Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing methods

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    "name": "Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing methods",
    "description": "Evidence-backed execution summary for Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing methods from Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing.",
    "totalTime": "PT81481M",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Materials and Methods",
        "text": "Human drNPCs were provided by New World Laboratories, Inc. (Laval, QC). drNPCs were directly differentiated from bone marrow somatic cells using transient transfection of the three factors Musashi-1 (Msi1), Neurogenin-2 (Ngn2), and methyl-CpG binding domain protein 2 (MBD2) and defined media containing NeuroCult-XF Proliferation medium (StemCell Technologies; Vancouver, BC) supplemented with epidermal growth factor (EGF) [20 ng/ml] (CellGenix; Freiburg, GER), fibroblast growth factor-2 (FGF-2) [50 ng/ml] (CellGenix), Valproic Acid [VPA, 1 mM] (Sigma-Aldrich; St. Louis, MO), and Noggin [20 ng/ml] (R&D Systems; Minneapolis, MN). VPA and Noggin were replaced by heparin [100 ng/ml] (Scientific Protein Laboratories) after 6 days of culture. Human fetal cortical and spinal NPCs were obtained from Clontech. Different lines of human iPSC-derived..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Biasing Human NPCs Toward an Oligodendrogenic Fate",
        "text": "We have optimized a general protocol to generate oNPCs from different lines of human PSC-derived, directly reprogrammed or fetal NPCs. The protocol attempts to mimic developmental cues to replicate neural tube patterning in vitro. In the first step, the NPCs were caudalized by culturing them on growth factor reduced Matrigel in DMEM/F12, supplemented with 0.1 µM retinoic acid (RA), B27 supplement (Life Technologies, Cat # 17504044), N2 supplement, and EGF (20 ng/ml) for 3 days. Cells underwent ventralization by treatment with 1 µM sonic hedgehog (Shh) agonist Purmorphamine (Millipore, Cat # 540220) for 5 days. EGF was replaced by FGF-2 (10 ng/ml) from the media for 3 days followed by the addition of 20 ng/ml PDGF-AA (Peptrotech 100-13A; Rocky Hill, NJ) for 14 days. The resulting cells were maintained on Laminin coated dishes in DMEM/F12, B27-A,..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Differentiation and Immunocytochemistry",
        "text": "In order to study the differentiation potential of cells in vitro, oNPCs were dissociated into single cells and plated on poly-ornithine/Laminin coated cover glasses (24 well plates: 4 × 10 3 cells/well). Cells were grown in Neurobasal medium (Thermo Fisher Scientific 21103049; Waltham, MA) supplemented with N2 (Thermo Fisher Scientific 17502048), B27 (Thermo Fisher Scientific 17504044), 0.1% fetal bovine serum, 10 µM Forskolin (Stem Cell Technologies 72112) and glutamax (Thermo Fisher Scientific 21103049) for an additional 10 days. Cells were fixed for 20 minutes with 4% paraformaldehyde in phosphate-buffered saline (PBS) and 40% sucrose at room temperature. Following fixation, cells were permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate in PBS for 5 minutes and then placed in blocking buffer (5% BSA) for 1 hour. Primary antibodies were diluted in..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "SCI Model",
        "text": "Female RNU rats (athymic nude rats; Crl:NIH- Foxn1 rnu; 12-week old; strain code 316; Charles River Laboratories, Wilmington, MA) were used in this study. The clip compression model of SCI has been characterized extensively and described previously. Under inhalational anesthesia using isoflurane (1%-2%) and a 1:1 mixture of O 2 /N 2 O, the rats underwent a T7-T9 laminectomy and received a 23 g clip (Walsh, Oakville, Ontario, Canada) compression injury for 1 minute at the T7 level of the spinal cord. Gel foam (Ferrosan, Denmark) was put on the spinal cord. Muscles were sutured, and the surgical wound was closed. Their bladders were manually expressed twice daily until the return of reflexive bladder control."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Cell Transplantation in the Spinal Cord",
        "text": "Nine days after injury, the rats were randomly divided into three groups: vehicle (injection of aCSF), control drNPC transplanted, and oNPCs transplanted rats. Ten rats were used per group. Under anesthesia, the spinal cord was reopened at the injury area and the rats were injected with a cell suspension of oNPCs. To prepare the cell suspension, a monolayer culture passage of eight cells was collected using Accutase. The cells were diluted in aCSF and used for cell transplantation. Using a Hamilton syringe connected to a 32-gauge metal needle and a stereotaxic injection system, a total volume of 8 µl of cell suspension, containing 4 × 10 5 live cells, was injected into the dorsal spinal cord. We injected the cells into the spinal cord at two sites 2 mm rostral and two sites 2 mm caudal to the lesional area on either side of the midline (0.6-0.8 mm). The injected..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Spinal Cord Tissue Processing and Lesion Morphometry",
        "text": "The animals were perfused with 0.1 M PBS, and fixed with 4% paraformaldehyde in the PBS. Postfixation was performed in the same fixative for 6 hours and 30% sucrose in 0.1 M PBS for 24 hours. After fixation, a 1.5 cm length of the spinal cord, centered at the injury site, was collected and sectioned axially at 30 µm. Serial sections were stained with the myelin-selective stain luxol fast blue (LFB) and hematoxylin and eosin (H&E) as described previously. Unbiased measurements were made with a Cavalieri volume probe using Stereo Investigator (MBF Bioscience, Williston, VT) for the area of total spinal cord, gray matter, cavity, and total lesion reported. The lesion area was identified as eosinophilic scar deposition with immune infiltrates and/or nonviable or anuclear host tissue. For white matter area quantification, the total area of the total spinal cord was subtracted..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "oNPC Transplantation is not Associated with Tumor Formation In Vivo After Long-Term Follow-Up",
        "text": "To evaluate the tumorigenicity of oNPCs, we transplanted cells into the intact spinal cords of NOD/SCID mice ( n = 3) and observed them over 150 days. The mice appeared healthy with no overt neurological deficits during the observational period. At 150 days post-transplantation, a histological analysis was conducted with immunohistochemistry for Ki67 and HuN showing colocalization in only 0.94 ± 0.47% of HuN + cells in the cord (Supporting Information Fig. and ). A subpopulation of the transplanted cells expressed Nestin, a marker for undifferentiated neural cells, but the distribution was sparse and tumor-like masses were not observed (Supporting Information Fig. and ). Moreover, H&E staining was performed at the injection site of cell transplantation, and formation of neural rosettes was not detected (Supporting Information Fig. and )."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Cell Transplantation into the Spinal Cord of NOD/SCID Mice",
        "text": "For the evaluation of safety, we transplanted drNPC-derived oNPCs or control unpatterned drNPCs from the same line as oNPCs (referred to as control NPCs or simply NPCs) into intact spinal cords of NOD/SCID mice (NOD/SCID mice; NOD.CB17 -Prkdc scid /NcrCrl) (18-22 g; 8-week old; strain code 394). The mice were anesthetized using isoflurane (1%-1.5%) and 1:1 mixture of O 2 /N 2 O. The spinal cord was exposed with a T10 laminectomy. A total volume of 4 µl of cell suspension, containing 2 × 10 5 live cells, was injected into the dorsal spinal cord. The injection was performed at four sites with 0.5 mm injected depth. To quantify Ki67 + cells in transplanted HuN + cells in the intact spinal cord, we randomly selected and captured 12 regions at × 63 magnification (numeric aperture)."
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        "@type": "HowToTool",
        "name": "Cell Transplantation in the Spinal Cord"
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        "@type": "HowToTool",
        "name": "Immunohistochemistry and Quantification in Spinal Cord Tissue Sections"
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      {
        "@type": "HowToTool",
        "name": "Immunohistochemistry and Quantification in Spinal Cord Tissue Sections"
      },
      {
        "@type": "HowToTool",
        "name": "Immuno-Electron Microscopy and its Quantification in Spinal Cord Tissue Sections"
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        "name": "Tail-Flick Test"
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        "@type": "HowToSupply",
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      {
        "@type": "HowToSupply",
        "name": "Biasing Human NPCs Toward an Oligodendrogenic Fate"
      },
      {
        "@type": "HowToSupply",
        "name": "Polymerase Chain Reaction"
      },
      {
        "@type": "HowToSupply",
        "name": "Differentiation and Immunocytochemistry"
      },
      {
        "@type": "HowToSupply",
        "name": "Cell Transplantation in the Spinal Cord"
      },
      {
        "@type": "HowToSupply",
        "name": "Spinal Cord Tissue Processing and Lesion Morphometry"
      },
      {
        "@type": "HowToSupply",
        "name": "Immuno-Electron Microscopy and its Quantification in Spinal Cord Tissue Sections"
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      "@type": "ScholarlyArticle",
      "headline": "Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing",
      "datePublished": "2018",
      "author": [
        {
          "@type": "Person",
          "name": "Narihito Nagoshi"
        },
        {
          "@type": "Person",
          "name": "Mohamad Khazaei"
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        {
          "@type": "Person",
          "name": "Jan-Eric Ahlfors"
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        {
          "@type": "Person",
          "name": "Christopher S. Ahuja"
        },
        {
          "@type": "Person",
          "name": "Satoshi Nori"
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        {
          "@type": "Person",
          "name": "Jian Wang"
        },
        {
          "@type": "Person",
          "name": "Shinsuke Shibata"
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        {
          "@type": "Person",
          "name": "Michael G. Fehlings"
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      "identifier": "10.1002/sctm.17-0269"
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