Journal: Antioxidants

Article Title: An In Vitro Oxidative Stress Model of the Human Inner Ear Using Human-Induced Pluripotent Stem Cell-Derived Otic Progenitor Cells

doi: 10.3390/antiox13111407

Figure Lengend Snippet: Derivation and characterization of OPCs from hiPSCs. ( a ) Scheme illustrating the generation of OPCs from hiPSCs and corresponding stages of human development. hiPSCs resemble the inner cell mass (ICM), while OPCs correspond to the otocyst stage. ( b ) Representative bright-field images of SK8-A hiPSCs on day 0 and derived OPCs on day 20. Scale bars, 100 µm. ( c ) qRT-PCR data showing fold changes in mRNA expression of otic-related markers ( PAX2 , PAX8 , and GATA3 ) in OPCs compared to hiPSCs. The expression levels in hiPSCs were set to 1. Mean ± SD; * p < 0.05; *** p < 0.001. ( d ) Representative immunocytochemistry images showing protein expression of otic lineage markers in OPCs. hiPSCs were used as negative controls to verify the absence of false-positive signals. Scale bars, 100 µm. Abbreviations: PAX2 , paired box 2; PAX8 , paired box 8; GATA3 , GATA binding protein 3. See “Statistical analysis and reproducibility” in for statistics and experimental information.

Article Snippet: The cells were blocked with 5% goat serum (Gibco, Grand Island, NY, USA, #PCN5000) or 5% normal horse serum (Abcam, Cambridge, UK, #ab7484) in PBST for 1 h. Subsequently, the cells were incubated overnight at 4 °C with the following primary antibodies diluted in 1% BSA in PBST: goat polyclonal IgG PAX2 (R&D Systems, Minneapolis, MN, USA; #AF3364; 1:50), rabbit polyclonal PAX8 (Abcam, Cambridge, UK; #AB97477; 1:200), rabbit monoclonal GATA3 (Cell Signaling Technology, Danvers, MA, USA; #5852; 1:800), and mouse monoclonal cytochrome c (Cell Signaling Technology, Danvers, MA, USA; #12963; 1:300).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Immunocytochemistry, Binding Assay

Journal: bioRxiv

Article Title: Descending locus coeruleus noradrenergic signaling to spinal astrocyte subset is required for stress-induced pain facilitation

doi: 10.1101/2024.11.14.623627

Figure Lengend Snippet: (A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of hM3Dq (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.

Article Snippet: Transverse spinal cord and brain sections (30 μm) were incubated in blocking solution (3% normal goat serum [#S-1000; Vector Laboratories] or normal donkey serum [#017-000-121; Jackson ImmunoResearch]) for 2 hours at room temperature and then incubated for 48 hours at 4°C with primary antibodies: polyclonal rabbit anti-tyrosine hydroxylase (TH; 1:1000; #AB152; Millipore); polyclonal sheep anti-TH (1:1000; #AB1542; Millipore); monoclonal mouse anti-noradrenaline transporter (NET; 1:2000; #NET05-2; Mab Technologies); polyclonal rabbit anti-green fluorescent protein (GFP; 1:1000; #598; MBL International); monoclonal rabbit anti-hemagglutinin (HA)-tag (1:1000; #3724; Cell Signaling); monoclonal rat anti-glial fibrillary acidic protein (GFAP; 1:2000; #13-0300; Invitrogen); polyclonal goat anti-SRY-related high-mobility group box 9 (SOX9; 1:1000; # AF3075; R&D Systems); polyclonal goat anti-paired box 2 (PAX2; 1:500; # AF3364; R&D Systems); monoclonal rat anti-mCherry (1:2000; #M11217; Thermo Fisher Scientific); polyclonal rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (pERK; 1:500; #9101; Cell Signaling); anti-isolectin B4 (IB4)-biotin conjugate (1:1000; # I21414; Thermo Fisher Scientific).

Techniques: Control, Knock-Out, Membrane, Expressing, Staining, Injection, Knockdown

Journal: Cell reports

Article Title: Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.

doi: 10.1016/j.celrep.2024.113892

Figure Lengend Snippet: Figure 1. Generation of NMOs from C9orf72-ALS patient and healthy control iPSCs (A) Schematic of iPSC induction into NMPs and NMOs within 50 days. (B) Representative images of cultures induced from C9n1 and C9n1 isogenic control (C9n1ISO) iPSCs on day 0. Scale bar, 200 mm. (C) Immunofluorescent images for NMPs identification by co-expressing Brachyury (BRA) (green) and SOX2 (magenta) in cultures induced from C9n1 and C9n1ISO iPSCs on day 0. Scale bar, 100 mm. (D) Bar plot shows the cell percentage of NMPs (BRA+/SOX2+), mesodermal progenitors (BRA+/SOX2–), and neural stem cells (BRA–/SOX2+) in cultures induced from C9n1 and C9n1ISO iPSCs on day 0. n = 3 individual cover slips. The data are shown as the mean ± SD. (E) Representative immunofluorescent images show the maturation of neuromuscular structure in C9n1/ISO and C9n6/ISO as well as C9n3 NMOs. The neuronal side was labeled with the antibodies against bIII-tubulin and the motor neuronal marker ISL1. The expression of MyHC indicated the SKM side. Scale bar, 200 mm. (F) Pax2+ and LIM1+ interneurons thatgrow inthe spinal cord were detected inthe neuronal side inC9n1/ISO and C9n6/ISO as wellas C9n3 NMOs. Scale bar, 100 mm. (G) Terminal Schwann cells (S100+) that cap the neurites and contact with aBTX+ postsynapsis were formed in both C9n1/ISO and C9n6/ISO as well as C9n3 NMOs. The identity of Schwann cells was also confirmed by myelin basic protein (MBP) together with Schwann cells (S100+) that cap aBTX+ postsynaptic sites. *Indicates the zoomed-in area. Scale bar, 50 mm. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies SOX2 Cell Signaling Technology Cat# 23064 RRID: AB_2714146 NANOG R&D system Cat#AF1997 RRID: AB_355097 OCT4 Abcam Cat# ab184665 RRID: AB_2895225 Brachyury R&D system Cat# AF2085 RRID: AB_2200235 SOX1 Abcam Cat# ab87775 RRID: AB_2616563 Pax3 DSHB Cat# AB_528426 SOX10 Cell Signaling Technology Cat# 89356 RRID: AB_2792980 ISL1 R&D system Cat# AF1837 RRID: AB_2126324 bIII-Tubulin Abcam Cat# ab18207 RRID: AB_444319 bIII-Tubulin Abcam Cat# ab7751 RRID: AB_306045 MyHC Sigma Aldrich Cat# M1570 RRID: AB_2147168 Pax2 R&D system Cat# AF3364 RRID: AB_10889828 LIM1 R&D system Cat# MAB2725 RRID: AB_2135636 Bassoon Abcam Cat# ab283680 GFAP R&D system Cat# MAB2594 RRID: AB_2109654 S100 Abcam Cat# ab52642 RRID: AB_882426 Myelin basic protein Abcam Cat# ab7349 RRID: AB_305869 P62 Abcam Cat# ab280086 RRID: AB_2923299 PolyGA Proteintech Cat# 24492-AP Goat anti-rabbit-Alexa 488 Abcam Cat# ab150077 RRID: AB_2630356 Goat anti-mouse-Alexa 555 Abcam Cat# ab150114 RRID: AB_2687594 Donkey anti-goat-Alexa 488 Abcam Cat# ab150129 RRID: AB_2687506 Donkey anti-rabbit-Alexa 555 Abcam Cat# ab150074 RRID: AB_2636997 Donkey anti–mouse-Alexa 647 Abcam Cat# ab150131 RRID: AB_2732857 Chemicals, peptides, and recombinant proteins mTeSRTM Plus Stemcell Tech.

Techniques: Control, Expressing, Labeling, Marker

Journal: Cell reports

Article Title: Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.

doi: 10.1016/j.celrep.2024.113892

Figure Lengend Snippet: Figure 1. Generation of NMOs from C9orf72-ALS patient and healthy control iPSCs (A) Schematic of iPSC induction into NMPs and NMOs within 50 days. (B) Representative images of cultures induced from C9n1 and C9n1 isogenic control (C9n1ISO) iPSCs on day 0. Scale bar, 200 mm. (C) Immunofluorescent images for NMPs identification by co-expressing Brachyury (BRA) (green) and SOX2 (magenta) in cultures induced from C9n1 and C9n1ISO iPSCs on day 0. Scale bar, 100 mm. (D) Bar plot shows the cell percentage of NMPs (BRA+/SOX2+), mesodermal progenitors (BRA+/SOX2–), and neural stem cells (BRA–/SOX2+) in cultures induced from C9n1 and C9n1ISO iPSCs on day 0. n = 3 individual cover slips. The data are shown as the mean ± SD. (E) Representative immunofluorescent images show the maturation of neuromuscular structure in C9n1/ISO and C9n6/ISO as well as C9n3 NMOs. The neuronal side was labeled with the antibodies against bIII-tubulin and the motor neuronal marker ISL1. The expression of MyHC indicated the SKM side. Scale bar, 200 mm. (F) Pax2+ and LIM1+ interneurons thatgrow inthe spinal cord were detected inthe neuronal side inC9n1/ISO and C9n6/ISO as wellas C9n3 NMOs. Scale bar, 100 mm. (G) Terminal Schwann cells (S100+) that cap the neurites and contact with aBTX+ postsynapsis were formed in both C9n1/ISO and C9n6/ISO as well as C9n3 NMOs. The identity of Schwann cells was also confirmed by myelin basic protein (MBP) together with Schwann cells (S100+) that cap aBTX+ postsynaptic sites. *Indicates the zoomed-in area. Scale bar, 50 mm. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies SOX2 Cell Signaling Technology Cat# 23064 RRID: AB_2714146 NANOG R&D system Cat#AF1997 RRID: AB_355097 OCT4 Abcam Cat# ab184665 RRID: AB_2895225 Brachyury R&D system Cat# AF2085 RRID: AB_2200235 SOX1 Abcam Cat# ab87775 RRID: AB_2616563 Pax3 DSHB Cat# AB_528426 SOX10 Cell Signaling Technology Cat# 89356 RRID: AB_2792980 ISL1 R&D system Cat# AF1837 RRID: AB_2126324 bIII-Tubulin Abcam Cat# ab18207 RRID: AB_444319 bIII-Tubulin Abcam Cat# ab7751 RRID: AB_306045 MyHC Sigma Aldrich Cat# M1570 RRID: AB_2147168 Pax2 R&D system Cat# AF3364 RRID: AB_10889828 LIM1 R&D system Cat# MAB2725 RRID: AB_2135636 Bassoon Abcam Cat# ab283680 GFAP R&D system Cat# MAB2594 RRID: AB_2109654 S100 Abcam Cat# ab52642 RRID: AB_882426 Myelin basic protein Abcam Cat# ab7349 RRID: AB_305869 P62 Abcam Cat# ab280086 RRID: AB_2923299 PolyGA Proteintech Cat# 24492-AP Goat anti-rabbit-Alexa 488 Abcam Cat# ab150077 RRID: AB_2630356 Goat anti-mouse-Alexa 555 Abcam Cat# ab150114 RRID: AB_2687594 Donkey anti-goat-Alexa 488 Abcam Cat# ab150129 RRID: AB_2687506 Donkey anti-rabbit-Alexa 555 Abcam Cat# ab150074 RRID: AB_2636997 Donkey anti–mouse-Alexa 647 Abcam Cat# ab150131 RRID: AB_2732857 Chemicals, peptides, and recombinant proteins mTeSRTM Plus Stemcell Tech.

Techniques: Control, Expressing, Labeling, Marker