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Volume 19, Issue 4, Pages 544-557 (October 2016)
Transplanted Human Stem Cell-Derived Interneuron Precursors Mitigate Mouse Bladder Dysfunction and Central Neuropathic Pain after Spinal Cord Injury Thomas M. Fandel, Alpa Trivedi, Cory R. Nicholas, Haoqian Zhang, Jiadong Chen, Aida F. Martinez, Linda J. Noble-Haeusslein, Arnold R. Kriegstein Cell Stem Cell Volume 19, Issue 4, Pages (October 2016) DOI: /j.stem Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 1 hESC-MGE Transplantation into the Uninjured Spinal Cord Does Not Alter Locomotor and Bladder Functions (A–D) Differentiation of MGE-like cells from hESCs. (A) hESC-derived spheroids expressing NKX2.1-GFP suggest ventral forebrain-type differentiation. Scale bar, 1 mm. (B) GFP+ cells co-express MGE progenitor cell marker OLIG2 and GABAergic neuronal marker DLX2 (blue, DAPI). Scale bar, 50 μm. See also Figure S1. (C) Representative FACS histogram shows NKX2.1-GFP at 6 weeks of differentiation (red, hESC-derived spheroids without the GFP reporter; n = 9 differentiation experiments). (D) Representative FACS histogram shows the GFP-high population stained with an APC-labeled neuronal lineage marker PSA-NCAM (red, isotype-APC control antibody; n = 9 differentiation experiments). (E–M) Functional results from mice transplanted with hESC-MGEs (low dose, n = 5; high dose, n = 5; and vehicle, n = 6) into the uninjured spinal cord. (E and F) Progression of locomotor function (E) and weight gain (F) over 6 months after transplantation are shown. (E) Averaged Basso Mouse Scale (BMS) score for both hind limbs is shown. (F) Relative (Rel.) changes of body weight compared to pre-transplantation weight are shown. (G–M) Urinary bladder function at 6 months post-transplantation is shown. (G–I) Representative cystometry recordings are from (G) vehicle-injected mice, (H) low-dose hESC-MGE-transplanted mice, and (I) high-dose hESC-MGE-transplanted mice. Arrows indicate micturition events. (J–M) Quantitation of cystometric outcomes compares vehicle-injected mice, low-dose hESC-MGE-transplanted mice, and high-dose hESC-MGE-transplanted mice. Results show maximal (Max.) voiding pressure (J), the number of non-voiding contractions (NVCs)/10 min (K), residual (Res.) urine (L), and voiding efficiency (M). See also Table S1. Data are presented as mean + SEM. Repeated-measure two-way ANOVA (E and F) and Kruskal-Wallis test (J–M) were performed. All data represent biological replicates. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 2 Transplanted hESC-MGEs Survive in the Uninjured Spinal Cord, Differentiating into GABAergic Neurons (A–E) Presented data are collected at 6 months post-transplantation. All data represent biological replicates. (A) Distribution pattern of hESC-MGEs in the uninjured spinal cord is shown. The cell transplantation group is subdivided into a low-dose group (n = 5, red) and a high-dose group (n = 5, black). The shaded area denotes the spread of transplantation sites. The graphs show the number of HNA+ cells along the rostro-caudal axis, where 0 represents the center of transplantation site, depicting human cell persistence of 23% ± 8% (low-dose group) and 30% ± 15% (high-dose group). Data are presented as mean + SEM. Cell distributions for individual animals are shown in Figure S1. (B–D) Percentages of NEUN and HNA double-positive cells (n = 5, each group) (B), OLIG2 and HNA double-positive cells (n = 3, each group) (C), and KI67 and HNA double-positive cells (n = 3, each group) (D) in the spinal cord of both low-dose and high-dose hESC-MGE-transplanted groups are shown. Data are presented as mean + SEM. (E) Each composite image is a representative confocal image that shows persisting hESC-MGEs (human nuclear antigen [HNA] in red) and other markers in green, as indicated on individual panels. Markers identify proliferating cells (KI67); MGE-derived GABAergic neurons (SOX6 and GABA); neurons (NEUN), MGE progenitors and/or oligodendroglia (OLIG2); human astrocytes (STEM123); immature neurons (DCX); and interneuron subtypes somatostatin (SST), calbindin (CB), calretinin (CR), and parvalbumin (PV). Scale bars, 50 μm. Arrowheads indicate double-positive cells. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 3 Timeline for Experimental Procedures using Spinal Cord-Injured Animals Schematic illustrates the chronological order of experiments for spinal cord injury (SCI), cell transplantation, and different functional assessments. See also Figure S2. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 4 Transplanted hESC-MGEs Survive and Migrate in the Injured Spinal Cord, Differentiating into GABAergic Neurons (A–C) Presented data are collected at 6 months post-transplantation (n = 13, biological replicates). (A) Migratory pattern of hESC-MGEs in the injured spinal cord. The graph shows the number of HNA+ cells along the rostro-caudal axis, depicting human cell persistence to be 106% ± 59%. Zero represents the center of the transplantation site and the arrow signifies the epicenter of SCI. The site of transplantation is 2 mm in length (shaded area) and 4–6 mm caudal to the site of injury. Negative numbers indicate distances rostral to the site of transplantation, and positive numbers indicate distance caudal to the site of transplantation. Cell distributions for individual animals are shown in Figure S3. Data are presented as mean + SEM. (B) Percentages of NEUN and HNA double-positive cells, SOX6 and HNA double-positive cells, OLIG2 and HNA double-positive cells, and KI67 and HNA double-positive cells in the injured spinal cord of hESC-MGE-transplanted animals are shown. Data are presented as mean + SEM. (C) Each composite image is a representative confocal microscopy image that shows persisting hESC-MGEs (HNA, red) and other markers in green as indicated on individual panels. Markers label proliferating cells (KI67); MGE-derived GABAergic neurons (SOX6 and GABA); neurons (NEUN); MGE progenitors and/or oligodendroglia (OLIG2); human astrocytes (STEM123); immature neurons (DCX); and interneuron subtypes somatostatin (SST), calbindin (CB), calretinin (CR), and parvalbumin (PV). Scale bars, 50 μm. Arrowheads indicate double-positive cells. Additional cell marker studies are shown in Figure S4. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 5 Transplanted hESC-MGEs Differentiate into Neurons that Are Functionally Integrated into the Injured Spinal Cord (A) Differential interference contrast microscopy image is superimposed with the fluorescence of NKX2.1-GFP. (B) Representative action potential firing pattern of hESC-MGE-derived neurons. See also Figure S6. (C) The hESC-derived neurons receive spontaneous synaptic currents. Recordings are from cells before (left), during the application of GABA receptor antagonist bicuculline methiodide (BMI, 20 μM) and the non-NMDA glutamate receptor antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 10 μM) (center), and after the washout phase (right). (D) Schematic diagram shows electrical stimulation of lateral spinal cord tracts. Stimulation evoked glutamatergic (cell 1) or GABAergic (cell 2) postsynaptic currents in hESC-MGE-derived neurons. (E) Post-immunostaining shows recorded neuron (neurobiotin) was double positive for NKX2.1-GFP and HNA. See also Figure S5. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 6 Transplanted hESC-MGEs Ameliorate SCI-Induced CNP and Strain-Related, Abnormal Over-Grooming Behavior (A and B) Progression of locomotor function (A) and weight gain (B) over 6 months following transplantation of hESC-MGEs or vehicle injection into the lumbar enlargement of the injured spinal cord (vehicle, n = 11 biological replicates; hESC-MGE, n = 17 biological replicates). The arrows indicate the event of cell transplantation (TX) at 2 weeks after SCI. (A) Averaged BMS score for both hind limbs is shown. (B) Relative (Rel.) changes of body weight compared to pre-transplantation weight are shown. Data are presented as mean + SEM. Repeated-measure two-way ANOVA was performed. (C–H) CNP as assessed by mechanical allodynia (von Frey test) (C–E) and thermal hyperalgesia (tail flick test) (F–H). (C) Mechanical (Mech.) threshold at 2 weeks post-SCI (pre-transplantation) compared to pre-injury (pre-inj) results (n = 23) is shown. (D) Mechanical threshold at 3 months post-transplantation (PTX) of hESC-MGEs or vehicle (Veh) injection into the injured spinal cord (vehicle, n = 11; hESC-MGE, n = 17) is shown. (E) Mechanical threshold at 6 months following hESC-MGE transplantation or vehicle injection into the injured spinal cord (vehicle, n = 8; hESC-MGE, n = 15) is shown. (F) Withdrawal (Withdr.) latency at 2 weeks post-injury (pre-transplantation) (n = 24) is shown. (G) Withdrawal latency at 3 months following hESC-MGE transplantation into the injured spinal cord (vehicle, n = 11; hESC-MGE, n = 17) is shown. (H) Withdrawal latency at 6 months following hESC-MGE transplantation into the injured spinal cord (vehicle, n = 10; hESC-MGE, n = 16) is shown. Data are presented as mean + SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, two-tailed paired t test (C and F; technical replicates); two-tailed Mann-Whitney tests (D, E, G, and H; biological replicates). (I–K) Mouse strain-related over-grooming at 6 months post-transplantation. Representative images show mice following hESC-MGE-transplantation (I) and vehicle injection (J) into the injured spinal cord. (K) Percentage of fur loss area in mice at 6 months after hESC-MGE transplantation (n = 17), vehicle injection (n = 9), and in age-matched, uninjured naive controls (n = 11; ∗p < 0.05, Kruskal-Wallis test, biological replicates). Data are presented as mean + SEM. See also Figure S7. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Figure 7 Transplanted hESC-MGEs Improve SCI-Induced Neurogenic Bladder Dysfunction (A–C) Representative spontaneous voiding patterns are from age-matched, uninjured naive controls (A); injured, vehicle-injected mice (B); and injured, hESC-MGE-transplanted mice (C). (D and E) Frequency distribution of various urine spot diameters at days 1 (D) and 2 (E) of testing, representing technical replicates, is shown. (F and G) The percentages of animals demonstrating voiding ability (F) and voiding spots >3 cm in diameter (G) are shown (vehicle, n = 9; hESC-MGE, n = 17; ∗p < 0.05 and ∗∗p < 0.01, two-tailed Fisher’s exact test). (H–J) Representative cystometry recordings are from age-matched naive controls (H), vehicle-injected mice (I), and hESC-MGE-transplanted mice (J). Arrows indicate micturition events. (K–N) Quantitations show cystometric outcomes comparing hESC-MGE (n = 17) to vehicle (n = 9) group presenting maximal (Max.) voiding pressure (K), the number of non-voiding contractions (NVCs)/10 min (L), residual (Res.) urine (M), and voiding efficiency (N). (O) Representative images show urinary bladders in each group. Scale bar, 2 mm. (P–R) Representative images of H&E-stained bladder wall histology for naive (P), vehicle (Q), and hESC-MGE (R) groups. The black lines denote the detrusor muscle. Scale bar, 200 μm. (S–U) Bladder weight relative to body weight (S), bladder circumference (T), and detrusor muscle area (U) comparing hESC-MGE (n = 17) to vehicle (n = 9) group. Results of age-matched naive control mice (n = 11) are shown as a solid red line with dashed black lines representing mean ± SEM. Other data are presented as mean + SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, two-tailed Mann-Whitney test). All data represent biological replicates. See also Figure S7 and Table S2. Cell Stem Cell , DOI: ( /j.stem ) Copyright © 2016 Elsevier Inc. Terms and Conditions
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Cell Stem Cell 2016 19, 544-557DOI: (10.1016/j.stem.2016.08.020)
Copyright © 2016 Elsevier Inc. Terms and Conditions
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