Research ArticleSpinal Cord Injury

GDNF rescues the fate of neural progenitor grafts by attenuating Notch signals in the injured spinal cord in rodents

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Science Translational Medicine  08 Jan 2020:
Vol. 12, Issue 525, eaau3538
DOI: 10.1126/scitranslmed.aau3538
  • Fig. 1 Transplanted NPCs preferentially differentiate to astrocytes in the injured spinal cord microenvironment.

    (A) In vivo differentiation profiles of hiPSC-NPCs were compared after transplantation into naïve or cervical level C6/C7 injured spinal cords. Representative images show that transplanted GFP+ cells in the spinal cord tissue differentiate into neuronal (Fox3+), astrocytic (GFAP+), and oligodendrocytic (APC+) fates at 8 weeks after transplantation. Scale bar, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (B) Quantification of the in vivo differentiation profile (means ± SEM, n  =  5; *P < 0.05, t test). (C) Quantitative real-time PCR analysis of gene expression in the cervical spinal cord 2 weeks after injury relative to the uninjured (naïve) cervical spinal cord. Data represent the mean log2 fold change ± SEM, (n = 3; *P < 0.05, one sample t test compared to baseline uninjured). (D) Representative immunostaining for Jagged1 in cervical spinal cord sections 2 weeks after injury. The right panel represents the white box in the left panel at a higher magnification. Scale bars, 500 μm (left) and 50 μm (right). (E) Immunohistochemical staining using anti-Jagged1 antibody on cross sections of an uninjured human cervical spinal cord obtained from a 48-year-old female donor (top) and an injured human cervical spinal cord obtained 8 months after injury from a 45-year-old female patient with a C5/C6 level injury as a result of a motor vehicle accident (bottom).

  • Fig. 2 GDNF counteracts injury-induced Notch activation.

    (A) Quantitative real-time PCR analysis of gene expression of different lines of hiPSC- or hESC-derived NPCs, as well as human fetal NPCs, treated with cleared SCI-h. Samples from each line were compared to corresponding NPC lines treated with Naïve-h. Gene expression values are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and presented as log2-transformed fold-change color-coded values. (B) Overlay histogram of intracellular staining flow cytometric analysis of Hes1 in hiPSC-NPCs treated with Naïve-h or SCI-h (total protein, 100 μg/ml). (C) Quantitative real-time PCR analysis of gene expression of hiPSC-NPCs treated with SCI-h and cotreated with different morphogens and differentiation factors for 1 week (rows). Samples were compared to no growth factor treatment. Gene expression values are normalized to GAPDH expression and presented as log2-transformed fold-change color-coded values. (D) Overlay histogram of flow cytometric analysis of intracellular Hes1 in hiPSC-NPCs treated with SCI-h and cotreated with GDNF (10 ng/ml; red) or BDNF (10 ng/ml; green). (E) Western blot analysis hiPSC-NPCs treated with Naïve-h or SCI-h for 1 week. Cleavage of Notch1 to Notch intracellular domain (NICD; arrowhead) fragments represents an indication for activation of Notch signaling. GAPDH was used as loading control.

  • Fig. 3 GDNF expression does not affect self-renewal of NPCs but biases NPC differentiation toward a neuronal cell fate.

    (A) The purified, monoclonal lines were expanded, and expression of GDNF was confirmed by a GDNF immunoassay (n = 3; **P < 0.01, t test). (B and C) Neurosphere forming assay for GDNF- and control-hiPSC-NPCs; cells were plated at a clonal density of 10 cells/μl. Scale bar, 200 μm (means ± SEM, n = 3; two-way ANOVA). (D) Effects of GDNF expression on the in vitro proliferation rate of hiPSC-NPCs. At each time point, a BrdU proliferation assay was performed to determine cell number (means ± SEM, n = 3; two-way repeated-measures ANOVA). (E and F) Representative image of in vitro differentiated hiPSC-NPCs and GDNF-hiPSC-NPCs that were stained for β-III-tubulin (β-III-Tub) (neuron marker), GFAP (astrocyte marker), or O1 (oligodendrocyte marker) and corresponding quantification (percentage of means ± SEM, n = 5; **P < 0.01, t test). Scale bar, 20 μm. (G to J) Patch-clamp analysis showing the Na+ current (G and H) and action potential firing (I and J) in neurons differentiated from hiPSC-NPCs and GDNF-hiPSC-NPCs.

  • Fig. 4 GDNF counteracts the effect of SCI-h on hiPSC-NPC fate determination via the DLK1 pathway.

    (A and B) hiPSC-NPC cultures were treated with a final concentration of homogenate (100 μg/ml) from injured (SCI-h) or naïve spinal cords (Naïve-h) in the absence of FGF and EGF to allow differentiation. Differentiated cells were stained for β-III-tubulin and GFAP, and the percentage of β-III-tubulin+ and GFAP+ cells was determined (means ± SEM, n = 5; *P < 0.05, **P < 0.01, one-way ANOVA). Scale bar, 20 μm. (C) Immunofluorescence staining of hiPSC-NPCs and GDNF-hiPSC-NPCs with anti-DLK1 antibody (red). Cell nuclei are labeled with DAPI. Scale bar, 20 μm. (D) Quantitative real-time PCR analysis of the expression profile of TFs involved in the differentiation of NPCs to neurons or astrocytes in cells treated with SCI-h relative to control hiPSC-NPCs treated with Naïve-h. Data represent the mean log2 fold change in gene expression relative to GAPDH ± SEM (n = 3; one-way ANOVA, *P < 0.05 compared to hiPSC-NPCs). (E and F) The CRISPR DLK1-KOs of hiPSC-NPCs and GDNF-hiPSC-NPCs were treated with SCI-h, and the differentiation profile was determined (means ± SEM, n = 5; one-way ANOVA, *P < 0.05 compared to the vehicle control group).

  • Fig. 5 GDNF-expressing NPCs demonstrate enhanced survival and increased DLK1 expression.

    (A) Representative images of GFP+-transplanted cells in longitudinal sections of the injured spinal cord. (B) Quantification of the survival or GFP+ cells at 8 weeks after transplantation into SCI (means ± SEM, n = 6; *P < 0.05, one-way ANOVA). (C) Representative confocal images of cervical spinal cord sections of rats transplanted with hiPSC-NPCs and GDNF-NPCs (green) at 8 weeks after transplantation stained with anti-DLK1 antibody (red). Scale bar, 20 μm. (D) Representative confocal images of cervical spinal cord sections of rats transplanted with hiPSC-NPCs and GDNF-hiPSC-NPCs (green) at 8 weeks after transplantation stained with anti-Hes1 antibody (red). Cell nuclei are labeled with DAPI. Scale bar, 20 μm.

  • Fig. 6 GDNF-expressing NPCs preferentially differentiate toward more neurons in vivo.

    (A) Representative images of transplanted NPCs differentiated to express markers of neurons (β-III-tubulin and Fox3), astrocytes (GFAP), early astrocytic differentiation (Aldh1L1), mature oligodendrocytes (APC), and immature oligodendrocytes (Olig2). Scale bar, 20 μm. (B to D) Quantitative analysis of trilineage differentiation profiles with specific markers (means ± SEM, n  =  10; one-way ANOVA, *P < 0.05 compared to hiPSC-NPCs).

  • Fig. 7 Graft-derived neuronal subtypes make synaptic connections with endogenous cells and enhance electric conduction.

    (A) Representative confocal images of cervical spinal cord sections of rats transplanted with GDNF-hiPSC-NPCs (green) at 8 weeks after transplantation stained with antibodies against neuronal subtype–specific TFs for motor neurons (Isl1 and Hb9), premotoneuron interneurons (FoxP1, Lhx3, and Chx10) and inhibitory interneurons (Pax2 and Gata3) 12 weeks after transplantation. Scale bar, 20 μm. (see fig. S9 for non–GDNF-expressing NPCs). (B) Transmission electron micrographs of spinal cord sections at the site of transplantation to show the formation of synapses between anti-GFP immunogold (black dots, white arrowheads)–labeled cells (pseudocolored to green) and endogenous axon terminals. Insets identify postsynaptic densities. The average diameter of gold particles was around 42.2 nm. Scale bars, 500 nm. (C) The number of synaptic densities per 100 μm2 of labeled cells and (D) the ratio of asymmetric/symmetric connections were quantified (means ± SEM, n  =  4; *P < 0.05, t test). (E) To test the effect of GDNF on electrical transmission, we analyzed electrically evoked compound action potential (CAP) transmission across the injury site (C5 to T1). Traces represent the average of six animals per group. (F). Quantification of CAP amplitude in animals translated hiPSC-NPCs at 8 weeks after transplantation (means ± SEM, n = 6; *P < 0.05, one-way ANOVA). (G) The conduction velocity was calculated as the recording distance (10 mm) divided by latency (t) (means ± SEM, n = 6; Kruskal-Wallis test and Dunn’s multiple comparisons test).

  • Fig. 8 GDNF-expressing NPCs contributed to better functional recovery than control NPCs.

    (A) The grip force exerted by the forelimbs was measured using a grip strength meter during the 8 weeks after transplantation. Each graph represents the force (in grams). (B) Global motor function was tested by each animal’s ability to sustain increasing degrees of incline with platforms ranging from 30° to 90°. An animal’s ability to tolerate larger incline angles is associated with better functional recovery. [for (A) and (B): means ± SEM, n = 10; two-way ANOVA with repeated measures, *P < 0.05 and **P < 0.01 for GDNF-hiPSC-NPCs compared to vehicle group, and # and × indicate P < 0.05 significance difference for GDNF-hiPSC-NPC-DLK1-KO and hiPSC-NPC groups, respectively, compared to vehicle group]. (C to F) Quantification of different forelimb gait parameters at 8 weeks after transplantation [means ± SEM, n = 6; Kruskal-Wallis test for (C), (D), and (F) and one-way ANOVA for (E), *P < 0.05 compared to vehicle group].

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/525/eaau3538/DC1

    Materials and Methods

    Fig. S1. Differentiation bias of transplanted human NPCs to astrocytes.

    Fig. S2. Increase in the amount proastrocytic differentiation factors after SCI.

    Fig. S3. Functional interaction network for differentially expressed genes in hiPSC-NPCs after treatment with GDNF.

    Fig. S4. Western blot confirmation of knocking out of DLK1 in hiPSC-NPCs.

    Fig. S5. Expression and secretion of GDNF into tissue from transplanted cells.

    Fig. S6. Reduced death in GDNF-expressing hiPSC-NPCs after exposure to injured spinal cord homogenate.

    Fig. S7. Quantification of DLK1, Hes1, and Ki67 staining.

    Fig. S8. Immunoelectron microscopy to analyze the myelination by transplanted cells.

    Fig. S9. Staining for neuron-specific subtypes and human cell–specific marker.

    Fig. S10. The effect of GDNF secretion from hiPSC-NPCs on host tissue preservation.

    Fig. S11. Evaluation of thermal and mechanical allodynia.

    Table S1. List of antibodies.

    Table S2. List of Applied Biosystems TaqMan probes used for qPCR.

    Table S3. List of growth factors and cytokines used for screening.

    Data file S1. Raw data.

    References (7685)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Differentiation bias of transplanted human NPCs to astrocytes.
    • Fig. S2. Increase in the amount proastrocytic differentiation factors after SCI.
    • Fig. S3. Functional interaction network for differentially expressed genes in hiPSC-NPCs after treatment with GDNF.
    • Fig. S4. Western blot confirmation of knocking out of DLK1 in hiPSC-NPCs.
    • Fig. S5. Expression and secretion of GDNF into tissue from transplanted cells.
    • Fig. S6. Reduced death in GDNF-expressing hiPSC-NPCs after exposure to injured spinal cord homogenate.
    • Fig. S7. Quantification of DLK1, Hes1, and Ki67 staining.
    • Fig. S8. Immunoelectron microscopy to analyze the myelination by transplanted cells.
    • Fig. S9. Staining for neuron-specific subtypes and human cell–specific marker.
    • Fig. S10. The effect of GDNF secretion from hiPSC-NPCs on host tissue preservation.
    • Fig. S11. Evaluation of thermal and mechanical allodynia.
    • Table S1. List of antibodies.
    • Table S2. List of Applied Biosystems TaqMan probes used for qPCR.
    • Table S3. List of growth factors and cytokines used for screening.
    • References (7685)

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    Other Supplementary Material for this manuscript includes the following:

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