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• W3182077185 abstract "•Neuromesodermal progenitor derivation of human spinal neurons as therapeutic cells•Neural ribbons bridge in vitro network formation and in vivo host transplantation•In vivo visualization of encapsulated graft placement with magnetic resonance imaging•Six-week viability of human neuronal networks with OPCs in rat contusion SCI To repair neural circuitry following spinal cord injury (SCI), neural stem cell (NSC) transplantation has held a primary focus; however, stochastic outcomes generate challenges driven in part by NSC differentiation and tumor formation. The recent ability to generate regionally specific neurons and their support cells now allows consideration of directed therapeutic approaches with pre-differentiated and networked spinal neural cells. Here, we form encapsulated, transplantable neuronal networks of regionally matched cervical spinal motor neurons, interneurons, and oligodendrocyte progenitor cells derived through trunk-biased neuromesodermal progenitors. We direct neurite formation in alginate-based neural ribbons to generate electrically active, synaptically connected networks, characterized by electrophysiology and calcium imaging before transplantation into rodent models of contused SCI for evaluation at 10-day and 6-week timepoints. The in vivo analyses demonstrate viability and retention of interconnected synaptic networks that readily integrate with the host parenchyma to advance goals of transplantable neural circuitry for SCI treatment. To repair neural circuitry following spinal cord injury (SCI), neural stem cell (NSC) transplantation has held a primary focus; however, stochastic outcomes generate challenges driven in part by NSC differentiation and tumor formation. The recent ability to generate regionally specific neurons and their support cells now allows consideration of directed therapeutic approaches with pre-differentiated and networked spinal neural cells. Here, we form encapsulated, transplantable neuronal networks of regionally matched cervical spinal motor neurons, interneurons, and oligodendrocyte progenitor cells derived through trunk-biased neuromesodermal progenitors. We direct neurite formation in alginate-based neural ribbons to generate electrically active, synaptically connected networks, characterized by electrophysiology and calcium imaging before transplantation into rodent models of contused SCI for evaluation at 10-day and 6-week timepoints. The in vivo analyses demonstrate viability and retention of interconnected synaptic networks that readily integrate with the host parenchyma to advance goals of transplantable neural circuitry for SCI treatment. Stem cell technologies remain at the forefront for therapeutic treatment of SCI, with the potential to address some of the complex spatiotemporal parameters surrounding injuries that include neuronal and glial cell death, destruction of axons, inflammation, and variations in anatomical context. Multipotent neural stem cells provide a pathway to neurogenesis as well as paracrine signaling that support regeneration and plasticity (Yu et al., 2012Yu L. Mahairaki V. Koliatsos V.E. Host induction by transplanted neural stem cells in the spinal cord: further evidence for an adult spinal cord neurogenic niche.Regen. Med. 2012; 7: 785-797Crossref PubMed Scopus (15) Google Scholar). However, once transplanted, neural stem cells are largely unregulated in cell division and differentiation, which creates stochastic outcomes that are a barrier to clinical implementation (Assinck et al., 2017Assinck P. Duncan G.J. Hilton B.J. Plemel J.R. Tetzlaff W. Cell transplantation therapy for spinal cord injury.Nat. Neurosci. 2017; 20: 637-647Crossref PubMed Scopus (329) Google Scholar). Nevertheless, stem cell technologies are evolving rapidly for SCI including realization of developmental principles to better anatomically match delivered therapeutic neural cells (Olmsted and Paluh, 2021Olmsted Z.T. Paluh J.L. Stem cell neurodevelopmental solutions for restorative treatments of the human trunk and spine.Front.Cell. Neurosci. 2021; 15: 667590Crossref PubMed Scopus (3) Google Scholar). The benefits of a neurodevelopmental approach are already being realized in groundbreaking advances to treat Parkinson’s disease (Barker et al., 2017Barker R.A. Parmer M. Studer L. Takahashi J. Human trials of stem cell-derived dopamine neurons for Parkinson’s disease: dawn of a new era.Cell Stem Cell. 2017; 21: 569-573Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). By focusing on a specific population of dopaminergic neurons in the substantia nigra of the midbrain that are affected (DeMaagd and Philip, 2015DeMaagd G. Philip A. Parkinson’s disease and its management: part 1: disease entity, risk factors, pathophysiology, clinical presentation, and diagnosis.P T. 2015; 40: 504-532PubMed Google Scholar), benefits for robust engraftment, in vivo neuronal maturation, and circuit repair are being realized (Kikuchi et al., 2017Kikuchi T. Morizane A. Doi D. Magotani H. Onoe H. Hayashi T. Mizuma H. Takara S. Takashi R. Inoue H. et al.Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model.Nature. 2017; 548: 592-596Crossref PubMed Scopus (293) Google Scholar; Xiong et al., 2021Xiong M. Tao Y. Gao Q. Feng B. Yan W. Zhou Y. Kotsonis T.A. Yuan T. You Z. Wu Z. et al.Human stem cell-derived neurons repair circuits and restore neural function.Cell Stem Cell. 2021; 28: 112-126.e6Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This approach has not yet been applied to treat acute traumatic insults, such as in SCI (Silva et al., 2014Silva N.A. Sousa N. Reis R.L. Salgado A.J. From basics to clinical: a comprehensive review on spinal cord injury.Prog. Neurobiol. 2014; 114: 25-27Crossref PubMed Scopus (421) Google Scholar) or stroke (Baker et al., 2019Baker E.W. Kinder H.A. West F.D. Neural stem cell therapy for stroke: a multimechanistic approach to restoring neurological function.Brain Behav. 2019; 9: e01214Crossref PubMed Scopus (29) Google Scholar), in which the spectrum of severity, anatomical distribution, and breadth of neural circuits affected within a single subject and between subjects will likely require patient-customized neuronal network therapies. Neuromesodermal progenitors are the embryological source of neurons and glia in the spinal cord (Henrique et al., 2015Henrique D. Abranches E. Verrier L. Storey K.G. Neuromesodermal progenitors and the making of the spinal cord.Development. 2015; 142: 2864-2875Crossref PubMed Scopus (136) Google Scholar), and are expected to be an ideal source for deriving therapeutic cells and networks for SCI. Spinal cord developmental principles and corresponding implementation into stem cell research has enabled the generation of cell types that are regionally matched to the site of injury (Gouti et al., 2014Gouti M. Tsakiridis A. Wymeersch F.J. Huang Y. Kleinjung J. Wilson V. Briscoe J. In vitro generation of neuromesodermal progenitors reveals distinct roles for Wnt signaling in the specification of spinal cord and paraxial mesoderm identity.PLoS Biol. 2014; 12: e1001937Crossref PubMed Scopus (171) Google Scholar; Sagner and Briscoe, 2019Sagner A. Briscoe J. Establishing neuronal diversity in the spinal cord: a time and a place.Development. 2019; 146: dev182154Crossref PubMed Scopus (58) Google Scholar). Importantly, the caudal neuromesodermal progenitor-derived cells represent the appropriate bifurcated developmental lineage for spinal neurons (Nedelec and Martinez-Arias, 2021Nedelec S. Martinez-Arias A. In vitro models of spinal motor circuit’s development in mammals: achievements and challenges.Curr. Opin. Neurobiol. 2021; 66: 240-249Crossref PubMed Scopus (2) Google Scholar; Wind et al., 2021Wind M. Gogolou A. Manipur I. Granata I. Butler L. Andrews P.W. Barbaric I. Ning K. Guarracino M.R. Placzek M. Tsakiridis A. Defining the signaling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives.Development. 2021; 148: dev194415Crossref PubMed Scopus (2) Google Scholar). This is a relatively recent finding and may be relevant even for neural stem cell studies in which the past use of a default CNS pathway with anterior telencephalic identity may have had an impact (Dulin et al., 2018Dulin J.N. Adler A.F. Kumamaru H. Poplawski G.H.D. Lee-Kubli C. Strobl H. Gibbs D. Kadoya K. Fawcett J.W. Lu P. Tuszynski M.H. Injured adult motor and sensory axons regenerate into appropriate organotypic domains of neural progenitor grafts.Nat. Commun. 2018; 9: 84Crossref PubMed Scopus (44) Google Scholar; Kumamaru et al., 2018Kumamaru H. Kadoya K. Adler A.F. Takashima Y. Graham L. Coppola G. Tuszynski M.H. Generation and post-injury integration of human spinal cord neural stem cells.Nat. Methods. 2018; 15: 723-731Crossref PubMed Scopus (55) Google Scholar). With up-to-date CNS developmental information applied to stem cell protocols, the reinvestigation into use of neurons in therapeutics for SCI is warranted. Combined with biomaterials (Katoh et al., 2019Katoh H. Yokota K. Fehlings M.G. Regeneration of spinal cord connectivity through stem cell transplantation and biomaterial scaffolds.Front.Cell. Neurosci. 2019; 13: 248Crossref PubMed Scopus (44) Google Scholar), there is a need to generate and test customized 3D neuronal networks for SCI as well as for a variety of in vitro and in vivo biomedical applications. We previously developed an improved alginate-based methodology for neural ribbon co-encapsulation of homotypic spinal cord neural stem cells and the microenvironment regulator chondroitinase ABC, designed to facilitate shipment of cells and remote SCI transplantation (Olmsted et al., 2020Olmsted Z.T. Stigliano C.S. Badri A. Zhang F. Williams A. Koffas M.A.G. Xie Y. Linhradt R.J. Cibelli J. Horner P.J. Paluh J.L. Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.Sci. Rep. 2020; 10: 12939Crossref PubMed Scopus (3) Google Scholar). Encapsulation of neural stem cells can provide neuroprotection of transplanted cells to improve survival (Tsintou et al., 2015Tsintou M. Dalamagkas K. Seifalian A.M. Advances in regenerative therapies for spinal cord injury: a biomaterials approach.Neural Regen. Res. 2015; 10: 726-742Crossref PubMed Scopus (82) Google Scholar; Ahuja and Fehlings, 2016Ahuja C.S. Fehlings M. Concise review: bridging the gap: novel neuroregenerative and neuroprotective strategies in spinal cord injury.Stem Cells Transl. Med. 2016; 5: 914-924Crossref PubMed Scopus (111) Google Scholar; Liu et al., 2018Liu S. Schackel T. Weidner N. Puttagunta R. Biomaterial-supported cell transplantation treatments for spinal cord injury: challenges and perspectives.FrontCell Neurosci. 2018; 11: 430Google Scholar; Olmsted et al., 2020Olmsted Z.T. Stigliano C.S. Badri A. Zhang F. Williams A. Koffas M.A.G. Xie Y. Linhradt R.J. Cibelli J. Horner P.J. Paluh J.L. Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.Sci. Rep. 2020; 10: 12939Crossref PubMed Scopus (3) Google Scholar). We extend this technology here as a platform for neural network formation in vitro. In this study, we generate neural ribbons of synaptically connected neuronal networks composed of functionally maturing caudal spinal motor neurons (SMNs), interneurons, and oligodendrocyte progenitor cells (OPCs) for in vitro analyses and demonstrate neuron retention in vivo following transplantation in a rat cervical hemicontusion SCI model. Our SMN-OPC neural ribbon-encapsulated networks demonstrate high synaptic density and the output of stereotypic firing in response to glutamate stimulation. When transplanted into the subacute phase hemicontusion injury microenvironment (15 days after injury) and analyzed by biomarker immunohistochemistry of spinal cord sections 10 days and 6 weeks after transplantation, we observe robust survival and engraftment. The presence of reduced cell numbers (~5,000 cells per animal) in neural ribbon grafts (versus traditional suspension methods that deliver up to 40x more cells) allows us to readily evaluate graft survival of OPCs, neurons, and retained synapses. Human neurons and OPCs graft along the parenchyma that borders the host injury cavity, intercalated with host tissue and co-localized with synaptic biomarkers. This study is the first to investigate the formation of pre-constructed human neuronal networks in an encapsulated ribbon format and use of these in a rat SCI model. We achieve neuron-OPC graft survival and integration without the use of additional injury site modifiers, such as chondroitinase ABC. SMNs were derived from the African American hiPSC line F3.5.2 (Chang et al., 2015Chang E.A. Tomov M.L. Suhr S.T. Luo J. Olmsted Z.T. Paluh J.L. Cibelli J. Derivation of ethnically diverse human induced pluripotent stem cell lines.Sci. Rep. 2015; 5: 15234Crossref PubMed Scopus (20) Google Scholar; Tomov et al., 2016Tomov M.L. Olmsted Z.T. Dogan H. Gongorurler E. Tsompana M. Otu H.H. Buck M. Chang E.-H. Cibelli J. Paluh J.L. Distinct and shared determinants of cardiomyocyte contractility in multi-lineage competent ethnically diverse iPSCs.Sci. Rep. 2016; 6: 37637Crossref PubMed Scopus (10) Google Scholar) in three stages referred to here as (1) induction, (2), patterning, and (3) neuronal maturation according to a protocol that we previously described (Figure 1A) (Olmsted et al., 2020Olmsted Z.T. Stigliano C.S. Badri A. Zhang F. Williams A. Koffas M.A.G. Xie Y. Linhradt R.J. Cibelli J. Horner P.J. Paluh J.L. Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.Sci. Rep. 2020; 10: 12939Crossref PubMed Scopus (3) Google Scholar). The hiPSCs were first used to generate trunk-biased neuromesodermal progenitors that are the developmental origin of the spinal cord in vivo (Figures 1B–1D) (Gouti et al., 2014Gouti M. Tsakiridis A. Wymeersch F.J. Huang Y. Kleinjung J. Wilson V. Briscoe J. In vitro generation of neuromesodermal progenitors reveals distinct roles for Wnt signaling in the specification of spinal cord and paraxial mesoderm identity.PLoS Biol. 2014; 12: e1001937Crossref PubMed Scopus (171) Google Scholar). Neuromesodermal progenitors were differentiated to spinal cord neuroectoderm using 100 nM retinoic acid and 200 nM Hh-Ag1.5, a potent sonic hedgehog agonist. Further exposure to retinoic acid and Hh-Ag1.5 in N2B27 basal medium generates motor neuron progenitors at high efficiency that mature to SMNs (Figure 1E). Over the course of differentiation, SMNs expressed a panel of canonical biomarkers including NKX6-1 (day 17), ISL1&2 (day 25), FOXP1 (day 28), HB9 (Mnx1, day 35), and choline acetyltransferase (ChAT, day 35) by immunofluorescence (Figure 1F). The differentiated cells also expressed generic biomarkers of neuronal compartments such as somatodendritic (MAP2), axonal (SMI312), and the pre-synaptic terminal (Synapsin 1, or SYN1). A list of all biomarkers used and their significance is provided (Table S1). Ventral and dorsal spinal interneurons and astrocytes that promote synaptogenesis are indispensable to normal neuronal circuit function in vivo. Since ventral spinal interneurons are patterned similarly to SMNs during development, we investigated the co-differentiation of both ventral and dorsal spinal interneurons in SMN cultures (Figures 1G and 1H), as well as astrocytes (Figure 1I). We provide representative images for CHX10 and PAX2 (Figure 1G) and quantified the proportion of nine generic subtypes of spinal neurons in day 58 cultures by immunofluorescence that are matched to the following biomarkers (Figure 1H): Nkx-2.2 (V3, ~2%), FOXP1+ISL1 (MN, ~52%), CHX10 (V2a, ~12%), GATA-3 (V2b, < 1%), PAX2 (V0/V1 or dI4/dI6, ~8%), LBX1 (dI4-dI6, 0%), TLX3 (dI3, ~1%), LHX9 (dI1, 0%), and BRN3A (sensory, ~25%). Low levels of LBX1, TLX3, and LHX9 in the presence of peripherin expression indicate that BRN3A+ cells were neural crest-derived sensory neurons that can be patterned through neuromesodermal progenitors (Frith et al., 2018Frith T.J.R. Granata I. Wind M. Stout E. Thompson O. Neumann K. Stavish D. Heath P.R. Ortmann D. Hackland J.O. et al.Human axial progenitors generate trunk neural crest cells in vitro.eLife. 2018; 7: e35786Crossref PubMed Scopus (36) Google Scholar). GFAP/CD44 astrocytes were not observed in SMN cultures until after day 40, shown here at day 58 (Figure 1I). In adherent cultures, INs developed in close proximity with SMNs, and this configuration was retained within neural ribbon-encapsulated constructs (Figure 1J). We describe neural ribbon encapsulation of differentiating neuronal cultures in the subsequent section (Figure 2). Neural ribbons generated by hydrogel encapsulation prior to day 40 do not contain astrocytes because these cells are not yet differentiated. We refer to the SMN cultures as simply “SMNs” throughout the manuscript. We applied an alginate hydrogel-based platform to reproducibly generate neural ribbons of encapsulated differentiating SMN cultures (Olmsted et al., 2020Olmsted Z.T. Stigliano C.S. Badri A. Zhang F. Williams A. Koffas M.A.G. Xie Y. Linhradt R.J. Cibelli J. Horner P.J. Paluh J.L. Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.Sci. Rep. 2020; 10: 12939Crossref PubMed Scopus (3) Google Scholar, previously designed for use with neural stem cells). Here, we further optimized neural ribbon methodologies in order to direct and image neuronal and glial cytoarchitecture configurations (Figure 2). Alginate biopolymer crosslinking as hydrogels included addition of Type 1 collagen as exogenous extracellular matrix to establish interpenetrating networks (IPNs) (Figure 2A). Sodium alginate (1.5% in NaCl) was further functionalized with bioactive peptide RGD that was used to promote cell adhesion and cell-cell interactions within neural ribbons (RGD-Alginate + Collagen, or RGD-Col). For immobilization, long-term culture and imaging, the extruded ribbons (Figure 2B) were rapidly formed by crosslinking in 100 mM CaCl2, rinsed, and embedded in fast-gelling collagen solution in the viewing area of a glass bottom dish. Cell-free and cell-laden ribbons each retained the circular cross-section and diameter of the needle tip template (60, 100, or 150 μm inner diameter) in CaCl2, but swelled by ~33% in culture medium. Neurite extension within 60 μm neural ribbons was constrained along the ribbon longitudinal axis by the addition of 4 μg/mL Aggrecan (chondroitin sulfate proteoglycan, CSPG) within the embedding medium. High CSPG concentrations can be inhibitory to axon penetration. Day 20 motor neuron progenitors were encapsulated as small triturated neurospheres (Figure 2C) or larger intact aggregates (Figure 2D) at 1 × 108 cells/ml concentration equivalent and cultured for up to 12 days. Cultures were transitioned to neuronal maturation medium at day 25 according to our protocol (Figure 1A). We achieved neurite anisotropic longitudinal alignment within neural ribbons and observed expression of lateral motor column SMN phenotypic biomarkers FOXP1 and ChAT, that co-localized with TUJ1 (Figure 2D). We detected pre- and post-synaptic proteins using SYN1 and PSD-95 biomarkers, respectively, as well-distributed puncta by day 10 after encapsulation (Figure 2E). Neural cell borders were visualized by co-staining with NCAM-1. OPCs facilitate neuronal damage recovery after SCI (Assinck et al., 2017Assinck P. Duncan G.J. Hilton B.J. Plemel J.R. Tetzlaff W. Cell transplantation therapy for spinal cord injury.Nat. Neurosci. 2017; 20: 637-647Crossref PubMed Scopus (329) Google Scholar), but are not co-differentiated with high efficiency by SMN protocols. We therefore generated hiPSC-derived OPCs and sorted them for co-culture with SMNs prior to neural ribbon co-encapsulation (Figure 3). OPC differentiation proceeds through spinal cord neuroectoderm (PAX6) and the motor progenitor spinal domain (Nestin/OLIG2), but is then specified by exposure to growth factors IGF1, FGF2, and PDGF-AA as well as thyroid hormone triiodothyronine (T3) at day 20 (Figure 3A) (Khazaei et al., 2017Khazaei M. Ahuja C.S. Fehlings M.G. Generation of oligodendrogenic spinal neural progenitor cells from human induced pluripotent stem cells.Curr. Protoc. Stem Cell Biol. 2017; 42: 2D.20.1-2D.20.14Crossref Scopus (12) Google Scholar). By omitting DAPT during the first five days of differentiation, OPCs are generated as indicated by Nkx-2.2 expression that defines the developing ventral OPCs, and ultimately the OPC-specific antigen O4 (Figure 3B). Efficiency of OPC differentiation was assessed by quantifying expression of the relevant biomarker versus DAPI at each important time point as follows: PAX6 (day 6, 1,023/1,1115 cells, 92%), OLIG2 (day 20, 879/977 cells, 90%), NKX-2.5 (day 25, 1,142/1,313 cells, 87%), O4 (day 36, 593/824 cells, 72%). To ensure the greatest reproducibility of in vivo data after CNS neural ribbon delivery, two additional purification stems were used; that is, magnetic cell sorting (MACS) separation of OPCs with the O4 antigen (Figures 3C and 3D), and SMN neurosphere suspension culture. For encapsulation, OPCs were generated from hiPSCs expressing GFP under the CAG promoter (Taylor et al., 2006Taylor L. Jones L. Tuszynski M.H. Blesch A. Neurotrophin-3 gradients established by lentiviral gene delivery promote short-distance axonal bridging beyond cellular grafts in the injured spinal cord.J. Neurosci. 2006; 26: 9713-9721Crossref PubMed Scopus (150) Google Scholar). Enriched GFP-OPCs were retained and mixed 1:5 with SMNs and co-cultured for 1 week in modified N2B27 bundling medium (Figure 3E). At this point, SMN-OPC co-cultures were encapsulated within 60 μm RGD-Col neural ribbons and embedded for longer term maintenance and imaging using GFP and SMI312 immunofluorescence, shown at day 7 after encapsulation (Figure 3F). Neural ribbon alginate hydrogels provide a platform for neural network formation and delivery in vivo and in cultured SMNs. To evaluate recovery and further maturation of SMNs following encapsulation, we released cells from encapsulation after 48 hr and then maintained neurons in the presence or absence of neurotrophic factors (NTFs) using slow release PLGA microbeads (Figure 4A). For encapsulation, 23 day motor neuron progenitors were passaged as aggregates and encapsulated in 60 μm neural ribbons (ProNova alginate + collagen, or NovaCol) lacking RGD peptide, or were used to generate motor neuron progenitor neurosphere cultures without encapsulation for comparison. Progenitor neural ribbons and neurospheres were cultured in suspension for 48 hr. On day 25, motor neuron progenitor aggregates were recovered from neural ribbons by dissolving alginate in 1.6% sodium citrate and seeded onto Matrigel-coated cover glass in parallel with non-encapsulated neurospheres. Cultures were further subject to SMN terminal differentiation and maintained up to day 52, analyzed at two general timepoints in differentiation that are days 32–35 and days 45-52. We provide examples of phase images of neuronal cultures (Figure 4B) and corresponding SMN biomarkers (Figure 4C) at the time that the whole-cell, patch clamp recordings were performed. Representative traces for the two timepoints are provided (Figure 4D). The recordings, in current-clamp mode, allowed us to analyze the passive and active membrane properties of SMNs at two different timepoints (total range: days 32-35 and 45-52) in naive conditions, in the presence of NTFs and in the presence of neural ribbon encapsulation (Figure 4E). The passive membrane properties (i.e. resting membrane potential, membrane resistance, membrane capacitance; Figure 4E, top) are not mediated by voltage- or ligand-gated ion channels. They describe how current flows through the membrane and how the membrane voltage responds to sub-threshold current step injections. The resting membrane potential ranged between −36 mV at d33-35 and −46 mV at d32-34 with neural ribbons (∗p = 0.023 by ANOVA). The membrane resistance (which describes how a current input can change the cell's voltage) differed between d32–34 and d51-52 only in the presence of NTFs (∗p = 0.048). The cell capacitance (which describes the ability of a cell to store electrical charges) was similar across all groups. These values are consistent with those obtained by Takazawa et al., 2012Takazawa T. Croft G.F. Amoroso M.W. Studer L. Wichterle H. MacDermott A.B. Maturation of spinal motor neurons derived from human embryonic stem cells.PLoS One. 2012; 7: e40154Crossref PubMed Scopus (51) Google Scholar working on SMNs derived from human embryonic stem cells (Takazawa et al., 2012Takazawa T. Croft G.F. Amoroso M.W. Studer L. Wichterle H. MacDermott A.B. Maturation of spinal motor neurons derived from human embryonic stem cells.PLoS One. 2012; 7: e40154Crossref PubMed Scopus (51) Google Scholar). Since the membrane resistance varies with the surface density of potassium leak channels, and the cell capacitance varies with the cell surface area, these findings indicate that the different culturing conditions do not exert a major and consistent interference with the expression of potassium leak channels and with cell size regulation in these cells. To study the active membrane properties of SMNs, we used DC current injections to maintain each cell at −65 mV, and applied short (5 ms) current steps to measure the rheobase, action potential (AP) threshold and peak (Figure 4E, bottom). On average, neurons fired APs in response to suprathreshold current injections, but not in all cases. The rheobase (i.e. the minimum current step amplitude required to evoke an AP) decreased at d32-34 after neural ribbon encapsulation. This effect was associated with a hyperpolarization of the AP threshold (∗p = 0.047), which also occurred between d32–35 and d45-52 SMN groups (∗p = 0.032). Together, the decreased rheobase and lower AP threshold suggest that neural ribbons increase the excitability of SMNs only in young cultures, not in older ones. This maturation trend has also been observed in developing SMNs (Gao and Ziskand-Conhaim, 1998Gao B.X. Ziskand-Conhaim L. Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons.J. Neurophysiol. 1998; 80: 3047-3061Crossref PubMed Scopus (142) Google Scholar). The AP peak was reduced between these SMN groups (∗p = 0.039), pointing to a potentially smaller driving force for Na+ (i.e. more hyperpolarized ENa) or different activation voltage for potassium channels after neural ribbon encapsulation. A similar effect was also detected between d32–34 and d51-52 cultures treated with growth factors (∗p = 0.047). As a consequence, the AP amplitude was slightly reduced at d51-52 in the presence of growth factors (∗p = 0.048). SMNs develop the ability to fire repetitively in response to sustained suprathreshold current steps as they mature. For this reason, we investigated whether a similar change occurred as SMNs matured, depending on the conditions in which they were maintained (Figure 4F). On average, SMNs fired multiple APs in response to suprathreshold, 500 ms long depolarizing current steps. The f/I plots showed similar trends across all groups, indicating that SMNs share the same ability to respond to sustained current injections, with pronounced accommodation among consecutive APs. We employed a microelectrode array (MEA) system in conjunction with calcium imaging to interrogate neuronal activity in neural ribbons (Figure 5). SMNs were encapsulated in RGD-Col neural ribbons at day 28 in differentiation and maintained in terminal differentiation medium. At day 35, we performed non-adherent, acute recordings in three separate neural ribbons positioned over hexagonal arrays of MEA electrodes and immobilized under a coverslip. Recordings were acquired before and after addition of 50 μM glutamate. Spiking activity was observed along the neural ribbon length, and was increased by glutamate stimulation in four of six electrodes (Figures 5A and 5B). Spike raster plots in the two most active electrodes (C1, D4) are provided (Figure 5C), indicating the time point of glutamate addition. To assess the degree of connectivity within the neural ribbons, we quantified both intra-electrode bursting activity in addition to network burst (NB) events and the percentage of spikes localized within a network burst (Figure 5D). The percent of spikes contained within NBs ranged from ~50 to 70%, where average NB duration was 1,011 ms. As additional validation of SMN spontaneous firing in neural ribbons, we performed live-cell calcium imaging with Fluo-4 AM on encapsulated neurons (60 μm RGD-Col), in which linear arrangements of cells were visible (Figure 5E). We also followed calcium signaling in aggregates that retain some of the neural ribbon shape during and after rapid alginate dissolution in 1.6% sodium citrate (Figure 5F) and observed retained rhythmic oscillations of somatic calcium transients (Figure 5G) with observable network bursting activity (Video S1). https://www.cell.com/cms/asset/b0f25195-ae87-4408-ad39-3d89febb32ae/mmc2.mp4Loading ... Download .mp4 (6.32 MB) Help with .mp4 files Video S1. Fluo-4 AM calcium imaging in aggregate collected from neural ribbon encapsulation. 20 frames/s. See also Figures 5F and 5G To optimize delivery and placement strategies with neural ribbons for the current study and to benefit future studies that may deliver multiple ribbons, we tested use of superparamagnetic iron oxide (SPIO) nanoparticles for tracking by magnetic resonance imaging (Figures 6A and 6B ). To track neural ribbon constructs and cells, we labeled day 29 neur" @default.
• W3182077185 created "2021-07-19" @default.
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• W3182077185 date "2021-08-01" @default.
• W3182077185 modified "2023-10-11" @default.
• W3182077185 title "Transplantable human motor networks as a neuron-directed strategy for spinal cord injury" @default.
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