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Abstract

Spinal cord injury (SCI) is associated with currently irreversible consequences in several functional components of the central nervous system. Despite the severity of injury, there remains no approved treatment to restore function. However, with a growing number of preclinical studies and clinical trials, cell transplantation has gained significant potential as a treatment for SCI. Researchers have identified several cell types as potential candidates for transplantation. To optimize successful functional outcomes after transplantation, one key factor concerns generating neuronal cells with regional and subtype specificity, thus calling on the developmental transcriptome patterning of spinal cord cells. A potential source of spinal cord cells for transplantation is the generation of exogenic neuronal progenitor cells via the emerging technologies of gene editing and blastocyst complementation. This review highlights the use of cell transplantation to treat SCI in the context of relevant developmental gene expression patterns useful for producing regionally specific exogenic spinal cells via in vitro differentiation and blastocyst complementation.

Keywords

spinal cord injury neural progenitor cells blastocyst complementation cell transplantation regenerative medicine

Introduction

The annual incidence of spinal cord injury (SCI) worldwide varies from 40 to 80 cases per million, with up to 90% of these cases the result of a traumatic cause¹. In the United States alone, there are roughly 54 cases per 1 million, or about 18,000 new cases each year, with a higher prevalence in men². In addition to the physical limitations that come with SCI, the associated socioeconomic burden can be crippling. Patients with SCI have estimated lifetime direct costs ranging from US$1.2 million to US$5 million, which does not include indirect costs, such as lost wages². However, this cost does vary tremendously depending on life expectancy and mortality rate. Persons with SCI have an estimated two to five times greater risk of mortality than the general population, with the first year after injury holding the greatest risk³. There are no existing cures for individuals with SCI, and while patients with incomplete injuries may experience limited improvement, full recovery after SCI is rare^4,5. The lack of available therapies along with the tremendous burden SCI has physically, economically, and emotionally highlights the need for efficacious and safe treatments for management of traumatic injury. Current experimental approaches for treatment have made use of neuron transplantation with the goal of restoring nervous system function after SCI. This review will summarize recent advances in the field of regenerative medicine on the generation and the potential use of exogenic neuronal cells to treat SCI. In addition, we will highlight the gene networks and expression patterns relevant to exogenic cell development for optimization of successful functional outcomes as a promising direction for future transplantation studies.

SCI Pathophysiology

SCI can present with a myriad of symptoms depending on the severity of the injury and its location along the spinal cord. Symptoms include partial or complete loss of motor and sensory function below the injury site, and can also affect regulatory systems that control bladder and bowel function, breathing, heart rate, and blood pressure¹. It is important to note that no two human cases are identical as each affected person presents with varied symptoms and potential for recovery. The initial traumatic event, or primary injury, is followed by several biological events termed the secondary injury. There are, however, three pathological stages that define the secondary injury in SCI: the acute phase (48 h), subacute phase (2–14 days), and chronic phase (more than 6 months)^4–7.

During the acute phase, disruption and swelling of the spinal cord occurs at the level of injury due to edema and intraspinal hemorrhage⁶. This disruption exposes the spinal cord to an influx of inflammatory cells and cytokines that cause necrotic cell death^7,8. Proinflammatory cytokines and peripheral inflammatory cells are recruited to the site of injury, and phagocytes rapidly remove debris. This process generates cytotoxic byproducts, which add to cell injury^9,10. The subacute stage of injury is primarily characterized by traumatic demyelination and further vascular compromise of the spinal cord⁶. Additional biochemical events, such as an accumulation of glutamate in extracellular compartments, contribute to excitotoxicity of surrounding neurons^11,12. At this point, many axons affected by the injury will begin to degenerate. Macrophages can also be seen actively ingesting fragments of myelin, and myelomalacia, or softening of the spinal cord, is typical^4,6. During the chronic phase, most of the necrotic tissue is eliminated and ingested by macrophages. The resulting cavities are filled with extracellular fluid, residual macrophages, and connective tissue, hindering cell regeneration, migration, and axon regrowth^13,14. SCI is also characterized by Wallerian degeneration, a process that involves the anterograde degeneration and pruning of axons and their myelin sheaths, beginning in the subacute phase and persistent throughout the chronic phase. In addition, an astroglial scar at the site of injury develops from proliferative astrocytes that form tightly interwoven glial fibers around the injury site^6,15.

The role of the glial scar is controversial, as initially it helps to reestablish the blood brain barrier and limit lesion size; however, later it appears to be detrimental to functional recovery by creating a barrier that prevents extensive axonal regrowth. It is also unclear the extent to which these degenerative processes have an endpoint or if they continue indefinitely, possibly due to chronic inflammation or autoimmunity. An understanding of the mechanisms of these pathophysiological events is essential, as potential therapies for SCI may target one or more of these processes. Furthermore, with the distinct progression of injury over time, it is crucial to optimize proposed therapies to the stage of SCI they will be administered.

Currently, there is no recognized treatment for the recovery of functional deficits after SCI. The current standard of care following SCI consists of a variety of management strategies, including intensive care unit admission for careful blood pressure control, optional use of corticosteroids, and early surgical intervention for decompression. Ultimately, many physicians emphasize the importance of early intervention. It has been suggested that if proper treatment is applied during the window immediately following the primary injury, the damage caused by secondary injury can be limited, thus improving functional outcomes^4,9,15. Although current clinical treatments are limited to those listed above, researchers have worked for decades to develop new and promising therapies. Most of these are considered “neuroprotective” and target acute and subacute injuries; very few experimental approaches have targeted chronic SCI. However, considering that the pathophysiology of chronic SCI is relatively static, re-establishing lost connections in the chronically injured spinal cord is a mechanistically plausible target^16,17. For both acute and chronic SCIs, a common strategy involves targeting regeneration of cells to restore lost connectivity, such as with cell transplantation, which is the focus of the remainder of this review. Cell replacement therapy, also referred to as cell transplantation, involves the transplantation of neural progenitor cells (NPCs), among other cell types, into the site of injury to restore function and connectivity. Numerous studies have demonstrated the safety and efficacy of this approach in animal models as well as its ability to support regeneration and functional recovery in early stage clinical trials. For the purposes of this review, we will be focusing on transplantation of NPCs, which are necessary for true neural cell replacement in the central nervous system (CNS). While there are reports of transdifferentiation from non-neural cell types to NPCs, this remains controversial, and thus will not be discussed further here.

Sources of Cells for Transplantation

Stem cells provide the ideal building blocks for cell replacement therapies because of their ability to self-renew and generate numerous cell types. Spinal neurons may be obtained from numerous stem cell sources, including fetal stem cells (FSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs). Several methods have been developed to generate spinal neurons in vitro from their corresponding stem cell progenitors. With advances in technology and methodologies allowing for a wide breadth of cell types to be produced, transplanting spinal progenitor cells as a therapeutic approach has risen in prominence, especially with respect to treating SCI.

Preclinical and clinical studies have utilized stem cells from multiple sources for SCI, all demonstrating varying degrees of success, with primary differences being in degree of pluripotency and methods of acquisition. ESCs are pluripotent stem cells originating from the inner cell mass of the mammalian blastocyst that can differentiate into cells from each of the three germ layers¹⁸. FSCs are multipotent stem cells originating from the fetus that develop into progenitor cells restricted to a single germ layer. Human iPSCs (hiPSCs) are generated by cellular reprogramming of adult somatic cells via transcription factors to reset cells to a pluripotent state¹⁸. While first accomplished through the use of viral transduction, which carries the risk of oncogene integration and tumor formation, several non-genome incorporating or nonviral delivery methods have since been developed that may be ideal for clinical application^19,20. Because hiPSCs are derived from the adult system and can be used autologously, this minimizes the need for immune suppression and greatly expands the pool of eligible recipients. MSCs are multipotent non-neural stem cells that may be obtained from adipose, bone marrow, and umbilical cord tissue²¹. Currently, MSCs are the most commonly studied for cell transplantation and have many ongoing clinical trials, likely because of their easy accessibility and capacity to be transplanted autologously. While predominantly used to produce mesoderm-derived tissues, such as bone, cartilage, and fat cells, there are also controversial reports of neural differentiation that will be highlighted below. Each of these sources has been used to generate neural or neural-like cells, making them prime candidates for use in spinal progenitor cell transplantation studies.

Advantages and limitations associated with each stem cell source must be considered when developing systems for their practical application. While ethical and immunological issues hamper the use of ESCs and FSCs in clinical settings, their application for SCI has been extensively studied in animal models²². To date, the most well-established protocols describing the generation of spinal neurons utilize cells derived from ESCs, providing a strong foundation for continued use²³. While region-specific FSC-derived and fetal tissue-derived NPCs have been demonstrated to integrate well with host tissue, limitations include issues of cellular heterogeneity with non-neural cells within the grafts, limited proliferation capability, immune rejection, and the need for fetal tissue²⁴. A major draw of the use of hiPSCs is their capacity to be transplanted autologously, although the current time requirements necessary to obtain donor samples, reprogram, differentiate, and perform quality control are prohibitive for acute and most subacute cases. Improved protocols for rapid induction and differentiation will be key for advancing application of autologous techniques²⁵. Matched samples from hiPSCs banks may provide a middle ground, providing a cheaper, off-the-shelf product, which would still require immune suppression, but with less likelihood of rejection. Finally, MSCs, though widely used, have shown conflicting evidence regarding their neural differentiation capacity in vivo, which would significantly affect their translational capability, especially when utilized in a chronic setting. Many researchers argue their effect is limited to neurotrophic and immunomodulatory support^26,27. An in-depth discussion of the history of MSC transplantation for SCI is beyond the scope of this review, therefore, we direct readers to Liau et al²¹, Rafiei et al²⁸, and Ruzicka et al²⁹ for further study of their preclinical and clinical applications. The next section will explore the current state of preclinical and clinical applications of stem cell-derived NPCs for cell transplantation in SCI.

Cell Replacement Therapy for SCI

Significant advances in the field of cell replacement therapy in the past 10 years have highlighted both the promise along with the future directions of this approach that may promote further functional benefit as a treatment after injury. The most commonly studied cell types for transplantation include NPCs, MSCs, oligodendrocyte progenitor cells (OPCs), and Schwann cells (SCs)^30–32. In general, these candidate cells can be subdivided into neuronal and non-neuronal types. While non-neuronal cells may provide some functional benefit, neuronal cells, such as NPCs, are advantageous by their additional ability to generate new neurons, astrocytes, and oligodendrocytes capable of replacing damaged or lost cells and may create a bridge across the injury site³³. The creation of neural relays between grafted cells and host neurons could be a vital component of restoration after injury. While current cell transplantation research provides promising results for injury improvement, a better understanding of the underlying mechanisms is needed. In this review, we focus on the transplantation of NPCs because of their potential for cell replacement, which is likely more applicable than other cell therapies to chronic SCI, an unmet public health need.

Numerous preclinical experiments have transplanted cells following SCI in a range of animal models, including mice, rats, minipigs, and nonhuman primates. These preclinical studies provide invaluable insight toward understanding the mechanism and therapeutic potential of cell replacement therapy, contributing to advancements in previous and ongoing clinical trials³⁴. Here, we briefly discuss functional outcomes and status of both preclinical and clinical studies using NPCs derived from fetal tissue, FSCs, ESCs, and iPSCs.

Preclinical NPC Transplantation Studies in Animal Models of SCI

Cell transplantation approaches in animal models of SCI have focused on the safety, efficacy, and extent of functional recovery in response to ESC-derived, FSC-derived, iPSC-derived, and fetal tissue-derived NPCs. These studies have demonstrated several mechanisms that promote nervous system recovery, including remyelination^35–44, axonal regeneration^{36–38,44–52}, tissue and nerve sparing^35–37,45, angiogenesis^36,37, and the release of neurotrophic factors^38,39 (Table 1). Significantly, many of these studies highlight the successful integration of transplanted exogenic neuronal cells as observed by cell survival, migration within host tissue, and synapse formation with host cells^{40–43,48–52,56,59–62}. These mechanisms may prove significant to restoring the cellular microenvironment at the site of injury by creating a growth permissive environment for axonal restoration and neural relay formation, ultimately promoting functional recovery. In fact, several of these studies highlight the recovery of motor function^{35,37–41,43–45,47,50–52,61} and reducing chronic neuropathic pain⁶³ in animal models of SCI. While motor and sensory recovery in animals with SCI was not a ubiquitously reported outcome, the studies that did report treatment-based functional recovery have emphasized the therapeutic benefits of cell replacement.

Table 1.

Common Mechanisms of Recovery After Cell Transplantation for SCI and the Associated Graft Cell Type.

Mechanisms of recovery	Associated transplant cell type	Reference
Neuroprotection	Neuron Astrocyte Oligodendrocyte	^{29,35–39,44–47,52,53}
Axon regeneration	Neuron	^{29,36,37,45,47,50–52,54,55}
Relay formation	Neuron	^{37,41–43,48,50–52,54–58},
Remyelination	Oligodendrocyte	^{35–38,40,42}
Angiogenesis	Astrocyte	^36,37,39,52
Immunomodulation	Astrocyte Microglia	^28,45

For neuroprotection, references were added based on evidence of tissue sparing, improved trophic support, decreased glial scarring, or less demyelination. It is important to note that neuroprotection can also contribute to the other mechanisms of recovery as well. For axon regeneration, references were added based on evidence of regrowth of severed or damaged host axons. For relay formation, references were added based on evidence of host to graft or graft to host synapse formation. For remyelination, references were added based on evidence that transplanted cells differentiated into oligodendrocytes and myelinated host axons. For angiogenesis, references were added based on evidence of new blood vessel formation. For immunomodulation, references were included based on evidence of altered immunological response, such as decreased microglial/macrophagic activity.

In addition to the successes of NPC transplantation in animal models of SCI, other factors also assist in enhancing functional outcomes. Co-transplantation of NPCs with other cells, such as macrophages⁶⁴, SCs⁶⁵, OPCs⁶⁶, or factors, such as Chondroitinase ABC⁴¹, has been shown to enhance cell integration and functional recovery. Designing regionally relevant neural cells for transplantation may promote further recovery, as regional specificity of ESC-derived neural cells has been shown to be a significant factor in creating a neural relay circuit for functional recovery⁵⁴. This was similarly observed with the transplantation of regionally specific iPSC-derived NPCs into the spinal cord⁵⁷. As such, highlighting the role of the regionally relevant microenvironment and cell populations should be a significant point of interest for transplantation-mediated SCI treatment approaches. The time, location, and manner in which cells are transplanted can also play a major role in their survival, integration, and ultimate efficacy. While the direct injection of cells at the site of injury has historically been the most common route of administration, this results in relatively poor survival and graft integration⁶⁷. The hostile microenvironment of the acute injury site may contribute to this, with subacute transplantation and the addition of grafts rostral and caudal to the injury improving these outcomes^68,69. The use of engineered biomaterials, including scaffolds, hydrogels, and nanoparticles also provides structural and nutrient support that enhances functional recovery after transplantation^70–72. Though these considerations are widespread within preclinical research, their adoption in clinical studies may be pivotal for translation.

Clinical Cell Transplantation Studies in Humans for SCI

Globally, a large number of clinical trials involving cell transplantation have been conducted at all stages of injury in humans³³. Several clinical trials have studied FSC-derived NPCs for transplantation after SCI. A phase I/II clinical trial conducted by Yonsei University Health System (KCT0000879) to evaluate the safety and efficacy of fetal telencephalon-derived NPCs in patients with cervical, motor complete SCI determined the treatment well-tolerated, safe, and neurologically beneficial, with about 25% of patients demonstrating improvement in the Abbreviated Injury Scale⁷³. StemCell Inc. also tested human FSCs for feasibility and safety after transplantation in a dose-escalation study, but the study was terminated early due to inability to reach sponsor-established benchmarks (NCT02163876, NCT01725880)^74–77. Finally, Neuralstem Inc. sponsored a phase I clinical trial (NCT01772810) to evaluate the safety and efficacy of fetal spinal cord-derived NPCs in 4 chronic SCI patients⁷⁸. Notably, this is the only spinal cord-derived human cell line to have received Food and Drug Administration (FDA) approval for transplantation in humans with SCI. Neurological assessments and electromyography revealed improvement in sensory and motor scores and increased muscle activity, suggesting renewed connectivity. However, this phase I study lacked a control group, and thus lacks the statistics required to determine the extent of treatment-based functional recovery⁷⁹. Phase I/II trials transplanting this cell line in amyotrophic lateral sclerosis (ALS) patients have also been completed, demonstrating safety but unclear functional effects (NCT01348451, NCT01730716)⁸⁰.

The only clinical trial involving transplantation of ESC-derived NPCs in humans is currently recruiting for a phase I/II trial for subacute SCI. S. Biomedics Co. intends to recruit five patients with C4–C7 complete SCI diagnosed as a grade A injury on the American Spinal Injury Association Impairment Scale. This trial (NCT04812431) will assess the efficacy and safety of polysialic acid neural cell adhesion molecule positive (PSA-NCAM+) NPCs derived from a human ESC line⁸¹. While this is the first study to transplant ESC-derived neuronal cells, the SCiSTAR trial, a clinical trial started by Geron Corporation and continued by Lineage Therapeutics, involved the transplantation of ESC-derived OPCs^82,83. Nearly, all patients (21 of 22) displayed at least one level of motor improvement, and seven patients showed two levels of motor improvement, which was sustained for at least 1 year post-transplantation⁸⁴. A follow-up of five patients at 10 years post-transplantation suggests that the treatment is well-tolerated, with no evidence of neurological deterioration or tumor formation at this time point⁸⁵. Lineage Therapeutics most recently announced its intent to initiate a new clinical trial with this cell line in both subacute and chronic SCI patients pending FDA clearance⁸⁶.

Despite the recent developments in generating and understanding iPSC-derived NPCs in vitro and their mechanistic effects in vivo over the past 20 years, its clinical translation to human SCI has yet to be fully explored. While hiPSC-based treatments for macular degeneration and Parkinson’s disease have been documented, no clinical trial using hiPSCs for SCI has been conducted^87,88. In fact, Japan is the first country to announce approval for a clinical trial using hiPSC-derived NPCs to treat subacute SCI⁸⁹; they intend to enroll a total of four participants and the first successful surgery was performed in December 2021⁹⁰. The Okano group proposes to assess the safety and efficacy of hiPSC transplantation in patients with complete cervical or thoracic injury. This trial marks an incredible advancement in the field and will likely shape future clinical trials in the rest of the world.

These clinical trials demonstrate the potential of using NPCs to treat SCI in humans. However, while improvement in motor function was reported by some trials, others have not been as successful or require further study to determine a conclusive outcome. Ultimately, no clinical trial has reached FDA approval for SCI treatment. As already mentioned, improvements in graft integration and functional recovery when cells are tailored to regionally relevant identities have been observed in multiple preclinical studies^54,57, and this may be key to drive translation within the field of cell replacement therapies.

Differentiation and Regional Specification of Spinal Neurons

Pluripotent stem cells require ex vivo differentiation to attain a neural lineage. Advancements in cell culture methodology, specifically in the field of cell transplantation over the last decade, have improved long-term survival, consistency, and replicability of differentiation protocols. This has included the implementation of defined reagents, feeder-free culture systems, and Good Cell and Tissue Culture Practice (GCCP) to guide the field closer to translation^91–93. While notable progress has been made, the inherent variability of cell culture and the vast assortment of utilized methodologies remain one of the biggest hurdles to achieving reproducibility.

Considerations for Growth Factor-Directed Differentiation

The culturing of stem cells in a dish itself possesses a stochasticity that contributes to inconsistent differentiation in even carefully controlled environments⁹⁴. Variable differentiation potential may be attributed to the process of reprogramming as well as the introduction of both genetic and epigenetic changes during culture, which are currently poorly understood^95–97. Additional sources of variation include the mode of culture, cell line differences, and a vast repository of matrices, small molecules, and techniques, many of which may be proprietary or susceptible to batch variability. Thus, a successful differentiation paradigm must both carefully select the conditions to produce the desired cell type and incorporate quality control checkpoints to make note of fluctuations in outcomes when all else is the same. Considerations for directed neuronal differentiation are discussed below, and a more detailed discussion of replicability and experimental design in cell culture experiments is provided by Chan and Teo⁹⁴.

Numerous methods for both 2D and 3D culture have been established. Culture in 2D involves the plating of adherent monolayers and is the traditional means of cell culture and differentiation. Advantages of this strategy include providing homogenous nutrient access to plated cells and enabling uniform proliferation. While single monolayers have historically been most common, micropatterning and sandwich culture are increasingly used to introduce more complex interactions to the 2D environment⁹⁸. However, cell fate following 2D differentiation often does not correspond to differentiation in vivo, with cell type composition varying significantly following transplantation⁹⁹. Culture in 3D provides a more complex environment that allows for greater emergence of signaling cascades, migration, and motility patterns¹⁰⁰. A growing number of groups have developed robust strategies for the generation of spheroids and organoids, which can be maintained in scaffolds, sheets, hydrogels, and within microfluidic devices⁹⁸. While these approaches produce heterogeneous populations of cells that more accurately reflect the in vivo microenvironment, they also tend to be more costly and require advanced culturing techniques that can be difficult to establish. This intricacy also leads to increased variability in outcomes that has made large-scale reproducibility difficult. Differentiation in both 2D and 3D have yielded important insights on relevant developmental pathways and the injury process, and care should be taken to determine which system will be most suitable based on the needs of a given experiment.

The use of various coating materials to provide an adhesive matrix for stem cells to grow on has been shown to influence both the efficiency of differentiation and the behavior of the terminally differentiated cells. These are generally intended to mimic the extracellular matrix (ECM) of native cells, which is known to play an important role in both the injured and diseased CNS¹⁰¹. Collagens, laminins, fibronectins, and poly-amino acids are among the most commonly used coating matrices. Laminin, a major component of ECM, has the greatest effect on NPC differentiation, expansion, and neurite outgrowth, and is often considered an essential ingredient in the coating process for both 2D and 3D applications¹⁰². For this reason, Matrigel is widely used for neuronal differentiation, however, its clinical relevance is impacted by its xenogeneic nature. Poly-amino acids, such as poly-D-lysine (PDL), poly-L-lysine (PLL), and poly-L-ornithine (PLO), are non-ECM proteins that promote cellular adhesion in 2D culture but provide poor support for neuronal maturation alone¹⁰³. Thus, many protocols call for a combination of matrix materials for optimal adhesion and differentiation. Determination of coating substrate must evaluate both efficiency and desired downstream application, as matrices may have differential effects on differentiation, migration, and maturation.

In addition to variations in the mode of cell culture, the means of differentiation may also vary substantially. Differentiation protocols themselves range from production of early progenitors to more fully developed postmitotic neurons. For this purpose, a variety of growth factors and neurotrophins have been documented to improve differentiation and transplantation outcomes¹⁰⁴. Differentiation of hiPSCs commonly begins with a neural induction stage, which has been reported both using small molecules for dual SMAD inhibition¹⁰⁵ and morphogen-free methods⁹². Following induction, cells may be maintained in an early neuroepithelial or neuromesodermal stage via application of fibroblast growth factors (FGFs) prior to subsequent differentiation into discrete lineages¹⁰⁶. Most protocols also use retinoic acid (RA) to promote neuralization and caudalization to a spinal identity. Subsequent regional specification to mature spinal interneuron subtypes involves both rostrocaudal and dorsoventral patterning, and is discussed in more detail below.

Considerations for Regional Specification of Spinal Neural Cell Types

There is profound cellular diversity within the spinal cord, and a growing body of research has revealed the importance of specific regional patterning of exogenous cells to the level and functional domain of the injury site prior to transplantation¹⁰⁷. To date, most applications of cell replacement therapy utilize cells that have been differentiated to a broad neural fate without consideration for the level of the neuraxis or the necessary sensorimotor components. This has contributed to low graft survival and failure of transplanted cells to integrate with host tissue, posing a major challenge in the application of cell replacement therapies. However, recent work has shown that NPC transplantation enables robust corticospinal regeneration and functional recovery only when the cells are patterned toward a caudalized fate⁵⁴. Furthermore, transplanted NPCs preferentially form connections with similar phenotypic regions^55,58. Thus, for the creation of clinically relevant models and treatments, it is of the utmost importance that scientists recapitulate appropriate characteristics at the area of injury. This degree of specialization will allow for targeted therapies that can focus on individual outcomes, including improvements in specific locomotor circuits, sensation, amelioration of pain, remyelination, and more.

Several protocols have been developed to generate regional subtypes of spinal neurons and interneurons in vitro, allowing for a wide range of positionally specified cells to be transplanted for SCI. While spinal caudalization of NPCs is increasingly common, tailoring of cells to a specific spinal level has been rare and requires precise coordination of FGFs and wingless-related integration (Wnt) agonists and antagonists¹⁰⁸. Great attention has been given to the use of motor neurons and ventral spinal interneurons for repair after injury, though the production and transplantation of dorsally specified interneurons remains an unmet need for meaningful sensory recovery. Ventral populations are widely attained by application of RA and sonic hedgehog (Shh) agonists. Derivation of V2a⁵⁸ and V3¹⁰⁹ interneuron populations is most commonly reported. Transplantation of ventral and motor neurons is conducted with the intention of restoring locomotion, autonomic function, and related motor activities depending on the level of injury—and many studies report success¹¹⁰. Dorsal specification requires application of bone morphogenetic proteins (BMPs) and only three protocols to date report generating these populations^111–113. Considering as many as 80% of SCI patients experience chronic pain and sensory impairment, the lack of research and attention on the development of these populations neglects the devastating impact of sensory dysfunction in traumatic SCI¹¹⁴. As a result, studies moving forward should consider rostrocaudal and dorsoventral neural identity as a significant factor with transplantation. These approaches rely on an understanding of the developmental neurobiology of the spinal cord and spinal neurons to identify relevant genes and signaling pathways for producing enriched and regionally specific cell populations.

Developmental Neurobiology of the Spinal Cord

The formation of the spinal cord during development is a process that initiates during gastrulation with the formation of the primitive streak and primitive node^115–118. Cells migrating through and from the primitive node will form the trilaminar embryonic disk composed of three germinal layers: endoderm, mesoderm, and ectoderm^118,119. The notochord, formed in the axial mesoderm, secretes growth factors to induce the creation of the neural plate through neuroectoderm, thus initiating primary neurulation^117,119. The neural plate then differentiates to create a neural groove surrounded by neural folds. These neural folds will fuse to form the neural tube, the precursor to both components of the CNS—the brain and the spinal cord¹¹⁶ (Fig. 1).

Figure 1.

Neural tube development. Schematic illustrating the development and differentiation of the neural tube—the precursor of the hindbrain and the spinal cord. Neural tube development begins with (A) the folding of the neural plate lateral edges. The neural folds fuse to form the neural tube. Developmental progression is characterized by (B) differentiation of the neural tube, maintaining dorsoventral polarity, to form the alar and basal plates. Continued differentiation of the neuroepithelial cells results in the formation of the marginal and mantle layers, which are the precursors to (C) the white and gray matter of the spinal cord, respectively.

After neural tube closure, the neural tube maintains a dorsoventral organizational polarity, where the dorsal and ventral parts of the neural tube are characterized by the presence of the roof and floor plates, respectively^120,121. Neuroepithelial cells near the neural canal differentiate and migrate laterally to form from the three central layers of the developing spinal cord: the ventricular layer, the mantle layer, and the marginal layer (Fig. 1B). Of these three layers, the marginal layer develops into the white matter of the spinal cord and the mantle layer develops into the gray matter. Further differentiation and dorsoventral polarization of the mantle layer leads to the formation of the alar and basal plates, the precursors to the dorsal and ventral horns of the spinal cord, respectively (Fig. 1C). Neuroepithelial cells of the alar plate will become sensory interneurons, whereas the cells of the basal plate will develop into the motor neurons of the developed spinal cord¹²².

In addition to dorsoventral polarity, rostrocaudal patterning is also a key factor in spinal cord development. Global rostrocaudal polarity is defined by five spinal segments: cervical, thoracic, lumbar, sacral, and coccygeal. Each spinal region is characterized by a number of extending nerve fibers from a specific dorsal root ganglion that innervate a single dermatome¹²³. Local rostrocaudal patterning is specific to the organization of spinal motor neurons and interneurons. Spinal motor neuron subtype specification along the rostrocaudal axis is particularly relevant for limb-specific motor control. Spinal interneuron patterning has recently been linked to the distribution of interneuron subtypes clustered by gene expression and contributes to both sensory and motor spinal networks. The combination of both motor and interneuron regional subtypes is of particular interest considering their role in central pattern generators (CPGs) of the spinal cord^124,125. Recapitulation of these developmental programs constitutes the first step in the generation of specific spinal populations.

Rostral–Caudal Patterning

During spinal cord development, the rostral and caudal identity of spinal cord cells is influenced by FGFs, RA, and growth differentiation factors (GDFs). Motor neuron identity along the rostral end of the neural tube is induced by a combination of high paraxial mesodermal RA expression and low caudal FGF8 and GDF11 expression^126,127 (Fig. 2A). These signaling gradients regulate Hox gene expression throughout the developing spinal cord (Fig. 2B). Hox genes are a family of transcription factors linked to anterior-posterior cell subtype specification throughout the CNS. Hox gene expression in developing spinal motor neurons follows a rostrocaudal organization^128,129. Specific Hox gene expression in turn aids in defining motor neuron columnar subtypes and regulating the motor neuron–muscle connection^130,131.

Figure 2.

Developmental rostral–caudal patterning of the spinal cord. Schematic demonstrating the rostrocaudal patterning of transcription factors and Hox genes involved in neuronal subtype specialization. (A) Several neuronal populations are also specialized along the rostrocaudal axis, influenced by gradients of RA and FGF/GDF expression. Within the motor neuron population generated from the pMN progenitor domain, there are subpopulations of motor neurons separated into six distinct motor columns: median motor column (MMC), hypaxial motor column (HMC), preganglionic motor column (PGC), lateral motor column (LMC), phrenic motor column (PMC), and spinal accessory column (SAC). (B) Hox gene expression along the rostrocaudal axis.

Spinal interneurons are similarly organized depending on rostrocaudal positioning. Several studies have implicated FGFs, RA, and Wnts in controlling the Hox gene expression of ventral interneurons. V0, V1, V2, and V3 interneurons have been shown to have differential gene expression along the rostrocaudal axis of the developing spinal cord, particularly subdivided at the brachial, thoracic, and lumbar levels^132,133.

The local distribution of spinal motor neurons and interneurons has been identified as a factor in the development of CPG circuits in the spinal cord¹²⁴. Considering the extensive role of spinal CPG networks for locomotion, the developmental rostrocaudal diversification of spinal motor neurons and interneurons has gained attention with the generation and transplantation of neurons and neural stem cells for nervous system repair after SCI. With the goal of cell transplantation studies being to restore lost function and repair neural circuits, the generation of neurons with regional specificity must be taken into consideration to account for pre-established local motor neuron–interneuron interactions along the rostrocaudal axis.

Dorsal–Ventral Patterning

Differences in form and function are most pronounced between the dorsal and ventral domains of the spinal cord, with dorsal progenitors generally giving rise to domains that process sensory input and ventral progenitors giving rise to domains that process motor output (Fig. 3). Readers may refer to Zholudeva et al¹⁰⁷, Sagner and Briscoe¹³⁴, and Hernandez-Miranda et al¹³⁵ for extended discussion of the developmental origins of these spinal neurons. Generation of ventral cell types requires activation of Shh signaling pathways. Shh is secreted by the floor plate during development and gives rise to all ventral progenitor domains in a concentration dependent fashion¹³⁵. Within the dorsal horn, a variety of spinal interneuron subtypes process sensory information from primary afferent inputs and modulate transmission to spinal motor circuits and supraspinal regions¹³⁴. However, very little progress has been made on the derivation of dorsal and sensory populations for transplantation in SCI. Early “Class A” dorsal progenitors arise via Wnt and BMP signaling¹³⁵. Late “Class B” dorsal progenitors arise independent of these signals, and the conditions driving their development in vitro have been more difficult to elucidate^136,137. To design protocols aimed at generating spinal neuronal cells with regionally relevant identity, the emerging technology of generating exogenic cells and organs via targeted gene editing, as well as blastocyst complementation may provide additional sources of authentic neuronal progenitor cells for transplantation for repair in SCI.

Figure 3.

Developmental dorsal–ventral patterning of the spinal cord. Wnt and BMP secreted from the roof plate and Shh secreted from the floor plate are the main factors influencing neuronal subtype specification along the dorsoventral gradients. The progenitor domains, pd1–pd6, p0–p3, and pMN, generate dorsal interneurons (dI1–dI6), ventral interneurons (V0–V3), and motor neurons (MN), respectively. Several progenitor and postmitotic markers have been identified for particular subpopulations of dorsal and ventral interneurons along the dorsoventral axis, acting as potential gene candidates for region-specific cell differentiation protocols.

Developmental Gene Networks for Exogenic Spinal Cell Specification and Differentiation

While several protocols have been established to generate different spinal neuron populations, the regional diversity of spinal neurons has yet to be fully recapitulated in vitro. However, the few protocols that have been developed to tailor neuronal differentiation toward specific subpopulations of spinal neurons have made use of relevant developmental cues and gene networks. Here, we discuss the subtype-specific genes that could be targeted to produce spinal neurons with regional specificity (Fig. 4).

Figure 4.

Generating neuronal progenitor subtypes for cell transplantation by temporal enhancement or suppression of key developmental pathways. Pluripotent stem cells (PSCs) must first undergo neural induction, traditionally to an early ectoderm phenotype, prior to further differentiation to specific neural types. Subsequent caudalization to a spinal identity is essential for production of dorsal, ventral, and motor domains. For generation of dorsal populations, TGF-β pathway agonists, such as BMPs, allow for maturation into dorsal interneurons upon extended culture. Particular networks of expression, such as Pouf41, Lbx1, and Tlx3, may be utilized for identification of specific interneuron subtypes. For generation of ventral motor and interneurons, sonic hedgehog (Shh) pathway agonists allow for maturation into motor neurons or ventral interneurons upon extended culture. Particular networks of expression, such as Neurog2, Hb9, and Isl1 for motor neurons and Chx10, Bhlhb5, and Prdm8 for ventral interneurons, may be utilized for identification of specific interneuron subtypes.

Motor Neurons

Motor neuron development is largely influenced by developmental gradients of RA, Shh, and FGF signaling^127,138. These signaling pathways influence the generation of the pMN progenitor domain where all spinal motor neurons arise. The pMN progenitor domain is characterized by the expression of homeodomain proteins PAX6, NKX6.1, and basic helix–loop–helix (bHLH) protein OLIG2^139–141 (Fig. 3). Motor neuron progenitors begin differentiation as a result of RA and OLIG2-mediated gene expression of GDE2¹⁴² and Neurog2¹⁴³, respectively. In particular, OLIG2 and Neurog2 promote transcription of downstream motor neuronal genes, such as homeobox gene Hb9, early in development^144–146. In addition, the ISL1–LHX3 complex has been shown to be significant in specifying motor neuronal fate over an interneuron fate^147,148.

Developmental motor neurons will adopt a fate dependent on the muscle it targets and its location along the spinal cord, thus relying on further differentiation cues along both the dorsoventral and rostrocaudal axes, as described above. Significantly, motor neurons follow a columnar organization, separating pools of motor neurons by their location and muscle targets. The four well-characterized motor columns are the median motor column (MMC), lateral motor column (LMC), hypaxial motor column (HMC), and the preganglionic column (PGC)¹³⁸ (Fig. 2A). MMC neurons are located all throughout the spinal cord and are characterized by the co-expression of Hb9, ISL1/2, OCT6, and LHX3/4^149,150. LMC neurons are found in the brachial and lumbar levels of the spinal cord, and are characterized by the expression of ISL2, FOXP1, and RALDH2¹³⁰. HMC neurons are located in the thoracic level of the spinal cord, and are known to express Hb9, ISL1, and ER81¹³⁰. PGC motor neurons are localized to the thoracic and upper-lumbar segments of the spinal cord and known to express SMAD1, NOS1, ZEB2, HNF6, and FOXP1^130,150,151. In addition to the MMC, LMC, HMC, and PGC, there are two additional, less-characterized motor columns—phrenic motor column (PMC) and spinal accessory column (SAC)—that warrant future research into the gene expression profiles of its neuronal populations.

Several protocols have been developed to generate motor neurons in vitro from ESCs and iPSCs. These protocols generally follow the processes of neural induction, differentiation, and maturation described above. Neural induction and differentiation are promoted by culturing the ESCs or iPSCs with BMP inhibitors, ROCK inhibitors, SMAD inhibitors, TGF-β inhibitors, Shh agonists, RA, and neurotrophic growth factors. Motor neuron differentiation is promoted by the presence of key developmental factors, such as Shh, RA, brain-derived neurotrophic factor (BDNF), and FGF. Subsequent motor neuron maturation involves several neurotrophic and growth factors^{91,105,152–162}. While these protocols have been shown to generate motor neurons in vitro, the majority of protocols generate neurons that are often immature, uncharacterized, or limited to the upper motor neuron subtype, therefore likely not locally relevant to an injury within the spinal cord. Considering the range of motor neuron subtypes throughout the spinal cord, each expressing different combinations of genes designed for specific muscle targeting, current studies have started to focus on generating subtype-specific motor neurons in vitro.

Spinal Interneurons

Dorsal interneurons

As described above, early dorsal spinal specification is driven largely by BMP and Wnt pathway signaling via secretion of factors from the roof plate. As cellular identity advances, a plethora of regulated gene networks coordinates the onset of distinct dorsal domains. Homeobox genes, including HOX genes and PAX genes, play a critical role in broad patterning of the spinal cord along the rostrocaudal and dorsoventral axes, respectively. For more precise dorsal specification, bHLH transcription factors contribute to development of distinct progenitor layers¹⁶³. Much of the work elucidating gene network expression in the spinal cord has been performed in chick and mouse knockout studies^164,165.

Class A dorsal progenitors, which are dependent on roof plate signaling for appropriate specification, predominantly express AtOH1 and Olig3. Multiple studies have confirmed Olig3 is necessary for the specification of class A fate^165–167. Class B dorsal progenitors rely more on a combination of homeobox and bHLH expression, including Ascl1, Pax7, and Ngn2. Combinatorial expression of multiple genes can be used to further classify distinct populations¹⁶⁸.

As progenitors further differentiate into a postmitotic state, gene expression undergoes a transition to six distinct interneuron populations. Class A progenitors give rise to interneuron population 1–3 (dI1–dI3) and class B progenitors give rise to interneuron populations 4–6 (dI4–dI6). In class A progenitors, AtOH1 and Olig3 expression gives way to Pou4f1 expression. Further specification can be seen in expression of Lhx9 by dI1s, FoxD3 by dI2s, and Tlx3 by dI3s. In class B progenitors, Ascl1 expression gives way to Lbx1 expression. Further specification can be seen in expression of Pax2 by dI4s, Lhx1/5 by dI5s, and co-expression of Pax2 and Bhlhb5 in dI6s. These cardinal classes have been defined by morphological, electrophysiological, and functional properties in addition to their distinct genetic profile¹⁰⁷. Subtype specific gene modification has proved valuable in generation of ventral populations, as described below, and future research may explore this possibility for dorsal interneuron specification.

With the limited work that has been done generating dorsal spinal interneuron populations, targeted gene editing to drive a dorsal phenotype has yet to be attempted. Upregulation of Wnt/β-catenin signaling pathways using exogenous factors has demonstrated strong results in the generation of class A progenitors, though a similar signaling cascade that regulates class B progenitors has yet to be identified.

Ventral interneurons

Ventral interneuron positioning is largely influenced by Hox gene expression, controlled by FGFs, RA, and Wnt signaling, and much like the dorsal interneurons, gene expression transitions as progenitors differentiate into the postmitotic stage, thus resulting in the four distinct cardinal classes (V0–V3). V0 interneurons arise from the dorsal–most ventral progenitor p0, and are defined by the expression of Dbx1¹⁶⁹, with postmitotic markers Evx1/2, Pax2, Lhx1/5. V1 identity arises in the p1 progenitor domain where the interneurons initially express Irx3 and Dbx2. Following maturation, these interneurons can be identified by the postmitotic marker En1. The p2 progenitor domain gives rise to V2 interneurons that express Irx3, Lhx3 and Foxn4^131,132. This cardinal class of interneurons is further subdivided into the well-characterized V2a and V2b interneurons. The segregation of these two subtypes is driven by Notch-Delta signaling followed by differential gene expression. V2a interneurons can be identified by their postmitotic marker Chx10, whereas V2b interneurons express Gata3. Finally, V3 interneurons arise from the p3 progenitor domain and largely express Nkx2.2 and Nhn3 along with the postmitotic marker, Sim1¹⁷⁰.

In the development of therapeutic cell transplantation models for SCI, the addition of regionally relevant cell specification procedures has enhanced recovery parameters after injury. V2a and V3 neurons have been generated in mouse and human stem cells with much success^170–173. V2a interneuron induction was first achieved using an in vitro protocol for differentiating mouse ESCs into interneurons through induction with RA, purmorphamine (Shh agonist), and DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester; Notch inhibitor). V2a enrichment in vitro was achieved by ectopic expression of puromycin N-acetyltransferase under the Chx10 promoter where puromycin resistance would be found in Chx10-expressing cells specifically. Considering that V2a interneurons are characterized by Chx10 expression during development, this protocol results in an interneuron population highly enriched with V2a interneurons. This differentiation protocol was also translated to hiPSCs, showing the robustness of this combination of morphogens and small molecules for developing V2a-enriched neuronal populations¹⁷². Similarly, the differentiation of V3 interneurons from mouse ESCs was achieved through lower concentrations of RA combined with longer exposure to a Shh agonist. The resulting population of interneurons displayed expression of V3 interneuron-specific genes Sim1 and Nkx2.2, suggesting a V3-enriched population of interneurons¹⁷¹. Although V2a and V3 interneurons have been generated in vitro, the other ventral neuron populations and subpopulations remain to be explored.

Generating Exogenic Neuronal Cells Via Blastocyst Complementation as a Source of Cells for Repair of SCI

Blastocyst Complementation

A recent novel approach in the field of regenerative medicine is the use of blastocyst complementation for the generation of exogenic organs and cells. The process of blastocyst complementation involves genetically engineering embryos from one organism to knockout specific target genes that are critical for cell specification, creating a developmental niche leading to agenesis of the cell type or organ of interest (Fig. 4, Table 2). Donor pluripotent stem cells from the same or different species are then injected into the modified blastocysts and transferred to pseudo-pregnant maternal surrogates. The healthy pluripotent stem cells injected can then occupy the empty niche and continue with normal development of the cells and organs of interest¹⁹⁴. It has been suggested that any cell type or organ has the potential to create developmental niches via specific target genes¹⁹⁵. This has shown to be the case with intraspecies and interspecies blastocyst complementation in recent years, with great success in generating a wide range of cells and organs, including inner ear sensory neurons, liver, forebrain, lymphocytes, kidney, pancreas, lung, thyroid, and eye^196–203.

Table 2.

Candidate Genes for Gene Knockout to Create a Niche for Blastocyst Complementation and Enhance Region-Specific Cell Subpopulations.

Niche	Genes for targeted knockout	Enhanced cell population after gene knockout and blastocyst complementation	Reference	Alternate genes
Dorsal spinal neurons	Gsh1/2, Ascl1	dI3–dI5 and dIL_A/dIL_B interneurons	Mizuguchi et al¹⁷⁴	Nmur2, Nmu, Tlx3, Prrxl1, Tac1, Lmx1b, Prkcg, Cacna2d1, Grp ¹⁷⁵
Dorsal spinal neurons	Pax3/7	Ventral En1⁺ cells	Mansouri and Gruss¹⁷⁶	Runx3¹⁷⁷, Er81¹⁷⁸
Ventral interneurons and motor spinal neurons	Dbx1	V1 interneurons	Pierani et al¹⁶⁹	Notch1¹⁷⁹, Hoxc8¹⁸⁰, SMN1¹⁸¹, FoxP1¹⁸²
	Nkx2.2/2.9	Motor neurons	Holz et al¹⁸³
	Nkx6.1	V1 interneurons	Sander et al¹⁸⁴
	Olig1/2	V2 interneurons and astrocytes	Zhou and Anderson¹⁴³
Oligodendrocytes	Olig1/2	V2 interneurons and astrocytes	Zhou and Anderson¹⁴³	Cx47 ¹⁸⁵
Astrocytes	Dbx1	Ventral p0-derived astrocytes	Sartoretti et al¹⁸⁶	SOD1¹⁸⁷, Act1¹⁸⁸
Astrocytes	Pax6	Immature astrocytes	Sakurai and Osumi¹⁸⁹	SOD1¹⁸⁷, Act1¹⁸⁸
Skeletal muscles	Pax3/7		Relaix et al¹⁹⁰	MSTN¹⁹¹, Zbed6¹⁹², IGF2¹⁹³

Additional knowledge is necessary to apply such technology to humans as there are still a number of barriers and challenges to overcome with this approach. A major concern involves the ethical debate surrounding human-animal chimeras, and the risk of humanization. However, a review of 150 peer-reviewed transplantation studies involving relatively high degree of neurological chimerism, found no evidence to suggest humanization of human-animal chimeras²⁰⁴. In addition, a survey measuring public readiness of human-animal chimeric research in the United States found that more than half of the population was accepting of blastocyst complementation research, making this approach all the more a viable option for future therapeutics²⁰⁵. There are also technical barriers, such as off-target chimerism within the brain and reproductive systems of human cells in host animals^204,205. Although there is no current research on the treatment of SCI with blastocyst complementation, this approach provides a promising future. Blastocyst complementation avoids the challenges associated with many other approaches, including the need for immunosuppression and risk of chemotherapy-associated toxicity (Fig. 5). While not yet attempted within the context of SCI, this novel approach, in theory, can be used to create human-animal chimeras to generate authentic exogenic spinal neurons for transplantation to restore function and connectivity following SCIs.

Figure 5.

Schematic of blastocyst complementation. Human pluripotent stem cells in vitro are injected into genetically engineered porcine blastocysts, which are then transferred to pseudo-pregnant dams. The chimeric blastocysts are left to develop to a fetal stage in which human neuronal stem/progenitor cells can be harvested or they can be left to mature into human organs to be harvested and processed for transplantation into patients.

Gene Editing for Blastocyst Complementation

A key step in generating exogenic cells via blastocyst complementation is the need to knockout specific genes in developing embryos to create a niche for injected wild-type stem cells to occupy and differentiate into the desired cell phenotype. Advances in genome editing with synthetic nucleases, such as transcription activator-like effector nucleases (TALEN), and CRISPR/Cas systems, are now being used to modify stem cell-derived populations for the production of relevant disease lines^206,207. These approaches are now being utilized to knockout genes with developing embryos as hosts for receiving injections of pluripotent stem cells to generate intraspecies and interspecies chimeras enabling the growth of specific organs and cells¹⁹⁵. The selection of appropriate genes for targeted knockout to create niches for producing chimeric animals for generating exogenic organs and cells is dependent on the understanding of the genes required for the developmental biology of the targeted organs and cells; see Table 2 for a comprehensive list of relevant spinal neuronal populations and corresponding target genes to knockout for generation of a developmental niche. It is important to consider, however, the potential limitation that presents with knocking out cardinal classes is the occurrence of divergence within the classes. For example, Hayashi et al²⁰⁸ demonstrate the divergence of Chx10 expression in V2a interneurons along the rostrocaudal axis that ultimately generates multiple subtypes of V2a interneurons. This divergence can be limiting for many current approaches in the regenerative medicine field. However, with blastocyst complementation, this challenge is less limiting since knocking out the cardinal class would ideally create the developmental niche that donor cells would take up completely. The donor cells would then grow and diverge normally during the developmental process, thus generating authentic exogenic neuronal populations that could be separately identified and used for region-specific cell transplantation within the spinal cord.

Ultimately, the goal is to identify the appropriate genes responsible for the cell or organ of interest, knock them out to create a developmental niche for the transplant of donor cells to grow said cell or organ of interest. Within the context of SCI, blastocyst complementation may be considered in the future for generating human-animal chimeras for a variety of spinal neuron populations.

Conclusion

SCI remains a significant area of study for treatment development. With the variety of therapeutic approaches available and studied to address SCI neuropathologies, progenitor cell transplantation has been at the forefront of several preclinical and clinical studies. The transplantation of progenitor cells has found various degrees of success in animal models of SCI and in human clinical trials. One key consideration with the generation of exogenic neuronal cells to optimize for success is the regional specification of cells for transplantation. Considering the importance of regional and subtype specificity during the development of neuronal cells of the spinal cord, similar considerations being made for cell transplantation may enhance therapeutic benefit. In fact, the adoption of regional specification in differentiation protocols has already improved transplantation outcomes and will continue to do so as techniques are refined and replicated. However, while distinct developmental pathways contribute to the production of these varied spinal cell types, their functional classification within either sensory or motor domains is an oversimplification. In truth, the spinal cord is a complex, interconnected system, and one domain cannot operate without the input from the other. Future work must consider application of both cell types in tandem to address the multifaceted nature of injury and recovery. Furthermore, the advent of blastocyst complementation to generate exogenic spinal neurons may provide regionally specific cells needed for appropriate neural connectivity and more robust functional recovery.

Footnotes

Acknowledgements

All figures were created with .

Author Contributions

Initial planning and drafting of this manuscript were completed by Dr Walter Low, Dr Ann Parr, Alex R., Anne H.-S., Madison W., and Zainab K. Significant writing, restructuring, editing, and reviewing were completed by Alex R., Anne H.-S., and Madison W. Figures were drafted and edited by Alex R., Anne H.-S., Madison W., and Jeffrey B. Final reviews and overviews were conducted by Dr Ann Parr and Dr Walter Low.

Ethical Approval

This study was approved by our institutional review board.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article. This work was supported by NIH grants R01 NS119297 and R01 AI173804, funds from the State of Minnesota Office of Higher Education Grant Program on Spinal Cord Injury and Traumatic Brain Injury Research, and the Suzanne M. Schwarz Fund.

ORCID iDs

Alex Roman

Walter C. Low

References

World Health Organization. Spinal cord injury; 2013. https://www.who.Int/news-room/fact-sheets/detail/spinal-cord-injury [accessed 2024 March 25].

National Spinal Cord Injury Statistical Center. Spinal cord injury facts and figures at a glance; 2020. doi:10.1080/10790268.2007.11753944.

World Health Organization, International Spinal Cord Society. International perspectives on spinal cord injury. Philadelphia (PA): Lippincott Williams and Wilkins; 2013.

Badhiwala

Ahuja

Fehlings

. Time is spine: a review of translational advances in spinal cord injury. J Neurosurg Spine. 2018;30(1):1–18. doi:10.3171/2018.9.SPINE18682.

Hodler

Kubik-Huch

DRA

von Schulthess

. Diseases of the brain, head and neck, spine 2020–2023. Springer; 2020. doi:10.1007/978-3-030-38490-6.

Kakulas

. Spinal cord injuries the facts of neuropathology: opportunities and limitations. Neuropsych Clin Neurosc Rep. 2019;1:1–5. doi:10.33702/cncnr.2019.1.1.1.

Lund

Ellman

Nissen

Nielsen

Jørgensen

Andersen

Gao

Brambilla

Degn

Clausen

, et al. The inflammatory response after moderate contusion spinal cord injury: a time study. Biology. 2022;11(6):939. doi:10.3390/biology11060939.

Pineau

Lacroix

. Proinflammatory cytokine synthesis in the injured mouse spinal cord: multiphasic expression pattern and identification of the cell types involved. J Comp Neurol. 2007;500(2):267–85. doi:10.1002/cne.21149.

Schwartz

Fehlings

. Chapter 14 secondary injury mechanisms of spinal cord trauma: a novel therapeutic approach for the management of secondary pathophysiology with the sodium channel blocker riluzole. Prog Brain Res. 2002;137:177–90. doi:10.1016/S0079-6123(02)37016-X.

10.

Hausmann

. Post-traumatic inflammation following spinal cord injury. Spinal Cord. 2003;41(7):369–78. doi:10.1038/sj.sc.3101483.

11.

Liu

Huang

Yang

Sun

Wang

Hang

. Necroptosis, a novel type of programmed cell death, contributes to early neural cells damage after spinal cord injury in adult mice. J Spinal Cord Med. 2015;38(6):745–53. doi:10.1179/2045772314Y.0000000224.

12.

Tator

Fehlings

. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg. 1991;75(1):15–26. doi:10.3171/jns.1991.75.1.0015.

13.

Milhorat

Capocelli

Jr Anzil

Kotzen

Milhorat

. Pathological basis of spinal cord cavitation in syringomyelia: analysis of 105 autopsy cases. J Neurosurg. 1995;82(5):802–12. doi:10.3171/jns.1995.82.5.0802.

14.

Norenberg

Smith

Marcillo

. The pathology of human spinal cord injury: defining the problems. J Neurotrauma. 2004;21(4):429–40. doi:10.1089/089771504323004575.

15.

Fehlings

Tetreault

Wilson

Kwon

Burns

Martin

Hawryluk

Harrop

. A clinical practice guideline for the management of acute spinal cord injury: introduction, rationale, and scope. Global Spine J. 2017;7(Suppl 3):84S–94S. doi:10.1177/2192568217703387.

16.

Yoshida

Tashiro

Nagoshi

Shinozaki

Shibata

Inoue

Ogawa

Shibata

Tsuji

Okano

Nakamura

. Chronic spinal cord injury regeneration with combined therapy comprising neural stem/progenitor cell transplantation, rehabilitation, and semaphorin 3A inhibitor. eNeuro. 2024;11(2):ENEURO.0378-23.2024. doi:10.1523/ENEURO.0378-23.2024.

17.

Lima

Monteiro

Salgado

Monteiro

Silva

. Pathophysiology and therapeutic approaches for spinal cord injury. Int J Mol Sci. 2022;23(22):13833. doi:10.3390/ijms232213833.

18.

Takahashi

Yamanaka

. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76. doi:10.1016/j.cell.2006.07.024.

19.

Chhabra

. Derivation of human induced pluripotent stem cell (iPSC) lines and mechanism of pluripotency: historical perspective and recent advances. Stem Cell Rev Rep. 2017;13(6):757–73. doi:10.1007/s12015-017-9766-9.

20.

González

Boué

Izpisúa Belmonte

. Methods for making induced pluripotent stem cells: reprogramming à la carte. Nat Rev Genet. 2011;12(4):231–42. doi:10.1038/nrg2937.

21.

Liau

Looi

Chia

Subramaniam

Law

. Treatment of spinal cord injury with mesenchymal stem cells. Cell Biosci. 2020;10(1):112. doi:10.1186/s13578-020-00475-3.

22.

Nakamura

Okano

. Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells. Cell Res. 2013;23(1):70–80. doi:10.1038/cr.2012.171.

23.

Fischer

Dulin

Lane

. Transplanting neural progenitor cells to restore connectivity after spinal cord injury. Nat Rev Neurosci. 2020;21(7):366–83. doi:10.1038/s41583-020-0314-2.

24.

Nambisan

. Stem cell research. In: Nambisan

, editor. An introduction to ethical, safety and intellectual property rights issues in biotechnology. Cambridge, MA: Academic Press; 2017, p. 55–81.

25.

Ortuño-Costela

MDC

Cerrada

García-López

Gallardo

. The challenge of bringing iPSCs to the patient. Int J Mol Sci. 2019;20(24):6305. doi:10.3390/ijms20246305.

26.

Iyer

Wilems

Sakiyama-Elbert

. Stem cells for spinal cord injury: strategies to inform differentiation and transplantation. Biotechnol Bioeng. 2017;114(2):245–59. doi:10.1002/bit.26074.

27.

Parr

Tator

Keating

. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant. 2007;40(7):609–19. doi:10.1038/sj.bmt.1705757.

28.

Rafiei Alavi

Madani Neishaboori

Hossein

Sarveazad

Yousefifard

. Efficacy of adipose tissue-derived stem cells in locomotion recovery after spinal cord injury: a systematic review and meta-analysis on animal studies. Syst Rev. 2021;10(1):213. doi:10.1186/s13643-021-01771-w.

29.

Ruzicka

Machova-Urdzikova

Gillick

Amemori

Romanyuk

Karova

Zaviskova

Dubisova

Kubinova

Murali

Sykova

, et al. A comparative study of three different types of stem cells for treatment of rat spinal cord injury. Cell Transplant. 2017;26(4):585–603. doi:10.3727/096368916X693671.

30.

Tetzlaff

Okon

Karimi-Abdolrezaee

Hill

Sparling

Plemel

Plunet

Tsai

Baptiste

Smithson

Kawaja

, et al. A systematic review of cellular transplantation therapies for spinal cord injury. J Neurotrauma. 2011;28(8):1611–82. doi:10.1089/neu.2009.1177.

31.

Kim

Hwang

Lee

Kim

. Stem cell-based cell therapy for spinal cord injury. Cell Transplant. 2007;16(4):355–64. doi:10.3727/000000007783464885.

32.

Huntemer-Silveira

Patil

Brickner

Parr

. Strategies for oligodendrocyte and myelin repair in traumatic CNS injury. Front Cell Neurosci. 2021;14:619707. https://www.frontiersin.org/articles/10.3389/fncel.2020.619707 [accessed 2024 March 25].

33.

Assinck

Duncan

Hilton

Plemel

Tetzlaff

. Cell transplantation therapy for spinal cord injury. Nat Neurosci. 2017;20(5):637–47. doi:10.1038/nn.4541.

34.

Yamazaki

Kawabori

Seki

Houkin

. Clinical trials of stem cell treatment for spinal cord injury. Int J Mol Sci. 2020;21(11):3994. doi:10.3390/ijms21113994.

35.

Salewski

Mitchell

Shen

Fehlings

. Transplantation of neural stem cells clonally derived from embryonic stem cells promotes recovery after murine spinal cord injury. Stem Cells Dev. 2015;24(1):36–50. doi:10.1089/scd.2014.0096.

36.

Kumagai

Okada

Yamane

Nagoshi

Kitamura

Mukaino

Tsuji

Fujiyoshi

Katoh

Okada

Shibata

, et al. Roles of ES cell-derived gliogenic neural stem/progenitor cells in functional recovery after spinal cord injury. PLoS ONE. 2009;4(11):e7706. doi:10.1371/journal.pone.0007706.

37.

Iwai

Shimada

Nishimura

Kobayashi

Itakura

Hori

Hikishima

Ebise

Negishi

Shibata

Habu

, et al. Allogeneic neural stem/progenitor cells derived from embryonic stem cells promote functional recovery after transplantation into injured spinal cord of nonhuman primates. Stem Cells Transl Med. 2015;4(7):708–19. doi:10.5966/sctm.2014-0215.

38.

Tsuji

Miura

Okada

Fujiyoshi

Mukaino

Nagoshi

Kitamura

Kumagai

Nishino

Tomisato

Higashi

, et al. Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury. Proc Natl Acad Sci U S A. 2010;107(28):12704–709. doi:10.1073/pnas.0910106107.

39.

Kobayashi

Okada

Itakura

Iwai

Nishimura

Yasuda

Nori

Hikishima

Konomi

Fujiyoshi

Tsuji

, et al. Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity. PLoS ONE. 2012;7(12):e52787. doi:10.1371/journal.pone.0052787.

40.

Salazar

Uchida

Hamers

Cummings

Anderson

. Human neural stem cells differentiate and promote locomotor recovery in an early chronic spinal coRd injury NOD-scid mouse model. PLoS ONE. 2010;5(8):e12272. doi:10.1371/journal.pone.0012272.

41.

Suzuki

Ahuja

Salewski

Satkunendrarajah

Nagoshi

Shibata

Fehlings

. Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury. PLoS ONE. 2017;12(8):e0182339. doi:10.1371/journal.pone.0182339.

42.

Cummings

Uchida

Tamaki

Salazar

Hooshmand

Summers

Gage

Anderson

. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A. 2005;102(39):14069–74. doi:10.1073/pnas.0507063102.

43.

Ogawa

Sawamoto

Miyata

Miyao

Watanabe

Nakamura

Bregman

Koike

Uchiyama

Toyama

Okano

. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res. 2002;69(6):925–33. doi:10.1002/jnr.10341.

44.

Iwanami

Kaneko

Nakamura

Kanemura

Mori

Kobayashi

Yamasaki

Momoshima

Ishii

Ando

Tanioka

, et al. Transplantation of human neural stem cells for spinal cord injury in primates. J Neurosci Res. 2005;80(2):182–90. doi:10.1002/jnr.20436.

45.

Cui

Y-F

J-C

Hargus

Jakovcevski

Schachner

Bernreuther

. Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery after spinal cord injury in mice. PLoS ONE. 2011;6(3):e17126. doi:10.1371/journal.pone.0017126.

46.

Vadivelu

Stewart

Horn

Liu

Silver

McDonald

. NG2+ progenitors derived from embryonic stem cells penetrate glial scar and promote axonal outgrowth into white matter after spinal cord injury. Stem Cells Transl Med. 2015;4(4):401–11. doi:10.5966/sctm.2014-0107.

47.

Yamane

Nakamura

Iwanami

Sakaguchi

Katoh

Yamada

Momoshima

Miyao

Ishii

Tamaoki

Nomura

, et al. Transplantation of galectin-1-expressing human neural stem cells into the injured spinal cord of adult common marmosets. J Neurosci Res. 2010;88(7):1394–405. doi:10.1002/jnr.22322.

48.

Yan

Welsh

Hatfield

Hazel

Johe

Koliatsos

. Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord. PLoS Med. 2007;4(2):e39. doi:10.1371/journal.pmed.0040039.

49.

Tarasenko

Gao

Nie

Johnson

Grady

Hulsebosch

McAdoo

. Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior. J Neurosci Res. 2007;85(1):47–57. doi:10.1002/jnr.21098.

50.

Wang

Graham

McHale

Gao

Brock

Blesch

Rosenzweig

Havton

Zheng

, et al. Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell. 2012;150(6):1264–73. doi:10.1016/j.cell.2012.08.020.

51.

Kumamaru

Kadoya

Adler

Takashima

Graham

Coppola

Tuszynski

. Generation and post-injury integration of human spinal cord neural stem cells. Nat Methods. 2018;15(9):723–31. doi:10.1038/s41592-018-0074-3.

52.

Nori

Okada

Yasuda

Tsuji

Takahashi

Kobayashi

Fujiyoshi

Koike

Uchiyama

Ikeda

Toyama

, et al. Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proc Natl Acad Sci U S A. 2011;108(40):16825–30. doi:10.1073/pnas.1108077108.

53.

Hwang

Kim

Lee

Joo

Suh-Kim

Sohn

Kim

. Transplantation of human neural stem cells transduced with Olig2 transcription factor improves locomotor recovery and enhances myelination in the white matter of rat spinal cord following contusive injury. BMC Neurosci. 2009;10(1):117. doi:10.1186/1471-2202-10-117.

54.

Dell’Anno

Wang

Onorati

Talpo

Sekine

Liu

Cafferty

WBJ

Sestan

Strittmatter

. Human neuroepithelial stem cell regional specificity enables spinal cord repair through a relay circuit. Nat Commun. 2018;9(1):3419. doi:10.1038/s41467-018-05844-8.

55.

Dulin

Adler

Kumamaru

Poplawski

GHD

Lee-Kubli

Strobl

Gibbs

Kadoya

Fawcett

Tuszynski

. Injured adult motor and sensory axons regenerate into appropriate organotypic domains of neural progenitor grafts. Nat Commun. 2018;9(1):84. doi:10.1038/s41467-017-02613-x.

56.

Strnadel

Carromeu

Bardy

Navarro

Platoshyn

Glud

Marsala

Kafka

Miyanohara

Kato

Tadokoro

, et al. Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs. Sci Transl Med. 2018;10(440):eaam6651. doi:10.1126/scitranslmed.aam6651.

57.

Kajikawa

Imaizumi

Shinozaki

Shibata

Shindo

Kitagawa

Shibata

Kamata

Kojima

Nagoshi

Matsumoto

, et al. Cell therapy for spinal cord injury by using human iPSC-derived region-specific neural progenitor cells. Molecular Brain. 2020;13(1):120. doi:10.1186/s13041-020-00662-w.

58.

Zholudeva

Iyer

Qiang

Spruance

Randelman

White

Bezdudnaya

Fischer

Sakiyama-Elbert

Lane

. Transplantation of neural progenitors and V2a interneurons after spinal cord injury. J Neurotrauma. 2018;35(24):2883–903. doi:10.1089/neu.2017.5439.

59.

Lepore

Fischer

. Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord. Exp Neurol. 2005;194(1):230–42. doi:10.1016/j.expneurol.2005.02.020.

60.

Lin

Lai

Shao

Chen

Lee

. The therapeutic effectiveness of delayed fetal spinal cord tissue transplantation on respiratory function following mid-cervical spinal cord injury. Neurotherapeutics. 2017;14(3):792–809. doi:10.1007/s13311-016-0509-4.

61.

Meng

Dong

Liu

Xue

Chen

. Co-transplantation of bFGF-expressing amniotic epithelial cells and neural stem cells promotes functional recovery in spinal cord-injured rats. Cell Biol Int. 2008;32(12):1546–58. doi:10.1016/j.cellbi.2008.09.001.

62.

Hooshmand

Sontag

Uchida

Tamaki

Anderson

Cummings

. Analysis of host-mediated repair mechanisms after human CNS-stem cell transplantation for spinal cord injury: correlation of engraftment with recovery. PLoS ONE. 2009;4(6):e5871. doi:10.1371/journal.pone.0005871.

63.

Hwang

Hahm

Choi

Park

Jeong

Yea

Kim

Hong

. Intrathecal transplantation of embryonic stem cell-derived spinal GABAergic neural precursor cells attenuates neuropathic pain in a spinal cord injury rat model. Cell Transplant. 2016;25(3):593–607. doi:10.3727/096368915X689460.

64.

Zhang

Zheng

Bian

Liu

Xue

Liu

Zhang

Song

Chung

, et al. Polarized macrophages have distinct roles in the differentiation and migration of embryonic spinal-cord-derived neural stem cells after grafting to injured sites of spinal cord. Mol Ther. 2015;23(6):1077–91. doi:10.1038/mt.2015.46.

65.

Niapour

Karamali

Nemati

Taghipour

Mardani

Nasr-Esfahani

Baharvand

. Cotransplantation of human embryonic stem cell-derived neural progenitors and Schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery. Cell Transplant. 2012;21(5):827–43. doi:10.3727/096368911X593163.

66.

Erceg

Ronaghi

Oria

Roselló

Aragó

Lopez

Radojevic

Moreno-Manzano

Rodríguez-Jiménez

Bhattacharya

Cordoba

, et al. Transplanted oligodendrocytes and motoneuron progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection. Stem Cells. 2010;28(9):1541–49. doi:10.1002/stem.489.

67.

Hill

Hurtado

Blits

Bahr

Wood

Bartlett Bunge

Oudega

. Early necrosis and apoptosis of Schwann cells transplanted into the injured rat spinal cord. Eur J Neurosci. 2007;26(6):1433–45. doi:10.1111/j.1460-9568.2007.05771.x.

68.

Karimi-Abdolrezaee

Eftekharpour

Wang

Morshead

Fehlings

. Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci. 2006;26(13):3377–89. doi:10.1523/JNEUROSCI.4184-05.2006.

69.

Parr

Kulbatski

Tator

. Transplantation of adult rat spinal cord stem/progenitor cells for spinal cord injury. J Neurotrauma. 2007;24(5):835–45. doi:10.1089/neu.2006.3771.

70.

Feng

Deng

Yong

Y-Y

J-M

Qin

D-L

Zhou

X-G

A-G

. The application of biomaterials in spinal cord injury. Int J Mol Sci. 2023;24(1):816. doi:10.3390/ijms24010816.

71.

Marquardt

Doulames

Wang

Dubbin

Suhar

Kratochvil

Medress

Plant

Heilshorn

. Designer, injectable gels to prevent transplanted Schwann cell loss during spinal cord injury therapy. Sci Adv. 2020;6(14):eaaz1039. doi:10.1126/sciadv.aaz1039.

72.

Olmsted

Stigliano

Badri

Zhang

Williams

Koffas

MAG

Xie

Linhardt

Cibelli

Horner

Paluh

. Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery. Sci Rep. 2020;10(1):12939. doi:10.1038/s41598-020-69274-7.

73.

Shin

Kim

Yoo

Kim

Yun

Lee

Jung

Hwang

Kim

Lee

Shin

, et al. Clinical trial of human fetal brain-derived neural stem/progenitor cell transplantation in patients with traumatic cervical spinal cord injury. Neural Plast. 2015;2015:630932. doi:10.1155/2015/630932.

74.

Levi

Okonkwo

Park

Jenkins

III Kurpad

Parr

Ganju

Aarabi

Kim

Casha

Fehlings

, et al. Emerging safety of intramedullary transplantation of human neural stem cells in chronic cervical and thoracic spinal cord injury. Neurosurgery. 2018;82(4):562–75. doi:10.1093/neuros/nyx250.

75.

Levi

Anderson

Okonkwo

Park

Bryce

Kurpad

Aarabi

Hsieh

Gant

. Clinical outcomes from a multi-center study of human neural stem cell transplantation in chronic cervical spinal cord injury. J Neurotrauma. 2019;36(6):891–902. doi:10.1089/neu.2018.5843.

76.

StemCells, Inc. A single-blind, randomized, parallel arm, phase II proof-of-concept study of the safety and efficacy of human central nervous system stem cells (HuCNS-SC) transplantation in cervical spinal cord injury (Clinical trial registration no. NCT02163876); 2016. https://clinicaltrials.Gov/ct2/show/NCT02163876 [accessed 2024 March 25].

77.

StemCells, Inc. Long-term follow-up (LTFU) study of the phase I/II safety and preliminary efficacy investigation of intramedullary spinal cord transplantation of HuCNS-SC® in subjects with thoracic (T2-T11) spinal cord trauma (Clinical trial registration no. NCT01725880); 2016. https://clinicaltrials.Gov/ct2/show/NCT01725880 [accessed 2024 March 25].

78.

Neuralstem Inc. A phase 1, open-label, single-site, safety study of human spinal cord-derived neural stem cell transplantation for the treatment of chronic SCI (Clinical trial registration no. NCT01772810); 2017. https://clinicaltrials.Gov/ct2/show/NCT01772810 [accessed 2024 March 25].

79.

Curtis

Martin

Gabel

Sidhu

Rzesiewicz

Mandeville

Gorp

Leerink

Tadokoro

Marsala

Jamieson

, et al. A first-in-human, phase I study of neural stem cell transplantation for chronic spinal cord injury. Cell Stem Cell. 2018;22(6):941–50.e6. Doi:10.1016/j.stem.2018.05.014.

80.

Glass

Hertzberg

Boulis

Riley

Federici

Polak

Bordeau

Fournier

Johe

Hazel

Cudkowicz

, et al. Transplantation of spinal cord–derived neural stem cells for ALS. Neurology. 2016;87(4):392–400. doi:10.1212/WNL.0000000000002889.

81.

S.Biomedics Co., Ltd. A single center, open label, single group, phase 1/2a clinical study to evaluate the safety and exploratory efficacy of transplantation therapy using PSA-NCAM(+) NPC derived from hESC line in AIS-A level of sub-acute SCI(from 7 to 60 days) (Clinical trial registration no. NCT04812431); 2020. https://clinicaltrials.Gov/ct2/show/NCT04812431 [accessed 2024 March 25].

82.

Lineage Cell Therapeutics, Inc. A phase 1 safety study of GRNOPC1 in patients with neurologically complete, subacute, spinal cord injury (Clinical trial registration no. NCT01217008); 2020. https://clinicaltrials.Gov/ct2/show/NCT01217008 [accessed 2024 March 25].

83.

Lineage Cell Therapeutics, Inc. A phase 1/2a dose escalation study of AST-OPC1 in subjects with subacute cervical spinal cord injury (Clinical trial registration no. NCT02302157); 2021. https://clinicaltrials.Gov/ct2/show/NCT02302157 [accessed 2024 March 25].

84.

Fessler

Ehsanian

Liu

Steinberg

Jones

Lebkowski

Wirth

McKenna

. A phase 1/2a dose-escalation study of oligodendrocyte progenitor cells in individuals with subacute cervical spinal cord injury. J Neurosurg Spine. 2022;37(6):812–20. doi:10.3171/2022.5.SPINE22167.

85.

McKenna

Ehsanian

Liu

Steinberg

Jones

Lebkowski

Wirth

Fessler

. Ten-year safety of pluripotent stem cell transplantation in acute thoracic spinal cord injury. J Neurosurg Spine. 2022;37(3):321–30. doi:10.3171/2021.12.SPINE21622.

86.

Lineage announces submission of OPC1 investigational new drug amendment for treatment of chronic and subacute spinal cord injury. Lineage Cell Therapeutics; 2023. https://investor.lineagecell.Com/news-releases/news-release-details/lineage-announces-submission-opc1-investigational-new-drug [accessed 2024 March 25].

87.

National Eye Institute (NEI). A phase I/IIa trial for autologous transplantation of induced pluripotent stem cell-derived retinal pigment epithelium for geographic atrophy associated with age-related macular degeneration (Clinical trial registration no. NCT04339764); 2022. https://clinicaltrials.Gov/ct2/show/NCT04339764 [accessed 2024 March 25].

88.

Takahashi

. IPS cell-based therapy for Parkinson’s disease: a Kyoto trial. Regen Ther. 2020;13:18–22. doi:10.1016/j.reth.2020.06.002.

89.

Sugai

Sumida

Shofuda

Yamaguchi

Tamura

Kohzuki

Abe

Shibata

Kamata

Ito

Okubo

, et al. First-in-human clinical trial of transplantation of iPSC-derived NS/PCs in subacute complete spinal cord injury: study protocol. Regen Ther. 2021;18:321–33. doi:10.1016/j.reth.2021.08.005.

90.

Inoue

Yamaguchi

CCJ

Ikeda

Okano

Kohyama

. Current status and prospects of regenerative medicine for spinal cord injury using human induced pluripotent stem cells: a review. Stem Cell Investig. 2023;10:6. doi:10.21037/sci-2022-037.

91.

Zhang

. Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc. 2009;4(9):1295–304. doi:10.1038/nprot.2009.127.

92.

Lippmann

Estevez-Silva

Ashton

. Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors. Stem Cells. 2014;32(4):1032–42. doi:10.1002/stem.1622.

93.

Walsh

Truong

Hill

Stoflet

Baden

Low

Keirstead

Dutton

Parr

. Defined culture conditions accelerate small-molecule-assisted neural induction for the production of neural progenitors from human-induced pluripotent stem cells. Cell Transplant. 2017;26(12):1890–902. doi:10.1177/0963689717737074.

94.

Chan

Teo

AKK

. Replicates in stem cell models—how complicated! Stem Cells. 2020;38(9):1055–59. doi:10.1002/stem.3237.

95.

Darabi

. Role of epigenetics in cellular reprogramming; from iPSCs to disease modeling and cell therapy. J Cell Biochem. 2022;123(2):147–54. doi:10.1002/jcb.30164.

96.

Liu

Zheng

Zhao

Yang

Tao

Xie

Qiu

Yang

. Chromosomal aberration arises during somatic reprogramming to pluripotent stem cells. Cell Div. 2020;15:12. doi:10.1186/s13008-020-00068-z.

97.

Liang

Zhang

. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell. 2013;13(2):149–59. doi:10.1016/j.stem.2013.07.001.

98.

Duval

Grover

Han

Mou

Pegoraro

Fredberg

Chen

. Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda). 2017;32(4):266–77. doi:10.1152/physiol.00036.2016.

99.

Joseph

Morrison

. Toward an understanding of the physiological function of mammalian stem cells. Dev Cell. 2005;9(2):173–83. doi:10.1016/j.devcel.2005.07.001.

100.

Astashkina

Mann

Grainger

. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacol Ther. 2012;134(1):82–106. doi:10.1016/j.pharmthera.2012.01.001.

101.

Sonbol

. Extracellular matrix remodeling in human disease. J Microsc Ultrastruct. 2018;6(3):123–28. doi:10.4103/JMAU.JMAU_4_18.

102.

Tavakoli

Derby

Serebryakova

Rao

Mattson

. Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Dev Biol. 2008;8(1):90. doi:10.1186/1471-213X-8-90.

103.

Liu

Pavathuparambil Abdul Manaph

Al-Hawwas

Bobrovskaya

Xiong

L-L

Zhou

X-F

. Coating materials for neural stem/progenitor cell culture and differentiation. Stem Cells Dev. 2020;29(8):463–74. doi:10.1089/scd.2019.0288.

104.

Louro

Pearse

. Stem and progenitor cell therapies: recent progress for spinal cord injury repair. Neurol Res. 2008;30(1):5–16. doi:10.1179/174313208X284070.

105.

Chambers

Fasano

Papapetrou

Tomishima

Sadelain

Studer

. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275–80. doi:10.1038/nbt.1529.

106.

Walsh

Truong

Nayak

Saldías Montivero

Low

Parr

Dutton

. Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population. Stem Cells. 2020;38(11):1400–408. doi:10.1002/stem.3260.

107.

Zholudeva

Abraira

Satkunendrarajah

McDevitt

Goulding

Magnuson

DSK

Lane

. Spinal interneurons as gatekeepers to neuroplasticity after injury or disease. J Neurosc. 2021;41(5):845–54. doi:10.1523/JNEUROSCI.1654-20.2020.

108.

Lippmann

Williams

Ruhl

Estevez-Silva

Chapman

Coon

Ashton

. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Reports. 2015;4(4):632–44. doi:10.1016/j.stemcr.2015.02.018.

109.

White

Sakiyama-Elbert

. Derivation of specific neural populations from pluripotent cells for understanding and treatment of spinal cord injury. Dev Dyn. 2019;248(1):78–87. doi:10.1002/dvdy.24680.

110.

Trawczynski

Liu

David

Fessler

. Restoring motor neurons in spinal cord injury with induced pluripotent stem cells. Front Cell Neurosci. 2019;13:369. https://www.frontiersin.org/articles/10.3389/fncel.2019.00369 [accessed 2024 March 25].

111.

Gupta

Sivalingam

Hain

Makkar

Sosa

Clark

Butler

. Deriving dorsal spinal sensory interneurons from human pluripotent stem cells. Stem Cell Reports. 2018;10(2):390–405. doi:10.1016/j.stemcr.2017.12.012.

112.

Huntemer-Silveira

Malone

Frie

Walsh

Parr

. Accelerated differentiation of human induced pluripotent stem cells into regionally specific dorsal and ventral spinal neural progenitor cells for application in spinal cord therapeutics. Front Neurosci. 2023;17:1251906. https://www.frontiersin.org/articles/10.3389/fnins.2023.1251906 [accessed 2024 March 25].

113.

Iyer

Shin

Cuskey

Tian

Nicol

Doersch

Seipel

McCalla

Roy

Ashton

. Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns. Sci Adv. 2022;8(39):eabn7430. doi:10.1126/sciadv.abn7430.

114.

Han

Kim

Abdi

Kim

. Stem cell therapy in pain medicine. Korean J Pain. 2019;32(4):245–55. doi:10.3344/kjp.2019.32.4.245.

115.

Mikawa

Poh

Kelly

Ishii

Reese

. Induction and patterning of the primitive streak, an organizing center of gastrulation in the amniote. Dev Dyn. 2004;229(3):422–32. doi:10.1002/dvdy.10458.

116.

Sadler

. Embryology of neural tube development. Am J Med Genet Part C Semin Med Genet. 2005;135(1):2–8. doi:10.1002/ajmg.c.30049.

117.

Naidich

Raybaud

. Embryogenesis of the spine and spinal cord. Rivista Neuroradiol. 1992;5(2):101–12. doi:10.1177/19714009920050S214.

118.

Muhr

Ackerman

. Embryology, gastrulation. StatPearls Publishing; 2022. http://www.ncbi.nlm.Nih.gov/books/NBK554394/ [accessed 2024 March 25].

119.

Rufener

Ibrahim

Parmar

. Imaging of congenital spine and spinal cord malformations. Neuroimag Clin. 2011;21(3):659–76. doi:10.1016/j.nic.2011.05.011.

120.

Ashwell

. Chapter 2—development of the spinal cord. In: The spinal cord. Academic Press; 2009, p. 8–16. doi:10.1016/B978-0-12-374247-6.50006-7.

121.

Shinozuka

Takada

. Morphological and functional changes of roof plate cells in spinal cord development. J Dev Biol. 2021;9(3):30. doi:10.3390/jdb9030030.

122.

ten Donkelaar

Yamada

Shiota

van der Vliet

. Overview of the development of the human brain and spinal cord. In: Clinical neuroembryology: development and developmental disorders of the human central nervous system. Springer; 2014, p. 1–52. doi:10.1007/978-3-642-54687-7_1.

123.

Konstantinidou

Silos-Santiago

Flaris

Snider

. Development of the primary afferent projection in human spinal cord. J Comp Neurol. 1995;354(1):1–12. doi:10.1002/cne.903540102.

124.

Guertin

. Central pattern generator for locomotion: anatomical, physiological, and pathophysiological considerations. Front Neurol. 2013;3:183. https://www.frontiersin.org/articles/10.3389/fneur.2012.00183 [accessed 2024 March 25].

125.

H-Y

. Development of the human spinal cord. In: Management and rehabilitation of spinal cord injuries. Springer Nature; 2022, p. 19–40. doi:10.1007/978-981-19-0228-4_2.

126.

Muhr

Graziano

Wilson

Jessell

Edlund

. Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos. Neuron. 1999;23(4):689–702. doi:10.1016/S0896-6273(01)80028-3.

127.

Liu

J-P

Laufer

Jessell

. Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron. 2001;32(6):997–1012. doi:10.1016/S0896-6273(01)00544-X.

128.

Cooper

Gentsch

Mitter

Bouissou

Healy

Rodriguez

Smith

Bernardo

. Rostrocaudal patterning and neural crest differentiation of human pre-neural spinal cord progenitors in vitro. Stem Cell Reports. 2022;17(4):894–910. doi:10.1016/j.stemcr.2022.02.018.

129.

Carpenter

. Hox genes and spinal cord development. Dev Neurosci. 2002;24(1):24–34. doi:10.1159/000064943.

130.

Dasen

De Camilli

Wang

Tucker

Jessell

. Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell. 2008;134(2):304–16. doi:10.1016/j.cell.2008.06.019.

131.

Francius

Harris

Rucchin

Hendricks

Stam

Barber

Kurek

Grosveld

Pierani

Goulding

Clotman

. Identification of multiple subsets of ventral interneurons and differential distribution along the rostrocaudal axis of the developing spinal cord. PLoS ONE. 2013;8(8):e70325. doi:10.1371/journal.pone.0070325.

132.

Niu

Alaynick

. Molecular and cellular development of spinal cord locomotor circuitry. Front Mol Neurosci. 2015;8:25. https://www.frontiersin.org/articles/10.3389/fnmol.2015.00025 [accessed 2024 March 25].

133.

Sweeney

Bikoff

Gabitto

Brenner-Morton

Baek

Yang

Tabak

Dasen

Kintner

Jessell

. Origin and segmental diversity of spinal inhibitory interneurons. Neuron. 2018;97(2):341–55.E3. Doi:10.1016/j.neuron.2017.12.029.

134.

Sagner

Briscoe

. Establishing neuronal diversity in the spinal cord: a time and a place. Development. 2019;146(22):dev182154. doi:10.1242/dev.182154.

135.

Hernandez-Miranda

Müller

Birchmeier

. The dorsal spinal cord and hindbrain: from developmental mechanisms to functional circuits. Develop Biol. 2017;432(1):34–42. doi:10.1016/j.ydbio.2016.10.008.

136.

Kim

Habiba

Doherty

Mills

Mercer

Huettner

. Regulation of mouse embryonic stem cell neural differentiation by retinoic acid. Develop Biol. 2009;328(2):456–71. doi:10.1016/j.ydbio.2009.02.001.

137.

Gupta

Yamauchi

Novitch

Butler

. Derivation of dorsal spinal sensory interneurons from human pluripotent stem cells. STAR Protoc. 2021;2(1):100319. doi:10.1016/j.xpro.2021.100319.

138.

Stifani

. Motor neurons and the generation of spinal motor neurons diversity. Front Cell Neurosci. 2014;8:293. https://www.frontiersin.org/articles/10.3389/fncel.2014.00293 [accessed 2024 March 25].

139.

Huettl

R-E

Eckstein

Stahl

Petricca

Ninkovic

Götz

Huber

. Functional dissection of the Pax6 paired domain: roles in neural tube patterning and peripheral nervous system development. Develop Biol. 2016;413(1):86–103. doi:10.1016/j.ydbio.2015.07.009.

140.

Liu

Qiu

Yang

. Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary in the spinal cord. Development. 2014;141(14):2875–84. doi:10.1242/dev.105387.

141.

De Marco Garcia

Jessell

. Early motor neuron pool identity and muscle nerve trajectory defined by postmitotic restrictions in Nkx6.1 activity. Neuron. 2008;57(2):217–31. doi:10.1016/j.neuron.2007.11.033.

142.

Rao

Sockanathan

. Transmembrane protein GDE2 induces motor neuron differentiation in vivo. Science. 2005;309(5744):2212–15. doi:10.1126/science.1117156.

143.

Zhou

Anderson

. The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. Cell. 2002;109(1):61–73. doi:10.1016/S0092-8674(02)00677-3.

144.

Lacomme

Liaubet

Pituello

Bel-Vialar

. NEUROG2 drives cell cycle exit of neuronal precursors by specifically repressing a subset of cyclins acting at the G1 and S phases of the cell cycle. Mol Cell Biol. 2012;32(13):2596–607. doi:10.1128/MCB.06745-11.

145.

Lee

S-K

Lee

Ruiz

Pfaff

. Olig2 and Ngn2 function in opposition to modulate gene expression in motor neuron progenitor cells. Genes Dev. 2005;19(2):282–94. doi:10.1101/gad.1257105.

146.

Scardigli

Schuurmans

Gradwohl

Guillemot

. Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord. Neuron. 2001;31(2):203–17. doi:10.1016/S0896-6273(01)00358-0.

147.

Erb

Lee

Yeon Seo

Lee

. The Isl1-Lhx3 complex promotes motor neuron specification by activating transcriptional pathways that enhance its own expression and formation. eNeuro. 2017;4(2):ENEURO.0349-16.2017. doi:10.1523/ENEURO.0349-16.2017.

148.

Thaler

Lee

S-K

Jurata

Gill

Pfaff

. LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell. 2002;110(2):237–49. doi:10.1016/S0092-8674(02)00823-1.

149.

Agalliu

Takada

Agalliu

McMahon

Jessell

. Motor neurons with axial muscle projections specified by Wnt4/5 signaling. Neuron. 2009;61(5):708–20. doi:10.1016/j.neuron.2008.12.026.

150.

Schaller

Buttigieg

Alory

Jacquier

Barad

Merchant

Gentien

de la Grange

Haase

. Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons. Proc Natl Acad Sci U S A. 2017;114(12):E2486–93. doi:10.1073/pnas.1615372114.

151.

Roy

Francius

Rousso

Seuntjens

Debruyn

Luxenhofer

Huber

Huylebroeck

Novitch

Clotman

. Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification. Development. 2012;139(17):3109–19. doi:10.1242/dev.078501.

152.

Boulting

Kiskinis

Croft

Amoroso

Oakley

Wainger

Williams

Kahler

Yamaki

Davidow

Rodolfa

, et al. A functionally characterized test set of human induced pluripotent stem cells. Nat Biotechnol. 2011;29(3):279–86. doi:10.1038/nbt.1783.

153.

Hester

Murtha

Song

Rao

Miranda

Meyer

Tian

Boulting

Schaffer

Zhu

Pfaff

, et al. Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes. Mol Ther. 2011;19(10):1905–12. doi:10.1038/mt.2011.135.

154.

Takazawa

Croft

Amoroso

Studer

Wichterle

Macdermott

. Maturation of spinal motor neurons derived from human embryonic stem cells. PLoS ONE. 2012;7(7):e40154. doi:10.1371/journal.pone.0040154.

155.

Wada

Honda

Minami

Tooi

Amagai

Nakatsuji

Aiba

. Highly efficient differentiation and enrichment of spinal motor neurons derived from human and monkey embryonic stem cells. PLoS ONE. 2009;4(8):e6722. doi:10.1371/journal.pone.0006722.

156.

Wichterle

Lieberam

Porter

Jessell

. Directed differentiation of embryonic stem cells into motor neurons. Cell. 2002;110(3):385–97. doi:10.1016/S0092-8674(02)00835-8.

157.

Shimojo

Onodera

Doi-Torii

Ishihara

Hattori

Miwa

Tanaka

Okada

Ohyama

Shoji

Nakanishi

, et al. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. Molecular Brain. 2015;8(1):79. doi:10.1186/s13041-015-0172-4.

158.

Zarnowska

Pankratz

Hansen

Pearce

Zhang

. Specification of motoneurons from human embryonic stem cells. Nat Biotechnol. 2005;23(2):215–21. doi:10.1038/nbt1063.

159.

Singh Roy

Nakano

Xuing

Kang

Nedergaard

Goldman

. Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells. Exp Neurol. 2005;196(2):224–34. doi:10.1016/j.expneurol.2005.06.021.

160.

Machado

Kanning

Kreis

Stevenson

Crossley

Nowak

Iacovino

Kyba

Chambers

Blanc

Lieberam

. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons. Development. 2014;141(4):784–94. doi:10.1242/dev.097188.

161.

Patani

Hollins

Wishart

Puddifoot

Alvarez

de Lera

Wyllie

Compston

Pedersen

Gillingwater

Hardingham

, et al. Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state. Nat Commun. 2011;2:214. doi:10.1038/ncomms1216.

162.

Amoroso

Croft

Williams

O’Keeffe

Carrasco

Davis

Roybon

Oakley

Maniatis

Henderson

Wichterle

. Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J Neurosci. 2013;33(2):574–86. doi:10.1523/JNEUROSCI.0906-12.2013.

163.

Helms

Johnson

. Specification of dorsal spinal cord interneurons. Curr Opin Neurobiol. 2003;13(1):42–49. doi:10.1016/S0959-4388(03)00010-2.

164.

Zechner

Müller

Wende

Walther

Taketo

Crenshaw

Treier

Birchmeier

. Bmp and Wnt/β-catenin signals control expression of the transcription factor Olig3 and the specification of spinal cord neurons. Develop Biol. 2007;303(1):181–90. doi:10.1016/j.ydbio.2006.10.045.

165.

Müller

Anlag

Wildner

Britsch

Treier

Birchmeier

. The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord. Genes Dev. 2005;19(6):733–43. doi:10.1101/gad.326105.

166.

Delile

Rayon

Melchionda

Edwards

Briscoe

Sagner

. Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord. Development. 2019;146(12):dev173807. doi:10.1242/dev.173807.

167.

Liu

Huang

Zheng

Takebayashi

Cao

Qiu

. Olig3 is not involved in the ventral patterning of spinal cord. PLoS ONE. 2014;9(10):e111076. doi:10.1371/journal.pone.0111076.

168.

Alaynick

Jessell

Pfaff

. SnapShot: spinal cord development. Cell. 2011;146(1):178.e171. Doi:10.1016/j.cell.2011.06.038.

169.

Pierani

Moran-Rivard

Sunshine

Littman

Goulding

Jessell

. Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1. Neuron. 2001;29(2):367–84. doi:10.1016/S0896-6273(01)00212-4.

170.

Sakiyama-Elbert

. Directed differentiation of V3 interneurons from mouse embryonic stem cells. Stem Cells Dev. 2015;24(22):2723–32. doi:10.1089/scd.2015.0122.

171.

Brown

Butts

McCreedy

Sakiyama-Elbert

. Generation of V2a interneurons from mouse embryonic stem cells. Stem Cells Dev. 2014;23(15):1765–76. doi:10.1089/scd.2013.0628.

172.

Butts

McCreedy

Martinez-Vargas

Mendoza-Camacho

Hookway

Gifford

Taneja

Noble-Haeusslein

McDevitt

. Differentiation of V2a interneurons from human pluripotent stem cells. Proc Natl Acad Sci U S A. 2017;114(19):4969–74. doi:10.1073/pnas.1608254114.

173.

Iyer

Huettner

Butts

Brown

Sakiyama-Elbert

. Generation of highly enriched V2a interneurons from mouse embryonic stem cells. Exp Neurol. 2016;277:305–16. doi:10.1016/j.expneurol.2016.01.011.

174.

Mizuguchi

Kriks

Cordes

Gossler

Goulding

. Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons. Nat Neurosci. 2006;9(6):770–78. doi:10.1038/nn1706.

175.

Lacroix-Fralish

Ledoux

Mogil

. The pain genes database: an interactive web browser of pain-related transgenic knockout studies. Pain. 2007;131(1–2):3.e1–e4. doi:10.1016/j.pain.2007.04.041.

176.

Mansouri

Gruss

. Pax3 and Pax7 are expressed in commissural neurons and restrict ventral neuronal identity in the spinal cord. Mech Dev. 1998;78(1–2):171–78. doi:10.1016/S0925-4773(98)00168-3.

177.

Inoue

Ozaki

Shiga

Ito

Masuda

Okado

Iseda

Kawaguchi

Ogawa

Bae

Yamashita

, et al. Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons. Nat Neurosci. 2002;5(10):946–54. doi:10.1038/nn925.

178.

Arber

Ladle

Lin

Frank

Jessell

. ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons. Cell. 2000;101(5):485–98. doi:10.1016/S0092-8674(00)80859-4.

179.

Yang

Tomita

Wines-Samuelson

Beglopoulos

Tansey

Kopan

Shen

. Notch1 signaling influences V2 interneuron and motor neuron development in the spinal cord. Dev Neurosci. 2006;28(1–2):102–17. doi:10.1159/000090757.

180.

Catela

Chen

Weng

Wen

Kratsios

. Control of spinal motor neuron terminal differentiation through sustained Hoxc8 gene activity. eLife. 2022;11:e70766. doi:10.7554/eLife.70766.

181.

Zhang

Pinto

Wan

Wang

Berg

Oliva

Singh

Dengler

Wei

Dreyfuss

. Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy. Proc Natl Acad Sci U S A. 2013;110(48):19348–53. doi:10.1073/pnas.1319280110.

182.

Rousso

Gaber

Wellik

Morrisey

Novitch

. Coordinated actions of the forkhead protein Foxp1 and Hox proteins in the columnar organization of spinal motor neurons. Neuron. 2008;59(2):226–40. doi:10.1016/j.neuron.2008.06.025.

183.

Holz

Kollmus

Ryge

Niederkofler

Dias

Ericson

Stoeckli

Kiehn

Arnold

. The transcription factors Nkx2.2 and Nkx2.9 play a novel role in floor plate development and commissural axon guidance. Development. 2010;137(24):4249–60. doi:10.1242/dev.053819.

184.

Sander

Paydar

Ericson

Briscoe

Berber

German

Jessell

Rubenstein

JLR

. Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates. Genes Dev. 2000;14(17):2134–39. doi:10.1101/gad.820400.

185.

Odermatt

Wellershaus

Wallraff

Seifert

Degen

Euwens

Fuss

Büssow

Schilling

Steinhäuser

Willecke

. Connexin 47 (Cx47)-deficient mice with enhanced green fluorescent protein reporter gene reveal predominant oligodendrocytic expression of Cx47 and display vacuolized myelin in the CNS. J Neurosci. 2003;23(11):4549–59. doi:10.1523/JNEUROSCI.23-11-04549.2003.

186.

Sartoretti

Campetella

Lanuza

. Dbx1 controls the development of astrocytes of the intermediate spinal cord by modulating Notch signaling. Development. 2022;149(15):dev200750. doi:10.1242/dev.200750.

187.

Chen

Qian

Chen

Blackbourn

Liu

Knobel

Ayala

Zhang

S-C

. Human-derived neural progenitors functionally replace astrocytes in adult mice. J Clin Invest. 2015;125(3):1033–42. doi:10.1172/JCI69097.

188.

Xiao

Zhang

Wang

Zhang

Rasouli

Casella

Ciric

Curtis

Rostami

Zhang

G-X

. CRISPR-mediated rapid generation of neural cell-specific knockout mice facilitates research in neurophysiology and pathology. Mol Ther Methods Clin Dev. 2021;20:755–64. doi:10.1016/j.omtm.2021.02.012.

189.

Sakurai

Osumi

. The neurogenesis-controlling factor, Pax6, inhibits proliferation and promotes maturation in murine astrocytes. J Neurosci. 2008;28(18):4604–12. doi:10.1523/JNEUROSCI.5074-07.2008.

190.

Relaix

Montarras

Zaffran

Gayraud-Morel

Rocancourt

Tajbakhsh

Mansouri

Cumano

Buckingham

. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J Cell Biol. 2006;172(1):91–102. doi:10.1083/jcb.200508044.

191.

Welle

Cardillo

Zanche

Tawil

. Skeletal muscle gene expression after myostatin knockout in mature mice. Physiol Genom. 2009;38(3):342–50. doi:10.1152/physiolgenomics.00054.2009.

192.

Liu

Wang

Tian

Wei

Cui

Jiang

Wang

. Effect of Zbed6 single-allele knockout on the growth and development of skeletal muscle in mice. Biology. 2023;12(2):325. doi:10.3390/biology12020325.

193.

Jeon

Carlborg

Törnsten

Giuffra

Amarger

Chardon

Andersson-Eklund

Andersson

Hansson

Lundström

Andersson

. A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus. Nat Genet. 1999;21(2):157–58. doi:10.1038/5938.

194.

Founta

K-M

Papanayotou

. In vivo generation of organs by blastocyst complementation: advances and challenges. Int J Stem Cells. 2022;15(2):113–21. doi:10.15283/ijsc21122.

195.

Crane

Aravalli

Asakura

Grande

Krishna

Carlson

Cheeran

Danczyk

Dutton

Hackett

, et al. Interspecies organogenesis for human transplantation. Cell Transplant. 2019;28(9–10):1091–105. doi:10.1177/0963689719845351.

196.

Steevens

Griesbach

You

Dutton

Low

Santi

. Generation of inner ear sensory neurons using blastocyst complementation in a Neurog1+/−−deficient mouse. Stem Cell Res Ther. 2021;12(1):122. doi:10.1186/s13287-021-02184-1.

197.

Ruiz-Estevez

Crane

Rodriguez-Villamil

Ongaratto

You

Steevens

Hill

Goldsmith

Webster

Sherry

Lim

, et al. Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs. Stem Cell Res Ther. 2021;12(1):292. doi:10.1186/s13287-021-02348-z.

198.

Chen

Lansford

Stewart

Young

Alt

. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc Natl Acad Sci U S A. 1993;90(10):4528. doi:10.1073/pnas.90.10.4528.

199.

Usui

Kobayashi

Yamaguchi

Knisely

Nishinakamura

Nakauchi

. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am J Pathol. 2012;180(6):2417–26. doi:10.1016/j.ajpath.2012.03.007.

200.

Kobayashi

Yamaguchi

Hamanaka

Kato-Itoh

Yamazaki

Ibata

Sato

Lee

Y-S

Usui

Knisely

Hirabayashi

, et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2012;142(5):787–99. doi:10.1016/j.cell.2010.07.039.

201.

Mori

Furuhashi

Danielsson

Hirata

Kakiuchi

Lin

Ohta

Riccio

Takahashi

Emala

, et al. Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells. Nat Med. 2019;25(11):1691–98. doi:10.1038/s41591-019-0635-8.

202.

Wen

Ustiyan

Wang

Guo

C-L

Kalin

Galvan

Weaver

Kalin

, et al. In vivo generation of lung and thyroid tissues from embryonic stem cells using blastocyst complementation. Am J Respir Crit Care Med. 2021;203(4):471–83. doi:10.1164/rccm.201909-1836OC.

203.

Chang

Liang

Dai

Chapdelaine-Williams

Andrews

Bronson

Schwer

Alt

. Neural blastocyst complementation enables mouse forebrain organogenesis. Nature. 2018;563(7729):126–30. doi:10.1038/s41586-018-0586-0.

204.

Crane

Voth

Shen

Low

. Concise review: human-animal neurological chimeras: humanized animals or human cells in an animal? Stem Cells. 2019;37(4):444–52. doi:10.1002/stem.2971.

205.

Crane

Shen

Brown

Cormack

Ruiz-Estevez

Voth

Sawai

Hatta

Fujita

Low

. The American public is ready to accept human-animal chimera research. Stem Cell Reports. 2020;15(4):804–10. doi:10.1016/j.stemcr.2020.08.018.

206.

Suzuki

Kim

Liu

G-H

Izpisua Belmonte

. A cut above the rest: targeted genome editing technologies in human pluripotent stem cells. J Biol Chem. 2014;289(8):4594–99. doi:10.1074/jbc.R113.488247.

207.

Ben Jehuda

Shemer

Binah

. Genome editing in induced pluripotent stem cells using CRISPR/Cas9. Stem Cell Rev Rep. 2018;14(3):323–36. doi:10.1007/s12015-018-9811-3.

208.

Hayashi

Hinckley

Driscoll

Moore

Levine

Hilde

Sharma

Pfaff

. Graded arrays of spinal and supraspinal V2a interneuron subtypes underlie forelimb and hindlimb motor control. Neuron. 2018;97(4):869–84.e5. Doi:10.1016/j.neuron.2018.01.023.

Cell Transplantation for Repair of the Spinal Cord and Prospects for Generating Region-Specific Exogenic Neuronal Cells

Abstract

Keywords

Introduction

SCI Pathophysiology

Sources of Cells for Transplantation

Cell Replacement Therapy for SCI

Preclinical NPC Transplantation Studies in Animal Models of SCI

Clinical Cell Transplantation Studies in Humans for SCI

Differentiation and Regional Specification of Spinal Neurons

Considerations for Growth Factor-Directed Differentiation

Considerations for Regional Specification of Spinal Neural Cell Types

Developmental Neurobiology of the Spinal Cord

Rostral–Caudal Patterning

Dorsal–Ventral Patterning

Developmental Gene Networks for Exogenic Spinal Cell Specification and Differentiation

Motor Neurons

Spinal Interneurons

Dorsal interneurons

Ventral interneurons

Generating Exogenic Neuronal Cells Via Blastocyst Complementation as a Source of Cells for Repair of SCI

Blastocyst Complementation

Gene Editing for Blastocyst Complementation

Conclusion

Footnotes

Acknowledgements

Author Contributions

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

Declaration of Conflicting Interests

Funding

ORCID iDs

References