarchitecture in a small number of cases. That study emphasized the key contribution of donor tissue age, and thus began a slow progression of work designed to establish the conditions required for tissue survival in brain-to-brain grafts. In the early twentieth century, transplant survival was poor, and true axonal integration of donor nervous tissue within the host brain was unforeseen. Wilfred LeGros Clark demonstrated in 1940 that neonatal and embryonic grafts survived better than postnatal tissues when transplanted into adult rabbit cortex and, importantly, showed that grafts obtained from neonatal donors were capable of developing characteristics that reflected a mature histological organization. However, at the time, medical leaders and researchers alike were still mired to the Cajal concept that neurons in the adult CNS were not capable of sustained growth or regeneration. Many neuroscientists believe that it was not until 1969, when Geoff Raisman demonstrated the phenomenon of axonal sprouting and new synapse formation following lesions of pathways projecting to the septum, that efforts to examine axonal growth potential and transplant connectivity began in earnest [12]. In the 1970s, Lars Olson and his colleagues in Lund, Sweden, grafted neural explants into the anterior chamber of the eye to characterize the process of axon growth between tissues. They showed that catecholaminecontaining cells from brainstem grafts could robustly innervate adjacent embryonic spinal cord tissues within a CNS environment [13]. With this model, they were able to more closely define the ideal embryonic ages for survival and growth of explants from different brain regions (reviewed in [14]). In 1972, Das and Altman demonstrated that embryonic-derived brain grafts were mitotically active and that mature neurons developed directly from immature ectodermal precursors following transplantation into adult cerebellum, suggesting that undifferentiated neural cells can retain progenitor properties in the host environment [15]. These early studies long predated the recent emphasis on “stem cell research” and demonstrated the tremendous potential of neural progenitors derived from appropriately aged donors to provide a viable source of fully differentiated graft tissues. The use of fetal tissue as a tool for examining neural repair was initially pursued in the brain, as Anders Björklund and his colleagues in Stockholm placed transplants from several regions of the neuraxis into the brains of adult rats as a tool for examining axonal growth mechanisms [16,17]. Indeed, grafts of midbrain substantia nigra placed into the denervated striatum of adult rats extended a plexus of dopaminergic axons from the grafted neurons to the host [18,19] (reviewed in [11]). Together, these studies established that transplants of embryonic neural tissues could survive for long periods

and extend axons with regional specificity into target adult host brain regions, and thus opened a new avenue of hope for patients with neurodegenerative diseases such as Parkinson’s disease or from damage following trauma. To place the time frame of these findings in perspective, this was just prior to the well-known landmark study in which Sam David and Albert Aguayo first combined peripheral nerve grafts with state-of-the-art axonal tract tracing techniques and proved that adult CNS neurons were also capable of sustaining long-distance axonal regeneration when exposed to a growth permissive environment [20]. Thus, the use of embryonic tissues as a research tool and potential therapy both for possible cell replacement and support of adult CNS host axon regeneration was widely embraced in the early 1980s as a conceivable goal for a wide range of neurological conditions [21].

1.2  Fetal Tissue Grafts Address Key Repair Objectives for SCI After it was established that CNS grafts could survive and axons would grow in the adult host CNS, the transplantation of embryonic tissue grafts into the spinal cord was pursued with excitement, with the ultimate goal of restoring function after SCI. A timeline of embryonic tissue transplantation in SCI (Figure 1) begins with the work of Nygren et al. [22], Nornes [23], and Commissiong [24–26], who adapted an approach similar to the Parkinson’s disease models above and placed neurotransmitter secreting cells from the brainstem into the lumbar region of the injured spinal cord after depletion of endogenous descending fibers with a complete midthoracic transection. Anatomical evidence confirmed that these grafted neurons could extend axons in both white and gray matter and form terminal arbors suggestive of synaptic integration. The anatomical specificity with which the axons found their targets was compelling; the new terminals were dense in dorsal and ventral horn and intermediate gray regions of the spinal cord [27], where they exhibited functional synaptic properties [28]. While biologically important, these results still remained unsatisfying to the SCI community, falling short of enabling restoration of voluntary motor control and/or normal sensation. For transplants to provide true functional repair potential, they would have to be placed into the site of injury, where they would have to survive and become integrated with neighboring host spinal circuitry [25]. Initial efforts to achieve graft survival in the lesion site of the injured spinal cord were not encouraging. Howard Nornes took great effort to examine the critical issue of the site of graft placement, and concluded that the brainstem grafts could not survive in the environment found at the injury itself [23]. A more thorough analysis

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FIGURE 1  The history of embryonic spinal cord transplantation research is divided into general approaches and illustrated with drawings as these evolved from the mid-1970s to the present. Each time window (vertical hash mark) indicates approximate years when the studies illustrated below the line were prominent. Descriptions of each approach are given below the line, followed by representative reference citations. Above the timeline are indicators (black triangles) of four clinical studies performed following extensive review in the United States between 1997 and the present. Inclusion criteria and basic design for the clinical studies were as follows: * ASIA A, C5-T11, transplant within 14 d after SCI; ** ASIA A, T3-T11, transplant 7–14 d after SCI; *** ASIA A, T3-T11, harvest 5 d after SCI, inject 24–40 d after SCI. Abbreviations in drawing section: FSC, fetal spinal cord; NT, neurotrophin; GF, growth factor; E, enzymes; NPCs, neural progenitor cells; NSCs, neural stem cells; ESCs, embryonic stem cells.

of survival conditions and complications in spinal cord lesions was extended by Gopal Das, who determined that with extreme care, embryonic cerebral cortical tissues could be placed into prepared partial spinal cord lesions, where they could integrate with adjacent gray matter, although fusion with adjacent white matter was inevitably prevented by the accumulation of reactive astrocytes at the interface. While a tremendous degree of insight regarding cellular reactions and vascularization was gained in these experiments using fetal cortex tissues, Dr. Das maintained that spinal cord or brainstem grafts had too poor an intrinsic growth potential and could not be successfully transplanted into a spinal cord lesion [29]. Astrocytes are specialized for maintaining the integrity of the CNS microenvironment, and after injury to the nervous system, they migrate to the lesion edge where they effectively isolate foreign tissues or grafts. In addition, injury to the meninges leads to invasion of mesenchymal cells from the periphery and formation of a basal lamina scar at the transition between the peripheral nervous system (PNS) and CNS. Therefore, the presence of regions of direct neuropil integration at the edges of the cortical grafts, regions lacking any astroglial intervention

or mesenchymal infiltration, was greatly encouraging for those studying repair strategies in SCI. These regions provided the possibility that if graft survival could be achieved, the embryonic tissue could directly integrate with the host spinal cord. In the early 1980s, the question of donor tissue age was revisited, and spinal cord pieces from embryonic day 14–15 (E14–E15) rats, slightly younger than those used previously, were found to be a viable source of graft cells, with sufficient growth potential to fill a carefully prepared cortical lesion site [30]. Most notably, the FSC donor tissues also showed extensive differentiation ability and formed grafts containing mature neurons, glia, and myelin with cytoarchitectural features resembling specific regions of the normal adult spinal cord. The reliability of graft survival in the injured spinal cord was then optimized by ensuring a well-vascularized and hemostatic cavity and by taking steps to minimize the invasion of peripheral mesenchymal cells into the graft site [31,32]. Over the next three decades, a stepwise progression of laboratory studies with intraspinal E14-derived FSC grafts served to extend the understanding of the capacity of these grafts to expand, differentiate, and form anatomical and functional connections with the injured adult spinal cord.

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1.3  FSC Transplants Contribute to Neuroprotection, Neuron Replacement, and Functional Integration across Species and Lesion Models FSC tissue transplantation provided, for the first time, an opportunity to envision the possible repair of damaged neural parenchyma of the spinal cord with healthy, mature, and anatomically differentiated spinal cord tissue. There were multiple possibilities for mechanisms of repair at the initial onset. The most obvious mechanism for recovery of function would be to restore continuity, either by providing a supportive tissue bridge for host axons to regenerate, or by serving as a propriospinal relay, with host and grafted neurons forming synaptic connections across the lesion site [31–33]. Over time, additional features and better understanding of FSC tissue suggested that these grafts could serve neuroprotective and neuromodulatory roles as well. Considerable knowledge of the potential for neural repair using FSC tissue began with studies carried out in newborn and adult rats. The earliest models of FSC graft function predicted that the grafts might support regeneration of long white matter ascending and descending axons. Unlike peripheral nerve grafts, the FSC graft neurons would theoretically integrate with the host tissue at both ends of a lesion without forming a characteristic CNS/PNS cell and extracellular matrix transition zone [30]. Indeed, embryonic grafts of hippocampal tissue had been shown to stimulate and support growth of transected host septohippocampal axons across a lesion to reinnervate the host hippocampus [34]. Thus, host ascending or descending axons might be able to cross from the host to graft and beyond into the distal host spinal cord to reinnervate their target nuclei. In addition, host neurons axotomized by the injury might survive better if they could grow into the parenchyma of the grafts. Initial proof-of-concept for the idea of bridging was obtained by Barbara Bregman, who showed that E14-FSC transplants placed into over-hemisection lesions in newborn rats could rescue injured rubrospinal neurons from lesion-induced atrophy [35] and could also enhance plasticity of descending serotonergic projections [36]. Then, in a complete transection injury model, serotonin immunohistochemistry revealed descending axons spanning the full length of the grafts and extending into the caudal spinal cord stump [37]. Behavioral studies demonstrated greater functional recovery in rats that received FSC grafts into hemisection lesions as newborns compared with controls [37–39]. Improved functional recovery was also shown later in rats that received grafts into cervical resection injuries as newborns, as well as in cats that received FSC grafts following compression injuries performed early after birth [40–42]. In no instance did E14-FSC grafts ever overgrow the initial volume of the lesion site or cause any histological or functional deficits in any studies.

Unlike the newborn hosts, long-tract axonal bridging does not occur when FSC grafts are placed into an injured adult rat spinal cord. Instead, the embryonic tissue provides a viable substrate for cellular interactions with host interneurons throughout the gray matter surrounding the site of injury. In the mature spinal cord, interneuronal circuits of the gray matter provide most of all ascending and descending integration and also mediate segmental function, pattern generation, and an extensive array of coordinated activity [5,8,43–46]. Indeed, while the large ventral motoneurons of the normal spinal cord are not found within mature E14-FSC grafts [30,32], the characteristic cellular features of dorsal and intermediate gray matter regions are well developed within the grafts [32,47]. When placed into acute resection lesions, the neuropil of FSC grafts fuses with that of the host gray matter and develops interface regions devoid of any intervening astrocytic or mesenchymal scar. In fact, FSC grafts that are placed into an established or chronic lesion site can even cause the reorganization of astrocyte processes and lead to the reduction of an existing glial border [33,48,49]. Axonal tracing studies of acute grafts revealed an extensive intragraft anatomical circuitry as well as evidence of ingrowth from ascending, descending, and local host axons into the grafts and outgrowth of graft-derived axons into the host spinal cord [32,50,51]. Ingrowth of specified populations of host axons was also demonstrated using immunohistochemical markers (Figure 2; see also [52,53]). In most cases, the length of axonal growth into and out of these grafts in adults is limited to a few millimeters, but axons can be followed across the interface to form distinct boutons directly adjacent to surrounding neurons. Host sensory fibers, including calcitonin gene-related peptide-containing axons, are an exception, as they can extend for several millimeters into the heart of FSC transplants in acute and delayed conditions, where they can form functional synapses [52–54]. With the recent interest in genetically labeled and modified mice, additional studies have also demonstrated that FSC grafts can be obtained from E12 mouse embryos, and these cells survive and extend axons after placement in mouse hemisection injuries [55,56]. Taken together, this body of work established as long as 15–20 years ago that intraspinal grafts, which are prepared from E14-FSC, are capable of forming, to a limited extent, all the necessary components of an integrated propriospinal relay when placed into prepared lesions of the adult rodent spinal cord (Figure 3). Clearly much has been learned about the cells of origin and the nature and limitations of these interactions since that time. These ideas are revisited later in this review. Extending the functional potential of FSC grafts soon demanded consideration of more clinically relevant contusion and compression injuries and nonrodent species. First, the use of FSC grafts was adapted to contusion

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FIGURE 2  Reconstructed drawing tube tracings of serial 50-μm thick sagittal sections showing immunocytochemically stained axons in an FSC graft at three months after injury and transplantation. Sections represent sequential drawings from the most lateral (A) to medial (I) extent of the hemicord graft, such that (A) is cut through the lateral host white matter and graft, (B)-(H) include host gray matter and white matter regions, and (I) includes a portion of the dorsal columns. Abbreviations: Ox, oxytocin, from hypothalamus; 5-HT, serotonin, from brainstem; CGRP, calcitonin gene related peptide, from peripheral afferents, or contained in motor neurons in (C) and (E). Scale = 1.0 mm.

injury lesions in rats. To prevent further damage with a resection, the donor tissues were isolated from E14FSC, mechanically dissociated in tissue culture medium, and then injected into the lesion cavity as a thick, cellular slurry. Survival of these grafts was not compromised by the preparation procedure, and perhaps initially surprisingly, the grafts developed and differentiated with regions of anatomically mature neuropil and routinely filled these extensive, cavitating lesions without any evidence or tendency for overgrowth. Axonal ingrowth and outgrowth was again evaluated in the contusion recipient tissues and was similar to that observed with the previous whole tissue grafts, and the grafts produced a highly vascularized and oxygen-rich environment [57,58]. Behavioral studies in the rat contusion model provided evidence of improvements in locomotor function in animals with E14-FSC grafts compared with animals that had no grafts [39,59]. Next, to identify and resolve the clinically relevant issues of scaling and allograft tissue rejection, FSC transplants were prepared from fetal cat spinal cord and injected into the site of acute and chronic compression injuries in adult cats. These cat feasibility studies lack

the numbers afforded by rodent experiments to evaluate population efficacy, but they confirmed that FSC grafts in a second species thrive, differentiate, and at least in one case, allowed a cat with an established and stable compression injury the ability to step over ground. Survival of these grafts was followed over the life of the host with magnetic resonance imaging and confirmed by detailed histological examination [57,60,61]. The withdrawal of immunosuppression led to graft rejection and subsequent loss of function, while no overt pathology was observed involving host tissue. Surprisingly, donor tissue rejection did not occur in all recipients following cessation of immunosuppression, indicating that immunological responses to intraspinal grafts of embryonic cells may be highly individualized. Next, additional benchtop studies were undertaken to characterize the immunological response to isograft and allografts of FSC tissue with and without systemic immunosuppression [62] and to identify the time course and growth potential of spinal cord tissue obtained from human FTCs when placed into the well-characterized rat lesion model [63]. These studies confirmed that human embryonic spinal cord could survive and show many

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FIGURE 3  Hypothetical relay circuitry established across an FSC transplant. Plausible neural integration would include (a) input neurons, (b) intrinsic graft neurons, and (c) output neurons. Rostral is depicted toward the left, caudal toward the right. The ascending relay (top) could support transmission of sensory information from peripheral receptors below the injury site to higher brain regions. Descending information can be transmitted from descending supraspinal fibers to motor units below the lesion. Finally, the grafts can establish an interneuronal relay for transmission of short and long propriospinal circuits.

of the established characteristics of survival and axon growth in an SCI model, with no evidence of overgrowth or immunological damage to the host tissue. Thus, nearly 15 years of careful and stepwise scientific work had been taken across several animal models and two species prior to consideration of human clinical studies [64]. After a full and open discussion of the scientific, clinical, and ethical concerns of the community, the first feasibility trial of in-human transplants of embryonic spinal cord tissue in the United States was performed into a small group of patients who had progressive syringomyelia, a unique clinical condition that worsens with time and requires surgical intervention as part of a normal treatment approach [65–67]. These subjects provide evidence of feasibility and safety of an FSC transplantation procedure, and some outcomes suggest that the approach has potential for benefit. Nevertheless, as a small experimental study, considerations of the changing clinical condition of individuals with syringomyelia [68] and continuing limitations of imaging methods to identify and follow transplant survival prevent definitive

conclusions to date regarding the extent or duration of graft integration or full evaluation of graft efficacy [1].

1.4  Combinations and Refined Grafting Approaches Enhance the Possibilities for Repair In the most recent decade, the potential of FSC tissues for neural repair has continually expanded to take advantage of the additive and synergistic effects of combination treatments. Numerous reports have established that the addition of exogenous neurotrophic factors, including nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), neurotrophin-3 (NT-3) and glial cell linederived neurotrophic factor (GDNF), enhance the axonal growth and functional capacity of FSC grafts in different models without altering the differentiation of the graft tissue (e.g., [69–73]). These factors serve to enhance survival of the graft tissue and can also increase plasticity of host fibers and growth of host axons into the graft. Likewise, axonal growth between host and graft can be enhanced with the addition of growth-promoting enzyme-directed

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2.  Alternative Cell Sources for Spinal Tissue Repair

therapies, including cyclic adenosine monophosphate amplification with phosphodiesterase inhibitors and manipulation of the extracellular matrix with chondroitinase [74,75]. Notably, because these trophic factors and many other agents bind to specific receptors or exert different effects on different neuronal and non-neuronal cells, they may influence specifically targeted host and graft neuronal populations or functions. The opportunities for combinations are far-reaching and the optimal combinations are still unresolved. Yet, as these basic studies with multiple in vivo models and therapies are used to direct applications for clinical SCI, the safety and mechanisms of action of combined approaches still must be carefully addressed and defined. As described below, the use of these factors and other modifications for preparation of immature cells prior to grafting has already been found to enhance the potential for cell survival, integration, and potential for repair.

2.  ALTERNATIVE CELL SOURCES FOR SPINAL TISSUE REPAIR Despite the promising potential for functional grafting and replacement of interneuronal circuitry with FSC tissues, there are substantial ethical and safety concerns that preclude the procurement and use of dissected fetal donor tissues for therapeutics [1,76,77]. Therefore, a number of research and translational teams are focused on identifying alternative cell sources for spinal cord reconstruction, including the use of non-neuronal cell grafts and several sources of neural progenitors.

2.1  Autologous Non-Neuronal Cell Grafts One appealing alternative approach for promoting repair after SCI is to harvest, expand, and purify or manipulate a renewable peripheral cell population from the patient’s own tissues for intraspinal autografts. Such cells cannot replace neurons, but they can be obtained with consent and thoroughly tested prior to grafting and can provide a permissive and supportive environment for axon regeneration to bridge a small lesion site. Most notably, these autologous cells would not require immunosuppressive therapy. Promising results such as bridging grafts have been reported using Schwann cells from peripheral nerve, ensheathing glia from olfactory bulb or nasal mucosa, fibroblasts, bone marrow-derived stem cells, adipose-derived cells, macrophages, and skin cells [78–89]. These cell types can each be expanded in vitro and also have a potential for controlled genetic manipulation prior to grafting to induce production of growth factors or adhesion molecules needed to enhance axonal regeneration [88,90–93]. Based on preclinical studies in rats, the US Food and Drug Administration (FDA) approved a phase I trial for

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patients with complete SCI using autologous macrophages, stimulated and expanded in vitro, and injected into the spinal cord at the caudal boundary of the magnetic resonance imaging-defined lesion site within 14 days of injury. Eight patients were enrolled and followed for one year in an escalating dose scheme from 2000 to 2002 [94]. A phase II trial was then initiated in 2003, and 50 patients were randomized into treatment and control groups. This efficacy study was stopped by the sponsor in 2006 due to financial limitations not related to the trial, but no differences were identified in the primary outcome measure [95,96]. Then, in 2012, the FDA approved a new experimental trial of autologous Schwann cell grafts for patients with complete SCI, in which Schwann cells will be harvested from the patient’s sural nerve five days after injury and, following expansion in vitro, will be injected into the lesion site at 24–40 days after injury [97]. This approach has been discussed in other reviews and elsewhere in this volume (reviewed in [98]), and while it is promising for influencing progression of the lesion and deriving future regenerative therapies, it does not directly influence the urgency of defining approaches for gray matter repair. It is useful to note that a generally similar autologous Schwann cell grafting paradigm has been completed on six patients with SCI to date in Tianjin, China, and while no control subjects have been included, a five-year follow-up report indicates no “immunological problems or severe adverse effects” [99]. An essential limitation of all of these non-neural bridging strategies for spinal cord regeneration remains the challenge of the formation of a CNS/PNS transition zone, where CNS glial cells prevent invasion of cells of peripheral origin [100]. Additional combination therapies and/or genetic manipulations will be required to enable regenerating axons to overcome the transition and re-enter the CNS. However, one important lesson that was first gleaned from these peripheral cell grafting studies is that the survival of many of these highly robust peripheral cell types is substantially enhanced by suspending the cells in an adhesive matrix, such as fibrin glue or Matrigel™ extracellular matrix preparations prior to injection [101–105], reviewed in [106]. Because long-term survival had not been a major difficulty for FSC grafts since the early 1980s, such matrix preparations have not been traditionally used or studied for potential enhancement of FSC integration.

2.2  Alternative Sources of Cells for Neuronal Replacement Grafts Alternative strategies for obtaining interneurons and supporting glial cells for spinal cord gray matter repair rely on the ability to expand limited precursor cells to obtain large numbers of neurons and glia, or the development

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and use of renewable cell lines. The current approaches can be divided into five general methods. These include: (1) expansion of partially differentiated neural and glial lineage restricted precursor cells derived from E14-FSC tissue (NPCs); (2) expansion and differentiation of pluripotent neural stem cells (NSCs) from neurospheres prepared from select regions of the embryonic or adult forebrain or spinal cord and maintained in mitogenic growth media; (3) use of mutant or induced neural tumor stem cell lines; (4) expansion of pluripotent, blastocyst-derived embryonic stem cells (ESCs) and subsequent differentiation to a neuronal or glial phenotype; and (5) genetic deprogramming and induction of pluripotent progenitors from autologous somatic cells (induced pluripotent stem cells; iPSCs) and differentiation to neurons and/or glial cells. These strategies are reviewed in greater detail elsewhere [1,98,107–109] and are described here only briefly, with an emphasis on what has been learned from this work with respect to survival and integration of grafted interneuronal and glial progenitors. 2.2.1  Embryonic Neural Progenitor Cells, or “NRPs and GRPs” NPCs were initially examined as a source of renewable, lineage-restricted CNS cells from the E14 spinal cord that likely represent the surviving starting cells for FSC transplants. Theele et al. [62] completed the first early time-course study of classic FSC graft tissue survival and growth and showed that under standard grafting conditions, only small clusters of immature FSC cells survive the first week of transplantation in close proximity to blood vessels, and these cells then continue to proliferate and are able to generate the full complex architecture of the mature spinal cord, including formation of neurons, astrocytes, and myelin. A decade later, Lepore and Fisher extended these findings [110,111]. They reasoned that the spinal cord cells present at this embryonic age included primarily undifferentiated progenitors and lineagerestricted neural and glial precursors and that these cells should be sufficient to generate spinal cord tissue grafts. By expanding these progenitors in culture, they could generate large numbers of cells from fewer donors, and the cell types could be separated and selected for different applications. They showed that dissociated E14 rat FSC cells could create renewable cells that would generate neurons, astrocytes, and oligodendrocytes. In initial studies, they and others had found that the isolated undifferentiated NSCs alone grafted into the brain and spinal cord showed poor survival and typically differentiated into glial cells [110,112,113]. Therefore, they sought to expand the remaining populations of partially differentiated, lineage-restricted precursors from these preparations. The defined culture conditions favored survival and self-renewal of progenitor cells (DMEM-F12 culture media with bovine serum albumin, Pen-Strep

antimicrobial, neural culture additives B27 and N2, and the growth factors, basic FGF (bFGF) and NT-3), and this media produced neural progenitors expressing the immature neural marker, nestin, as well as neuronalrestricted precursors (NRPs; expressing embryonic neural cell adhesion molecule (eNCAM)) and glial lineagerestricted progenitors (GRPs; expressing the surface marker A2B5), with the absence of cells with undifferentiated stem cell or mature neuronal or glia markers. While the exact ratio of the progenitor cell types in these preparations is influenced by subtle variations in growth and maintenance factors and number of passages, the resulting grafts are safe and show no tumorigenic characteristics. Further purification can be done using antibodies recognizing eNCAM to isolate the NRPs [112] or A2B5 to isolate the GRPs [114–116], but the greatest potential for functional integration and interactions with host interneuronal circuitry is now known to be found with use of the combined cell populations, collectively referred to as NRP/GRPs. Results across laboratories suggest that NRP/GRPs share many similarities to FSC transplants in rats and in cats [117], including the potential for introducing new interneuronal constituents and providing a substrate for modified function through axonal growth between graft and host [118]. A few studies have reported improved functional recovery after SCI following transplantation of these NRP/GRP grafts [119,120]. In a direct comparison study using cells from rats genetically labeled with alkaline phosphatase, Lepore and Fischer showed that the expanded NRP/GRP grafts had greater cell survival at four days after grafting than those from the acutely prepared FSC dissociates. In addition, the use of the genetic label enabled visualization of NRP/ GRPs and suggested that these cells migrated farther from the lesion cavity as well [111]. These results suggest that the culture procedure itself facilitates the survival and growth of the E14 derived progenitors. Notably, these and most of the subsequent studies with a wide range of neural cell sources have incorporated the use of cocktails of growth factor and proliferation media in the cell isolation and expansion steps, which enhances the survival of the grafted cells when placed into the hostile, inflammatory, or poorly vascularized environment of an SCI site. Furthermore, as this field has evolved, it is increasingly clear that while large numbers of the more proliferative glial precursors can be obtained from limited source material and maintained in vitro, the ability to derive large numbers of appropriately mixed NRP/ GRP cells will rely either on alternative preparation methods or the availability of fetal source tissues. 2.2.2  NSCs from Embryonic or Adult Brain or Spinal Cord The second approach for generating sources of neural progenitor cells is the in vitro expansion of pluripotent

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NSCs. NSCs can be obtained from tissue explants taken from neurogenic regions of embryonic or adult brain or spinal cord [121–126]. Unlike the NRP/GRP preparations, however, these cells are not maintained in two-dimensional cultures in differentiation media, but instead are maintained and expanded in culture as proliferating cell clusters called neurospheres. Neurospheres are expanded and remain undifferentiated in the presence of potent ectodermal mitogens, usually epidermal growth factor (EGF) and/or bFGF. Subsequent removal of the growth factors from the media and/or plating the neurospheres on dishes with adhesive extracellular matrix proteins induces their differentiation into neurons, astrocytes, and oligodendrocytes. Adding differentiation factors to the media can cause further specification of cell fate [127,128]. The most remarkable feature of transplants derived from NSCs is the wide range of cellular potential that can be achieved, and in some conditions, these cells can indeed replace astrocytes, oligodendrocytes, and many different types of neurons when grafted into the injured spinal cord. For example, Cummings and Anderson et al. have examined NSCs derived from embryonic human brain that are predifferentiated to a neural lineage fate in vitro. Such cells can survive and migrate extensively from the site of injection, where they primarily differentiate to form oligodendrocytes and neurons and appear to remyelinate axons in subacute and chronic SCI rodent models [129,130]. Similarly, Karimi-Abdolrezaee et al. have used adult subventricular zone-derived neurospheres to promote remyelination when grafted into the injured spinal cord and infused in vivo with EGF/FGF2 and plateletderived growth factor alpha (PDGFa), to enhance survival and maintain an oligodendroglial fate [131,132]. However, to date, the specific methods for expanding and differentiating these cells is highly variable across research labs and companies interested in the potential of NSCs. Furthermore, following transplantation into the injured spinal cord, the differentiation fate, patterns of migration, and functional effects of these cell preparations are as highly varied as would be expected from the range of starting populations (reviewed in [98]). Notably, the grafts examined to date do not appear to develop into what looks like mature differentiated spinal cord tissue, and there is no consensus to date about how to use NSCs to achieve intraspinal grafts of mature neural tissues that would be both safe and suitable for functional interneuronal replacement strategies. However, the great breadth of work with a wide range of NSC approaches and strategies continues to contribute to a huge explosion in empirical experience and understanding of how different growth and grafting conditions as well as different lesion environments can influence survival, differentiation, and integration of neural cell grafts. Thus, if we learn that NSCs are not ideally suited

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for large-scale interneuron circuit replacement, these lessons will remain valuable as those approaches are developed. 2.2.3  Immortalized Neuronal Cell Lines Another source of renewable cells for neuronal replacement include those obtained from spontaneous or induced neuronal tumor cell lines or by experimental induction of oncogenes or proliferation genes such as c-myc into neuroepithelial precursors (reviewed in [1,133,134]). A number of neuroblast tumor lines have arisen spontaneously, but generally, these have not been explored for replacement therapy due to their uncontrolled proliferation. Embryonal carcinoma cell lines can be differentiated to a postmitotic neuronal fate with combinations of retinoic acid and mitotic inhibitors (P19 and NTera-2 cells, for example). These cells have been grafted into both intact and injured spinal cord, where they maintain neuronal characteristics and generate extensive axonal outgrowth, as described in detail elsewhere [135]. Alternatively, somatic cells can be experimentally immortalized by genetic introduction of oncogenes to induce a wide range of neuronal and multipotent neural precursors. The combined characteristics of potential tumor reversion and failure to integrate with host neuropil have reduced the potential excitement of this approach for future therapies, but it is likely that further work with inducible genetic programming and induced pluripotent cell research (below) will promote revisiting this approach. 2.2.4  ESC Derived NSC ESCs are truly pluripotent cells that are obtained from early blastocyts and are capable of generating all of the cells of the body (reviewed in [107–109]). The cells are typically propagated as embryoid bodies in the presence of leukemia inhibitory factor, then expanded in proliferation media, and finally driven to a neural lineage cell fate with retinoic acid or combinations of selected growth factors. Unless differentiation is complete prior to grafting, the pluripotency and mitogenicity of many of these cell preparations can lead to the formation of teratomas when placed into the adult brain or spinal cord [136]. Nevertheless, a number of studies have used such cells that have been driven along a neural or oligodendroglial lineage, with several reports of functional recovery attributed to neurotrophic or neuroprotective actions, synaptic activity, or remyelination [137–140]. In 2010, Geron, Inc. completed all preclinical requirements for their ES-derived oligodendrocyte progenitor cells and initiated an FDA approved phase I/II clinical trial for patients with SCI [141]. However, the details of cell preparation remain proprietary, and the trial was halted due to financial constraints. Considerable communication between researchers and better characterization of

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the cell preparations still remain as formidable hurdles to be overcome before the potential for neural tissue replacement with stem cells can be fully understood and realized. 2.2.5  Induced Pluripotent Stem Cells (iPSCs) Perhaps the most exciting prospect for neuronal replacement strategies lies in the recent approach for obtaining autologous neural progenitors, by inducing the dedifferentiation of mature somatic cells with genetic reprogramming, followed by expansion and differentiation into neural specific precursors or NPCs prior to transplantation. The obvious appeal is the potential to prepare cell grafts of neurons and glia without the use of embryonic or fetal starting material and avoiding the need for immunosuppression [142–144]. The initial discovery by Takahashi and Yamanaka that somatic cells could be dedifferentiated [145] has driven extensive work based initially on overexpression of four key developmental oncogenes, which together convert a differentiated fibroblast to a pluripotent stem cell. The risks in applying this approach to generate graft tissue are considerable, because the foreign genes must first be incorporated safely into the starting cells and then those cells must be expanded and reprogramming must be complete to avoid any residual tumorigenicity [146,147]. The extent of tumor growth from iPSCderived NSCs depends in part on the type of starting somatic cell as well as the induction and differentiation events. Recently, different passaging conditions and combinations of oncogenes and growth factors have implicated the potential for less risky genetic manipulation [143]. While it is still far from being a feasible clinical approach for SCI [108,148], future therapies using iPSCs are undoubtedly forthcoming. Basic research studies are essential to clarify key questions about the genetic control of cell division and differentiation and to define the regulatory steps needed to minimize tumorigenicity.

3.  REVISITING FSC AND NEURAL PROGENITOR TRANSPLANTATION TO ESTABLISH FUNCTIONAL RELAYS As illustrated in the timeline (Figure 1), a relative gap occurred from around 2002 to 2010 in the publication of research studies with grafted FSC tissues. This period coincides with knowledge of the limitations of graft/ host axonal integration and the search for alternative cell sources for neural grafts. The identification of optimal conditions for alternative cell-based strategies during this time has provided a number of key insights that can be applied to enhance cell survival and axonal integration of grafted neural progenitor cells. In addition, an important shift has finally begun to focus the goals of

neuronal progenitor therapies from that of addressing recovery of locomotion in models of white matter repair to identifying appropriate donor cells and individualized methods for integration.

3.1  Enhanced Cell Survival and Robust Graft Axonal Outgrowth In a recent series of experiments, Lu et al. [149] demonstrated an advance in graft integration and potential for interneuronal replacement strategies by directly combining the knowledge and tools from many strategies, old and new, to enhance the survival and growth of axons from intraspinal FSC grafts. The key objective was to develop a neuronal replacement therapy that would be robust enough to survive and integrate with host tissues when placed into an established lesion after a complete spinal cord transection. The approach began with E14-FSC tissue from rats with a reporter gene expressing green fluorescent protein. A simple trypsinized cell suspension did not survive in the poorly vascularized complete transection site, so they created conditions to enhance survival and induce growth and differentiation of the cells beginning at the time of grafting. The cells were embedded in a fibrin matrix, which enhances survival, as was shown exquisitely with autologous somatic cell graft studies above. The matrix included a “cocktail of growth factors” that was carefully vetted and chosen at doses to enable maximal proliferation (EGF and FGF2), and enhance survival and differentiation (BDNF, NT-3, PDGFa, insulin-like growth factor 1 (IGF-1), acidic fibroblast growth factor (aFGF), glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), and a calpain inhibitor). Many of these support factors have been described above as supplements to FSC grafts in vivo or included as potent survival and differentiation factors for NRP/GRPs, NSCs, or ESCs. To further establish the effectiveness of this strategy, they then used the exact same growth conditions to support grafted NSCs obtained from human FSC tissue and maintained by NeuroStem, Inc. and also grafted human ES-derived NSCs produced from the US stem cell line HUES7. The cells survived well, with extensive growth from graft to host and synapse formation in both host and graft. The extent of cell survival and outgrowth within the first few days after grafting strongly suggests that an improvement in growth state of the donor cells compared with those prepared in tissue culture medium alone [62,110]. These grafts supported functional improvements that were eliminated following retransection, but further work and control experiments are still necessary to define the relative contributions of the fibrin matrix and growth cocktail alone in tissue sparing and recovery and to determine the long-term consequences of growth factor-induced plasticity in the

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References

host on the functional changes. Importantly, a careful analysis of the anatomical characteristics of these grafts confirmed that the grafts indeed form regionally mature spinal cord neuropil, and yet none of these grafts served as a bridge for axon growth across the site of an SCI and back into the host CNS.

3.2  Functional Integration of an Interneuronal Relay in Cervical Spinal Cord The functional abilities and needs of the SCI population are highly individualized with regard to the spinal level of injury, severity of injury, concomitant complications such as pain and autonomic dysfunction, and degree of independence. As basic scientists move forward, it is important to consider a personalized approach to future neural replacement strategies. FSC and neural progenitor grafts offer a unique opportunity to select neuronal populations for integration with select interneuronal regions. One spinal cord region that relies on highly active gray matter circuits is the phrenic interneuron pool. These cervical interneurons are directly coupled to phrenic motoneurons controlling the diaphragm [7,150]. Modulation and plasticity of these interneuron pools could restore control of phrenic activity in respiratory function after cervical SCI. To determine whether different donor subpopulations could have different effects on the respiratory interneuron circuits, White et al. [151] prepared FSC grafts selected specifically from dorsal or ventral E14 spinal cord strips and placed them into hemisection lesions in lower cervical spinal cord. In this comparison, they found that all of the FSC grafts integrated with the brainstem phrenic interneurons, and they identified differences in inspiration responses to respiratory challenge between the dorsal and ventral tissue recipients. Transynaptic neuronal tracing with pseudorabies virus confirmed that primary and secondary interneurons from the graft innervated the phrenic nerve, and suggest that the graft neurons indeed influenced functional drive of the diaphragm. Future refinements of donor cell populations may enable optimal repair of different interneuronal functions such as those mediated by local pattern generators in cervical and lumbar spinal cord regions.

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cells differentiate to form mature neurons and glial cells with complex regional specification. The grafts integrate with the host spinal cord and form functional synapses and through a variety of mechanisms can contribute to improvements in functional recovery. While not covered here, recent studies have also demonstrated additional roles of neural progenitor grafts for neurotrophic and immunomodulatory therapies (see [107]). Moving forward, it is important to continue to drive and support multiple lines of laboratory research that will accomplish key preclinical objectives. First, a strong effort is necessary to identify suitable autologous tissue sources and methods for the safe derivation of neural progenitors for grafting. In addition, further delineation of specific neural and glial cell types will allow the design of individual patient therapies to address the wide range of SCI conditions present in the clinical population. Third, basic studies must continue to identify and enable appropriate growth and guidance cues for directional growth of axons both toward and from specific host target sites. Finally, complementary strategies must use the potential of endogenous repair mechanisms and rehabilitation to drive the intrinsic neuroplasticity of the surviving injured neurons and axons within the spinal cord. With clinical insight and open collaborations, these lines of research will synergize to move functional neural repair toward a foreseeable clinical reality for the SCI community.

References









4. SUMMARY



This historical review of experimental progress in FSC transplantation over the last few decades demonstrates that neural and glial progenitors from embryonic mammalian spinal cord tissue are indeed well suited to meet the objectives of a cellular repair strategy for SCI. Specifically, FSC-derived progenitors survive and fill the lesion site in a wide range of injury types and species. These





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V.  TRANSPLANTATION-MEDIATED NEURAL REGENERATION