scholarly journals Neuromesodermal Progenitors Advance Network Formation of Spinal Neurons and Support Cells in Neural Ribbons In Vitro and Unprotected Survival in a Rat Subacute Contusion Model

2020 ◽  
Author(s):  
Zachary T. Olmsted ◽  
Cinzia Stigliano ◽  
Annalisa Scimemi ◽  
Brandon Marzullo ◽  
Tatiana Wolfe ◽  
...  
Keyword(s):  
Stem Cell ◽  
Neuronal Networks ◽  
Network Formation ◽  
Functional Evaluation ◽  
Human Stem Cell ◽  
Cell Type ◽  
Spinal Neurons ◽  
Cord Injury

AbstractImproved human stem cell interventions to treat CNS trauma requires continued expansion of in vitro models and delivery platforms to fill gaps in analysis and treatment. Transplanted neural stem cells (NSCs) face unique, multi-faceted challenges beyond survival that include differentiation, maturation, and integration into a complex cytokine-releasing microenvironment that impinges on a multipotent cell type. Alternate strategies to transplant neurons and neuronal networks deserve reevaluation, particularly since novel differentiation protocols mimicking region-specific developmental and positional cues have recently emerged. To investigate transplantation of neurons and their early networks, we generate in vitro neural ribbons containing spinal neurons and support cells anatomically matched for cervical spinal cord injury (SCI). These glutamate-responsive, electrically-active neural ribbons apply a new hiPSC differentiation strategy transiting through neuromesodermal progenitors (NMps) to derive developmentally relevant spinal motor neurons (SMNs), interneurons (INs), and oligodendrocyte progenitor cells (OPCs). Bioinformatic profiling validates region-specific identities. Neurons and neuronal networks are functionally evaluated for action potential firing, calcium signaling, population activity, and synaptogenesis. NMp-derived neurons survive in vivo within the subacute phase hemi-contusion injury cavity when delivered either as free suspension or as encapsulated networks of pre-formed CNS cytoarchitectures. Delivery as encapsulated networks further supports survival of lower cell numbers and rapid graft penetration into host tissue. Neural network ribbons therefore provide a novel intermediary approach between cell suspensions and complex organoids for investigating network formation and early transplantation events with hiPSC-derived neurons, providing flexibility to rapidly tune cell type(s), cell ratios, and traceable biomarkers.Significance StatementIn the two decades since human stem cell technologies have emerged, the challenge has remained to improve the developmentally relevant derivation of therapeutic cells. The ability to now generate anatomically matched neurons for SCI necessitates a re-evaluation of these cells and their networks in vitro and in vivo. In this study, we apply developmental cues via neuromesodermal progenitors to generate spinal neurons from hiPSCs. Genetic and functional evaluation of these cells as in vitro neuronal networks, due to their capacity to survive and graft effectively within the rat subacute contusion cavity, offer novel approaches for customizing SCI transplantation. This work demonstrates a strategy to develop transplantable, chemically-responsive networks linking in vitro models with injury customization towards improved in vivo outcomes.

scholarly journals A human stem cell-derived neurosensory-epithelial circuitry on-a-chip to model herpes simplex virus reactivation

2020 ◽  
Author(s):  
Pietro Giuseppe Mazzara ◽  
Elena Criscuolo ◽  
Marco Rasponi ◽  
Luca Massimino ◽  
Sharon Muggeo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Sensory Neurons ◽  
Viral Latency ◽  
Human Stem Cell ◽  
Cell Type ◽  
Viral Pathogens ◽  
Simplex Virus

AbstractBoth emerging viruses and well-known viral pathogens endowed with neurotropism can either impair directly the neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, the current lack of an in vitro system modeling the connectivity between human neurons and peripheral tissues excludes the analysis of viral latency and reactivation and the assessment of natural/artificial induced anti-viral immunity. In this study, we developed the first stable topographic neurosensory-epithelial connection on-a-chip using human stem cell derived dorsal root ganglia (DRG) sensory neurons. Bulk and single cell transcriptomics showed that different combinations of key receptors for Herpes Simplex Virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of the HSV infectivity faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides for the first time a powerful in vitro platform to model viral latency and reactivation of human viral pathogens.

scholarly journals Recombinant human stem cell factor, a c-kit ligand, stimulates hematopoiesis in primates

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 1975-1980 ◽  
Author(s):  
RG Andrews ◽  
GH Knitter ◽  
SH Bartelmez ◽  
KE Langley ◽  
D Farrar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Absolute Number ◽  
Human Stem Cell ◽  
Clinical Role ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Human Stem Cell Factor

Abstract Recombinant human stem cell factor (SCF) is homologous with recombinant rat SCF (rrSCF) and is a ligand for c-kit. We determined the influence of SCF on hematopoiesis in vitro and in vivo in baboons. In vitro, SCF alone stimulated little growth of hematopoietic colony-forming cells from baboon marrow, but did increase the number of colonies formed in response to erythropoietin (Epo), interleukin-3 (IL-3), and granulocyte- macrophage colony-stimulating factor (GM-CSF). In vivo, SCF caused an increase in the peripheral blood of the number of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In marrow, it caused an increase in marrow cellularity and in the absolute number of colony-forming unit-granulocyte-monocyte (CFU-GM) and burst- forming unit-erythroid (BFU-E) in marrow following infusion of SCF. The in vivo stimulation of multiple lymphohematopoietic lineages corroborates previous in vitro studies and suggests a potentially important clinical role for SCF.

Fluorescent Magnetic Iron Oxide NanoparticleEncapsulated Protein Hydrogel Against Doxorubicin-Associated Cardiotoxicity and for Enhanced Cardiomyocyte Survival

2020 ◽  
Vol 16 (6) ◽  
pp. 922-930
Author(s):  
Pingshuan Dong ◽  
Honglei Wang ◽  
Shiying Xing ◽  
Xuming Yang ◽  
Shaoxin Wang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tube Formation ◽  
Human Stem Cell ◽  
Silk Sericin ◽  
Treatment Agent ◽  
Induced Apoptosis ◽  
Cardiomyocyte Survival ◽  
Dual Staining

Doxorubicin (DOX) is a widely used and effective anticancer drug. However, it shows high cardiotoxicity in several patients. The exact biological mechanisms of DOX-induced cardiotoxicity remain unclear. In the present study, we developed and assessed novel injectable hydrogel matrices combined with nanoparticles and secretome biomolecules to reduce DOXinduced cytotoxicity in human stem cell-derived cardiomyocytes. A Fe2O3 nanoparticle-loaded biocompatible silk sericin nanocomposite form was fabricated and used as an injectable carrier for secretome for in vivo cardiomyocyte metabolism. The formulated hydrogels carrying secretome were analyzed in vitro for proliferation, migration, and tube formation of human stem cell-derived cardiomyocytes. Biological analyses revealed that the secretome-encapsulated florescent Fe3O2 Silk sericin (Sec@MSS) hydrogel markedly reduced calcein-PI dual staining in cardiomyocytes, revealing significantly induced apoptosis. Furthermore, we evaluated the mitochondrial membrane potential for DOX and Sec@MSS hydrogel, and demonstrated apoptosis of the cardiomyocytes in the DOX-alone and Sec@MSS groups. However, the cardiotoxicity of Sec@MSS sericin was much lower than that in the DOX group, and was further evaluated via VEGFR and TUNEL analyses. The results indicate that Sec@MSS hydrogel might serve as an effective treatment agent in cardiac diseases in the future.

Synaptic Connectivity in Cultured Hypothalamic Neuronal Networks

1997 ◽  
Vol 77 (6) ◽  
pp. 3218-3225 ◽  
Author(s):  
Thomas H. Müller ◽  
D. Swandulla ◽  
H. U. Zeilhofer
Keyword(s):  
Neuronal Networks ◽  
Network Formation ◽  
Inhibitory Neurons ◽  
Synaptic Connectivity ◽  
Synaptic Connections ◽  
Cellular Mechanisms ◽  
Patch Clamp Technique ◽  
Novel Approach

Müller, Thomas H., D. Swandulla, and H. U. Zeilhofer. Synaptic connectivity in cultured hypothalamic neuronal networks. J. Neurophysiol. 77: 3218–3225, 1997. We have developed a novel approach to analyze the synaptic connectivity of spontaneously active networks of hypothalamic neurons in culture. Synaptic connections were identified by recording simultaneously from pairs of neurons using the whole cell configuration of the patch-clamp technique and testing for evoked postsynaptic current responses to electrical stimulation of one of the neurons. Excitatory and inhibitory responses were distinguished on the basis of their voltage and time dependence. The distribution of latencies between presynaptic stimulation and postsynaptic response showed multiple peaks at regular intervals, suggesting that responses via both monosynaptic and polysynaptic paths were recorded. The probability that an excitatory event is transmitted to another excitatory neuron and results in an above-threshold stimulation was found to be only one in three to four. This low value indicates that in addition to evoked synaptic responses other sources of excitatory drive must contribute to the spontaneous activity observed in these networks. The various types of synaptic connections (excitatory and inhibitory, monosynaptic, and polysynaptic) were counted, and the observations analyzed using a probabilistic model of the network structure. This analysis provides estimates for the ratio of inhibitory to excitatory neurons in the network (1:1.5) and for the ratio of postsynaptic cells receiving input from a single GABAergic or glutamatergic neuron (3:1). The total number of inhibitory synaptic connections was twice that of excitatory connections. Cell pairs mutually connected by an excitatory and an inhibitory synapse occurred significantly more often than predicted by a random process. These results suggests that the formation of neuronal networks in vitro is controlled by cellular mechanisms that favor inhibitory connections in general and specifically enhance the formation of reciprocal connections between pairs of excitatory and inhibitory neurons. These mechanisms may contribute to network formation and function in vivo.

scholarly journals P.182 Self-Assembling Peptide Biomaterial to Optimize Human Stem Cell-Based Regeneration of the Injured Spinal Cord

2021 ◽  
Vol 48 (s3) ◽  
pp. S72-S72
Author(s):  
C Ahuja ◽  
M Khazaei ◽  
M Fehlings
Keyword(s):  
Spinal Cord ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Post Transplantation ◽  
Self Assembling ◽  
Promising Therapeutic Approach ◽  
Cord Injury ◽  
Induced Pluripotent

Background: Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) are a promising therapeutic approach to regenerate after spinal cord injury (SCI) as they can differentiate to myelinating oligodendrocytes, synaptically-active neurons, and supportive astrocytes. Unfortunately, most chronically injured patients develop ex vacuo microcystic cavitations which prevent regenerative cell migration and neurite outgrowth. QL6 is a novel, pH-neutral, biomaterial which can self-assembles into a supportive extracellular matrix (ECM)-like matrix in vivo. This work assesses QL6’s ability to support hiPS-NSC-based regeneration. Methods:In Vitro:hiPS-NSCs were extensively characterized by EDTA assay, qPCR, and immunocytochemistry(ICC), electron microscopy(EM) and neurosphere formation assays. In Vivo:Immunodeficient rats received clinically-relevant chronic C6-7 injuries. Animals were randomized: (1)vehicle, (2)hiPS-NSCs, (3)QL6, (4)QL6+hiPS-NSCs. All rats underwent treadmill rehabilitation and behavioural testing. A subset underwent single-cell RNA sequencing(scRNAseq). Results: hiPS-NSCs proliferated robustly on QL6(Ki67+/DAPI+; 29%vs6%; p<0.01). EDTA assay showed hiPS-NSC binding to QL6 to be driven by calcium-independent mechanisms. Importantly, QL6 enhanced adherent neurosphere formation. EM-imaging provided the first images of the hiPS-NSC/QL6 interaction. Behavioural assessments demonstrate synergistic improvements with combinatorial treatment. High-throughput scRNAseq differential gene expression analyses suggest QL6 is altering lineage signalling in the human graft post-transplantation. Conclusions: This work provides key proof-of-concept data that QL6 can support translationally-relevant human iPS-NSCs in traumatic SCI.

scholarly journals Recombinant human stem cell factor, a c-kit ligand, stimulates hematopoiesis in primates

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 1975-1980 ◽  
Author(s):  
RG Andrews ◽  
GH Knitter ◽  
SH Bartelmez ◽  
KE Langley ◽  
D Farrar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Absolute Number ◽  
Human Stem Cell ◽  
Clinical Role ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Human Stem Cell Factor

Recombinant human stem cell factor (SCF) is homologous with recombinant rat SCF (rrSCF) and is a ligand for c-kit. We determined the influence of SCF on hematopoiesis in vitro and in vivo in baboons. In vitro, SCF alone stimulated little growth of hematopoietic colony-forming cells from baboon marrow, but did increase the number of colonies formed in response to erythropoietin (Epo), interleukin-3 (IL-3), and granulocyte- macrophage colony-stimulating factor (GM-CSF). In vivo, SCF caused an increase in the peripheral blood of the number of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In marrow, it caused an increase in marrow cellularity and in the absolute number of colony-forming unit-granulocyte-monocyte (CFU-GM) and burst- forming unit-erythroid (BFU-E) in marrow following infusion of SCF. The in vivo stimulation of multiple lymphohematopoietic lineages corroborates previous in vitro studies and suggests a potentially important clinical role for SCF.

scholarly journals Neural Stem Cell-Conditioned Medium Suppresses Inflammation and Promotes Spinal Cord Injury Recovery

2017 ◽  
Vol 26 (3) ◽  
pp. 469-482 ◽  
Author(s):  
Zhijian Cheng ◽  
Dale B. Bosco ◽  
Li Sun ◽  
Xiaoming Chen ◽  
Yunsheng Xu ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Stem Cell ◽  
Conditioned Medium ◽  
Neural Stem Cell ◽  
Therapeutic Potential ◽  
Secondary Injury ◽  
Cord Injury

Spinal cord injury (SCI) causes functional impairment as a result of the initial injury followed by secondary injury mechanism. SCI provokes an inflammatory response that causes secondary tissue damage and neurodegeneration. While the use of neural stem cell (NSC) engraftment to mitigate secondary injury has been of interest to many researchers, it still faces several limitations. As such, we investigated if NSC-conditioned medium (NSC-M) possesses therapeutic potential for the treatment of SCI. It has been proposed that many of the beneficial effects attributed to stem cell therapies are due to secreted factors. Utilizing primary cell culture and murine models of SCI, we determined that systemic treatment with NSC-M was able to significantly improve motor function and lesion healing. In addition, NSC-M demonstrated significant anti-inflammatory potential in vitro and in vivo, reducing inflammatory cytokine expression in both activated macrophages and injured spinal cord tissues. NSC-M was also able to reduce the expression of inducible nitric oxide synthase (iNOS) within the spleen of injured animals, indicating an ability to reduce systemic inflammation. Thus, we believe that NSC-M offers a possible alternative to direct stem cell engraftment for the treatment of SCI.

scholarly journals Xenogeneic expression of human stem cell factor in transgenic mice mimics codominant c-kit mutations

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3203-3211 ◽  
Author(s):  
MK Majumdar ◽  
ET Everett ◽  
X Xiao ◽  
R Cooper ◽  
K Langley ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transgenic Mice ◽  
Stem Cell Factor ◽  
Coat Color ◽  
Protein Product ◽  
Cell Surface Expression ◽  
Human Stem Cell ◽  
Human Stem Cell Factor

Mutations of c-kit, which encodes a transmembrane receptor tyrosine kinase, have been identified in mice by abnormal coat color, anemia, and germ cell defects. Mice heterozygous for mutations of c-kit have a white forehead blaze and a white ventral spot, leading these mutants to be termed dominant White spotting (W). We have previously demonstrated that the membrane-associated isoform of human stem cell factor (hSCF220, the ligand for c-kit) is inefficiently processed in murine stromal cell transfectants. Thus, in murine cell lines analyzed in vitro, hSCF220 transfectants present SCF as a membrane restricted protein in contrast to the murine SCF220 cDNA protein product, which is slowly cleaved and secreted. We show here that transgenic mice expressing the human SCF220 isoform in vivo display a phenotype indistinguishable from some alleles of W. Specifically, hSCF220- expressing transgenic mice display a prominent forehead blaze and a white ventral spot. Generations of doubly heterozygous animals that carry both a mutated c-kit allele and the hSCF220 transgene display a more severe coat color abnormality. This phenotype appears to be due to occupancy of murine c-kit by human SCF and diminished cell surface expression of endogenous murine SCF. Normal signaling events that lead to cell survival or proliferation appear to be disrupted in vivo in these transgenic mice.

scholarly journals Mesenchymal Stem Cell-Derived Exosomes Reduce A1 Astrocytes via Downregulation of Phosphorylated NFκB P65 Subunit in Spinal Cord Injury

2018 ◽  
Vol 50 (4) ◽  
pp. 1535-1559 ◽  
Author(s):  
Lin Wang ◽  
Shuang Pei ◽  
Linlin Han ◽  
Bin Guo ◽  
Yanfei Li ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Nuclear Translocation ◽  
Neuroprotective Effects ◽  
Vitro Assay ◽  
Cord Injury

Background/Aims: Neurotoxic A1 astrocytes are induced by inflammation after spinal cord injury (SCI), and the inflammation-related Nuclear Factor Kappa B (NFκB) pathway may be related to A1-astrocyte activation. Mesenchymal stem cell (MSC) transplantation is a promising therapy for SCI, where transplanted MSCs exhibit anti-inflammatory effects by downregulating proinflammatory factors, such as Tumor Necrosis Factor (TNF)-α and NFκB. MSC-exosomes (MSC-exo) reportedly mimic the beneficial effects of MSCs. Therefore, in this study, we investigated whether MSCs and MSC-exo exert inhibitory effects on A1 astrocytes and are beneficial for recovery after SCI. Methods: The effects of MSC and MSC-exo on SCIinduced A1 astrocytes, and the potential mechanisms were investigated in vitro and in vivo using immunofluorescence and western blot. In addition, we assessed the histopathology, levels of proinflammatory cytokines and locomotor function to verify the effects of MSC and MSC-exo on SCI rats. Results: MSC or MSC-exo co-culture reduced the proportion of SCIinduced A1 astrocytes. Intravenously-injected MSC or MSC-exo after SCI significantly reduced the proportion of A1 astrocytes, the percentage of p65 positive nuclei in astrocytes, and the percentage of TUNEL-positive cells in the ventral horn. Additionally, we observed decreased lesion area and expression of TNFα, Interleukin (IL)-1α and IL-1β, elevated expression of Myelin Basic Protein (MBP), Synaptophysin (Syn) and Neuronal Nuclei (NeuN), and improved Basso, Beattie & Bresnahan (BBB) scores and inclined-plane-test angle. In vitro assay showed that MSC and MSC-exo reduced SCI-induced A1 astrocytes, probably via inhibiting the nuclear translocation of the NFκB p65. Conclusion: MSC and MSC-exo reduce SCI-induced A1 astrocytes, probably via inhibiting nuclear translocation of NFκB p65, and exert antiinflammatory and neuroprotective effects following SCI, with the therapeutic effect of MSCexo comparable with that of MSCs when applied intravenously.

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